Bacterial cellulose hydrolysis in anaerobic environmental subsystems--Clostridium thermocellum and Clostridium stercorarium, thermophilic plant-fiber degraders.

PubMed

Zverlov, Vladimir V; Schwarz, Wolfgang H

2008-03-01

Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are extremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum, an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellulose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from C. thermocellum cultures grown on cellulose, cellobiose, barley beta-1,3-1,4-glucan, or a mixture of xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur in at least one preparation. A number of enzymatic components had not been identified previously. The proportion of components changes if C. thermocellum is grown on different substrates. Mutants of C. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The characteristics of these mutants provide strong evidence of the positive effect of complex (cellulosome) formation on hydrolysis of crystalline cellulose.

Complex carbohydrates reduce the frequency of antagonistic interactions among bacteria degrading cellulose and xylan.

PubMed

Deng, Yi-Jie; Wang, Shiao Y

2017-03-01

Bacterial competition for resources is common in nature but positive interactions among bacteria are also evident. We speculate that the structural complexity of substrate might play a role in mediating bacterial interactions. We tested the hypothesis that the frequency of antagonistic interactions among lignocellulolytic bacteria is reduced when complex polysaccharide is the main carbon source compared to when a simple sugar such as glucose is available. Results using all possible pairwise interactions among 35 bacteria isolated from salt marsh detritus showed that the frequency of antagonistic interactions was significantly lower on carboxymethyl cellulose (CMC)-xylan medium (7.8%) than on glucose medium (15.5%). The two interaction networks were also different in their structures. Although 75 antagonistic interactions occurred on both media, there were 115 that occurred only on glucose and 20 only on CMC-xylan, indicating that some antagonistic interactions were substrate specific. We also found that the frequency of antagonism differed among phylogenetic groups. Gammaproteobacteria and Bacillus sp. were the most antagonistic and they tended to antagonize Bacteroidetes and Actinobacteria, the most susceptible groups. Results from the study suggest that substrate complexity affects how bacteria interact and that bacterial interactions in a community are dynamic as nutrient conditions change. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cellular automata modeling depicts degradation of cellulosic material by a cellulase system with single-molecule resolution.

PubMed

Eibinger, Manuel; Zahel, Thomas; Ganner, Thomas; Plank, Harald; Nidetzky, Bernd

2016-01-01

Enzymatic hydrolysis of cellulose involves the spatiotemporally correlated action of distinct polysaccharide chain cleaving activities confined to the surface of an insoluble substrate. Because cellulases differ in preference for attacking crystalline compared to amorphous cellulose, the spatial distribution of structural order across the cellulose surface imposes additional constraints on the dynamic interplay between the enzymes. Reconstruction of total system behavior from single-molecule activity parameters is a longstanding key goal in the field. We have developed a stochastic, cellular automata-based modeling approach to describe degradation of cellulosic material by a cellulase system at single-molecule resolution. Substrate morphology was modeled to represent the amorphous and crystalline phases as well as the different spatial orientations of the polysaccharide chains. The enzyme system model consisted of an internally chain-cleaving endoglucanase (EG) as well as two processively acting, reducing and non-reducing chain end-cleaving cellobiohydrolases (CBHs). Substrate preference (amorphous: EG, CBH II; crystalline: CBH I) and characteristic frequencies for chain cleavage, processive movement, and dissociation were assigned from biochemical data. Once adsorbed, enzymes were allowed to reach surface-exposed substrate sites through "random-walk" lateral diffusion or processive motion. Simulations revealed that slow dissociation of processive enzymes at obstacles obstructing further movement resulted in local jamming of the cellulases, with consequent delay in the degradation of the surface area affected. Exploiting validation against evidence from atomic force microscopy imaging as a unique opportunity opened up by the modeling approach, we show that spatiotemporal characteristics of cellulose surface degradation by the system of synergizing cellulases were reproduced quantitatively at the nanometer resolution of the experimental data. This in turn gave useful prediction of the soluble sugar release rate. Salient dynamic features of cellulose surface degradation by different cellulases acting in synergy were reproduced in simulations in good agreement with evidence from high-resolution visualization experiments. Due to the single-molecule resolution of the modeling approach, the utility of the presented model lies not only in predicting system behavior but also in elucidating inherently complex (e.g., stochastic) phenomena involved in enzymatic cellulose degradation. Thus, it creates synergy with experiment to advance the mechanistic understanding for improved application.

Impact of pretreated Switchgrass and biomass carbohydrates on Clostridium thermocellum ATCC 27405 cellulosome composition: a quantitative proteomic analysis.

PubMed

Raman, Babu; Pan, Chongle; Hurst, Gregory B; Rodriguez, Miguel; McKeown, Catherine K; Lankford, Patricia K; Samatova, Nagiza F; Mielenz, Jonathan R

2009-01-01

Economic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes. In this study, we used quantitative proteomics (multidimensional LC-MS/MS and (15)N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum ATCC 27405 was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to (15)N labeled cellulosome samples isolated from crystalline cellulose-grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 16 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass-grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose-grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass, in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline. Overall, the quantitative results suggest a coordinated substrate-specific regulation of cellulosomal subunit composition in C. thermocellum to better suit the organism's needs for growth under different conditions. To date, this study provides the most comprehensive comparison of cellulosomal compositional changes in C. thermocellum in response to different carbon sources. Such studies are vital to engineering a strain that is best suited to grow on specific substrates of interest and provide the building blocks for constructing designer cellulosomes with tailored enzyme composition for industrial ethanol production.

Metadata Analysis of Phanerochaete chrysosporium Gene Expression Data Identified Common CAZymes Encoding Gene Expression Profiles Involved in Cellulose and Hemicellulose Degradation.

PubMed

Kameshwar, Ayyappa Kumar Sista; Qin, Wensheng

2017-01-01

In literature, extensive studies have been conducted on popular wood degrading white rot fungus, Phanerochaete chrysosporium about its lignin degrading mechanisms compared to the cellulose and hemicellulose degrading abilities. This study delineates cellulose and hemicellulose degrading mechanisms through large scale metadata analysis of P. chrysosporium gene expression data (retrieved from NCBI GEO) to understand the common expression patterns of differentially expressed genes when cultured on different growth substrates. Genes encoding glycoside hydrolase classes commonly expressed during breakdown of cellulose such as GH-5,6,7,9,44,45,48 and hemicellulose are GH-2,8,10,11,26,30,43,47 were found to be highly expressed among varied growth conditions including simple customized and complex natural plant biomass growth mediums. Genes encoding carbohydrate esterase class enzymes CE (1,4,8,9,15,16) polysaccharide lyase class enzymes PL-8 and PL-14, and glycosyl transferases classes GT (1,2,4,8,15,20,35,39,48) were differentially expressed in natural plant biomass growth mediums. Based on these results, P. chrysosporium, on natural plant biomass substrates was found to express lignin and hemicellulose degrading enzymes more than cellulolytic enzymes except GH-61 (LPMO) class enzymes, in early stages. It was observed that the fate of P. chrysosporium transcriptome is significantly affected by the wood substrate provided. We believe, the gene expression findings in this study plays crucial role in developing genetically efficient microbe with effective cellulose and hemicellulose degradation abilities.

Extracellular lignase: a key to enhanced cellulose utilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hira, A.; Barnett, S.M.; Shieh, C.H.

1978-01-01

An alternate approach to the conventional chemical processing of lignin, a potential renewable resource, is enzymic conversion. Biodegradation of wood, a lignin-cellulose complex, is accomplished naturally by various enzymes of microbial origin. Extracellular lignases have been isolated from pure cultures of Polyporus versicolor, Phanerochaete chrysosporium, and Pleurotus ostreatus. The isolated enzyme systems from these organisms have shown substrate specificity for guaiacol and hydroquinone and yielded a positive syringaldazine test. A commercial lignin was degraded by the enzyme system.

Characterization of the cellulose-degrading bacterium NCIMB 10462

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dees, C.; Scott, T.C.; Phelps, T.J.

The gram-negative cellulase-producing bacterium NCIMB 10462 has been previously named Pseudomonas fluorescens subsp. or var. cellulose. Because of renewed interest in cellulose-degrading bacteria for use in the bioconversion of cellulose to chemical feed stocks and fuels, we re-examined the characteristics of this microorganism to determine its true metabolic potential. Metabolic and physical characterization of NCIMB 10462 revealed that this is an alkalophilic, non-fermentative, gram-negative, oxidase-positive, motile, cellulose-degrading bacterium. The aerobic substrate utilization profile of this bacterium has few characteristics consistent with a classification of P. fluorescens and a very low probability match with the genus Sphingomonas. However, total lipid analysismore » did not reveal that any sphingolipid bases are produced by this bacterium. NCIMB 10462 grows best aerobically, but also grows well in complex media under reducing conditions. NCIMB 10462 grows slowly under anaerobic conditions on complex media, but growth on cellulosic media occurred only under aerobic conditions. Total fatty acid analysis (MIDI) of NCIMB 10462 failed to group this bacterium with a known pseudomonas species. However, fatty acid analysis of the bacteria when grown at temperatures below 37{degrees}C suggest that the organism is a pseudomonad. Since a predominant characteristic of this bacterium is its ability to degrade cellulose, we suggest that it be called Pseudomonas cellulosa.« less

Conversion of Lignocellulosic Biomass to Nanocellulose: Structure and Chemical Process

PubMed Central

Lee, H. V.; Hamid, S. B. A.; Zain, S. K.

2014-01-01

Lignocellulosic biomass is a complex biopolymer that is primary composed of cellulose, hemicellulose, and lignin. The presence of cellulose in biomass is able to depolymerise into nanodimension biomaterial, with exceptional mechanical properties for biocomposites, pharmaceutical carriers, and electronic substrate's application. However, the entangled biomass ultrastructure consists of inherent properties, such as strong lignin layers, low cellulose accessibility to chemicals, and high cellulose crystallinity, which inhibit the digestibility of the biomass for cellulose extraction. This situation offers both challenges and promises for the biomass biorefinery development to utilize the cellulose from lignocellulosic biomass. Thus, multistep biorefinery processes are necessary to ensure the deconstruction of noncellulosic content in lignocellulosic biomass, while maintaining cellulose product for further hydrolysis into nanocellulose material. In this review, we discuss the molecular structure basis for biomass recalcitrance, reengineering process of lignocellulosic biomass into nanocellulose via chemical, and novel catalytic approaches. Furthermore, review on catalyst design to overcome key barriers regarding the natural resistance of biomass will be presented herein. PMID:25247208

The inhibition of hemicellulosic sugars on cellulose hydrolysis are highly dependant on the cellulase productive binding, processivity, and substrate surface charges.

PubMed

Zhai, Rui; Hu, Jinguang; Saddler, Jack N

2018-06-01

In this study, the influence of major hemicellulosic sugars (mannose and xylose) on cellulose hydrolysis and major enzyme activities were evaluated by using both commercial enzyme cocktail and purified cellulase monocomponents over a "library" of cellulosic substrates. Surprisingly, the results showed that unlike glucose, mannose/xylose did not inhibit individual cellulase activities but significantly decreased their hydrolytic performance on cellulose substrates. When various enzyme-substrate interactions (e.g. adsorption/desorption, productive binding, and processive moving) were evaluated, it appeared that these hemicellulosic sugars significantly reduced the productive binding and processivity of Cel7A, which in turn limited cellulase hydrolytic efficacy. Among a range of major cellulose characteristics (e.g. crystallinity, degree of polymerization, accessibility, and surface charges), the acid group content of the cellulosic substrates seemed to be the main driver that determined the extent of hemicellulosic sugar inhibition. Our results provided new insights for better understanding the sugar inhibition mechanisms of cellulose hydrolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluations of cellulose accessibilities of lignocelluloses by solute exclusion and protein adsorption techniques

Treesearch

Q.Q. Wang; Z. He; Z. Zhu; Y.-H.P. Zhang; Y. Ni; X.L. Luo; J.Y. Zhu

2012-01-01

Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by drying the never dried pretreated sample and a set of differently pretreated lodgepople pine substrates, were evaluated using solute exclusion and protein adsorption methods. Direct measurements of cellulase adsorption onto cellulose surface of the set of pretreated substrates were...

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis.

PubMed

Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

2013-10-29

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200-300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis

PubMed Central

Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

2013-01-01

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200–300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP. PMID:24127606

Reliable simultaneous zymographic method of characterization of cellulolytic enzymes from fungal cellulase complex.

PubMed

Dojnov, Biljana; Grujić, Marica; Vujčić, Zoran

2015-08-01

A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4-methylumbelliferyl-β-d-cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling of competitive mutualistic relationships. Application to cellulose degradation by Streptomyces sp. strains.

PubMed

Thierie, Jacques; Penninckx, Michel J

2007-12-01

A "cascade" model depicts microbial degradation of a complex nutrient/substrate through a succession of intermediate compounds. Each stage is characterized by a particular species producing a typical degradation enzyme induced by its own degradation product. The final compound of the cascade consists of a single assimilable substrate used by all species. This results in a competition situation, whereas the contribution of all strains to the production of a complete set of efficient enzymes generates a mutualistic relationship. The model was shown to be appropriate to describe degradation of cellulose by a consortium of Streptomyces sp. strains. The simplicity and the model capacity for generalization are promising and could be used for various degradation processes both at laboratory and environmental scales.

Metagenomic discovery of biomass-degrading genes and genomes from cow rumen.

PubMed

Hess, Matthias; Sczyrba, Alexander; Egan, Rob; Kim, Tae-Wan; Chokhawala, Harshal; Schroth, Gary; Luo, Shujun; Clark, Douglas S; Chen, Feng; Zhang, Tao; Mackie, Roderick I; Pennacchio, Len A; Tringe, Susannah G; Visel, Axel; Woyke, Tanja; Wang, Zhong; Rubin, Edward M

2011-01-28

The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.

Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency*

PubMed Central

Eibinger, Manuel; Ganner, Thomas; Bubner, Patricia; Rošker, Stephanie; Kracher, Daniel; Haltrich, Dietmar; Ludwig, Roland; Plank, Harald; Nidetzky, Bernd

2014-01-01

Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization. PMID:25361767

A novel proteomics sample preparation method for secretome analysis of Hypocrea jecorina growing on insoluble substrates.

PubMed

Bengtsson, Oskar; Arntzen, Magnus Ø; Mathiesen, Geir; Skaugen, Morten; Eijsink, Vincent G H

2016-01-10

Analysis of the secretomes of filamentous fungi growing on insoluble lignocellulosic substrates is of major current interest because of the industrial potential of secreted fungal enzymes. Importantly, such studies can help identifying key enzymes from a large arsenal of bioinformatically detected candidates in fungal genomes. We describe a simple, plate-based method to analyze the secretome of Hypocrea jecorina growing on insoluble substrates that allows harsh sample preparation methods promoting desorption, and subsequent identification, of substrate-bound proteins, while minimizing contamination with non-secreted proteins from leaking or lysed cells. The validity of the method was demonstrated by comparative secretome analysis of wild-type H.jecorina strain QM6a growing on bagasse, birch wood, spruce wood or pure cellulose, using label-fee quantification. The proteomic data thus obtained were consistent with existing data from transcriptomics and proteomics studies and revealed clear differences in the responses to complex lignocellulosic substrates and the response to pure cellulose. This easy method is likely to be generally applicable to filamentous fungi and to other microorganisms growing on insoluble substrates. Copyright © 2015 Elsevier B.V. All rights reserved.

Peptide-cellulose conjugates for protease point of care diagnostics and treatment

USDA-ARS?s Scientific Manuscript database

Peptide-cellulose conjugates containing Human Neutrophil Elastase substrate sequences with both colorimetric and fluorometric signal molecules have been synthesized on a variety of cellulosic and nanocellulosic substrates including cotton and wood nanocrystals, wood nanocomposites, cotton-based aero...

Determination of the cellulase activity distribution in Clostridium thermocellum and Caldicellulosiruptor obsidiansis cultures using a fluorescent substrate

DOE PAGES

Morrell-Falvey, Jennifer L.; Elkins, James G.; Wang, Zhi-Wu

2015-05-30

This study took advantage of resorufin cellobioside as a fluorescent substrate to determine the distribution of cellulase activity in cellulosic biomass fermentation systems. Cellulolytic biofilms were found to express nearly four orders greater cellulase activity compared to planktonic cultures of Clostridium thermocellum and Caldicellulosiruptor obsidiansis, which can be primarily attributed to the high cell concentration and surface attachment. The formation of biofilms results in cellulases being secreted close to their substrates, which appears to be an energetically favorable stategy for insoluble substrate utilization. For the same reason, cellulases should be closely associated with the surfaces of suspended cell in solublemore » substrate-fed culture, which has been verified with cellobiose-fed cultures of C. thermocellum and C. obsidiansis. This study addressed the importance of cellulase activity distribution in cellulosic biomass fermentation, and provided theoretical foundation for the leading role of biofilm in cellulose degradation. System optimization and reactor designs that promote biofilmformation in cellulosic biomass hydrolysismay promise an improved cellulosic biofuel process.« less

A kinetic study of Trichoderma reesei Cel7B catalyzed cellulose hydrolysis.

PubMed

Song, Xiangfei; Zhang, Shujun; Wang, Yefei; Li, Jingwen; He, Chunyan; Yao, Lishan

2016-06-01

One prominent feature of Trichoderma reesei (Tr) endoglucanases catalyzed cellulose hydrolysis is that the reaction slows down quickly after it starts (within minutes). But the mechanism of the slowdown is not well understood. A structural model of Tr- Cel7B catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. The 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. This slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amount of reducing sugars released after 24h by Cel7B in phosphoric acid swollen cellulose, Avicel and filter paper cellulose hydrolysis is correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ∼15-35% more reducing sugars than the wild type (WT) Cel7B in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose. The activity of Tr- Cel7B against the insoluble substrate cellulose is determined by the enzyme's capability in hydrolyzing the soluble substrate. Copyright © 2016 Elsevier Inc. All rights reserved.

Investigating Commercial Cellulase Performances Toward Specific Biomass Recalcitrance Factors Using Reference Substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Xiaohui; Bowden, Mark E.; Engelhard, Mark H.

Three commercial cellulase preparations, Novozymes Cellic® Ctec2, Dupont Accellerase® 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulose enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulose performance. While the degree of polymerization of cellulose in the reference substrates didmore » not present a major recalcitrance factor to Novozymes Cellic® Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.« less

Phylogenetic diversity of carbohydrate degrading culturable bacteria from Mandovi and Zuari estuaries, Goa, west coast of India

NASA Astrophysics Data System (ADS)

Khandeparker, Rakhee; Verma, Preeti; Meena, Ram M.; Deobagkar, Deepti D.

2011-12-01

Coastal and estuarine waters are highly productive and dynamic ecosystems. The complex carbohydrate composition of the ecosystem would lead to colonisation of microbial communities with abilities to produce an array of complex carbohydrate degrading enzymes. We have examined the abundance and phylogenetic diversity of culturable bacteria with abilities to produce complex carbohydrate degrading enzymes in the Mondovi and Zuari eustauri. It was interesting to note that 65% of isolated bacteria could produce complex carbohydrate degrading enzymes. A majority of these bacteria belonged to Bacillus genera followed by Vibrio, Marinobacter, Exiquinobacterium, Alteromonas, Enterobacter and Aeromonas. Most abundant bacterial genus to degrade hemicellulose and cellulose were Bacillus and Vibrio respectively. Most abundant bacterial genus to degrade hemicellulose and cellulose were Bacillus and Vibrio respectively. It was seen that 46% of Bacillus had ability to degrade both the substrate while only 14% of Vibrio had bifunctionality.

The type of carbohydrates specifically selects microbial community structures and fermentation patterns.

PubMed

Chatellard, Lucile; Trably, Eric; Carrère, Hélène

2016-12-01

The impact on dark fermentation of seven carbohydrates as model substrates of lignocellulosic fractions (glucose, cellobiose, microcrystalline cellulose, arabinose, xylose, xylan and wheat straw) was investigated. Metabolic patterns and bacterial communities were characterized at the end of batch tests inoculated with manure digestate. It was found that hydrogen production was linked to the sugar type (pentose or hexose) and the degree of polymerisation. Hexoses produced less hydrogen, with a specific selection of lactate-producing bacterial community structures. Maximal hydrogen production was five times higher on pentose-based substrates, with specific bacterial community structures producing acetate and butyrate as main metabolites. Low hydrogen amounts accumulated from complex sugars (cellulose, xylan and wheat straw). A relatively high proportion of the reads was affiliated to Ruminococcaceae suggesting an efficient hydrolytic activity. Knowing that the bacterial community structure is very specific to a particular substrate offers new possibilities to design more efficient H 2 -producing biological systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Taxonomic characterization of the cellulose-degrading bacterium NCIB 10462

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dees, C.; Ringleberg, D.; Scott, T.C.

The gram negative cellulase-producing bacterium NCIB 10462 has been previously named Pseudomonas fluorescens subsp. or var. cellulosa. Since there is renewed interest in cellulose-degrading bacteria for use in bioconversion of cellulose to chemical feed stocks and fuels, we re-examined the characteristics of this microorganism to determine its proper taxonomic characterization and to further define it`s true metabolic potential. Metabolic and physical characterization of NCIB 10462 revealed that this was an alkalophilic, non-fermentative, gram negative, oxidase positive, motile, cellulose-degrading bacterium. The aerobic substrate utilization profile of this bacterium was found to have few characteristics consistent with a classification of P. fluorescensmore » with a very low probability match with the genus Sphingomonas. Total lipid analysis did not reveal that any sphingolipid bases are produced by this bacterium. NCIB 10462 was found to grow best aerobically but also grows well in complex media under reducing conditions. NCIB 10462 grew slowly under full anaerobic conditions on complex media but growth on cellulosic media was found only under aerobic conditions. Total fatty acid analysis (MIDI) of NCIB 10462 failed to group this bacterium with a known pseudomonas species. However, fatty acid analysis of the bacteria when grown at temperatures below 37{degrees}C suggest that the organism is a pseudomonad. Since a predominant characteristic of this bacterium is it`s ability to degrade cellulose, we suggest it be called Pseudomonas cellulosa.« less

A biomolecular recognition approach for the functionalization of cellulose with gold nanoparticles.

PubMed

Almeida, A; Rosa, A M M; Azevedo, A M; Prazeres, D M F

2017-09-01

Materials with new and improved functionalities can be obtained by modifying cellulose with gold nanoparticles (AuNPs) via the in situ reduction of a gold precursor or the deposition or covalent immobilization of pre-synthesized AuNPs. Here, we present an alternative biomolecular recognition approach to functionalize cellulose with biotin-AuNPs that relies on a complex of 2 recognition elements: a ZZ-CBM3 fusion that combines a carbohydrate-binding module (CBM) with the ZZ fragment of the staphylococcal protein A and an anti-biotin antibody. Paper and cellulose microparticles with AuNPs immobilized via the ZZ-CBM3:anti-biotin IgG supramolecular complex displayed an intense red color, whereas essentially no color was detected when AuNPs were deposited over the unmodified materials. Scanning electron microscopy analysis revealed a homogeneous distribution of AuNPs when immobilized via ZZ-CBM3:anti-biotin IgG complexes and aggregation of AuNPs when deposited over paper, suggesting that color differences are due to interparticle plasmon coupling effects. The approach could be used to functionalize paper substrates and cellulose nanocrystals with AuNPs. More important, however, is the fact that the occurrence of a biomolecular recognition event between the CBM-immobilized antibody and its specific, AuNP-conjugated antigen is signaled by red color. This opens up the way for the development of simple and straightforward paper/cellulose-based tests where detection of a target analyte can be made by direct use of color signaling. Copyright © 2017 John Wiley & Sons, Ltd.

How does cellulosome composition influence deconstruction of lignocellulosic substrates in Clostridium (Ruminiclostridium) thermocellum DSM 1313?

PubMed

Yoav, Shahar; Barak, Yoav; Shamshoum, Melina; Borovok, Ilya; Lamed, Raphael; Dassa, Bareket; Hadar, Yitzhak; Morag, Ely; Bayer, Edward A

2017-01-01

Bioethanol production processes involve enzymatic hydrolysis of pretreated lignocellulosic biomass into fermentable sugars. Due to the relatively high cost of enzyme production, the development of potent and cost-effective cellulolytic cocktails is critical for increasing the cost-effectiveness of bioethanol production. In this context, the multi-protein cellulolytic complex of Clostridium ( Ruminiclostridium ) thermocellum, the cellulosome, was studied here. C. thermocellum is known to assemble cellulosomes of various subunit (enzyme) compositions, in response to the available carbon source. In the current study, different carbon sources were used, and their influence on both cellulosomal composition and the resultant activity was investigated. Glucose, cellobiose, microcrystalline cellulose, alkaline-pretreated switchgrass, alkaline-pretreated corn stover, and dilute acid-pretreated corn stover were used as sole carbon sources in the growth media of C. thermocellum strain DSM 1313. The purified cellulosomes were compared for their activity on selected cellulosic substrates. Interestingly, cellulosomes derived from cells grown on lignocellulosic biomass showed no advantage in hydrolyzing the original carbon source used for their production. Instead, microcrystalline cellulose- and glucose-derived cellulosomes were equal or superior in their capacity to deconstruct lignocellulosic biomass. Mass spectrometry analysis revealed differential composition of catalytic and structural subunits (scaffoldins) in the different cellulosome samples. The most abundant catalytic subunits in all cellulosome types include Cel48S, Cel9K, Cel9Q, Cel9R, and Cel5G. Microcrystalline cellulose- and glucose-derived cellulosome samples showed higher endoglucanase-to-exoglucanase ratios and higher catalytic subunit-per-scaffoldin ratios compared to lignocellulose-derived cellulosome types. The results reported here highlight the finding that cellulosomes derived from cells grown on glucose and microcrystalline cellulose are more efficient in their action on cellulosic substrates than other cellulosome preparations. These results should be considered in the future development of C. thermocellum -based cellulolytic cocktails, designer cellulosomes, or engineering of improved strains for deconstruction of lignocellulosic biomass.

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

DOE PAGES

Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; ...

2014-10-21

Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel)more » initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.« less

Processive Endoglucanases Mediate Degradation of Cellulose by Saccharophagus degradans▿ †

PubMed Central

Watson, Brian J.; Zhang, Haitao; Longmire, Atkinson G.; Moon, Young Hwan; Hutcheson, Steven W.

2009-01-01

Bacteria and fungi are thought to degrade cellulose through the activity of either a complexed or a noncomplexed cellulolytic system composed of endoglucanases and cellobiohydrolases. The marine bacterium Saccharophagus degradans 2-40 produces a multicomponent cellulolytic system that is unusual in its abundance of GH5-containing endoglucanases. Secreted enzymes of this bacterium release high levels of cellobiose from cellulosic materials. Through cloning and purification, the predicted biochemical activities of the one annotated cellobiohydrolase Cel6A and the GH5-containing endoglucanases were evaluated. Cel6A was shown to be a classic endoglucanase, but Cel5H showed significantly higher activity on several types of cellulose, was the highest expressed, and processively released cellobiose from cellulosic substrates. Cel5G, Cel5H, and Cel5J were found to be members of a separate phylogenetic clade and were all shown to be processive. The processive endoglucanases are functionally equivalent to the endoglucanases and cellobiohydrolases required for other cellulolytic systems, thus providing a cellobiohydrolase-independent mechanism for this bacterium to convert cellulose to glucose. PMID:19617364

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose

PubMed Central

Edwards, J. Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle nee; French, Alfred D.; Condon, Brian D.

2018-01-01

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis–Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding. PMID:29534033

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose.

PubMed

Edwards, J Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle Nee; French, Alfred D; Condon, Brian D

2018-03-13

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis-Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity ( K m ) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased K m observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency ( k cat / K m ), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding.

Assembly of synthetic cellulose I.

PubMed

Lee, J H; Brown, R M; Kuga, S; Shoda, S; Kobayashi, S

1994-08-02

Cellulose microfibrils with an electron diffraction pattern characteristic of crystalline native cellulose I have been assembled abiotically by means of a cellulase-catalyzed polymerization of beta-cellobiosyl fluoride substrate monomer in acetonitrile/acetate buffer. Substantial purification of the Trichoderma viride cellulase enzyme was found to be essential for the formation of the synthetic cellulose I allomorph. Assembly of synthetic cellulose I appears to be a result of a micellar aggregation of the partially purified enzyme and the substrate in an organic/aqueous solvent system favoring the alignment of glucan chains with the same polarity and extended chain conformation, resulting in crystallization to form the metastable cellulose I allomorph.

Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability.

PubMed

Moraïs, Sarah; Stern, Johanna; Kahn, Amaranta; Galanopoulou, Anastasia P; Yoav, Shahar; Shamshoum, Melina; Smith, Matthew A; Hatzinikolaou, Dimitris G; Arnold, Frances H; Bayer, Edward A

2016-01-01

The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.Graphical abstractConversion of conventional designer cellulosomes into thermophilic designer cellulosomes.

Natural cellulose fiber as substrate for supercapacitor.

PubMed

Gui, Zhe; Zhu, Hongli; Gillette, Eleanor; Han, Xiaogang; Rubloff, Gary W; Hu, Liangbing; Lee, Sang Bok

2013-07-23

Cellulose fibers with porous structure and electrolyte absorption properties are considered to be a good potential substrate for the deposition of energy material for energy storage devices. Unlike traditional substrates, such as gold or stainless steel, paper prepared from cellulose fibers in this study not only functions as a substrate with large surface area but also acts as an interior electrolyte reservoir, where electrolyte can be absorbed much in the cellulose fibers and is ready to diffuse into an energy storage material. We demonstrated the value of this internal electrolyte reservoir by comparing a series of hierarchical hybrid supercapacitor electrodes based on homemade cellulose paper or polyester textile integrated with carbon nanotubes (CNTs) by simple solution dip and electrodeposited with MnO2. Atomic layer deposition of Al2O3 onto the fiber surface was used to limit electrolyte absorption into the fibers for comparison. Configurations designed with different numbers of ion diffusion pathways were compared to show that cellulose fibers in paper can act as a good interior electrolyte reservoir and provide an effective pathway for ion transport facilitation. Further optimization using an additional CNT coating resulted in an electrode of paper/CNTs/MnO2/CNTs, which has dual ion diffusion and electron transfer pathways and demonstrated superior supercapacitive performance. This paper highlights the merits of the mesoporous cellulose fibers as substrates for supercapacitor electrodes, in which the water-swelling effect of the cellulose fibers can absorb electrolyte, and the mesoporous internal structure of the fibers can provide channels for ions to diffuse to the electrochemical energy storage materials.

Effects of reactive Mn(III)-oxalate complexes on structurally intact plant cell walls

NASA Astrophysics Data System (ADS)

Summering, J. A.; Keiluweit, M.; Goni, M. A.; Nico, P. S.; Kleber, M.

2011-12-01

Lignin components in the in plant litter are commonly assumed to have longer residence times in soil than many other compounds, which are supposedly, more easily degradable. The supposed resistance of lignin compounds to decomposition is generally attributed to the complex chain of biochemical steps required to create footholds in the non-porous structure of ligno-cellulose in cell walls. Interestingly, Mn(III) complexes have shown the ability to degrade ligno-cellulose. Mn(III) chelated by ligands such as oxalate are soluble oxidizers with a high affinity for lignin structures. Here we determined (i) the formation and decay kinetics of the Mn(III)-oxalate complexes in aqueous solution and (ii) the effects that these complexes have on intact ligno-cellulose. UV/vis spectroscopy and iodometric titrations confirmed the transient nature of Mn(III)-oxalate complexes with decay rates being in the order of hours. Zinnia elegans tracheary elements - a model ligno-cellulose substrate - were treated with Mn(III)-oxalate complexes in a newly developed flow-through reactor. Soluble decomposition products released during the treatment were analyzed by GC/MS and the degree of cell integrity was measured by cell counts, pre- and post-treatment counts indicate a decrease in intact Zinnia elegans as a result of Mn(III)-treatment. GC/MS results showed the release of a multitude of solubilized lignin breakdown products from plant cell walls. We conclude that Mn(III)-oxalate complexes have the ability to lyse intact plant cells and solubilize lignin. Lignin decomposition may thus be seen as resource dependent, with Mn(III) a powerful resource that should be abundant in terrestrial characterized by frequent redox fluctuations.

Microwave flexible transistors on cellulose nanofibrillated fiber substrates

Treesearch

Jung-Hun Seo; Tzu-Hsuan Chang; Jaeseong Lee; Ronald Sabo; Weidong Zhou; Zhiyong Cai; Shaoqin Gong; Zhenqiang Ma

2015-01-01

In this paper, we demonstrate microwave flexible thin-film transistors (TFTs) on biodegradable substrates towards potential green portable devices. The combination of cellulose nanofibrillated fiber (CNF) substrate, which is a biobased and biodegradable platform, with transferrable single crystalline Si nanomembrane (Si NM), enables the realization of truly...

[Influence of the cycle number in processing of cellulose from waste paper on its ability to hydrolysis by cellulases].

PubMed

Morozova, V V; Semenova, M V; Rozhkova, A M; Kondrat'eva, E G; Okunev, O N; Bekkarevich, A O; Novozhilov, E V; Sinitsin, A P

2010-01-01

Hydrolytic ability of laboratory enzyme preparations from fungus of the Penicillium genus was investigated using kraft pulp from nonbleached softwood and bleached hardwood cellulose as substrates. The enzyme preparations were shown to efficiently hydrolyze both softwood and hardwood cellulose. The yields of glucose and reducing sugars were 24-36 g/l and 27-37 g/l from 100 g/l of dry substrate in 48 h, respectively, and depended on the number of substrate grinding cycles.

Highly transparent, low-haze, hybrid cellulose nanopaper as electrodes for flexible electronics

Treesearch

Xuezhu Xu; Jian Zhou; Long Jiang; Gilles Lubineau; Tienkhee Ng; Boon S. Ooi; Hsien-Yu Liao; Chao Shen; Long Chen; Junyong Zhu

2016-01-01

Paper is an excellent candidate to replace plastics as a substrate for flexible electronics due to its low cost, renewability and flexibility. Cellulose nanopaper (CNP), a new type of paper made of nanosized cellulose fibers, is a promising substrate material for transparent and flexible electrodes due to its potentially high transparency and high mechanical strength....

Microbial ecology of anaerobic digesters: the key players of anaerobiosis.

PubMed

Ali Shah, Fayyaz; Mahmood, Qaisar; Maroof Shah, Mohammad; Pervez, Arshid; Ahmad Asad, Saeed

2014-01-01

Anaerobic digestion is the method of wastes treatment aimed at a reduction of their hazardous effects on the biosphere. The mutualistic behavior of various anaerobic microorganisms results in the decomposition of complex organic substances into simple, chemically stabilized compounds, mainly methane and CO2. The conversions of complex organic compounds to CH4 and CO2 are possible due to the cooperation of four different groups of microorganisms, that is, fermentative, syntrophic, acetogenic, and methanogenic bacteria. Microbes adopt various pathways to evade from the unfavorable conditions in the anaerobic digester like competition between sulfate reducing bacteria (SRB) and methane forming bacteria for the same substrate. Methanosarcina are able to use both acetoclastic and hydrogenotrophic pathways for methane production. This review highlights the cellulosic microorganisms, structure of cellulose, inoculum to substrate ratio, and source of inoculum and its effect on methanogenesis. The molecular techniques such as DGGE (denaturing gradient gel electrophoresis) utilized for dynamic changes in microbial communities and FISH (fluorescent in situ hybridization) that deal with taxonomy and interaction and distribution of tropic groups used are also discussed.

Microbial Ecology of Anaerobic Digesters: The Key Players of Anaerobiosis

PubMed Central

Ali Shah, Fayyaz; Mahmood, Qaisar; Maroof Shah, Mohammad; Pervez, Arshid; Ahmad Asad, Saeed

2014-01-01

Anaerobic digestion is the method of wastes treatment aimed at a reduction of their hazardous effects on the biosphere. The mutualistic behavior of various anaerobic microorganisms results in the decomposition of complex organic substances into simple, chemically stabilized compounds, mainly methane and CO2. The conversions of complex organic compounds to CH4 and CO2 are possible due to the cooperation of four different groups of microorganisms, that is, fermentative, syntrophic, acetogenic, and methanogenic bacteria. Microbes adopt various pathways to evade from the unfavorable conditions in the anaerobic digester like competition between sulfate reducing bacteria (SRB) and methane forming bacteria for the same substrate. Methanosarcina are able to use both acetoclastic and hydrogenotrophic pathways for methane production. This review highlights the cellulosic microorganisms, structure of cellulose, inoculum to substrate ratio, and source of inoculum and its effect on methanogenesis. The molecular techniques such as DGGE (denaturing gradient gel electrophoresis) utilized for dynamic changes in microbial communities and FISH (fluorescent in situ hybridization) that deal with taxonomy and interaction and distribution of tropic groups used are also discussed. PMID:24701142

Steam pretreatment of agricultural residues facilitates hemicellulose recovery while enhancing enzyme accessibility to cellulose.

PubMed

Chandra, Richard P; Arantes, Valdeir; Saddler, Jack

2015-06-01

The origins of lignocellulosic biomass and the pretreatment used to enhance enzyme accessibility to the cellulosic component are known to be strongly influenced by various substrate characteristics. To assess the impact that fibre properties might have on enzymatic hydrolysis, seven agricultural residues were characterised before and after steam pretreatment using a single pretreatment condition (190°C, 5min, 3% SO2) previously shown to enhance fractionation and hydrolysis of the cellulosic component of corn stover. When the fibre length, width and coarseness, viscosity, water retention value and cellulose crystallinity were monitored, no clear correlation was observed between any single substrate characteristic and the substrate's ease of enzymatic hydrolysis. However, the amount of hemicellulose that was solubilised during pretreatment correlated (r(2)=0.98) with the effectiveness of enzyme hydrolysis of each pretreated substrate. Simons's staining, to measure the cellulose accessibility, showed good correlation (r(2)=0.83) with hemicellulose removal and the extent of enzymatic hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cellulosic ethanol production via consolidated bioprocessing by a novel thermophilic anaerobic bacterium isolated from a Himalayan hot spring.

PubMed

Singh, Nisha; Mathur, Anshu S; Tuli, Deepak K; Gupta, Ravi P; Barrow, Colin J; Puri, Munish

2017-01-01

Cellulose-degrading thermophilic anaerobic bacterium as a suitable host for consolidated bioprocessing (CBP) has been proposed as an economically suited platform for the production of second-generation biofuels. To recognize the overall objective of CBP, fermentation using co-culture of different cellulolytic and sugar-fermenting thermophilic anaerobic bacteria has been widely studied as an approach to achieving improved ethanol production. We assessed monoculture and co-culture fermentation of novel thermophilic anaerobic bacterium for ethanol production from real substrates under controlled conditions. In this study, Clostridium sp. DBT-IOC-C19, a cellulose-degrading thermophilic anaerobic bacterium, was isolated from the cellulolytic enrichment cultures obtained from a Himalayan hot spring. Strain DBT-IOC-C19 exhibited a broad substrate spectrum and presented single-step conversion of various cellulosic and hemicellulosic substrates to ethanol, acetate, and lactate with ethanol being the major fermentation product. Additionally, the effect of varying cellulose concentrations on the fermentation performance of the strain was studied, indicating a maximum cellulose utilization ability of 10 g L -1 cellulose. Avicel degradation kinetics of the strain DBT-IOC-C19 displayed 94.6% degradation at 5 g L -1 and 82.74% degradation at 10 g L -1 avicel concentration within 96 h of fermentation. In a comparative study with Clostridium thermocellum DSM 1313, the ethanol and total product concentrations were higher by the newly isolated strain on pretreated rice straw at an equivalent substrate loading. Three different co-culture combinations were used on various substrates that presented two-fold yield improvement than the monoculture during batch fermentation. This study demonstrated the direct fermentation ability of the novel thermophilic anaerobic bacteria on various cellulosic and hemicellulosic substrates into ethanol without the aid of any exogenous enzymes, representing CBP-based fermentation approach. Here, the broad substrate utilization spectrum of isolated cellulolytic thermophilic anaerobic bacterium was shown to be of potential utility. We demonstrated that the co-culture strategy involving novel strains is efficient in improving ethanol production from real substrate.

Microbial Cellulose Utilization: Fundamentals and Biotechnology

PubMed Central

Lynd, Lee R.; Weimer, Paul J.; van Zyl, Willem H.; Pretorius, Isak S.

2002-01-01

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts. PMID:12209002

Chapter 2.3 Cellulose Nanofibril Composite Substrates for Flexible Electronics

Treesearch

Ronald Sabo; Jung-Hun Seo; Zhenqiang Ma

2013-01-01

Flexible electronics have a large number of potential applications, including malleable displays and wearable computers. Current research into high-speed, flexible electronic substrates uses plastics for the flexible substrate, but these plastics typically have drawbacks, such as high thermal expansion coefficients. Transparent films made from cellulose...

Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies.

PubMed

Fong, Monica; Berrin, Jean-Guy; Paës, Gabriel

2016-01-01

Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant substrates, the role of individual chemical and structural features affecting enzyme-binding properties is also difficult to decipher. We have designed and used lignified model assemblies of plant cell walls as templates to characterize binding properties of multi-modular cellulases. These three-dimensional assemblies are modulated in their composition using the three principal polymers found in secondary plant cell walls (cellulose, hemicellulose, and lignin). Binding properties of enzymes are obtained from the measurement of their mobility that depends on their interactions with the polymers and chemical motifs of the assemblies. The affinity of the multi-modular GH45 cellulase was characterized using a statistical analysis to determine the role played by each assembly polymer. Presence of hemicellulose had much less impact on affinity than cellulose and model lignin. Depending on the number of CBMs appended to the cellulase catalytic core, binding properties toward cellulose and lignin were highly contrasted. Model assemblies bring new insights into the molecular determinants that are responsible for interactions between enzymes and substrate without the need of complex analysis. Consequently, we believe that model bioinspired assemblies will provide relevant information for the design and optimization of enzyme cocktails in the context of biorefineries.

Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

DOE PAGES

O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

2016-12-22

Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

Structure and engineering of celluloses.

PubMed

Pérez, Serge; Samain, Daniel

2010-01-01

This chapter collates the developments and conclusions of many of the extensive studies that have been conducted on cellulose, with particular emphasis on the structural and morphological features while not ignoring the most recent results derived from the elucidation of unique biosynthetic pathways. The presentation of structural and morphological data gathered together in this chapter follows the historical development of our knowledge of the different structural levels of cellulose and its various organizational levels. These levels concern features such as chain conformation, chain polarity, chain association, crystal polarity, and microfibril structure and organization. This chapter provides some historical landmarks related to the evolution of concepts in the field of biopolymer science, which parallel the developments of novel methods for characterization of complex macromolecular structures. The elucidation of the different structural levels of organization opens the way to relating structure to function and properties. The chemical and biochemical methods that have been developed to dissolve and further modify cellulose chains are briefly covered. Particular emphasis is given to the facets of topochemistry and topoenzymology where the morphological features play a key role in determining unique physicochemical properties. A final chapter addresses what might be considered tomorrow's goal in amplifying the economic importance of cellulose in the context of sustainable development. Selected examples illustrate the types of result that can be obtained when cellulose fibers are no longer viewed as inert substrates, and when the polyhydroxyl nature of their surfaces, as well as their entire structural complexity, are taken into account. Copyright © 2010 Elsevier Inc. All rights reserved.

Gas-phase surface esterification of cellulose microfibrils and whiskers.

PubMed

Berlioz, Sophie; Molina-Boisseau, Sonia; Nishiyama, Yoshiharu; Heux, Laurent

2009-08-10

A new and highly efficient synthetic method has been developed for the surface esterification of model cellulosic substrates of high crystallinity and accessibility, namely, freeze-dried tunicin whiskers and bacterial cellulose microfibrils dried by the critical point method. The reaction, which is based on the gas-phase action of palmitoyl chloride, was monitored by solid-state CP-MAS (13)C NMR. It was found that the grafting density not only depended on the experimental conditions, but also on the nature and conditioning of the cellulose samples. The structural and morphological modifications of the substrates at various degrees of grafting were revealed by scanning electron microscopy and X-ray diffraction analysis. These characterizations indicated that the esterification proceeded from the surface of the substrate to their crystalline core. Hence, for moderate degree of substitution, the surface was fully grafted whereas the cellulose core remained unmodified and the original fibrous morphology maintained. An almost total esterification could be achieved under certain conditions, leading to highly substituted cellulose esters, presenting characteristic X-ray diffraction patterns.

Physicochemical structural changes of cellulosic substrates during enzymatic saccharification

DOE PAGES

Meng, Xianzhi; Yoo, Chang Geun; Li, Mi; ...

2016-12-30

Enzymatic hydrolysis represents one of the major steps and barriers in the commercialization process of converting cellulosic substrates into biofuels and other value added products. It is usually achieved by a synergistic action of enzyme mixture typically consisting of multiple enzymes such as glucanase, cellobiohydrolase and β-glucosidase with different mode of actions. Due to the innate biomass recalcitrance, enzymatic hydrolysis normally starts with an initial fast rate of hydrolysis followed by a rapid decrease of rate toward the end of hydrolysis. With majority of literature studies focusing on the effect of key substrate characteristics on the initial rate or finalmore » yield of enzymatic hydrolysis, information about physicochemical structural changes of cellulosic substrates during enzymatic hydrolysis is still quite limited. Consequently, what slows down the reaction rate toward the end of hydrolysis is not well understood. Lastly, this review highlights recent advances in understanding the structural changes of cellulosic substrates during the hydrolysis process, to better understand the fundamental mechanisms of enzymatic hydrolysis.« less

Physicochemical structural changes of cellulosic substrates during enzymatic saccharification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meng, Xianzhi; Yoo, Chang Geun; Li, Mi

Enzymatic hydrolysis represents one of the major steps and barriers in the commercialization process of converting cellulosic substrates into biofuels and other value added products. It is usually achieved by a synergistic action of enzyme mixture typically consisting of multiple enzymes such as glucanase, cellobiohydrolase and β-glucosidase with different mode of actions. Due to the innate biomass recalcitrance, enzymatic hydrolysis normally starts with an initial fast rate of hydrolysis followed by a rapid decrease of rate toward the end of hydrolysis. With majority of literature studies focusing on the effect of key substrate characteristics on the initial rate or finalmore » yield of enzymatic hydrolysis, information about physicochemical structural changes of cellulosic substrates during enzymatic hydrolysis is still quite limited. Consequently, what slows down the reaction rate toward the end of hydrolysis is not well understood. Lastly, this review highlights recent advances in understanding the structural changes of cellulosic substrates during the hydrolysis process, to better understand the fundamental mechanisms of enzymatic hydrolysis.« less

Visualizing cellulase activity.

PubMed

Bubner, Patricia; Plank, Harald; Nidetzky, Bernd

2013-06-01

Commercial exploitation of lignocellulose for biotechnological production of fuels and commodity chemicals requires efficient-usually enzymatic-saccharification of the highly recalcitrant insoluble substrate. A key characteristic of cellulose conversion is that the actual hydrolysis of the polysaccharide chains is intrinsically entangled with physical disruption of substrate morphology and structure. This "substrate deconstruction" by cellulase activity is a slow, yet markedly dynamic process that occurs at different length scales from and above the nanometer range. Little is currently known about the role of progressive substrate deconstruction on hydrolysis efficiency. Application of advanced visualization techniques to the characterization of enzymatic degradation of different celluloses has provided important new insights, at the requisite nano-scale resolution and down to the level of single enzyme molecules, into cellulase activity on the cellulose surface. Using true in situ imaging, dynamic features of enzyme action and substrate deconstruction were portrayed at different morphological levels of the cellulose, thus providing new suggestions and interpretations of rate-determining factors. Here, we review the milestones achieved through visualization, the methods which significantly promoted the field, compare suitable (model) substrates, and identify limiting factors, challenges and future tasks. Copyright © 2013 Wiley Periodicals, Inc.

Purification and properties of an alkaline ribonuclease from the hepatic cytosol fraction of bullfrog, Rana catesbeiana.

PubMed

Nagano, H; Kiuchi, H; Abe, Y; Shukuya, R

1976-07-01

In the hepatic cytosol fraction of bullfrog, Rana catesbeiana, an alkaline RNase [EC 3.1.4.22] exists in two forms. One is the free form of RNase, which elutes from a carboxymethyl-cellulose column at a concentration of 0.2 M NaC1. The other is a masked or latent form (RNase-RNase inhibitor complex) which is not adsorbed on the carboxymethyl-cellulose column and which can be converted to the free form of RNase by the addition of p-chloromercuribenzoate. Electrophoretically pure RNase was obtained by the following procedure. The unadsorbed fraction of hepatic cytosol on a column of carboxymethyl-cellulose was treated with p-chloromercuribenzoate and then applied to a second carboxymethyl-cellulose column. The molar weight of RNase was determined to be approximately 12,000 by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From the results of gel filtration, the molecular weight of the RNase-RNase inhibitor complex was 130,000. The RNase hydrolyzed poly C, poly U, and poly I, but not poly A or poly G. When poly C was used as a substrate, 2',3'-cyclic CMP as an intermediate and 3'-CMP as a final product were identified. The results of amino acid analysis indicated the presence of an unusual component. The general properties of the RNase and the RNase-RNase inhibitor complex are also reported.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

Electricity production and microbial biofilm characterization in cellulose-fed microbial fuel cells.

PubMed

Ren, Z; Steinberg, L M; Regan, J M

2008-01-01

Converting biodegradable materials into electricity, microbial fuel cells (MFCs) present a promising technology for renewable energy production in specific applications. Unlike typical soluble substrates that have been used as electron donors in MFC studies, cellulose is unique because it requires a microbial consortium that can metabolize both an insoluble electron donor (cellulose) and electron acceptor (electrode). In this study, electricity generation and the microbial ecology of cellulose-fed MFCs were analyzed using a defined co-culture of Clostridium cellulolyticum and Geobacter sulfurreducens. Fluorescent in situ hybridization and quantitative PCR showed that when particulate MN301 cellulose was used as sole substrate, most Clostridium cells were found adhered to cellulose particles in suspension, while most Geobacter cells were attached to the electrode. By comparison, both bacteria resided in suspension and biofilm samples when soluble carboxymethyl cellulose was used. This distinct function-related distribution of the bacteria suggests an opportunity to optimize reactor operation by settling cellulose and decanting supernatant to extend cellulose hydrolysis and improve cellulose-electricity conversion. (c) IWA Publishing 2008.

Enhanced the enzymatic hydrolysis efficiency of wheat straw after combined steam explosion and laccase pretreatment.

PubMed

Qiu, Weihua; Chen, Hongzhang

2012-08-01

Laccase, capable of selectively degrading lignin while keeping cellulose intact, has been widely applied for the modification and bio-bleaching of pulp. In this study Sclerotium sp. laccase (MSLac) was employed in combination with steam explosion to evaluate the effect of this treatment on cellulose hydrolysis. Combined steam explosion with laccase pretreatment enhanced the cellulose conversion rate of wheat straw no matter in the case of successive (MSLac-Cel) and simultaneous (MSLac+Cel) MSLac and cellulase hydrolysis. The highest cellulose conversion rate of 84.23% was obtained when steam-exploded wheat straw (SEWS) (1.3 MPa, 5 min) was treated by MSLac+Cel at a laccase loading of 0.55 U g(-1) substrate. FT-IR and SEM analyses indicated that MSLac oxidized the phenol and changed electron configuration of the ring, which contributed to loosening the compact wrap of lignin-carbohydrate complex and consequently enhancing the enzymatic hydrolysis efficiency of cellulose. This article provided a promising method for lignocellulose bio-pretreatment. Copyright © 2012 Elsevier Ltd. All rights reserved.

Physical and chemical differences between one-stage and two-stage hydrothermal pretreated hardwood substrates for use in cellulosic ethanol production

DOE PAGES

Guilliams, Andrew; Pattathil, Sivakumar; Willies, Deidre; ...

2016-02-03

Here, there are many different types of pretreatment carried out to prepare cellulosic substrates for fermentation. In this study, one- and two-stage hydrothermal pretreatment were carried out to determine their effects on subsequent fermentations. The two substrates were found to behave differently during fermentation. The two substrates were then characterized using physical and chemical parameters.

Alternative Environmental Roles for Cellulose Produced by Acetobacter xylinum

PubMed Central

Williams, W. Scott; Cannon, Robert E.

1989-01-01

The cellulose-producing bacterium Acetobacter xylinum has been considered a strict aerobe, and it has been suggested that the function of cellulose is to hold cells in an aerobic environment. In this study, we showed that A. xylinum is capable of growing microaerophilically. Cellulose pellicles provided significant protection to A. xylinum cells from the killing effects of UV light. In experiments measuring colonization by A. xylinum, molds, and other bacteria on pieces of apple, cellulose pellicles enhanced colonization of A. xylinum on the substrate and provided protection from competitors which use the same substrate as a source of nutrients. Cellulose pellicles produced by A. xylinum may have multiple functions in the growth and survival of the organism in nature. PMID:16348023

Single-cell protein from waste cellulose

NASA Technical Reports Server (NTRS)

Dunlap, C. E.; Callihan, C. D.

1973-01-01

The recycle, reuse, or reclamation of single cell protein from liquid and solid agricultural waste fibers by a fermentation process is reported. It is shown that cellulose comprises the bulk of the fibers at 50% to 55% of the dry weight of the refuse and that its biodegradability is of prime importance in the choice of a substrate. The application of sodium hydroxide followed by heat and pressure serves to de-polymerize and disrupt lignin structure while swelling the cellulose to increase water uptake and pore volume. Some of the lignin, hemi-celluloses, ash, and cellulose of the material is hydrolized and solubilized. Introduction of microorganisms to the substrate fibers mixed with nutrients produces continuous fermentation of cellulose for further protein extraction and purification.

Fungal Taxa Target Different Carbon Substrates in Harvard Forest Soils

NASA Astrophysics Data System (ADS)

Hanson, C. A.; Allison, S. D.; Wallenstein, M. D.; Mellilo, J. M.; Treseder, K. K.

2006-12-01

The mineralization of soil organic carbon is a major component of the global carbon cycle and is largely controlled by soil microbial communities. However, little is known about the functional roles of soil microbes or whether different microbial taxa target different carbon substrates under natural conditions. To examine this possibility, we assessed the community composition of active fungi by using a novel nucleotide analog technique in soils from the Harvard Forest. We hypothesized that fungal community composition would shift in response to the addition of different substrates and that specific fungal taxa would respond differentially to particular carbon sources. To test this hypothesis, we added a nucleotide analog probe directly to soils in conjunction with one of five carbon compounds of increasing recalcitrance: glycine, sucrose, cellulose, tannin-protein complex, and lignin. During 48 hour incubations, the nucleotide analog was incorporated into newly replicated DNA of soil organisms that proliferated following the addition of the substrates. In this way, we labeled the DNA of microbes that respond to a particular carbon source. Labeled DNA was isolated and fungal Internal Transcribed Spacer (ITS) regions of ribosomal DNA (rDNA) were sequenced and analyzed to identify active fungi to near-species resolution. Diversity analyses at the ≥97% sequence similarity level indicated that taxonomic richness was greater under cellulose (Shannon Index: 3.23 ± 0.11 with ± 95% CI) and lignin (2.87 ± 0.15) additions than the other treatments (2.34 ± 0.16 to 2.64 ± 0.13). In addition, community composition of active fungi shifted under glycine, sucrose, and cellulose additions. Specifically, the community under glycine was significantly different from communities under control, cellulose, and tannin-protein (P<0.05). Additionally, the sucrose and cellulose communities were marginally different from the control community (P = 0.059 and 0.054, respectively) and each other (P = 0.058). Together these results support our hypothesis that fungal communities change in response to different carbon sources. We found 11 fungal operational taxonomic units (OTUs) whose relative abundances differed at least marginally significantly among substrates. One OTU related to Mortierella increased in abundance under cellulose, but was absent or rare under the other substrates. Another OTU related to an unidentified Basidiomycete was only present under lignin addition, while yet another OTU closely related to Mortierella macrocystis greatly increased in abundance under tannin-protein and slightly increased in response to lignin and sucrose. This confirms our hypothesis that particular taxa respond differently to specific carbon substrates and suggests that some fungal taxa may specialize in the break-down of particular carbon sources in soils. Overall, our results imply that microbes have varying roles in the mineralization of soil carbon, and thus microbial community composition may be an important control over ecosystem carbon dynamics and storage, especially in relation to global change.

Cytosolic invertase contributes to the supply of substrate for cellulose biosynthesis in developing wood.

PubMed

Rende, Umut; Wang, Wei; Gandla, Madhavi Latha; Jönsson, Leif J; Niittylä, Totte

2017-04-01

Carbon for cellulose biosynthesis is derived from sucrose. Cellulose is synthesized from uridine 5'-diphosphoglucose (UDP-glucose), but the enzyme(s) responsible for the initial sucrose cleavage and the source of UDP-glucose for cellulose biosynthesis in developing wood have not been defined. We investigated the role of CYTOSOLIC INVERTASEs (CINs) during wood formation in hybrid aspen (Populus tremula × tremuloides) and characterized transgenic lines with reduced CIN activity during secondary cell wall biosynthesis. Suppression of CIN activity by 38-55% led to a 9-13% reduction in crystalline cellulose. The changes in cellulose were reflected in reduced diameter of acid-insoluble cellulose microfibrils and increased glucose release from wood upon enzymatic digestion of cellulose. Reduced CIN activity decreased the amount of the cellulose biosynthesis precursor UDP-glucose in developing wood, pointing to the likely cause of the cellulose phenotype. The findings suggest that CIN activity has an important role in the cellulose biosynthesis of trees, and indicate that cellulose biosynthesis in wood relies on a quantifiable UDP-glucose pool. The results also introduce a concept of altering cellulose microfibril properties by modifying substrate supply to cellulose biosynthesis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Integrated cellulosic enzymes hydrolysis and fermentative advanced yeast bioconversion solution ready for biomass biorefineries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-05-04

These are slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme,more » as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Production of succinic acid from oil palm empty fruit bunch cellulose using Actinobacillus succinogenes

NASA Astrophysics Data System (ADS)

Pasma, Satriani Aga; Daik, Rusli; Maskat, Mohamad Yusof

2013-11-01

Succinic acid is a common metabolite in plants, animals and microorganisms. It has been used widely in agricultural, food and pharmaceutical industries. Enzymatic hydrolysate glucose from oil palm empty fruit bunch (OPEFB) cellulose was used as a substrate for succinic acid production using Actinobacillus succinogenes. Using cellulose extraction from OPEFB can enhance the production of glucose as a main substrate for succinic acid production. The highest concentration of glucose produced from enzymatic hydrolysis is 167 mg/mL and the sugar recovery is 0.73 g/g of OPEFB. By optimizing the culture medium for succinic acid fermentation with enzymatic hydrolysate of OPEFB cellulose, the nitrogen sources could be reduced to just only 2.5 g yeast extract and 2.5 g corn step liquor. Batch fermentation was carried out using enzymatic hydrolysate of OPEFB cellulose with yeast extract, corn steep liquor and the salts mixture, 23.5 g/L succinic acid was obtained with consumption of 72 g/L glucose in enzymatic hydrolysate of OPEFB cellulose at 38 hours and 37°C. This study suggests that enzymatic hydrolysate of OPEFB cellulose maybe an alternative substrate for the efficient production of succinic acid by Actinobacillus succinogenes.

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility.

PubMed

Jeoh, Tina; Ishizawa, Claudia I; Davis, Mark F; Himmel, Michael E; Adney, William S; Johnson, David K

2007-09-01

Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.

Form and Function of Clostridium thermocellum Biofilms

PubMed Central

Dumitrache, Alexandru; Allen, Grant; Liss, Steven N.; Lynd, Lee R.

2013-01-01

The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h−1) 12-fold higher than the bacterium's maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion. PMID:23087042

Form and function of Clostridium thermocellum biofilms.

PubMed

Dumitrache, Alexandru; Wolfaardt, Gideon; Allen, Grant; Liss, Steven N; Lynd, Lee R

2013-01-01

The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h(-1)) 12-fold higher than the bacterium's maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion.

ZnO nanostructures directly grown on paper and bacterial cellulose substrates without any surface modification layer.

PubMed

Costa, Saionara V; Gonçalves, Agnaldo S; Zaguete, Maria A; Mazon, Talita; Nogueira, Ana F

2013-09-21

In this report, hierarchical ZnO nano- and microstructures were directly grown for the first time on a bacterial cellulose substrate and on two additional different papers by hydrothermal synthesis without any surface modification layer. Compactness and smoothness of the substrates are two important parameters that allow the growth of oriented structures.

Drying of Pigment-Cellulose Nanofibril Substrates

PubMed Central

Timofeev, Oleg; Torvinen, Katariina; Sievänen, Jenni; Kaljunen, Timo; Kouko, Jarmo; Ketoja, Jukka A.

2014-01-01

A new substrate containing cellulose nanofibrils and inorganic pigment particles has been developed for printed electronics applications. The studied composite structure contains 80% fillers and is mechanically stable and flexible. Before drying, the solids content can be as low as 20% due to the high water binding capacity of the cellulose nanofibrils. We have studied several drying methods and their effects on the substrate properties. The aim is to achieve a tight, smooth surface keeping the drying efficiency simultaneously at a high level. The methods studied include: (1) drying on a hot metal surface; (2) air impingement drying; and (3) hot pressing. Somewhat surprisingly, drying rates measured for the pigment-cellulose nanofibril substrates were quite similar to those for the reference board sheets. Very high dewatering rates were observed for the hot pressing at high moisture contents. The drying method had significant effects on the final substrate properties, especially on short-range surface smoothness. The best smoothness was obtained with a combination of impingement and contact drying. The mechanical properties of the sheets were also affected by the drying method and associated temperature. PMID:28788220

Enzyme Kinetics Experiment with the Multienzyme Complex Viscozyme L and Two Substrates for the Accurate Determination of Michaelian Parameters

ERIC Educational Resources Information Center

Guerra, Nelson Pérez

2017-01-01

A laboratory experiment in which students study the kinetics of the Viscozyme-L-catalyzed hydrolysis of cellulose and starch comparatively was designed for an upper-division biochemistry laboratory. The main objective of this experiment was to provide an opportunity to perform enhanced enzyme kinetics data analysis using appropriate informatics…

Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks

PubMed Central

Aebersold, Mathias J.; Thompson-Steckel, Greta; Joutang, Adriane; Schneider, Moritz; Burchert, Conrad; Forró, Csaba; Weydert, Serge; Han, Hana; Vörös, János

2018-01-01

Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such in vitro bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of in vitro assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days in vitro at densities ranging from 50,000 cells/cm2 down to isolated cells at 1,000 cells/cm2. Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development. PMID:29535595

Secretomic survey of Trichoderma harzianum grown on plant biomass substrates.

PubMed

Gómez-Mendoza, Diana Paola; Junqueira, Magno; do Vale, Luis Henrique Ferreira; Domont, Gilberto Barbosa; Ferreira Filho, Edivaldo Ximenes; Sousa, Marcelo Valle de; Ricart, Carlos André Ornelas

2014-04-04

The present work aims at characterizing T. harzianum secretome when the fungus is grown in synthetic medium supplemented with one of the four substrates: glucose, cellulose, xylan, and sugarcane bagasse (SB). The characterization was done by enzymatic assays and proteomic analysis using 2-DE/MALDI-TOF and gel-free shotgun LC-MS/MS. The results showed that SB induced the highest cellulolytic and xylanolytic activities when compared with the other substrates, while remarkable differences in terms of number and distribution of protein spots in 2-DE gels were also observed among the samples. Additionally, treatment of the secretomes with PNGase F revealed that most spot trails in 2-DE gels corresponded to N-glycosylated proteoforms. The LC-MS/MS analysis of the samples identified 626 different protein groups, including carbohydrate-active enzymes and accessory, noncatalytic, and cell-wall-associated proteins. Although the SB-induced secretome displayed the highest cellulolytic and xylanolytic activities, it did not correspond to a higher proteome complexity because CM-cellulose-induced secretome was significantly more diverse. Among the identified proteins, 73% were exclusive to one condition, while only 5% were present in all samples. Therefore, this study disclosed the variation of T. harzianum secretome in response to different substrates and revealed the diversity of the fungus enzymatic toolbox.

Spectrophotometric determination of substrate-borne polyacrylamide.

PubMed

Lu, Jianhang; Wu, Laosheng

2002-08-28

Polyacrylamides (PAMs) have wide application in many industries and in agriculture. Scientific research and industrial applications manifested a need for a method that can quantify substrate-borne PAM. The N-bromination method (a PAM analytical technique based on N-bromination of amide groups and spectrophotometric determination of the formed starch-triiodide complex), which was originally developed for determining PAM in aqueous solutions, was modified to quantify substrate-borne PAM. In the modified method, the quantity of substrate-borne PAM was converted to a concentration of starch-triiodide complex in aqueous solution that was then measured by spectrophotometry. The method sensitivity varied with substrates due to sorption of reagents and reaction intermediates on the substrates. Therefore, separate calibration for each substrate was required. Results from PAM samples in sand, cellulose, organic matter burnt soils, and clay minerals showed that this method had good accuracy and reproducibility. The PAM recoveries ranged from 95.8% to 103.7%, and the relative standard deviations (n = 4) were <7.5% in all cases. The optimum range of PAM in each sample is 10-80 microg. The technique can serve as an effective tool in improving PAM application and facilitating PAM-related research.

Microwave-assisted ionic liquid-mediated rapid catalytic conversion of non-edible lignocellulosic Sunn hemp fibres to biofuels.

PubMed

Paul, Souvik Kumar; Chakraborty, Saikat

2018-04-01

Sunn hemp fibre - a cellulose-rich crystalline non-food energy crop, containing 75.6% cellulose, 10.05% hemicellulose, 10.32% lignin, with high crystallinity (80.17%) and degree of polymerization (650) - is identified as a new non-food substrate for lignocellulosic biofuel production. Microwave irradiation is employed to rapidly rupture the cellulose's glycosidic bonds and enhance glucose yield to 78.7% at 160 °C in only 46 min. The reactants - long-chain cellulose, ionic liquid, transition metal catalyst, and water - form a polar supramolecular complex that rotates under the microwave's alternating polarity and rapidly dissipates the electromagnetic energy through molecular collisions, thus accelerating glycosidic bond breakage. In 46 min, 1 kg of Sunn hemp fibres containing 756 g of cellulose produces 595 g of glucose at 160 °C, and 203 g of hydroxymethyl furfural (furanic biofuel precursor) at 180 °C. Yeast mediated glucose fermentation produces 75.6% bioethanol yield at 30 °C, and the ionic liquid is recycled for cost-effectiveness. Copyright © 2018 Elsevier Ltd. All rights reserved.

Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

PubMed

Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

2018-02-02

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Highly hydrophobic biopolymers prepared by the surface pentafluorobenzoylation of cellulose substrates.

PubMed

Cunha, Ana G; Freire, Carmen S R; Silvestre, Armando J D; Pascoal Neto, Carlos; Gandini, Alessandro; Orblin, Elina; Fardim, Pedro

2007-04-01

New highly hydrophobic/lipophobic biopolymers were prepared by the controlled heterogeneous pentafluorobenzoylation of cellulose substrates, i.e., plant and bacterial cellulose fibers. The characterization of the modified fibers was performed by elemental analysis, FTIR spectroscopy, X-ray diffraction, thermogravimetry, and surface analysis (XPS, ToF-SIMS, and contact angle measurements). The degree of substitution of the ensuing pentafluorobenzoylated fibers ranged from 0.014 to 0.39. The hydrolytic stability of these perfluorinated cellulose derivatives was also evaluated and showed that they were quite water stable, although of course the fluorinated moieties could readily be removed by hydrolysis in an aqueous alkaline medium.

The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brunecky, Roman; Donohoe, Bryon S.; Yarbrough, John M.

The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systemsmore » employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Furthermore, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.« less

The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose

DOE PAGES

Brunecky, Roman; Donohoe, Bryon S.; Yarbrough, John M.; ...

2017-08-29

The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systemsmore » employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Furthermore, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.« less

Methods and compositions for simultaneous saccharification and fermentation

DOEpatents

Ingram, Lonnie O'Neal; Zhou, Shengde

2006-04-11

The invention provides compositions and methods for the synergistic degradation of oligosaccharides by endoglucanases. The invention further provides recombinant host cells containing one or more genes encoding endoglucanses which are capable of the synergistic degradation of oligosaccharides. Preferred host cells of the invention are ethanologenic and capable of carrying out simultaneous saccharification and fermentation resulting in the production of ethanol from complex cellulose substrates.

Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction

PubMed Central

Conway, Jonathan M.; McKinley, Bennett S.; Seals, Nathaniel L.; Hernandez, Diana; Khatibi, Piyum A.; Poudel, Suresh; Giannone, Richard J.; Hettich, Robert L.; Williams-Rhaesa, Amanda M.; Lipscomb, Gina L.; Adams, Michael W. W.

2017-01-01

ABSTRACT The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization. IMPORTANCE The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii, the nuanced, substrate-specific in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in C. bescii, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile. PMID:28986379

Investigating commercial cellulase performances toward specific biomass recalcitrance factors using reference substrates.

PubMed

Ju, Xiaohui; Bowden, Mark; Engelhard, Mark; Zhang, Xiao

2014-05-01

Three commercial cellulase preparations, Novozymes Cellic(®) Ctec2, Dupont Accellerase(®) 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulase enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulase performance. While the degree of polymerization of cellulose in the reference substrates did not present a major recalcitrance factor to Novozymes Cellic(®) Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.

Vertical Integration of Biomass Saccharification of Enzymes for Sustainable Cellulosic Biofuel Production in a Biorefinery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-05-09

These are a set of slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Fluorescent cellulose microfibrils as substrate for the detection of cellulase activity.

PubMed

Helbert, William; Chanzy, Henri; Husum, Tommy Lykke; Schülein, Martin; Ernst, Steffen

2003-01-01

To devise a sensitive cellulase assay based on substrates having most of the physical characteristics of native cellulose, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was used as a grafting agent to prepare suspensions of fluorescent microfibrils from bacterial cellulose. These suspensions were digested by a series of commercially relevant cellulases from Humicola insolens origin: cloned Cel6B and Cel 45A as well as crude H. insolens complex. The digestion induced the release of fluorescent cellodextrins as well as reducing sugars. After adequate centrifugation, these soluble products were analyzed as a function of grafting content, digestion time, and cellulase characteristics. The resulting data allowed the grafting conditions to be optimized in order to maximize the quantity of soluble products and therefore to increase the sensitivity of the detection. A comparison between the amount of released fluorescence and that of released reducing sugar allowed the differentiation between processive exo and endo cellulase activities. The casting of films of DTAF-grafted microfibrils at the bottom of the microwell titer plates also led to sensitive cellulase detection. As these films kept their integrity and remained firmly glued to the well bottom during the digestion time, they are tailored made for a full automation of the cellulases testing.

Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

PubMed Central

Hetzler, Stephan; Bröker, Daniel

2013-01-01

The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml−1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step. PMID:23793636

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

PubMed Central

Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.; Park, Sunkyu; Kim, Seong H.

2015-01-01

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlate with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. It was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component. PMID:26463274

Deactivation of Cellulase at the Air-Liquid Interface Is the Main Cause of Incomplete Cellulose Conversion at Low Enzyme Loadings.

PubMed

Bhagia, Samarthya; Dhir, Rachna; Kumar, Rajeev; Wyman, Charles E

2018-01-22

Amphiphilic additives such as bovine serum albumin (BSA) and Tween have been used to improve cellulose hydrolysis by cellulases. However, there has been a lack of clarity to explain their mechanism of action in enzymatic hydrolysis of pure or low-lignin cellulosic substrates. In this work, a commercial Trichoderma reesei enzyme preparation and the amphiphilic additives BSA and Tween 20 were applied for hydrolysis of pure Avicel cellulose. The results showed that these additives only had large effects on cellulose conversion at low enzyme to substrate ratios when the reaction flasks were shaken. Furthermore, changes in the air-liquid interfacial area profoundly affected cellulose conversion, but surfactants reduced or prevented cellulase deactivation at the air-liquid interface. Not shaking the flasks or adding low amounts of surfactant resulted in near theoretical cellulose conversion at low enzyme loadings given enough reaction time. At low enzyme loadings, hydrolysis of cellulose in lignocellulosic biomass with low lignin content suffered from enhanced enzyme deactivation at the air-liquid interface.

Surface-structured bacterial cellulose with guided assembly-based biolithography (GAB).

PubMed

Bottan, Simone; Robotti, Francesco; Jayathissa, Prageeth; Hegglin, Alicia; Bahamonde, Nicolas; Heredia-Guerrero, José A; Bayer, Ilker S; Scarpellini, Alice; Merker, Hannes; Lindenblatt, Nicole; Poulikakos, Dimos; Ferrari, Aldo

2015-01-27

A powerful replica molding methodology to transfer on-demand functional topographies to the surface of bacterial cellulose nanofiber textures is presented. With this method, termed guided assembly-based biolithography (GAB), a surface-structured polydimethylsiloxane (PDMS) mold is introduced at the gas-liquid interface of an Acetobacter xylinum culture. Upon bacterial fermentation, the generated bacterial cellulose nanofibers are assembled in a three-dimensional network reproducing the geometric shape imposed by the mold. Additionally, GAB yields directional alignment of individual nanofibers and memory of the transferred geometrical features upon dehydration and rehydration of the substrates. Scanning electron and atomic force microscopy are used to establish the good fidelity of this facile and affordable method. Interaction of surface-structured bacterial cellulose substrates with human fibroblasts and keratinocytes illustrates the efficient control of cellular activities which are fundamental in skin wound healing and tissue regeneration. The deployment of surface-structured bacterial cellulose substrates in model animals as skin wound dressing or body implant further proves the high durability and low inflammatory response to the material over a period of 21 days, demonstrating beneficial effects of surface structure on skin regeneration.

Rapid Cellulose-Mediated Microwave Sintering for High-Conductivity Ag Patterns on Paper.

PubMed

Jung, Sunshin; Chun, Su Jin; Shon, Chae-Hwa

2016-08-10

Cellulose-based paper is essential in everyday life, but it also has further potentials for use in low-cost, printable, disposable, and eco-friendly electronics. Here, a method is developed for the cellulose-mediated microwave sintering of Ag patterns on conventional paper, in which the paper plays a significant role both as a flexible insulating substrate for the conductive Ag pattern and as a lossy dielectric media for rapid microwave heating. The anisotropic dielectric properties of the cellulose fibers mean that a microwave electric field applied parallel to the paper substrate provides sufficient heating to produce Ag patterns with a conductivity 29-38% that of bulk Ag in a short period of time (∼1 s) at 250-300 °C. Significantly, there is little thermal degradation of the substrate during this process. The microwave-sintered Ag patterns exhibit good mechanical stability against 10 000 bending cycles and can be easily soldered with lead-free solder. Therefore, cellulose-mediated microwave sintering presents a promising means of achieving short processing times and high electrical performance in flexible paper electronics.

UO(2) 2+ speciation determines uranium toxicity and bioaccumulation in an environmental Pseudomonas sp. isolate.

PubMed

Vanengelen, Michael R; Field, Erin K; Gerlach, Robin; Lee, Brady D; Apel, William A; Peyton, Brent M

2010-04-01

In the present study, experiments were performed to investigate how representative cellulosic breakdown products, when serving as growth substrates under aerobic conditions, affect hexavalent uranyl cation (UO(2) (2+)) toxicity and bioaccumulation within a Pseudomonas sp. isolate (designated isolate A). Isolate A taken from the Cold Test Pit South (CTPS) region of the Idaho National Laboratory (INL), Idaho Falls, ID, USA. The INL houses low-level uranium-contaminated cellulosic material and understanding how this material, and specifically its breakdown products, affect U-bacterial interactions is important for understanding UO(2) (2+) fate and mobility. Toxicity was modeled using a generalized Monod expression. Butyrate, dextrose, ethanol, and lactate served as growth substrates. The potential contribution of bicarbonate species present in high concentrations was also investigated and compared with toxicity and bioaccumulation patterns seen in low-bicarbonate conditions. Isolate A was significantly more sensitive to UO(2) (2+) and accumulated significantly more UO(2) (2+) in low-bicarbonate concentrations. In addition, UO(2) (2+) growth inhibition and bioaccumulation varied depending on the growth substrate. In the presence of high bicarbonate concentrations, sensitivity to UO(2) (2+) inhibition was greatly mitigated, and did not vary between the four substrates tested. The extent of UO(2) (2+) accumulation was also diminished. The observed patterns were related to UO(2) (2+) aqueous complexation, as predicted by MINTEQ (ver. 2.52) (Easton, PA, USA). In the low- bicarbonate medium, the presence of positively charged and unstable UO(2) (2+)-hydroxide complexes explained both the greater sensitivity of isolate A to UO(2) (2+), and the ability of isolate A to accumulate significant amounts of UO(2) (2+). The exclusive presence of negatively charged and stable UO(2) (2+)-carbonate complexes in the high bi-carbonate medium explained the diminished sensitivity of isolate A to UO(2) (2+) toxicity, and limited ability of isolate A to accumulate UO(2) (2+). (c) 2010 SETAC.

Pigment-cellulose nanofibril composite and its application as a separator-substrate in printed supercapacitors

NASA Astrophysics Data System (ADS)

Torvinen, Katariina; Lehtimäki, Suvi; Keränen, Janne T.; Sievänen, Jenni; Vartiainen, Jari; Hellén, Erkki; Lupo, Donald; Tuukkanen, Sampo

2015-11-01

Pigment-cellulose nanofibril (PCN) composites were manufactured in a pilot line and used as a separator-substrate in printed graphene and carbon nanotube supercapacitors. The composites consisted typically of 80% pigment and 20% cellulose nanofibrils (CNF). This composition makes them a cost-effective alternative as a substrate for printed electronics at high temperatures that only very special plastic films can nowadays stand. The properties of these substrates can be varied within a relatively large range by the selection of raw materials and their relative proportions. A semi-industrial scale pilot line was successfully used to produce smooth, flexible, and nanoporous composites, and their performance was tested in a double functional separator-substrate element in supercapacitors. The nanostructural carbon films printed on the composite worked simultaneously as high surface area active electrodes and current collectors. Low-cost supercapacitors made from environmentally friendly materials have significant potential for use in flexible, wearable, and disposable low-end products. [Figure not available: see fulltext.

Recyclable organic solar cells on cellulose nanocrystal substrates

PubMed Central

Zhou, Yinhua; Fuentes-Hernandez, Canek; Khan, Talha M.; Liu, Jen-Chieh; Hsu, James; Shim, Jae Won; Dindar, Amir; Youngblood, Jeffrey P.; Moon, Robert J.; Kippelen, Bernard

2013-01-01

Solar energy is potentially the largest source of renewable energy at our disposal, but significant advances are required to make photovoltaic technologies economically viable and, from a life-cycle perspective, environmentally friendly, and consequently scalable. Cellulose nanomaterials are emerging high-value nanoparticles extracted from plants that are abundant, renewable, and sustainable. Here, we report on the first demonstration of efficient polymer solar cells fabricated on optically transparent cellulose nanocrystal (CNC) substrates. The solar cells fabricated on the CNC substrates display good rectification in the dark and reach a power conversion efficiency of 2.7%. In addition, we demonstrate that these solar cells can be easily separated and recycled into their major components using low-energy processes at room temperature, opening the door for a truly recyclable solar cell technology. Efficient and easily recyclable organic solar cells on CNC substrates are expected to be an attractive technology for sustainable, scalable, and environmentally-friendly energy production. PMID:23524333

Recyclable organic solar cells on cellulose nanocrystal substrates.

PubMed

Zhou, Yinhua; Fuentes-Hernandez, Canek; Khan, Talha M; Liu, Jen-Chieh; Hsu, James; Shim, Jae Won; Dindar, Amir; Youngblood, Jeffrey P; Moon, Robert J; Kippelen, Bernard

2013-01-01

Solar energy is potentially the largest source of renewable energy at our disposal, but significant advances are required to make photovoltaic technologies economically viable and, from a life-cycle perspective, environmentally friendly, and consequently scalable. Cellulose nanomaterials are emerging high-value nanoparticles extracted from plants that are abundant, renewable, and sustainable. Here, we report on the first demonstration of efficient polymer solar cells fabricated on optically transparent cellulose nanocrystal (CNC) substrates. The solar cells fabricated on the CNC substrates display good rectification in the dark and reach a power conversion efficiency of 2.7%. In addition, we demonstrate that these solar cells can be easily separated and recycled into their major components using low-energy processes at room temperature, opening the door for a truly recyclable solar cell technology. Efficient and easily recyclable organic solar cells on CNC substrates are expected to be an attractive technology for sustainable, scalable, and environmentally-friendly energy production.

Combined enzyme mediated fermentation of cellulose and xylose to ethanol by Schizosaccharomyces pombe, cellulase, [beta]-glucosidase, and xylose isomerase

DOEpatents

Lastick, S.M.; Mohagheghi, A.; Tucker, M.P.; Grohmann, K.

1994-12-13

A process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol; loading the substrates with the fermentation mix composed of yeast, enzymes and substrates; fermenting the loaded substrates and enzymes under anaerobic conditions at a pH range of between about 5.0 to about 6.0 and at a temperature range of between about 35 C to about 40 C until the fermentation is completed, the xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast through means of the anaerobic fermentation; and recovering the ethanol. 2 figures.

Empirical evaluation of inhibitory product, substrate, and enzyme effects during the enzymatic saccharification of lignocellulosic biomass.

PubMed

Smith, Benjamin T; Knutsen, Jeffrey S; Davis, Robert H

2010-05-01

The cellulose hydrolysis kinetics during batch enzymatic saccharification are typified by a rapid initial rate that subsequently decays, resulting in incomplete conversion. Previous studies suggest that changes associated with the solution, substrate, or enzymes may be responsible. In this work, kinetic experiments were conducted to determine the relative magnitude of these effects. Pretreated corn stover (PCS) was used as a lignocellulosic substrate likely to be found in a commercial saccharification process, while Avicel and Kraft lignin were used to create model substrates. Glucose inhibition was observed by spiking the reaction slurry with glucose during initial-rate experiments. Increasing the glucose concentration from 7 to 48 g/L reduced the cellulose conversion rate by 94%. When product sugars were removed using ultrafiltration with a 10 kDa membrane, the glucose-based conversion increased by 9.5%. Reductions in substrate reactivity with conversion were compared directly by saccharifying PCS and Avicel substrates that had been pre-reacted to different conversions. Reaction of substrate with a pre-conversion of 40% resulted in about 40% reduction in the initial rate of saccharification, relative to fresh substrate with identical cellulose concentration. Overall, glucose inhibition and reduced substrate reactivity appear to be dominant factors, whereas minimal reductions of enzyme activity were observed.

Cellulose microfibril structure: inspirations from plant diversity

NASA Astrophysics Data System (ADS)

Roberts, A. W.

2018-03-01

Cellulose microfibrils are synthesized at the plasma membrane by cellulose synthase catalytic subunits that associate to form cellulose synthesis complexes. Variation in the organization of these complexes underlies the variation in cellulose microfibril structure among diverse organisms. However, little is known about how the catalytic subunits interact to form complexes with different morphologies. We are using an evolutionary approach to investigate the roles of different catalytic subunit isoforms in organisms that have rosette-type cellulose synthesis complexes.

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlatemore » with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. It was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component.« less

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlatemore » with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. Moreover, it was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component.« less

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

DOE PAGES

Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.; ...

2015-10-14

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlatemore » with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. Moreover, it was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component.« less

Colloid, adhesive and release properties of nanoparticular ternary complexes between cationic and anionic polysaccharides and basic proteins like bone morphogenetic protein BMP-2.

PubMed

Petzold, R; Vehlow, D; Urban, B; Grab, A L; Cavalcanti-Adam, E A; Alt, V; Müller, M

2017-03-01

Herein we describe an interfacial local drug delivery system for bone morphogenetic protein 2 (BMP-2) based on coatings of polyelectrolyte complex (PEC) nanoparticles (NP). The application horizon is the functionalization of bone substituting materials (BSM) used for the therapy of systemic bone diseases. Nanoparticular ternary complexes of cationic and anionic polysaccharides and BMP-2 or two further model proteins, respectively, were prepared in dependence of the molar mixing ratio, pH value and of the cationic polysaccharide. As further proteins chymotrypsin (CHY) and papain (PAP) were selected, which served as model proteins for BMP-2 due to similar isoelectric points and molecular weights. As charged polysaccharides ethylenediamine modified cellulose (EDAC) and trimethylammonium modified cellulose (PQ10) were combined with cellulose sulphatesulfate (CS). Mixing diluted cationic and anionic polysaccharide and protein solutions according to a slight either anionic or cationic excess charge colloidal ternary dispersions formed, which were cast onto germanium model substrates by water evaporation. Dynamic light scattering (DLS) demonstrated, that these dispersions were colloidally stable for at least one week. Fourier Transform Infrared (FTIR) showed, that the cast protein loaded PEC NP coatings were irreversibly adhesive at the model substrate in contact to HEPES buffer and solely CHY, PAP and BMP-2 were released within long-term time scale. Advantageously, out of the three proteins BMP-2 showed the smallest initial burst and the slowest release kinetics and around 25% of the initial BMP-2 content were released within 14days. Released BMP-2 showed significant activity in the myoblast cells indicating the ability to regulate the formation of new bone. Therefore, BMP-2 loaded PEC NP are suggested as novel promising tool for the functionalization of BSM used for the therapy of systemic bone diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Efficient recyclable organic solar cells on cellulose nanocrystal substrates with a conducting polymer top electrode deposited by film-transfer lamination

Treesearch

Yinhua Zhou; Talha M. Khan; Jen-Chieh Liu; Canek Fuentes-Hernandez; Jae Won Shim; Ehsan Najafabadi; Jeffrey P. Youngblood; Robert J. Moon; Bernard Kippelen

2014-01-01

We report on efficient solar cells on recyclable cellulose nanocrystal (CNC) substrates with a new device structure wherein polyethylenimine-modified Ag is used as the bottom electron-collecting electrode and high-conductivity poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS, PH1000) is used as the semitransparent top holecollecting electrode. The...

The productive cellulase binding capacity of cellulosic substrates.

PubMed

Karuna, Nardrapee; Jeoh, Tina

2017-03-01

Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases. Productive cellulase binding to cellulose results in hydrolysis and can be quantified by measuring hydrolysis rates. In this study, we measured the productive Trichoderma reesei Cel7A (TrCel7A) binding capacity of five cellulosic substrates from different sources and processing histories. Swollen filter paper and bacterial cellulose had higher productive binding capacities of ∼6 µmol/g while filter paper, microcrystalline cellulose, and algal cellulose had lower productive binding capacities of ∼3 µmol/g. Swelling and regenerating filter paper using phosphoric acid increased the initial accessibility of the reducing ends to TrCel7A from 4 to 6 µmol/g. Moreover, this increase in initial productive binding capacity accounted in large part for the difference in the overall digestibility between filter paper and swollen filter paper. We further demonstrated that an understanding of how the productive binding capacity declines over the course of the hydrolysis reaction has the potential to predict overall saccharification time courses. Biotechnol. Bioeng. 2017;114: 533-542. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Preparation and activity of bubbling-immobilized cellobiase within chitosan-alginate composite.

PubMed

Wang, Fang; Su, Rong-Xin; Qi, Wei; Zhang, Ming-Jia; He, Zhi-Min

2010-01-01

Cellobiase can hydrolyze cellobiose into glucose; it plays a key role in the process of cellulose hydrolysis by reducing the product inhibition. To reuse the enzyme and improve the economic value of cellulosic ethanol, cellobiase was immobilized using sodium alginate and chitosan as carriers by the bubbling method. The immobilization conditions were optimized as follows: enzyme loading of 100 U cellobiase/g carrier, 30 min immobilization, 3.5 wt% sodium alginate, 0.25 wt% chitosan, and 2 wt% calcium chloride. Compared to free enzyme, the immobilized cellobiase had a decreased apparent K(m) and the maximum activity at a lower pH, indicating its higher acidic and thermal stability. The immobilized cellobiase was further tested in the hydrolysis of cellobiose and various cellulosic substrates (microcrystalline cellulose, filter paper, and ammonia-pretreated corn cobs). Together with cellulases, the immobilized cellobiase converted the cellulosic substrates into glucose with the rate and extent similar to the free enzyme.

Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hiras, Jennifer; Wu, Yu -Wei; Deng, Kai

Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacterium Thermobispora bispora that were highly abundant in the most active consortium. Among the cellulasesmore » from T. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite of T. bispora hydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered.« less

Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

DOE PAGES

Hiras, Jennifer; Wu, Yu -Wei; Deng, Kai; ...

2016-08-23

Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacterium Thermobispora bispora that were highly abundant in the most active consortium. Among the cellulasesmore » from T. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite of T. bispora hydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered.« less

Modeling enzymatic hydrolysis of lignocellulosic substrates using confocal fluorescence microscopy I: filter paper cellulose.

PubMed

Luterbacher, Jeremy S; Moran-Mirabal, Jose M; Burkholder, Eric W; Walker, Larry P

2015-01-01

Enzymatic hydrolysis is one of the critical steps in depolymerizing lignocellulosic biomass into fermentable sugars for further upgrading into fuels and/or chemicals. However, many studies still rely on empirical trends to optimize enzymatic reactions. An improved understanding of enzymatic hydrolysis could allow research efforts to follow a rational design guided by an appropriate theoretical framework. In this study, we present a method to image cellulosic substrates with complex three-dimensional structure, such as filter paper, undergoing hydrolysis under conditions relevant to industrial saccharification processes (i.e., temperature of 50°C, using commercial cellulolytic cocktails). Fluorescence intensities resulting from confocal images were used to estimate parameters for a diffusion and reaction model. Furthermore, the observation of a relatively constant bound enzyme fluorescence signal throughout hydrolysis supported our modeling assumption regarding the structure of biomass during hydrolysis. The observed behavior suggests that pore evolution can be modeled as widening of infinitely long slits. The resulting model accurately predicts the concentrations of soluble carbohydrates obtained from independent saccharification experiments conducted in bulk, demonstrating its relevance to biomass conversion work. © 2014 Wiley Periodicals, Inc.

Engineering Cellulases for Biorefinery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-06-27

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Thermostable Cellulases: Why & How?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-04-19

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Plasma-enhanced synthesis of green flame retardant cellulosic materials

NASA Astrophysics Data System (ADS)

Totolin, Vladimir

The natural fiber-containing fabrics and composites are more environmentally friendly, and are used in transportation (automobiles, aerospace), military applications, construction industries (ceiling paneling, partition boards), consumer products, etc. Therefore, the flammability characteristics of the composites based on polymers and natural fibers play an important role. This dissertation presents the development of plasma assisted - green flame retardant coatings for cellulosic substrates. The overall objective of this work was to generate durable flame retardant treatment on cellulosic materials. In the first approach sodium silicate layers were pre-deposited onto clean cotton substrates and cross linked using low pressure, non-equilibrium oxygen plasma. A statistical design of experiments was used to optimize the plasma parameters. The modified cotton samples were tested for flammability using an automatic 45° angle flammability test chamber. Aging tests were conducted to evaluate the coating resistance during the accelerated laundry technique. The samples revealed a high flame retardant behavior and good thermal stability proved by thermo-gravimetric analysis. In the second approach flame retardant cellulosic materials have been produced using a silicon dioxide (SiO2) network coating. SiO 2 network armor was prepared through hydrolysis and condensation of the precursor tetraethyl orthosilicate (TEOS), prior coating the substrates, and was cross linked on the surface of the substrates using atmospheric pressure plasma (APP) technique. Due to protection effects of the SiO2 network armor, the cellulosic based fibers exhibit enhanced thermal properties and improved flame retardancy. In the third approach, the TEOS/APP treatments were extended to linen fabrics. The thermal analysis showed a higher char content and a strong endothermic process of the treated samples compared with control ones, indicating a good thermal stability. Also, the surface analysis proved the existence of the silica-based coatings on all treated cellulosic substrates after intense ultrasound washes. The results obtained in this work allow us to conclude that silica-based coatings used in conjunction with plasma processes have high potential to obtain green flame retardant cellulosic materials with potential applications in the development of upholstered furniture, clothing and military applications.

An in-depth understanding of biomass recalcitrance using natural poplar variants as the feedstock

DOE PAGES

Meng, Xianzhi; Pu, Yunqiao; Yoo, Chang Geun; ...

2016-12-12

Here, in an effort to better understand the biomass recalcitrance, six natural poplar variants were selected as feedstocks based on previous sugar release analysis. Compositional analysis and physicochemical characterizations of these poplars were performed and the correlations between these physicochemical properties and enzymatic hydrolysis yield were investigated. Gel permeation chromatography (GPC) and 13C solid state NMR were used to determine the degree of polymerization (DP) and crystallinity index (CrI) of cellulose, and the results along with the sugar release study indicated that cellulose DP likely played a more important role in enzymatic hydrolysis. Simons’ stain revealed that the accessible surface area of substrate significantly varied among these variants from 17.3 to 33.2 mg gmore » $$–1\\atop{biomass}$$ as reflected by dye adsorption, and cellulose accessibility was shown as one of the major factors governing substrates digestibility. HSQC and 31P NMR analysis detailed the structural features of poplar lignin variants. Overall, cellulose relevant factors appeared to have a stronger correlation with glucose release, if any, than lignin structural features. Lignin structural features, such as a phenolic hydroxyl group and the ratio of syringyl and guaiacyl (S/G), were found to have a more convincing impact on xylose release. Low lignin content, low cellulose DP, and high cellulose accessibility generally favor enzymatic hydrolysis; however, recalcitrance cannot be simply judged on any single substrate factor.« less

An in-depth understanding of biomass recalcitrance using natural poplar variants as the feedstock

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meng, Xianzhi; Pu, Yunqiao; Yoo, Chang Geun

Here, in an effort to better understand the biomass recalcitrance, six natural poplar variants were selected as feedstocks based on previous sugar release analysis. Compositional analysis and physicochemical characterizations of these poplars were performed and the correlations between these physicochemical properties and enzymatic hydrolysis yield were investigated. Gel permeation chromatography (GPC) and 13C solid state NMR were used to determine the degree of polymerization (DP) and crystallinity index (CrI) of cellulose, and the results along with the sugar release study indicated that cellulose DP likely played a more important role in enzymatic hydrolysis. Simons’ stain revealed that the accessible surface area of substrate significantly varied among these variants from 17.3 to 33.2 mg gmore » $$–1\\atop{biomass}$$ as reflected by dye adsorption, and cellulose accessibility was shown as one of the major factors governing substrates digestibility. HSQC and 31P NMR analysis detailed the structural features of poplar lignin variants. Overall, cellulose relevant factors appeared to have a stronger correlation with glucose release, if any, than lignin structural features. Lignin structural features, such as a phenolic hydroxyl group and the ratio of syringyl and guaiacyl (S/G), were found to have a more convincing impact on xylose release. Low lignin content, low cellulose DP, and high cellulose accessibility generally favor enzymatic hydrolysis; however, recalcitrance cannot be simply judged on any single substrate factor.« less

Xylanase supplementation on enzymatic saccharification of dilute acid pretreated poplars at different severities

Treesearch

Chao Zhang; Xinshu Zhuang; Zhao Jiang Wang; Fred Matt; Franz St. John; J.Y. Zhu

2013-01-01

Three pairs of solid substrates from dilute acid pretreatment of two poplar wood samples were enzymatically hydrolyzed by cellulase preparations supplemented with xylanase. Supplementation of xylanase improved cellulose saccharification perhaps due to improved cellulose accessibility by xylan hydrolysis. Total xylan removal directly affected enzymatic cellulose...

Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hiras, Jennifer; Wu, Yu-Wei; Deng, Kai

ABSTRACT Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacteriumThermobispora bisporathat were highly abundant in the most active consortium. Among the cellulases fromT. bispora,more » the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite ofT. bisporahydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered. IMPORTANCECellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose generated communities whose soluble enzymes exhibit differential abilities to hydrolyze crystalline cellulose. Comparative proteomics of these communities identified a protein of glycoside hydrolase family 12 (GH12), a family of proteins previously observed to primarily hydrolyze soluble substrates, as a candidate that accounted for some of the differences in hydrolytic activities. Heterologous expression confirmed that the GH12 protein identified by proteomics was active on crystalline cellulose and hydrolyzed cellulose by a random mechanism, in contrast to most cellulases that act on the crystalline polymer in a processive mechanism.« less

Thermal stress in flexible interdigital transducers with anisotropic electroactive cellulose substrates

NASA Astrophysics Data System (ADS)

Yoon, Sean J.; Kim, Jung Woong; Kim, Hyun Chan; Kang, Jinmo; Kim, Jaehwan

2017-12-01

Thermal stress in flexible interdigital transducers a reliability concern in the development of flexible devices, which may lead to interface delamination, stress voiding and plastic deformation. In this paper, a mathematical model is presented to investigate the effect of material selections on the thermal stress in interdigital transducers. We modified the linear relationships in the composite materials theory with the effect of high curvature, anisotropic substrate and small substrate thickness. We evaluated the thermal stresses of interdigital transducers, fabricated with various electrodes, insulators and substrate materials for the comparison. The results show that, among various insulators, organic polymer developed the highest stress level while oxide showed the lowest stress level. Aluminium shows a higher stress level and curvature as an electrode than gold. As substrate materials, polyimide and electroactive cellulose show similar stress levels except the opposite sign convention to each other. Polyimide shows positive curvatures while electroactive cellulose shows negative curvatures, which is attributed to the stress and thermal expansion state of the metal/insulator composite. The results show that the insulator is found to be responsible for the confinement across the metal lines while the substrate is responsible for the confinement along the metal lines.

Comparative study on gamma irradiation and cold plasma pretreatment for a cellulosic substrate modification with phenolic compounds

NASA Astrophysics Data System (ADS)

Irimia, Anamaria; Ioanid, Ghiocel Emil; Zaharescu, Traian; Coroabă, Adina; Doroftei, Florica; Safrany, Agnes; Vasile, Cornelia

2017-01-01

The efficiency of the activation of the cellulose/chitin mix substrate by cold plasma or γ-radiation exposure in order to modify it with bioactive compounds was studied. The eugenol or vegetable oils such as grape seed oil and rosehip seed oil have been grafted onto activated substrate. The examination of modified cellulose/chitin mix substrate by ATR-FTIR spectroscopy, X-ray photoelectron spectroscopy and scanning electron microscopy confirms that the structural and morphological changes took place in both cases. The grafting degrees of the surface layer estimated from XPS data varied from 31.1% to 58.7% for air cold plasma activation and from 9.7% to 22.8% for γ-irradiation treatment. They depend both on bioactive compound used and procedure of substrate activation. Higher grafting degree are obtain by using vegetable oils than in the case of modification with eugenol and the air cold plasma activation seems to be much efficient than γ-irradiation. By grafting the polymeric substrate with bioactive compounds, antimicrobial and antioxidant properties have been conferred. Such materials can be considered promising for food packaging applications and medical textiles and also the applied procedures are environmental friendly ones.

The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kruus, K.; Wu, J.H.D.; Lua, A.C.

1995-09-26

Enzymatic cellulose degradation is a heterogeneous reaction requiring binding of soluble cellulase molecules to the solid substrate. Based on our studies of the cellulase complex of Clostridium thermocellum (the cellulosome), we have previously proposed that such binding can be brought about by a special {open_quotes}anchorage subunit.{close_quotes} In this {open_quotes}anchor-enzyme{close_quotes} model, CipA (a major subunit of the cellulosome) enhances the activity of CelS (the most abundant catalytic subunit of the cellulosome) by anchoring it to the cellulose surface. We have subsequently reported that CelS contains a conserved duplicated sequence at its C terminus and the CipA contains nine repeated sequences withmore » a cellulose binding domain (CBD) in between the second and third repeats. In this work, we reexamined the anchor-enzyme mechanism by using recombinant CelS (rCelS) and various CipA domains, CBD, R3 (the repeat next to CBD), and CBD/R3, expressed in Escherichia coli. As analyzed by non-denaturing gel electrophoresis, rCelS, through its conserved duplicated sequence, formed a stable complex with R3 or CBD/R3 but not with CBD. Although R3 or CBD alone did not affect the binding of rCelS to cellulose, such binding was dependent on CBD/R3, indicating the anchorage role of CBD/R3. Such anchorage apparently increased the rCelS activity toward crystalline cellulose. These results substantiate the proposed anchor-enzyme model and the expected roles of individual CipA domains and the conserved duplicated sequence of CelS.« less

Highly transparent, low-haze, hybrid cellulose nanopaper as electrodes for flexible electronics.

PubMed

Xu, Xuezhu; Zhou, Jian; Jiang, Long; Lubineau, Gilles; Ng, Tienkhee; Ooi, Boon S; Liao, Hsien-Yu; Shen, Chao; Chen, Long; Zhu, J Y

2016-06-16

Paper is an excellent candidate to replace plastics as a substrate for flexible electronics due to its low cost, renewability and flexibility. Cellulose nanopaper (CNP), a new type of paper made of nanosized cellulose fibers, is a promising substrate material for transparent and flexible electrodes due to its potentially high transparency and high mechanical strength. Although CNP substrates can achieve high transparency, they are still characterized by high diffuse transmittance and small direct transmittance, resulting in high optical haze of the substrates. In this study, we proposed a simple methodology for large-scale production of high-transparency, low-haze CNP comprising both long cellulose nanofibrils (CNFs) and short cellulose nanocrystals (CNCs). By varying the CNC/CNF ratio in the hybrid CNP, we could tailor its total transmittance, direct transmittance and diffuse transmittance. By increasing the CNC content, the optical haze of the hybrid CNP could be decreased and its transparency could be increased. The direct transmittance and optical haze of the CNP were 75.1% and 10.0%, respectively, greatly improved from the values of previously reported CNP (31.1% and 62.0%, respectively). Transparent, flexible electrodes were fabricated by coating the hybrid CNP with silver nanowires (AgNWs). The electrodes showed a low sheet resistance (minimum 1.2 Ω sq(-1)) and a high total transmittance (maximum of 82.5%). The electrodes were used to make a light emitting diode (LED) assembly to demonstrate their potential use in flexible displays.

Comparative analysis of the digestibility of sewage fine sieved fraction and hygiene paper produced from virgin fibers and recycled fibers.

PubMed

Ghasimi, Dara S M; Zandvoort, Marcel H; Adriaanse, Michiel; van Lier, Jules B; de Kreuk, Merle

2016-07-01

Sewage fine sieved fraction (FSF) is a heterogeneous substrate consisting of mainly toilet paper fibers sequestered from municipal raw sewage by a fine screen. In earlier studies, a maximum biodegradation of 62% and 57% of the sewage FSF was found under thermophilic (55°C) and mesophilic (35°C) conditions, respectively. In order to research this limited biodegradability of sewage FSF, this study investigates the biodegradation of different types of cellulosic fibers-based hygiene papers including virgin fibers based toilet paper (VTP), recycled fiber based toilet paper (RTP), virgin pulp for paper production (VPPP) as a raw material, as well as microcrystalline cellulose (MCC) as a kind of fiberless reference material. The anaerobic biodegradation or digestibility tests were conducted under thermophilic and mesophilic conditions. Results of the experiments showed different biomethane potential (BMP) values for each tested cellulose fiber-based substrate, which might be associated with the physical characteristics of the fibers, type of pulping, presence of lignin encrusted fibers, and/or the presence of additive chemicals and refractory compounds. Higher hydrolysis rates (Kh), higher specific methane production rates (SMPR) and shorter required incubation times to achieve 90% of the BMP (t90%CH4), were achieved under thermophilic conditions for all examined substrates compared to the mesophilic ones. Furthermore, the biodegradability of all employed cellulose fiber-based substrates was in the same range, 38-45%, under both conditions and less than the observed FSF biodegradability, i.e. 57-62%. MCC achieved the highest BMP and biodegradability, 86-91%, among all cellulosic substrates. Copyright © 2016 Elsevier Ltd. All rights reserved.

The role of inoculum's origin on the methane yield of different substrates in biochemical methane potential (BMP) tests.

PubMed

Koch, Konrad; Lippert, Thomas; Drewes, Jörg E

2017-11-01

The impact of the inoculum's origin on the methane yield in Biochemical Methane Potential (BMP) tests was investigated. The three most commonly applied inocula were chosen, originating from (i) a digester of a wastewater treatment plant, (ii) an agricultural biogas plant treating manure and energy crops, and (iii) a biowaste treatment plant. The performance of each inoculum was tested with four different substrates, namely sewage sludge, dried whole crop maize, food waste, and microcrystalline cellulose as a typical reference material. The results revealed that the choice of inoculum had no significant impact on the specific methane yield of the tested substrates except for cellulose. Still, the specific methane production rate was significantly influenced by the choice of the inoculum especially for sewage sludge, but also for food waste and cellulose, whereas it became clear that an inoculum adapted to a substrate is beneficial for a speedy digestion. Copyright © 2017 Elsevier Ltd. All rights reserved.

Utilization of agricultural wastes for production of ethanol. Progress report, October 1979-May 1980

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, B.

1980-05-01

The project proposes to develop methods to utilize agricultural wastes, especially cottonseed hulls and peanut shells to produce ethanol. Initial steps will involve development of methods to break down cellulose to a usable form of substrates for chemical or biological digestion. The process of ethanol production will consist of (a) preparatory step to separate fibrous (cellulose) and non-fibrous (non-cellulosic compounds). The non-cellulosic residues which may include grains, fats or other substrates for alcoholic fermentation. The fibrous residues will be first pre-treated to digest cellulose with acid, alkali, and sulfur dioxide gas or other solvents. (b) The altered cellulose will bemore » digested by suitable micro-organisms and cellulose enzymes before alcoholic fermentation. The digester and fermentative unit will be specially designed to develop a prototype for pilot plant for a continuous process. The first phase of the project will be devoted toward screening of a suitable method for cellulose modification, separation of fibrous and non-fibrous residues, the micro-organism and enzyme preparations. Work is in progress on: the effects of various microorganisms on the degree of saccharification; the effects of higher concentrations of acids, alkali, and EDTA on efficiency of microbial degradation; and the effects of chemicals on enzymatic digestion.« less

Application of the 2-cyanoacetamide method for spectrophotometric assay of cellulase enzyme activity

USDA-ARS?s Scientific Manuscript database

Cellulose is the most abundant form of carbon on the planet. Breakdown of cellulose microfibrils in the plant cell wall is a means by which microbes gain ingress into their respective hosts. Cellulose degradation is also important for global carbon recycling and is the primary substrate for producti...

The structures of native celluloses, and the origin of their variability

Treesearch

R. H. Atalla

1999-01-01

The structures of native celluloses have traditionally been presented in terms of two-domain models consisting of crystalline and non-crystalline fractions. Such models have been of little help in advancing understanding of enzyme-substrate interactions. In this report we first address issues that complicate characterization of the structure of native celluloses...

Recombinant hosts suitable for simultaneous saccharification and fermentation

DOEpatents

Ingram, Lonnie O'Neal; Zhou, Shengde

2007-06-05

The invention provides recombinant host cells containing at least one heterologous polynucleotide encoding a polysaccharase under the transcriptional control of a surrogate promoter capable of increasing the expression of the polysaccharase. In addition, the invention further provides such hosts with genes encoding secretory protein/s to facilitate the secretion of the expressed polysaccharase. Preferred hosts of the invention are ethanologenic and capable of carrying out simultaneous saccharification fermentation resulting in the production of ethanol from complex cellulose substrates.

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

PubMed

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-12-09

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A*

PubMed Central

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-01-01

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. PMID:27780868

Functional analysis of the Glucan Degradation Locus (GDL) in Caldicellulosiruptor bescii reveals essential roles of component glycoside hydrolases in plant biomass deconstruction.

PubMed

Conway, Jonathan M; McKinley, Bennett S; Seals, Nathaniel L; Hernandez, Diana; Khatibi, Piyum A; Poudel, Suresh; Giannone, Richard J; Hettich, Robert L; Williams-Rhaesa, Amanda M; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

2017-10-06

The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but one that can be exploited for conversion of lignocellulosic feedstocks into bio-based fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The Glucan Degradation Locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative post-translational modifying enzymes, in addition to multi-domain, multi-functional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation, as compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GHs in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extent to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomasses (switchgrass or poplar) was examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture and not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization. Importance The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures between 70-78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii , the nuanced, substrate-specific, in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multi-domain cellulases in C. bescii , working in conjunction with the aggregate, secreted enzyme inventory, as the key to the plant biomass degradation ability by this extreme thermophile. Copyright © 2017 American Society for Microbiology.

Enzymatic Hydrolysis of Cellulosic Materials to Fermentable Sugars for the Production of Ethanol

DTIC Science & Technology

1980-10-12

Pretreatment . • . . • . . . . . • . . . 19 5. Enzyme Production (Prepilot Scale) • . • ·. • • . . . . . • • • • 29 6. Saccharification (Prepilot...hour hydrolysis of 15% substrate. TASK II 1. Poplar shavings were compression mill pretreated most effectively at an initial moisture content of 12...concentration, pretreatment of.cellulose substrates, glucose syrup concentration, temperature, acidity, residence time, recovery of enzymes, fungi, glucose

Cellobiohydrolase (CBH) Activity Assays.

PubMed

Sharma, Hem Kanta; Qin, Wensheng; Xu, Chunbao Charles

2018-01-01

Cellulosic biomass is the most abundant biopolymer on the earth. It has great potential to quench the thirst of liquid energy by producing biofuels and thus help to mitigate human reliance on fossil fuels. Although several cellulase activity assay methods have been used to disintegrate the glycosidic bonds, the appropriate selection of substrates and synergistic involvement of multiple enzymes in hydrolytic activity is not yet fully understood. The proper quantification of hydrolytic enzymes and hydrolysates is challenging because of the heterogeneity of cellulose, changes in enzyme-substrate ratio and the presence of some inhibitory compounds like cellobiose and cellodextran. In the glycosyl hydrolase (GH) family, cellobiohydrolase (CBH) is expected to disrupt the crystalline cellulose and release the sugar molecules. Several methods have been proposed for CBH assay with slight modification in substrate and quantification of hydrolysates. However, the Avicel method is still considered as the most promising and efficient hydrolytic technique so far. The most commonly used CBH assays including Avicel and other recent methods for proper quantification are outlined in this chapter. Also a qualitative screening of CBH producing bacteria using carboxymethyl cellulose (CMC) agar plates is described.

Saccharification of rice straw by cellulase from a local Trichoderma harzianum SNRS3 for biobutanol production.

PubMed

Rahnama, Nooshin; Foo, Hooi Ling; Abdul Rahman, Nor Aini; Ariff, Arbakariya; Md Shah, Umi Kalsom

2014-12-12

Rice straw has shown to be a promising agricultural by-product in the bioconversion of biomass to value-added products. Hydrolysis of cellulose, a main constituent of lignocellulosic biomass, is a requirement for fermentable sugar production and its subsequent bioconversion to biofuels such as biobutanol. The high cost of commercial enzymes is a major impediment to the industrial application of cellulases. Therefore, the use of local microbial enzymes has been suggested. Trichoderma harzianum strains are potential CMCase and β-glucosidase producers. However, few researches have been reported on cellulase production by T. harzianum and the subsequent use of the crude cellulase for cellulose enzymatic hydrolysis. For cellulose hydrolysis to be efficiently performed, the presence of the whole set of cellulase components including exoglucanase, endoglucanase, and β-glucosidase at a considerable concentration is required. Biomass recalcitrance is also a bottleneck in the bioconversion of agricultural residues to value-added products. An effective pretreatment could be of central significance in the bioconversion of biomass to biofuels. Rice straw pretreated using various concentrations of NaOH was subjected to enzymatic hydrolysis. The saccharification of rice straw pretreated with 2% (w/v) NaOH using crude cellulase from local T. harzianum SNRS3 resulted in the production of 29.87 g/L reducing sugar and a yield of 0.6 g/g substrate. The use of rice straw hydrolysate as carbon source for biobutanol fermentation by Clostridium acetobutylicum ATCC 824 resulted in an ABE yield, ABE productivity, and biobutanol yield of 0.27 g/g glucose, 0.04 g/L/h and 0.16 g/g glucose, respectively. As a potential β-glucosidase producer, T. harzianum SNRS3 used in this study was able to produce β-glucosidase at the activity of 173.71 U/g substrate. However, for cellulose hydrolysis to be efficient, Filter Paper Activity at a considerable concentration is also required to initiate the hydrolytic reaction. According to the results of our study, FPase is a major component of cellulose hydrolytic enzyme complex system and the reducing sugar rate-limiting enzyme. Our study revealed that rice straw hydrolysate served as a potential substrate for biobutanol production and FPase is a rate-limiting enzyme in saccharification.

Molecular mechanism of lytic polysaccharide monooxygenases.

PubMed

Hedegård, Erik Donovan; Ryde, Ulf

2018-04-21

The lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes that can enhance polysaccharide depolymerization through an oxidative mechanism and hence boost generation of biofuel from e.g. cellulose. By employing density functional theory in a combination of quantum mechanics and molecular mechanics (QM/MM), we report a complete description of the molecular mechanism of LPMOs. The QM/MM scheme allows us to describe all reaction steps with a detailed protein environment and we show that this is necessary. Several active species capable of abstracting a hydrogen from the substrate have been proposed previously and starting from recent crystallographic work on a substrate-LPMO complex, we investigate previously suggested paths as well as new ones. We describe the generation of the reactive intermediates, the abstraction of a hydrogen atom from the polysaccharide substrate, as well as the final recombination step in which OH is transferred back to the substrate. We show that a superoxo [CuO 2 ] + complex can be protonated by a nearby histidine residue (suggested by recent mutagenesis studies and crystallographic work) and, provided an electron source is available, leads to formation of an oxyl-complex after cleavage of the O-O bond and dissociation of water. The oxyl complex either reacts with the substrate or is further protonated to a hydroxyl complex. Both the oxyl and hydroxyl complexes are also readily generated from a reaction with H 2 O 2 , which was recently suggested to be the true co-substrate, rather than O 2 . The C-H abstraction by the oxyl and hydroxy complexes is overall favorable with activation barriers of 69 and 94 kJ mol -1 , compared to the much higher barrier (156 kJ mol -1 ) obtained for the copper-superoxo species. We obtain good structural agreement for intermediates for which structural data are available and the estimated reaction energies agree with experimental rate constants. Thus, our suggested mechanism is the most complete to date and concur with available experimental evidence.

Examining an underappreciated control on lignin decomposition in soils? Effects of reactive manganese species on intact plant cell walls

NASA Astrophysics Data System (ADS)

Keiluweit, M.; Bougoure, J.; Pett-Ridge, J.; Kleber, M.; Nico, P. S.

2011-12-01

Lignin comprises a dominant proportion of carbon fluxes into the soil (representing up to 50% of plant litter and roots). Two lines of evidence suggest that manganese (Mn) acts as a strong controlling factor on the residence time of lignin in soil ecosystems. First, Mn content is highly correlated with litter decomposition in temperate and boreal forest soil ecosystems and, second, microbial agents of lignin degradation have been reported to rely on reactive Mn(III)-complexes to specifically oxidize lignin. However, few attempts have been made to isolate the mechanisms responsible for the apparent Mn-dependence of lignin decomposition in soils. Here we tested the hypothesis that Mn(III)-oxalate complexes may act as a perforating 'pretreatment' for structurally intact plant cell walls. We propose that these diffusible oxidizers are small enough to penetrate and react with non-porous ligno-cellulose in cell walls. This process was investigated by reacting single Zinnia elegans tracheary elements with Mn(III)-oxalate complexes in a continuous flow-through microreactor. The uniformity of cultured tracheary elements allowed us to examine Mn(III)-induced changes in cell wall chemistry and ultrastructure on the micro-scale using fluorescence and electron microscopy as well as synchrotron-based infrared and X-ray spectromicroscopy. Our results show that Mn(III)-complexes substantially oxidize specific lignin components of the cell wall, solubilize decomposition products, severely undermine the cell wall integrity, and cause cell lysis. We conclude that Mn(III)-complexes induce oxidative damage in plant cell walls that renders ligno-cellulose substrates more accessible for microbial lignin- and cellulose-decomposing enzymes. Implications of our results for the rate limiting impact of soil Mn speciation and availability on litter decomposition in forest soils will be discussed.

A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants.

PubMed Central

Amor, Y; Haigler, C H; Johnson, S; Wainscott, M; Delmer, D P

1995-01-01

Sucrose synthase (SuSy; EC 2.4.1.13; sucrose + UDP reversible UDPglucose + fructose) has always been studied as a cytoplasmic enzyme in plant cells where it serves to degrade sucrose and provide carbon for respiration and synthesis of cell wall polysaccharides and starch. We report here that at least half of the total SuSy of developing cotton fibers (Gossypium hirsutum) is tightly associated with the plasma membrane. Therefore, this form of SuSy might serve to channel carbon directly from sucrose to cellulose and/or callose synthases in the plasma membrane. By using detached and permeabilized cotton fibers, we show that carbon from sucrose can be converted at high rates to both cellulose and callose. Synthesis of cellulose or callose is favored by addition of EGTA or calcium and cellobiose, respectively. These findings contrast with the traditional observation that when UDPglucose is used as substrate in vitro, callose is the major product synthesized. Immunolocalization studies show that SuSy can be localized at the fiber surface in patterns consistent with the deposition of cellulose or callose. Thus, these results support a model in which SuSy exists in a complex with the beta-glucan synthases and serves to channel carbon from sucrose to glucan. Images Fig. 1 Fig. 3 Fig. 4 PMID:7568131

Cellulose Triacetate Dielectric Films For Capacitors

NASA Technical Reports Server (NTRS)

Yen, Shiao-Ping S.; Jow, T. Richard

1994-01-01

Cellulose triacetate investigated for use as dielectric material in high-energy-density capacitors for pulsed-electrical-power systems. Films of cellulose triacetate metalized on one or both sides for use as substrates for electrodes and/or as dielectrics between electrodes in capacitors. Used without metalization as simple dielectric films. Advantages include high breakdown strength and self-healing capability.

Single-molecule Imaging Analysis of Elementary Reaction Steps of Trichoderma reesei Cellobiohydrolase I (Cel7A) Hydrolyzing Crystalline Cellulose Iα and IIII*

PubMed Central

Shibafuji, Yusuke; Nakamura, Akihiko; Uchihashi, Takayuki; Sugimoto, Naohisa; Fukuda, Shingo; Watanabe, Hiroki; Samejima, Masahiro; Ando, Toshio; Noji, Hiroyuki; Koivula, Anu; Igarashi, Kiyohiko; Iino, Ryota

2014-01-01

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes. PMID:24692563

Processing and fractional utilization of lignocellulosic substrates by "pure" and "natural and defined mixed" culture C.TM1, C.SA IV and rumen mixed culture consortia CD.

PubMed

Sankar, M; Chandra, T S

2003-01-01

A detailed analysis was made of chemical fractions of common agro-residues before and after pretreatment (alkali and hydrogen peroxide), and the selective utilization of components such as WSS, EBS, TSS, lignin, cellulose and hemicellulose by pure and mixed cultures of cellulolytic and xylanolytic Clostridia was monitored and correlated with the organisms' enzyme activity. For all cultures pretreatment gave higher utilization of hemicellulose and cellulose fractions; hydrogen peroxide pretreatment was more effective than NaOH treatment. Lignin utilization was not very significant even on pretreatment. C.TM1 and C.SA IV utilized hemicellulose and cellulose better than mixed cultures in selected substrates. These results help to determine the substrate composition, pretreatment conditions and enzyme system of the organism needed when designing an inoculum for agricultural waste treatment processes such as composting or biogas generation.

Biocompatible 3D SERS substrate for trace detection of amino acids and melamine

NASA Astrophysics Data System (ADS)

Satheeshkumar, Elumalai; Karuppaiya, Palaniyandi; Sivashanmugan, Kundan; Chao, Wei-Ting; Tsay, Hsin-Sheng; Yoshimura, Masahiro

2017-06-01

A novel, low-cost and biocompatible three-dimensional (3D) substrate for surface-enhanced Raman spectroscopy (SERS) is fabricated using gold nanoparticles (AuNPs) loaded on cellulose paper for detection of amino acids and melamine. Dysosma pleiantha rhizome (Dp-Rhi) capped AuNPs (Dp-Rhi_AuNPs) were prepared by in situ using aqueous extract of Dp-Rhi and in situ functionalized Dp-Rhi on AuNPs surface was verified by Fourier transform infrared spectroscopy and zeta potentials analysis shows a negative (- 18.4 mV) surface charges, which confirm that presence of Dp-Rhi on AuNPs. The biocompatibility of Dp-Rhi_AuNPs is also examined by cell viability of FaDu cells using MTS assay and compared to control group. In conclusion, the SERS performance of AuNPs@cellulose paper substrates were systematically demonstrated and examined with different excitation wavelengths (i.e. 532, 632.8 and 785 nm lasers) and the as-prepared 3D substrates provided an enhancement factor approaching 7 orders of magnitude compared with conventional Raman intensity using para-nitrothiophenol (p-NTP), para-aminothiophenol (p-ATP) and para-mercaptobenzoic acid (p-MBA) as probe molecules. The strong electromagnetic effect was generated at the interface of AuNPs and pre-treated roughened cellulose paper is also investigated by simulation in which the formation of possible Raman hot-spot zone in fiber-like microstructure of cellulose paper decorated with AuNPs. Notably, with optimized condition of as-prepared 3D AuNPs@cellulose paper is highly sensitive in the SERS detection of aqueous tyrosine (10- 10 M) and melamine (10- 9 M).

Improvement of pesticide adsorption capacity of cellulose fibre by high-energy irradiation-initiated grafting of glycidyl methacrylate

NASA Astrophysics Data System (ADS)

Takács, Erzsébet; Wojnárovits, László; Koczog Horváth, Éva; Fekete, Tamás; Borsa, Judit

2012-09-01

Cellulose as a renewable raw material was used for preparation of adsorbent of organic impurities in wastewater treatment. Hydrophobic surface of cellulose substrate was developed by grafting glycidyl methacrylate in simultaneous grafting using gamma irradiation initiation. Water uptake of cellulose significantly decreased while adsorption of phenol and a pesticide molecule (2,4-dichlorophenoxyacetic acid: 2,4-D) increased upon grafting. Adsorption equilibrium data fitted the Freundlich isotherm for both solutes.

Homofermentative production of optically pure L-lactic acid from xylose by genetically engineered Escherichia coli B.

PubMed

Zhao, Jinfang; Xu, Liyuan; Wang, Yongze; Zhao, Xiao; Wang, Jinhua; Garza, Erin; Manow, Ryan; Zhou, Shengde

2013-06-07

Polylactic acid (PLA), a biodegradable polymer, has the potential to replace (at least partially) traditional petroleum-based plastics, minimizing "white pollution". However, cost-effective production of optically pure L-lactic acid is needed to achieve the full potential of PLA. Currently, starch-based glucose is used for L-lactic acid fermentation by lactic acid bacteria. Due to its competition with food resources, an alternative non-food substrate such as cellulosic biomass is needed for L-lactic acid fermentation. Nevertheless, the substrate (sugar stream) derived from cellulosic biomass contains significant amounts of xylose, which is unfermentable by most lactic acid bacteria. However, the microorganisms that do ferment xylose usually carry out heterolactic acid fermentation. As a result, an alternative strain should be developed for homofermentative production of optically pure L-lactic acid using cellulosic biomass. In this study, an ethanologenic Escherichia coli strain, SZ470 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA), was reengineered for homofermentative production of L-lactic acid from xylose (1.2 mole xylose = > 2 mole L-lactic acid), by deleting the alcohol dehydrogenase gene (adhE) and integrating the L-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici. The resulting strain, WL203, was metabolically evolved further through serial transfers in screw-cap tubes containing xylose, resulting in the strain WL204 with improved anaerobic cell growth. When tested in 70 g L-1 xylose fermentation (complex medium), WL204 produced 62 g L-1 L-lactic acid, with a maximum production rate of 1.631 g L-1 h-1 and a yield of 97% based on xylose metabolized. HPLC analysis using a chiral column showed that an L-lactic acid optical purity of 99.5% was achieved by WL204. These results demonstrated that WL204 has the potential for homofermentative production of L-lactic acid using cellulosic biomass derived substrates, which contain a significant amount of xylose.

Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid

DOE PAGES

Shiga, Tânia M.; Xiao, Weihua; Yang, Haibing; ...

2017-12-27

The crystallinity of cellulose is a principal factor limiting the efficient hydrolysis of biomass to fermentable sugars or direct catalytic conversion to biofuel components. We evaluated the impact of TFA-induced gelatinization of crystalline cellulose on enhancement of enzymatic digestion and catalytic conversion to biofuel substrates. Low-temperature swelling of cotton linter cellulose in TFA at subzero temperatures followed by gentle heating to 55 degrees C dissolves the microfibril structure and forms composites of crystalline and amorphous gels upon addition of ethanol. The extent of gelatinization of crystalline cellulose was determined by reduction of birefringence in darkfield microscopy, loss of X-ray diffractability,more » and loss of resistance to acid hydrolysis. Upon freeze-drying, an additional degree of crystallinity returned as mostly cellulose II. Both enzymatic digestion with a commercial cellulase cocktail and maleic acid/AlCl3-catalyzed conversion to 5-hydroxymethylfurfural and levulinic acid were markedly enhanced with the low-temperature swollen cellulose. Only small improvements in rates and extent of hydrolysis and catalytic conversion were achieved upon heating to fully dissolve cellulose. Low-temperature swelling of cellulose in TFA substantially reduces recalcitrance of crystalline cellulose to both enzymatic digestion and catalytic conversion. In a closed system to prevent loss of fluorohydrocarbons, the relative ease of recovery and regeneration of TFA by distillation makes it a potentially useful agent in large-scale deconstruction of biomass, not only for enzymatic depolymerization but also for enhancing rates of catalytic conversion to biofuel components and useful bio-products.« less

Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiga, Tânia M.; Xiao, Weihua; Yang, Haibing

The crystallinity of cellulose is a principal factor limiting the efficient hydrolysis of biomass to fermentable sugars or direct catalytic conversion to biofuel components. We evaluated the impact of TFA-induced gelatinization of crystalline cellulose on enhancement of enzymatic digestion and catalytic conversion to biofuel substrates. Low-temperature swelling of cotton linter cellulose in TFA at subzero temperatures followed by gentle heating to 55 degrees C dissolves the microfibril structure and forms composites of crystalline and amorphous gels upon addition of ethanol. The extent of gelatinization of crystalline cellulose was determined by reduction of birefringence in darkfield microscopy, loss of X-ray diffractability,more » and loss of resistance to acid hydrolysis. Upon freeze-drying, an additional degree of crystallinity returned as mostly cellulose II. Both enzymatic digestion with a commercial cellulase cocktail and maleic acid/AlCl3-catalyzed conversion to 5-hydroxymethylfurfural and levulinic acid were markedly enhanced with the low-temperature swollen cellulose. Only small improvements in rates and extent of hydrolysis and catalytic conversion were achieved upon heating to fully dissolve cellulose. Low-temperature swelling of cellulose in TFA substantially reduces recalcitrance of crystalline cellulose to both enzymatic digestion and catalytic conversion. In a closed system to prevent loss of fluorohydrocarbons, the relative ease of recovery and regeneration of TFA by distillation makes it a potentially useful agent in large-scale deconstruction of biomass, not only for enzymatic depolymerization but also for enhancing rates of catalytic conversion to biofuel components and useful bio-products.« less

Biodegradation of the alkaline cellulose degradation products generated during radioactive waste disposal.

PubMed

Rout, Simon P; Radford, Jessica; Laws, Andrew P; Sweeney, Francis; Elmekawy, Ahmed; Gillie, Lisa J; Humphreys, Paul N

2014-01-01

The anoxic, alkaline hydrolysis of cellulosic materials generates a range of cellulose degradation products (CDP) including α and β forms of isosaccharinic acid (ISA) and is expected to occur in radioactive waste disposal sites receiving intermediate level radioactive wastes. The generation of ISA's is of particular relevance to the disposal of these wastes since they are able to form complexes with radioelements such as Pu enhancing their migration. This study demonstrates that microbial communities present in near-surface anoxic sediments are able to degrade CDP including both forms of ISA via iron reduction, sulphate reduction and methanogenesis, without any prior exposure to these substrates. No significant difference (n = 6, p = 0.118) in α and β ISA degradation rates were seen under either iron reducing, sulphate reducing or methanogenic conditions, giving an overall mean degradation rate of 4.7 × 10(-2) hr(-1) (SE ± 2.9 × 10(-3)). These results suggest that a radioactive waste disposal site is likely to be colonised by organisms able to degrade CDP and associated ISA's during the construction and operational phase of the facility.

Crystal structure and genetic modifications of FI-CMCase from Aspergillus aculeatus F-50.

PubMed

Huang, Jian-Wen; Liu, Weidong; Lai, Hui-Lin; Cheng, Ya-Shan; Zheng, Yingying; Li, Qian; Sun, Hong; Kuo, Chih-Jung; Guo, Rey-Ting; Chen, Chun-Chi

2016-09-16

Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in many commercial applications. Endo-1,4-β-d-glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of 1,4-β-glycosidic bonds of the cellulose main chain to cleave cellulose into smaller fragments, is the key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase), which is classified into the glycoside hydrolase (GH) family 12, was demonstrated to be effectively expressed in the industrial strain Pichia pastoris. Here, the crystal structure and complex structures of P. pastoris-expressed FI-CMCase were solved to high resolution. The overall structure is analyzed and compared to other GH12 members. In addition, the substrate-surrounding residues were engineered to search for variants with improved enzymatic activity. Among 14 mutants constructed, one with two-fold increase in protein expression was identified, which possesses a potential to be further developed as a commercial enzyme product. Copyright © 2016 Elsevier Inc. All rights reserved.

Towards an Integrated QR Code Biosensor: Light-Driven Sample Acquisition and Bacterial Cellulose Paper Substrate.

PubMed

Yuan, Mingquan; Jiang, Qisheng; Liu, Keng-Ku; Singamaneni, Srikanth; Chakrabartty, Shantanu

2018-06-01

This paper addresses two key challenges toward an integrated forward error-correcting biosensor based on our previously reported self-assembled quick-response (QR) code. The first challenge involves the choice of the paper substrate for printing and self-assembling the QR code. We have compared four different substrates that includes regular printing paper, Whatman filter paper, nitrocellulose membrane and lab synthesized bacterial cellulose. We report that out of the four substrates bacterial cellulose outperforms the others in terms of probe (gold nanorods) and ink retention capability. The second challenge involves remote activation of the analyte sampling and the QR code self-assembly process. In this paper, we use light as a trigger signal and a graphite layer as a light-absorbing material. The resulting change in temperature due to infrared absorption leads to a temperature gradient that then exerts a diffusive force driving the analyte toward the regions of self-assembly. The working principle has been verified in this paper using assembled biosensor prototypes where we demonstrate higher sample flow rate due to light induced thermal gradients.

Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

PubMed Central

Verastegui, Y.; Cheng, J.; Engel, K.; Kolczynski, D.; Mortimer, S.; Lavigne, J.; Montalibet, J.; Romantsov, T.; Hall, M.; McConkey, B. J.; Rose, D. R.; Tomashek, J. J.; Scott, B. R.

2014-01-01

ABSTRACT Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. PMID:25028422

Thermostable Cellulases: Why & How?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-03-24

These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Development of a Commerical Enzyme System for Lignocellulosic Biomass Saccharification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-02-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Highly Efficient Thermostable DSM Cellulases: Why & How?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-04-26

These are the slides from this presentation. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase componentmore » enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Fully Integrated Lignocellulosic Biorefinery with Onsite Production of Enzymes and Yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-06-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates.

PubMed

Oliveira, G S; Ulhoa, C J; Silveira, M H L; Andreaus, J; Silva-Pereira, I; Poças-Fonseca, M J; Faria, F P

2013-01-01

Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl β-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.

The Use of Cellulose Nanocrystals for Potential Application in Topical Delivery of Hydroquinone.

PubMed

Taheri, Azade; Mohammadi, Mina

2015-07-01

Nanotechnology-based drug delivery systems can enhance drug permeation through the skin and improve the drug stability. The biodegradability and biocompatibility of cellulose nanocrystals have made these nanoparticles good candidates to use in biomedical applications. The hyperpigmentation is a common skin disorder that could be caused by number of reasons such as sun exposure and pregnancy. Hydroquinone could inhibit the production of melanin and eliminate the discolorations of skin. This study is aimed at introducing cellulose nanocrystals as suitable carriers for drug delivery to skin. Prepared cellulose nanocrystals were characterized by dynamic light scattering and atomic force microscopy. The size of cellulose nanocrystals determined using dynamic light scattering was 301 ± 10 nm. Hydroquinone-cellulose nanocrystal complex was prepared by incubating of hydroquinone solution in cellulose nanocrystals suspension. The size of hydroquinone-cellulose nanocrystal complex determined using dynamic light scattering was 310 ± 10 nm. The hydroquinone content of the hydroquinone-cellulose complex was determined using UV/vis spectroscopy. Hydroquinone was bound to cellulose nanocrystals representing 79.3 ± 2% maximum binding efficiency when 1.1 mg hydroquinone was added to 1 mL of cellulose nanocrystals suspension (2 mg cellulose nanocrystal). The hydroquinone-cellulose nanocrystal complex showed an approximately sustained release profile of hydroquinone. Approximately, 80% of bound hydroquinone released in 4 h. © 2014 John Wiley & Sons A/S.

Plant cellulose synthesis: CESA proteins crossing kingdoms.

PubMed

Kumar, Manoj; Turner, Simon

2015-04-01

Cellulose is a biopolymer of considerable economic importance. It is synthesised by the cellulose synthase complex (CSC) in species ranging from bacteria to higher plants. Enormous progress in our understanding of bacterial cellulose synthesis has come with the recent publication of both the crystal structure and biochemical characterisation of a purified complex able to synthesis cellulose in vitro. A model structure of a plant CESA protein suggests considerable similarity between the bacterial and plant cellulose synthesis. In this review article we will cover current knowledge of how plant CESA proteins synthesise cellulose. In particular the focus will be on the lessons learned from the recent work on the catalytic mechanism and the implications that new data on cellulose structure has for the assembly of CESA proteins into the large complex that synthesis plant cellulose microfibrils. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Improvement of the enzymatic hydrolysis of furfural residues by pretreatment with combined green liquor and ethanol organosolv.

PubMed

Yu, Hailong; Xing, Yang; Lei, Fuhou; Liu, Zhiping; Liu, Zuguang; Jiang, Jianxin

2014-09-01

Furfural residues (FRs) were pretreated with ethanol and a green liquor (GL) catalyst to produce fermentable sugar. Anthraquinone (AQ) was used as an auxiliary reagent to improve delignification and reduce cellulose decomposition. The results showed that 42.7% of lignin was removed and 96.5% of cellulose was recovered from substrates pretreated with 1.0 mL GL/g of dry substrate and 0.4% (w/w) AQ at 140°C for 1h. Compared with raw material, ethanol-GL pretreatment of FRs increased the glucose yield from 69.0% to 85.9% after 96 h hydrolysis with 18 FPU/g-cellulose for cellulase, 27 CBU/g-cellulose for β-glucosidase. The Brauner-Emmett-Teller surface area was reduced during pretreatment, which did not inhibit the enzymatic hydrolysis. Owing to the reduced surface area, the unproductive binding of cellulase to lignin was decreased, thus improving the enzymatic hydrolysis. The degree of polymerization of cellulose from FRs was too low to be a key factor for improving enzymatic hydrolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Purification and characterization of a cellulolytic multienzyme complex produced by Neocallimastix patriciarum J11.

PubMed

Wang, Hui-Chang; Chen, Yo-Chia; Hseu, Ruey-Shyang

2014-08-22

Understanding the roles of the components of the multienzyme complex of the anaerobial cellulase system, acting on complex substrates, is crucial to the development of efficient cellulase systems for industrial applications such as converting lignocellulose to sugars for bioethanol production. In this study, we purified the multienzyme complex of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The multienzyme complex is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight of these constituents have demonstrated β-glucanase activity on zymogram analysis. The multienzyme complex contained scaffoldings that respond to the gathering of the cellulolytic components. The levels and subunit ratio of the multienzyme complex from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the complexes were consistent. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through liquid chromatography/mass spectrometry, revealing that at least three types of cellulase, including one endoglucanase and two exoglucanases, could be found in the multienzyme complex of N. patriciarum J11. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the multienzyme complex produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase. Copyright © 2014 Elsevier Inc. All rights reserved.

Temperature sensitivity of organic substrate decay varies with pH

NASA Astrophysics Data System (ADS)

Min, K.; Lehmeier, C.; Ballantyne, F.; Billings, S. A.

2012-12-01

Cellulose is the most abundant biopolymer in soils and globally ubiquitous. It serves as a primary carbon source for myriad microbes able to release cellulases which cleave the cellulose into smaller molecules. For example, β-glucosidase, one type of cellulase, breaks down a terminal β-glycosidic bond of cellulose. The carbon of the liberated glucose becomes available for microbial uptake, after which it can then be mineralized and returned to the atmosphere via heterotrophic respiration. Thus, exoenzymes play an important role in the global cycling of carbon. Numerous studies suggest that global warming potentially increases the rate at which β-glucosidase breaks down cellulose, but it is not known how pH of the soil solution influences the effect of temperature on cellulose decomposition rates; this is important given the globally wide range of soil pH. Using fluorescence enzyme assay techniques, we studied the effect of temperature and pH on the reaction rate at which purified β-Glucosidase decays β-D-cellobioside (a compound often employed to simulate cellulose). We evaluated the temperature sensitivity of this reaction at five temperatures (5, 10, 15, 20, and 25°C) and six pH values (3.5, 4.5, 5.5, 6.5, 7.5, and 8.5)encompassing the naturally occurring range in soils, in a full-factorial design. First, we determined Vmax at 25°C and pH 6.5, standard conditions for measuring enzyme activities in many studies. The Vmax was 858.65 μmol h-1mg-1and was achieved at substrate concentration of 270 μM. At all pH values, the reaction rate slowed down at lower temperatures; at a pH of 3.5, no enzymatic activity was detected. The enzyme activity was significantly different between pH 4.5 and higher pHs. For example, enzyme reactivity at pH 4.5 was significantly lower than that at 7.5 at 20 and 25°C (Bonferroni-corrected P =0.0006, 0.0004, respectively), but not at lower temperatures. Similarly, enzyme reactivity at pH 4.5 was lower than that at pH 8.5 at 10, 15, and 25°C (P=0.0009, 0.0007, 0.0005, respectively), with a near-significant trend at 20°C (P=0.0023), and exhibited a nearly significant depression in response to temperature at 25°C compared to that at pH 6.5 (P=0.0015). Our results suggest that exoenzymatic cellulose decomposition with warming may be more enhanced in soil systems exhibiting higher pH. This work highlights the importance of soil solution pH as a driver of temperature sensitivity of substrate decay, and adds a level of complexity for developing accurate predictions of soil carbon cycling with climate change.

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates.

PubMed

Yang, Bin; Wyman, Charles E

2006-07-05

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis. (c) 2006 Wiley Periodicals, Inc.

Multi-Mode Binding of Cellobiohydrolase Cel7A from Trichoderma reesei to Cellulose

PubMed Central

Jalak, Jürgen; Väljamäe, Priit

2014-01-01

Enzymatic hydrolysis of recalcitrant polysaccharides like cellulose takes place on the solid-liquid interface. Therefore the adsorption of enzymes to the solid surface is a pre-requisite for catalysis. Here we used enzymatic activity measurements with fluorescent model-substrate 4-methyl-umbelliferyl-β-D-lactoside for sensitive monitoring of the binding of cellobiohydrolase TrCel7A from Trichoderma reesei to bacterial cellulose (BC). The binding at low nanomolar free TrCel7A concentrations was exclusively active site mediated and was consistent with Langmuir's one binding site model with K d and A max values of 2.9 nM and 126 nmol/g BC, respectively. This is the strongest binding observed with non-complexed cellulases and apparently represents the productive binding of TrCel7A to cellulose chain ends on the hydrophobic face of BC microfibril. With increasing free TrCel7A concentrations the isotherm gradually deviated from the Langmuir's one binding site model. This was caused by the increasing contribution of lower affinity binding modes that included both active site mediated binding and non-productive binding with active site free from cellulose chain. The binding of TrCel7A to BC was found to be only partially reversible. Furthermore, the isotherm was dependent on the concentration of BC with more efficient binding observed at lower BC concentrations. The phenomenon can be ascribed to the BC concentration dependent aggregation of BC microfibrils with concomitant reduction of specific surface area. PMID:25265511

Direct transfer of graphene onto flexible substrates.

PubMed

Martins, Luiz G P; Song, Yi; Zeng, Tingying; Dresselhaus, Mildred S; Kong, Jing; Araujo, Paulo T

2013-10-29

In this paper we explore the direct transfer via lamination of chemical vapor deposition graphene onto different flexible substrates. The transfer method investigated here is fast, simple, and does not require an intermediate transfer membrane, such as polymethylmethacrylate, which needs to be removed afterward. Various substrates of general interest in research and industry were studied in this work, including polytetrafluoroethylene filter membranes, PVC, cellulose nitrate/cellulose acetate filter membranes, polycarbonate, paraffin, polyethylene terephthalate, paper, and cloth. By comparing the properties of these substrates, two critical factors to ensure a successful transfer on bare substrates were identified: the substrate's hydrophobicity and good contact between the substrate and graphene. For substrates that do not satisfy those requirements, polymethylmethacrylate can be used as a surface modifier or glue to ensure successful transfer. Our results can be applied to facilitate current processes and open up directions for applications of chemical vapor deposition graphene on flexible substrates. A broad range of applications can be envisioned, including fabrication of graphene devices for opto/organic electronics, graphene membranes for gas/liquid separation, and ubiquitous electronics with graphene.

Advanced Materials From Fungal Mycelium: Fabrication and Tuning of Physical Properties

NASA Astrophysics Data System (ADS)

Haneef, Muhammad; Ceseracciu, Luca; Canale, Claudio; Bayer, Ilker S.; Heredia-Guerrero, José A.; Athanassiou, Athanassia

2017-01-01

In this work is presented a new category of self-growing, fibrous, natural composite materials with controlled physical properties that can be produced in large quantities and over wide areas, based on mycelium, the main body of fungi. Mycelia from two types of edible, medicinal fungi, Ganoderma lucidum and Pleurotus ostreatus, have been carefully cultivated, being fed by two bio-substrates: cellulose and cellulose/potato-dextrose, the second being easier to digest by mycelium due to presence of simple sugars in its composition. After specific growing times the mycelia have been processed in order to cease their growth. Depending on their feeding substrate, the final fibrous structures showed different relative concentrations in polysaccharides, lipids, proteins and chitin. Such differences are reflected as alterations in morphology and mechanical properties. The materials grown on cellulose contained more chitin and showed higher Young’s modulus and lower elongation than those grown on dextrose-containing substrates, indicating that the mycelium materials get stiffer when their feeding substrate is harder to digest. All the developed fibrous materials were hydrophobic with water contact angles higher than 120°. The possibility of tailoring mycelium materials’ properties by properly choosing their nutrient substrates paves the way for their use in various scale applications.

Surface Plasmon Resonance Imaging of the Enzymatic Degradation of Cellulose Microfibrils

NASA Astrophysics Data System (ADS)

Reiter, Kyle; Raegen, Adam; Clarke, Anthony; Lipkowski, Jacek; Dutcher, John

2012-02-01

As the largest component of biomass on Earth, cellulose represents a significant potential energy reservoir. Enzymatic hydrolysis of cellulose into fermentable sugars, an integral step in the production of biofuel, is a challenging problem on an industrial scale. More efficient conversion processes may be developed by an increased understanding of the action of the cellulolytic enzymes involved in cellulose degradation. We have used our recently developed quantitative, angle-scanning surface plasmon resonance imaging (SPRi) device to study the degradation of cellulose microfibrils upon exposure to cellulosic enzymes. In particular, we have studied the action of individual enzymes, and combinations of enzymes, from the Hypocrea Jecorina cellulase system on heterogeneous, industrially-relevant cellulose substrates. This has allowed us to define a characteristic time of action for the enzymes for different degrees of surface coverage of the cellulose microfibrils.

Biodegradation of high molecular weight lignin under sulfate reducing conditions: lignin degradability and degradation by-products.

PubMed

Ko, Jae-Jung; Shimizu, Yoshihisa; Ikeda, Kazuhiro; Kim, Seog-Ku; Park, Chul-Hwi; Matsui, Saburo

2009-02-01

This study is designed to investigate the biodegradation of high molecular weight (HMW) lignin under sulfate reducing conditions. With a continuously mesophilic operated reactor in the presence of co-substrates of cellulose, the changes in HMW lignin concentration and chemical structure were analyzed. The acid precipitable polymeric lignin (APPL) and lignin monomers, which are known as degradation by-products, were isolated and detected. The results showed that HMW lignin decreased and showed a maximum degradation capacity of 3.49 mg/l/day. APPL was confirmed as a polymeric degradation by-product and was accumulated in accordance with HMW lignin reduction. We also observed non-linear accumulation of aromatic lignin monomers such as hydrocinnamic acid. Through our experimental results, it was determined that HMW lignin, when provided with a co-substrate of cellulose, is biodegraded through production of APPL and aromatic monomers under anaerobic sulfate reducing conditions with a co-substrate of cellulose.

Polypyrrole Coated Cellulosic Substrate Modified by Copper Oxide as Electrode for Nitrate Electroreduction

NASA Astrophysics Data System (ADS)

Hamam, A.; Oukil, D.; Dib, A.; Hammache, H.; Makhloufi, L.; Saidani, B.

2015-08-01

The aim of this work is to synthesize polypyrrole (PPy) films on nonconducting cellulosic substrate and modified by copper oxide particles for use in the nitrate electroreduction process. Firstly, the chemical polymerization of polypyrrole onto cellulosic substrate is conducted by using FeCl3 as an oxidant and pyrrole as monomer. The thickness and topography of the different PPy films obtained were estimated using a profilometer apparatus. The electrochemical reactivity of the obtained electrodes was tested by voltamperometry technique and electrochemical impedance spectroscopy. Secondly, the modification of the PPy film surface by incorporation of copper oxide particles is conducted by applying a galvanostatic procedure from a CuCl2 solution. The SEM, EDX and XRD analysis showed the presence of CuO particles in the polymer films with dimensions less than 50 nm. From cyclic voltamperometry experiments, the composite activity for the nitrate electroreduction reaction was evaluated and the peak of nitrate reduction is found to vary linearly with initial nitrate concentration.

Lauren Magnusson | NREL

Science.gov Websites

fermentation of an insoluble cellulosic substrate under continuous culture conditions, and that C. thermocellum lignocellulosic biomass Fermentation of waste green algal cell mass for hydrogen production Education M.S , 2005 Featured Publications "Continuous hydrogen production during fermentation of α-cellulose by

A coarse-grained model for synergistic action of multiple enzymes on cellulose

DOE PAGES

Asztalos, Andrea; Daniels, Marcus; Sethi, Anurag; ...

2012-08-01

In this study, degradation of cellulose to glucose requires the cooperative action of three classes of enzymes, collectively known as cellulases. Endoglucanases randomly bind to cellulose surfaces and generate new chain ends by hydrolyzing -1,4-D-glycosidic bonds. Exoglucanases bind to free chain ends and hydrolyze glycosidic bonds in a processive manner releasing cellobiose units. Then, -glucosidases hydrolyze soluble cellobiose to glucose. Optimal synergistic action of these enzymes is essential for efficient digestion of cellulose. Experiments show that as hydrolysis proceeds and the cellulose substrate becomes more heterogeneous, the overall degradation slows down. As catalysis occurs on the surface of crystalline cellulose,more » several factors affect the overall hydrolysis. Therefore, spatial models of cellulose degradation must capture effects such as enzyme crowding and surface heterogeneity, which have been shown to lead to a reduction in hydrolysis rates. As a result, we present a coarse-grained stochastic model for capturing the key events associated with the enzymatic degradation of cellulose at the mesoscopic level. This functional model accounts for the mobility and action of a single cellulase enzyme as well as the synergy of multiple endo- and exo-cellulases on a cellulose surface. The quantitative description of cellulose degradation is calculated on a spatial model by including free and bound states of both endo- and exo-cellulases with explicit reactive surface terms (e.g., hydrogen bond breaking, covalent bond cleavages) and corresponding reaction rates. The dynamical evolution of the system is simulated by including physical interactions between cellulases and cellulose. In conclusion, our coarse-grained model reproduces the qualitative behavior of endoglucanases and exoglucanases by accounting for the spatial heterogeneity of the cellulose surface as well as other spatial factors such as enzyme crowding. Importantly, it captures the endo-exo synergism of cellulase enzyme cocktails. This model constitutes a critical step towards testing hypotheses and understanding approaches for maximizing synergy and substrate properties with a goal of cost effective enzymatic hydrolysis.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE PAGES

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko; ...

2018-03-23

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

PubMed

Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

2018-01-01

Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii 's utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.

Mechanics of Cellulose Synthase Complexes in Living Plant Cells

NASA Astrophysics Data System (ADS)

Zehfroosh, Nina; Liu, Derui; Ramos, Kieran P.; Yang, Xiaoli; Goldner, Lori S.; Baskin, Tobias I.

The polymer cellulose is one of the major components of the world's biomass with unique and fascinating characteristics such as its high tensile strength, renewability, biodegradability, and biocompatibility. Because of these distinctive aspects, cellulose has been the subject of enormous scientific and industrial interest, yet there are still fundamental open questions about cellulose biosynthesis. Cellulose is synthesized by a complex of transmembrane proteins called ``Cellulose Synthase A'' (CESA) in the plasma membrane. Studying the dynamics and kinematics of the CESA complex will help reveal the mechanism of cellulose synthesis and permit the development and validation of models of CESA motility. To understand what drives these complexes through the cell membrane, we used total internal reflection fluorescence microscopy (TIRFM) and variable angle epi-fluorescence microscopy to track individual, fluorescently-labeled CESA complexes as they move in the hypocotyl and root of living plants. A mean square displacement analysis will be applied to distinguish ballistic, diffusional, and other forms of motion. We report on the results of these tracking experiments. This work was funded by NSF/PHY-1205989.

Wax transfer printing to enable robust barrier definition in devices based on non-standard porous materials

NASA Astrophysics Data System (ADS)

To, Anthony; Downs, Corey; Fu, Elain

2017-05-01

Wax printing has become a common method of fabricating channels in cellulose-based microfluidic devices. However, a limitation of wax printing is that it is restricted to relatively thin, smooth substrates that are compatible with processing by a commercial wax printer. In the current report, we describe a simple patterning method that extends the utility of wax printers for creating hydrophobic barriers on non-standard porous substrates via a process called wax transfer printing. We demonstrate the use of multiple wax transfer cycles to create well-defined, robust, and reproducible barriers in a thick cellulose substrate that is not compatible with feeding through a wax printer. We characterize the method for (i) wax spreading within the substrate as a function of heating time, (ii) the ability to create functional barriers in a substrate, and (iii) reproducibility in line width.

The correlation between cellulose allomorphs (I and II) and conversion after removal of hemicellulose and lignin of lignocellulose.

PubMed

Song, Yanliang; Zhang, Jingzhi; Zhang, Xu; Tan, Tianwei

2015-10-01

H2SO4, NaOH and H3PO4 were applied to decompose lignocellulose samples (giant reeds, pennisetum and cotton stalks) to investigate the correlation between cellulose allomorphs (cellulose I and II) and conversion of cellulose. The effect of removal of hemicellulose and lignin on the surface morphology, crystallinity index (CrI), cellulose allomorphs (cellulose I and II), and enzymatic hydrolysis under different pretreatments was also studied. CrI caused by H3PO4 pretreatment reached 11.19%, 24.93% and 8.15% for the three samples, respectively. Corn stalk showed highest conversion of cellulose among three samples, irrespective of the pretreatment used. This accounted for the widely use of corn stalk as the renewable crop substrate to synthesize biofuels like ethanol. CrI of cellulose I (CrI-I) negatively affects cellulose conversion but CrI of cellulose II (CrI-II) positively affects cellulose conversion. It contributes to make the strategy to transform cellulose I to cellulose II and enhancing enzymatic hydrolysis of lignocellulose. Copyright © 2015 Elsevier Ltd. All rights reserved.

IMPACTS OF BIOFILM FORMATION ON CELLULOSE FERMENTATION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leschine, Susan

2009-10-31

This project addressed four major areas of investigation: i) characterization of formation of Cellulomonas uda biofilms on cellulose; ii) characterization of Clostridium phytofermentans biofilm development; colonization of cellulose and its regulation; iii) characterization of Thermobifida fusca biofilm development; colonization of cellulose and its regulation; and iii) description of the architecture of mature C. uda, C. phytofermentans, and T. fusca biofilms. This research is aimed at advancing understanding of biofilm formation and other complex processes involved in the degradation of the abundant cellulosic biomass, and the biology of the microbes involved. Information obtained from these studies is invaluable in the developmentmore » of practical applications, such as the single-step bioconversion of cellulose-containing residues to fuels and other bioproducts. Our results have clearly shown that cellulose-decomposing microbes rapidly colonize cellulose and form complex structures typical of biofilms. Furthermore, our observations suggest that, as cells multiply on nutritive surfaces during biofilms formation, dramatic cell morphological changes occur. We speculated that morphological changes, which involve a transition from rod-shaped cells to more rounded forms, might be more apparent in a filamentous microbe. In order to test this hypothesis, we included in our research a study of biofilm formation by T. fusca, a thermophilic cellulolytic actinomycete commonly found in compost. The cellulase system of T. fusca has been extensively detailed through the work of David Wilson and colleagues at Cornell, and also, genome sequence of a T. fusca strain has been determine by the DOE Joint Genome Institute. Thus, T. fusca is an excellent subject for studies of biofilm development and its potential impacts on cellulose degradation. We also completed a study of the chitinase system of C. uda. This work provided essential background information for understanding how C. uda colonizes and degrades insoluble substrates. Major accomplishments of the project include: • Development of media containing dialysis tubing (described by the manufacturer as “regenerated cellulose”) as sole carbon and energy source and a nutritive surface for the growth of cellulolytic bacteria, and development of various microscopic methods to image biofilms on dialysis tubing. • Demonstration that cultures of C. phytofermentans, an obligate anaerobe, C. uda, a facultative aerobe, and T. fusca, a filamentous aerobe, formed microbial communities on the surface of dialysis tubing, which possessed architectural features and functional characteristics typical of biofilms. • Demonstration that biofilm formation on the nutritive surface, cellulose, involves a complex developmental processes, including colonization of dialysis tubing, formation of cell clusters attached to the nutritive surface, cell morphological changes, formation of complex structures embedded in extracellular polymeric matrices, and dispersal of biofilm communities as the nutritive surface is degraded. • Determination of surface specificity and regulatory aspects of biofilm formation by C. phytofermentans, C. uda, and T. fusca. • Demonstration that biofilm formation by T. fusca forms an integral part of the life cycle of this filamentous cellulolytic bacterium, including studies on the role of mycelial pellet formation in the T. fusca life cycle and a comparison of mycelial pellets to surface-attached T. fusca biofilms. • Characterization of T. fusca biofilm EPS, including demonstration of a functional role for EPS constituents. • Correlation of T. fusca developmental life cycle and cellulase gene expression.« less

Structure of the cellulose synthase complex of Gluconacetobacter hansenii at 23.4 Å resolution

DOE PAGES

Du, Juan; Vepachedu, Venkata; Cho, Sung Hyun; ...

2016-05-23

Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsDmore » in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 angstrom for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. Furthermore, the results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.« less

Structure of the cellulose synthase complex of Gluconacetobacter hansenii at 23.4 Å resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Juan; Vepachedu, Venkata; Cho, Sung Hyun

Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsDmore » in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 angstrom for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. Furthermore, the results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.« less

Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution

PubMed Central

Du, Juan; Vepachedu, Venkata; Cho, Sung Hyun; Kumar, Manish; Nixon, B. Tracy

2016-01-01

Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose. PMID:27214134

Removal of slowly biodegradable COD in combined thermophilic UASB and MBBR systems.

PubMed

Ji, M; Yu, J; Chen, H; Yue, P L

2001-09-01

Starch, cellulose and polyvinyl alcohol (PVA) are common substrates of the slowly biodegradable COD (SBCOD) in industrial wastewaters. Removal of the individual and mixed SbCOD substrates was investigated in a combined system of thermophilic upflow anaerobic sludge blanket (TUASB) reactor (55 degrees C) and aerobic moving bed biofilm reactor (MBBR). The removal mechanisms of the three SBCOD substrates were quite different. Starch-COD was almost equally utilized and removed in the two reactors. Cellulose-COD was completely (97-98%) removed from water in the TUASB reactor by microbial entrapment and sedimentation of the cellulose fibers. PVA alone was hardly biodegraded and removed by the combined reactors. However, PVA-COD could be removed to some extent in a binary solution of starch (77%) plus PVA (23%). The PVA macromolecules in the binary solution actually affected the microbial activity in the TUASB reactor resulting accumulation of volatile fatty acids, which shifted the overall COD removal from the TUASB to the MBBR reactor where SBCOD including PVA-COD was removed. Since the three SBCOD substrates were removed by different mechanisms, the combined reactors showed a better and more stable performance than individual reactors.

Direct transfer of graphene onto flexible substrates

PubMed Central

Martins, Luiz G. P.; Song, Yi; Zeng, Tingying; Dresselhaus, Mildred S.; Kong, Jing; Araujo, Paulo T.

2013-01-01

In this paper we explore the direct transfer via lamination of chemical vapor deposition graphene onto different flexible substrates. The transfer method investigated here is fast, simple, and does not require an intermediate transfer membrane, such as polymethylmethacrylate, which needs to be removed afterward. Various substrates of general interest in research and industry were studied in this work, including polytetrafluoroethylene filter membranes, PVC, cellulose nitrate/cellulose acetate filter membranes, polycarbonate, paraffin, polyethylene terephthalate, paper, and cloth. By comparing the properties of these substrates, two critical factors to ensure a successful transfer on bare substrates were identified: the substrate’s hydrophobicity and good contact between the substrate and graphene. For substrates that do not satisfy those requirements, polymethylmethacrylate can be used as a surface modifier or glue to ensure successful transfer. Our results can be applied to facilitate current processes and open up directions for applications of chemical vapor deposition graphene on flexible substrates. A broad range of applications can be envisioned, including fabrication of graphene devices for opto/organic electronics, graphene membranes for gas/liquid separation, and ubiquitous electronics with graphene. PMID:24127582

Evaluation of four ionic liquids for pretreatment of lignocellulosic biomass.

PubMed

Gräsvik, John; Winestrand, Sandra; Normark, Monica; Jönsson, Leif J; Mikkola, Jyri-Pekka

2014-04-30

Lignocellulosic biomass is highly recalcitrant and various pretreatment techniques are needed to facilitate its effective enzymatic hydrolysis to produce sugars for further conversion to bio-based chemicals. Ionic liquids (ILs) are of interest in pretreatment because of their potential to dissolve lignocellulosic materials including crystalline cellulose. Four imidazolium-based ionic liquids (ILs) ([C=C2C1im][MeCO2], [C4C1im][MeCO2], [C4C1im][Cl], and [C4C1im][HSO4]) well known for their capability to dissolve lignocellulosic species were synthesized and then used for pretreatment of substrates prior to enzymatic hydrolysis. In order to achieve a broad evaluation, seven cellulosic, hemicellulosic and lignocellulosic substrates, crystalline as well as amorphous, were selected. The lignocellulosic substrates included hybrid aspen and Norway spruce. The monosaccharides in the enzymatic hydrolysate were determined using high-performance anion-exchange chromatography. The best results, as judged by the saccharification efficiency, were achieved with [C4C1im][Cl] for cellulosic substrates and with the acetate-based ILs for hybrid aspen and Norway spruce. After pretreatment with acetate-based ILs, the conversion to glucose of glucan in recalcitrant softwood lignocellulose reached similar levels as obtained with pure crystalline and amorphous cellulosic substrates. IL pretreatment of lignocellulose resulted in sugar yields comparable with that obtained with acidic pretreatment. Heterogeneous dissolution with [C4C1im][HSO4] gave promising results with aspen, the less recalcitrant of the two types of lignocellulose included in the investigation. The ability of ILs to dissolve lignocellulosic biomass under gentle conditions and with little or no by-product formation contributes to making them highly interesting alternatives for pretreatment in processes where high product yields are of critical importance.

Evaluation of four ionic liquids for pretreatment of lignocellulosic biomass

PubMed Central

2014-01-01

Background Lignocellulosic biomass is highly recalcitrant and various pretreatment techniques are needed to facilitate its effective enzymatic hydrolysis to produce sugars for further conversion to bio-based chemicals. Ionic liquids (ILs) are of interest in pretreatment because of their potential to dissolve lignocellulosic materials including crystalline cellulose. Results Four imidazolium-based ionic liquids (ILs) ([C=C2C1im][MeCO2], [C4C1im][MeCO2], [C4C1im][Cl], and [C4C1im][HSO4]) well known for their capability to dissolve lignocellulosic species were synthesized and then used for pretreatment of substrates prior to enzymatic hydrolysis. In order to achieve a broad evaluation, seven cellulosic, hemicellulosic and lignocellulosic substrates, crystalline as well as amorphous, were selected. The lignocellulosic substrates included hybrid aspen and Norway spruce. The monosaccharides in the enzymatic hydrolysate were determined using high-performance anion-exchange chromatography. The best results, as judged by the saccharification efficiency, were achieved with [C4C1im][Cl] for cellulosic substrates and with the acetate-based ILs for hybrid aspen and Norway spruce. After pretreatment with acetate-based ILs, the conversion to glucose of glucan in recalcitrant softwood lignocellulose reached similar levels as obtained with pure crystalline and amorphous cellulosic substrates. IL pretreatment of lignocellulose resulted in sugar yields comparable with that obtained with acidic pretreatment. Heterogeneous dissolution with [C4C1im][HSO4] gave promising results with aspen, the less recalcitrant of the two types of lignocellulose included in the investigation. Conclusions The ability of ILs to dissolve lignocellulosic biomass under gentle conditions and with little or no by-product formation contributes to making them highly interesting alternatives for pretreatment in processes where high product yields are of critical importance. PMID:24779378

Evidence for the identity and some comparative properties of alpha-ketoglutarate and 2-keto-4-hydroxyglutarate dehydrogenase activity.

PubMed

Gupta, S C; Dekker, E E

1980-02-10

Enzyme preparations of pig heart and Escherichia coli are shown to catalyze a NAD+- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate. Several independent lines of evidence support the conclusion that this hydroxyketo acid is a substrate for the well known alpha-ketoglutarate dehydrogenase complex of the citric acid cycle. The evidence includes (a) a constant ratio of specific activity values for the two substrates through several steps of purification, (b) identical elution profiles from a calcium phosphate gel-cellulose column and a constant ratio of specific activity toward the two substrates throughout the activity peak, (c) identical inactivation curves in controlled heat denaturation studies, (d) the same pH activity curves, (e) no effect on the oxidation of either keto acid by repeated freezing and thawing of dehydrogenase preparations, and (f) the same activity pattern when the E. coli complex is distributed into several fractions by sucrose density gradient centrifugation. Additionally, the same cofactors are required for maximal activity and glyoxylate inhibits the oxidation of either substrate noncompetitively. Ferricyanide-linked oxidation of 2-keto-4-hydroxyglutarate yields malate as the product and a 1:2:1 stoichiometric relationship is obtained between the amount of hydroxyketo acid oxidized, ferricyanide reduced, and malate formed.

Enhancement of D-lactic acid production from a mixed glucose and xylose substrate by the Escherichia coli strain JH15 devoid of the glucose effect.

PubMed

Lu, Hongying; Zhao, Xiao; Wang, Yongze; Ding, Xiaoren; Wang, Jinhua; Garza, Erin; Manow, Ryan; Iverson, Andrew; Zhou, Shengde

2016-02-19

A thermal tolerant stereo-complex poly-lactic acid (SC-PLA) can be made by mixing Poly-D-lactic acid (PDLA) and poly-L-lactic acid (PLLA) at a defined ratio. This environmentally friendly biodegradable polymer could replace traditional recalcitrant petroleum-based plastics. To achieve this goal, however, it is imperative to produce optically pure lactic acid isomers using a cost-effective substrate such as cellulosic biomass. The roadblock of this process is that: 1) xylose derived from cellulosic biomass is un-fermentable by most lactic acid bacteria; 2) the glucose effect results in delayed and incomplete xylose fermentation. An alternative strain devoid of the glucose effect is needed to co-utilize both glucose and xylose for improved D-lactic acid production using a cellulosic biomass substrate. A previously engineered L-lactic acid Escherichia coli strain, WL204 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA ΔadhE, ΔldhA::ldhL), was reengineered for production of D-lactic acid, by replacing the recombinant L-lactate dehydrogenase gene (ldhL) with a D-lactate dehydrogenase gene (ldhA). The glucose effect (catabolite repression) of the resulting strain, JH13, was eliminated by deletion of the ptsG gene which encodes for IIBC(glc) (a PTS enzyme for glucose transport). The derived strain, JH14, was metabolically evolved through serial transfers in screw-cap tubes containing glucose. The evolved strain, JH15, regained improved anaerobic cell growth using glucose. In fermentations using a mixture of glucose (50 g L(-1)) and xylose (50 g L(-1)), JH15 co-utilized both glucose and xylose, achieving an average sugar consumption rate of 1.04 g L(-1)h(-1), a D-lactic acid titer of 83 g L(-1), and a productivity of 0.86 g L(-1) h(-1). This result represents a 46 % improved sugar consumption rate, a 26 % increased D-lactic acid titer, and a 48 % enhanced productivity, compared to that achieved by JH13. These results demonstrated that JH15 has the potential for fermentative production of D-lactic acid using cellulosic biomass derived substrates, which contain a mixture of C6 and C5 sugars.

A kinetic study on microwave-assisted conversion of cellulose and lignocellulosic waste into hydroxymethylfurfural/furfural.

PubMed

da Silva Lacerda, Viviane; López-Sotelo, Juan Benito; Correa-Guimarães, Adriana; Hernández-Navarro, Salvador; Sánchez-Bascones, Mercedes; Navas-Gracia, Luis M; Martín-Ramos, Pablo; Pérez-Lebeña, Eduardo; Martín-Gil, Jesús

2015-03-01

Native cellulose, lignocellulosic materials from Brazil (carnauba palm leaves and macauba pulp and shell) and pine nut shell from Spain have been studied as substrates for the production of HMF and furfural in a conventional microwave oven. In order to promote the dissolution of native cellulose, several ionic liquids, catalysts, organic solvents and water doses have been assessed. The most suitable mixture (5mL of choline chloride/oxalic acid, 2mL of sulfolane, 2mL of water, 0.02g of TiO2 and 0.1g of substrate) has been chosen to conduct kinetic studies at different reaction times (5-60min) and various temperatures (120-200°C) and to evaluate the best conditions for HMF+furfural production according to Seaman's model. The best production yields of HMF+furfural have been attained for native cellulose, with a yield of 53.24% when an ultrasonic pretreatment was used prior to a microwave treatment with stirring. Copyright © 2014 Elsevier Ltd. All rights reserved.

The effect of deuteration on the structure of bacterial cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bali, Garima; Foston, Marcus; O'Neill, Hugh Michael

2013-01-01

ABSTRACT In vivo generated deuterated bacterial cellulose, cultivated from 100% deuterated glycerol in D2O medium, was analyzed for deuterium incorporation by ionic liquid dissolution and 2H and 1H nuclear magnetic resonance (NMR). A solution NMR method of the dissolved cellulose was used to determine that this bacterial cellulose had 85 % deuterium incorporation. Acetylation and 1H and 2H NMR of deuterated bacterial cellulose indicated near equal deuteration at all sites of the glucopyranosyl ring except C-6 which was partly deuterated. Despite the high level of deuterium incorporation there were no significant differences in the molecular and morphological properties were observedmore » for the deuterated and protio bacterial cellulose samples. The highly deuterated bacterial cellulose presented here can be used as a model substrate for studying cellulose biopolymer properties via future small angle neutron scattering (SANS) studies.« less

High brightness phosphorescent organic light emitting diodes on transparent and flexible cellulose films.

PubMed

Purandare, Sumit; Gomez, Eliot F; Steckl, Andrew J

2014-03-07

Organic light-emitting diodes (OLED) were fabricated on flexible and transparent reconstituted cellulose obtained from wood pulp. Cellulose is naturally available, abundant, and biodegradable and offers a unique substrate alternative for the fabrication of flexible OLEDs. Transparent cellulose material was formed by dissolution of cellulose in an organic solvent (dimethyl acetamide) at elevated temperature (165 °C) in the presence of a salt (LiCl). The optical transmission of 40-μm thick transparent cellulose sheet averaged 85% over the visible spectrum. High brightness and high efficiency thin film OLEDs were fabricated on transparent cellulose films using phosphorescent Ir(ppy)3 as the emitter material. The OLEDs achieved current and luminous emission efficiencies as high as 47 cd A(-1) and 20 lm W(-1), respectively, and a maximum brightness of 10,000 cd m(-2).

High brightness phosphorescent organic light emitting diodes on transparent and flexible cellulose films

NASA Astrophysics Data System (ADS)

Purandare, Sumit; Gomez, Eliot F.; Steckl, Andrew J.

2014-03-01

Organic light-emitting diodes (OLED) were fabricated on flexible and transparent reconstituted cellulose obtained from wood pulp. Cellulose is naturally available, abundant, and biodegradable and offers a unique substrate alternative for the fabrication of flexible OLEDs. Transparent cellulose material was formed by dissolution of cellulose in an organic solvent (dimethyl acetamide) at elevated temperature (165 °C) in the presence of a salt (LiCl). The optical transmission of 40-μm thick transparent cellulose sheet averaged 85% over the visible spectrum. High brightness and high efficiency thin film OLEDs were fabricated on transparent cellulose films using phosphorescent Ir(ppy)3 as the emitter material. The OLEDs achieved current and luminous emission efficiencies as high as 47 cd A-1 and 20 lm W-1, respectively, and a maximum brightness of 10 000 cd m-2.

Fabrication of cellulose/graphene paper as a stable-cycling anode materials without collector.

PubMed

Zhang, Chunliang; Cha, Ruitao; Yang, Luming; Mou, Kaiwen; Jiang, Xingyu

2018-03-15

Flexible and foldable devices attract substantial attention in low-cost electronics. Among the flexible substrate materials, paper has several attractive advantages. In our study, we fabricate cellulose/graphene paper by wet end formation (papermaking). The cationic polyacrylamide remarkably improve the retention ratio of graphene of cellulose/graphene slurry. Besides, cellulose/graphene paper exhibits well mechanical properties such as its flexibility and folding endurance. And we replace copper foil collector with cellulose/graphene paper in lithium-ion batteries without collector, and investigate its electrochemical properties. The obtained results show that cellulose/graphene paper presents excellent charge-discharge stability after 1600th cycles as the anode of lithium-ion batteries. These advantages highlight the potential applications of cellulose/graphene paper as anode materials for lithium-ion batteries. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nanomechanical Sensing of Biological Interfacial Interactions

NASA Astrophysics Data System (ADS)

Du, Wenjian

Cellulose is the most abundant biopolymer on earth. Cellulase is an enzyme capable of converting insoluble cellulose into soluble sugars. Cellulosic biofuel produced from such fermentable simple sugars is a promising substitute as an energy source. However, its economic feasibility is limited by the low efficiency of the enzymatic hydrolysis of cellulose by cellulase. Cellulose is insoluble and resistant to enzymatic degradation, not only because the beta-1,4-glycosidic bonds are strong covalent bonds, but also because cellulose microfibrils are packed into tightly bound, crystalline lattices. Enzymatic hydrolysis of cellulose by cellulase involves three steps--initial binding, decrystallization, and hydrolytic cleavage. Currently, the mechanism for the decrystallization has not yet been elucidated, though it is speculated to be the rate-limiting step of the overall enzymatic activity. The major technical challenge limiting the understanding of the decrystallization is the lack of an effective experimental approach capable of examining the decrystallization, an interfacial enzymatic activity on solid substrates. The work presented develops a nanomechanical sensing approach to investigate both the decrystallization and enzymatic hydrolytic cleavage of cellulose. The first experimental evidence of the decrystallization is obtained by comparing the results from native cellulase and non-hydrolytic cellulase. Surface topography has been applied to examine the activities of native cellulase and non-hydrolytic cellulase on cellulose substrate. The study demonstrates additional experimental evidence of the decrystallization in the hydrolysis of cellulose. By combining simulation and monitoring technology, the current study also investigates the structural changes of cellulose at a molecular level. In particular, the study employs cellulose nanoparticles with a bilayer structure on mica sheets. By comparing results from a molecular dynamic simulation and the distance between cellulose layers monitored by means of the atomic force microscopy (AFM), the current study shows that water molecules can efficiently reduce the energy required for separating two layers of cellulose bilayers during hydration of cellulose bilayer nanoparticles. The findings of the study contribute to explicating the mechanism of cellulose the decrystallization, a free-energetically unfavorable process, through enzymatic hydrolysis of cellulase. The study also investigates the application of a cell-based microcantilever sensor to monitor the real-time ligand-induced response of living cells. These nanomechanical approaches offer unique perspectives on the interfacial activities of biological molecules.

Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose.

PubMed

Selig, Michael J; Viamajala, Sridhar; Decker, Stephen R; Tucker, Melvin P; Himmel, Michael E; Vinzant, Todd B

2007-01-01

Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these "lignin" droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 degrees C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 degrees C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.

Solid state fermentation for production of microbial cellulases: Recent advances and improvement strategies.

PubMed

Behera, Sudhanshu S; Ray, Ramesh C

2016-05-01

Lignocellulose is the most plentiful non-food biomass and one of the most inexhaustible renewable resources on the planet, which is an alternative sustainable energy source for the production of second generation biofuels. Lignocelluloses are composed of cellulose, hemicellulose and lignin, in which the sugar polymers account for a large portion of the biomass. Cellulases belong to the glycoside hydrolase family and catalyze the hydrolysis of glyosidic linkages depolymerizing cellulose to fermentable sugars. They are multi-enzymatic complex proteins and require the synergistic action of three key enzymes: endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.176) (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21) for the depolymerization of cellulose to glucose. Solid state fermentation, which holds growth of microorganisms on moist solid substrates in the absence of free flowing water, has gained considerable attention of late due its several advantages over submerged fermentation. The review summarizes the critical analysis of recent literature covering production of cellulase in solid state fermentation using advance technologies such as consolidated bioprocessing, metabolic engineering and strain improvement, and circumscribes the strategies to improve the enzyme yield. Copyright © 2016. Published by Elsevier B.V.

Conversion of cellulosic materials into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma spp. under SHF and SSF processes.

PubMed

Faria, Nuno Torres; Santos, Marisa; Ferreira, Carla; Marques, Susana; Ferreira, Frederico Castelo; Fonseca, César

2014-11-04

Mannosylerythritol lipids (MEL) are glycolipids with unique biosurfactant properties and are produced by Pseudozyma spp. from different substrates, preferably vegetable oils, but also sugars, glycerol or hydrocarbons. However, solvent intensive downstream processing and the relatively high prices of raw materials currently used for MEL production are drawbacks in its sustainable commercial deployment. The present work aims to demonstrate MEL production from cellulosic materials and investigate the requirements and consequences of combining commercial cellulolytic enzymes and Pseudozyma spp. under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes. MEL was produced from cellulosic substrates, Avicel® as reference (>99% cellulose) and hydrothermally pretreated wheat straw, using commercial cellulolytic enzymes (Celluclast 1.5 L® and Novozyme 188®) and Pseudozyma antarctica PYCC 5048(T) or Pseudozyma aphidis PYCC 5535(T). The strategies included SHF, SSF and fed-batch SSF with pre-hydrolysis. While SSF was isothermal at 28°C, in SHF and fed-batch SSF, yeast fermentation was preceded by an enzymatic (pre-)hydrolysis step at 50°C for 48 h. Pseudozyma antarctica showed the highest MEL yields from both cellulosic substrates, reaching titres of 4.0 and 1.4 g/l by SHF of Avicel® and wheat straw (40 g/l glucan), respectively, using enzymes at low dosage (3.6 and 8.5 FPU/gglucan at 28°C and 50°C, respectively) with prior dialysis. Higher MEL titres were obtained by fed-batch SSF with pre-hydrolysis, reaching 4.5 and 2.5 g/l from Avicel® and wheat straw (80 g/l glucan), respectively. This work reports for the first time MEL production from cellulosic materials. The process was successfully performed through SHF, SSF or Fed-batch SSF, requiring, for maximal performance, dialysed commercial cellulolytic enzymes. The use of inexpensive lignocellulosic substrates associated to straightforward downstream processing from sugary broths is expected to have a great impact in the economy of MEL production for the biosurfactant market, inasmuch as low enzyme dosage is sufficient for good systems performance.

Cellulose microfibril deposition: coordinated activity at the plant plasma membrane.

PubMed

Lindeboom, J; Mulder, B M; Vos, J W; Ketelaar, T; Emons, A M C

2008-08-01

Plant cell wall production is a membrane-bound process. Cell walls are composed of cellulose microfibrils, embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose synthase complexes into the plasma membrane. These complexes, the nanomachines that produce the cellulose microfibrils, move inside the plasma membrane leaving the cellulose microfibrils in their wake. Cellulose microfibril angle is an important determinant of cell development and of tissue properties and as such relevant for the industrial use of plant material. Here, we provide an integrated view of the events taking place in the not more than 100 nm deep area in and around the plasma membrane, correlating recent results provided by the distinct field of plant cell biology. We discuss the coordinated activities of exocytosis, endocytosis, and movement of cellulose synthase complexes while producing cellulose microfibrils and the link of these processes to the cortical microtubules.

Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose.

PubMed

Olsen, Johan P; Alasepp, Kadri; Kari, Jeppe; Cruys-Bagger, Nicolaj; Borch, Kim; Westh, Peter

2016-06-01

The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have implemented an electrochemical biosensor assay for product inhibition studies of cellulases acting on their natural substrate, cellulose. Using this method we measured the hydrolytic rate of Cel7A as a function of both product (inhibitor) concentration and substrate load. This data enabled analyses along the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing rate of the reverse reaction, lowering the net hydrolytic velocity as product concentrations increase. Strictly this is not a case of inhibition, as no catalytically inactive is formed. The other mechanism that matched the kinetic data was noncompetitive inhibition with an inhibition constant of 490 ± 40 μM. Noncompetitive inhibition implies that the inhibitor binds with comparable strength to either free enzyme or an enzymesubstrate complex, that is, that association between enzyme and substrate has no effect on the binding of the inhibitor. This mechanism is rarely observed, but we argue, that the special architecture of Cel7A with numerous subsites for binding of both substrate and product could give rise to a true noncompetitive inhibition mechanism. Biotechnol. Bioeng. 2016;113: 1178-1186. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Inkjet Printed Surface Enhanced Raman Spectroscopy Array on Cellulose Paper

PubMed Central

Yu, Wei W.; White, Ian M.

2011-01-01

A novel, ultra low-cost surface enhanced Raman spectroscopy (SERS) substrate has been developed by modifying the surface chemistry of cellulose paper and patterning nanoparticle arrays, all with a consumer inkjet printer. Micro/nanofabrication of SERS substrates for on-chip chemical and biomolecular analysis has been under intense investigation. However, the high cost of producing these substrates and the limited shelf life severely limit their use, especially for routine laboratory analysis and for point-of-sample analysis in the field. Paper-based microfluidic biosensing systems have shown great potential as low-cost disposable analysis tools. In this work, this concept is extended to SERS-based detection. Using an inexpensive consumer inkjet printer, cellulose paper substrates are modified to be hydrophobic in the sensing regions. Synthesized silver nanoparticles are printed onto this hydrophobic paper substrate with microscale precision to form sensing arrays. The hydrophobic surface prevents the aqueous sample from spreading throughout the paper and thus concentrates the analyte within the sensing region. A SERS fingerprint signal for Rhodamine 6G dye was observed for samples with as low as 10 femtomoles of analyte in a total sample volume of 1 μL. This extraordinarily simple technique can be used to construct SERS microarrays immediately before sample analysis, enabling ultra low-cost chemical and biomolecular detection in the lab as well as in the field at the point of sample collection. PMID:21058689

The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum.

PubMed

Srinivasan, S; Griffiths, K R; McGuire, V; Champion, A; Williams, K L; Alexander, S

2000-08-01

The terminal event of spore differentiation in the cellular slime mould Dictyostelium discoideum is the assembly of the spore coat, which surrounds the dormant amoeba and allows the organism to survive during extended periods of environmental stress. The spore coat is a polarized extracellular matrix composed of glycoproteins and cellulose. The process of spore coat formation begins by the regulated secretion of spore coat proteins from the prespore vesicles (PSVs). Four of the major spore coat proteins (SP96, PsB/SP85, SP70 and SP60) exist as a preassembled multiprotein complex within the PSVs. This complete complex has an endogenous cellulose-binding activity. Mutant strains lacking either the SP96 or SP70 proteins produce partial complexes that do not have cellulose-binding activity, while mutants lacking SP60 produce a partial complex that retains this activity. Using a combination of immunofluorescence microscopy and biochemical methods we now show that the lack of cellulose-binding activity in the SP96 and SP70 mutants results in abnormally assembled spore coats and spores with greatly reduced viability. In contrast, the SP60 mutant, in which the PsB complex retains its cellulose-binding activity, produces spores with apparently unaltered structure and viability. Thus, it is the loss of the cellulose-binding activity of the PsB complex, rather than the mere loss of individual spore coat proteins, that results in compromised spore coat structure. These results support the idea that the cellulose-binding activity associated with the complete PsB complex plays an active role in the assembly of the spore coat.

Recyclable organic solar cells on cellulose nanocrystal substrates

Treesearch

Yinhua Zhou; Canek Fuentes-Hernandez; Talha M. Khan; Jen-Chieh Liu; James Hsu; Jae Won Shim; Amir Dindar; Jeffrey P. Youngblood; Robert J. Moon; Bernard Kippelen

2013-01-01

Solar energy is potentially the largest source of renewable energy at our disposal, but significant advances are required to make photovoltaic technologies economically viable and, from a life-cycle perspective, environmentally friendly, and consequently scalable. Cellulose nanomaterials are emerging high-value nanoparticles extracted from plants that are abundant,...

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production

Treesearch

Dana J. Wolbach; Alan Kuo; Trey K. Sato; Katlyn M. Potts; Asaf A. Salamov; Kurt M. LaButti; Hui Sun; Alicia Clum; Jasmyn L. Pangilinan; Erika A. Lindquist; Susan Lucas; Alla Lapidus; Mingjie Jin; Christa Gunawan; Venkatesh Balan; Bruce E. Dale; Thomas W. Jeffries; Robert Zinkel; Kerrie W. Barry; Igor V. Grigoriev; Audrey P. Gasch

2011-01-01

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative...

Production of single cell protein from cellulose wastes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Humphrey, A.E.; Moreira, A.; Armiger, W.

1977-01-01

Experiments made with a thermophilic Actinomyces that utilizes cellulose for growth are summarized. The organism, identified as a Thermoactinomyces sp., is a highly filamentous fungi. Although initial work was done with feedlot wastes, the variability of the data made it necessary to work on a uniform cellulose substrate Avicel. A probable mechanism of cellulose degradation by this fungi is suggested. Preliminary results are encouraging, but high growth rate must be maintained if a high cell yield is to be achieved. Both glucose and oxygen-limited growth were encountered; it is not known if these were coincidental or not. (JSR)

Cellulose Nanofiber Composite Substrates for Flexible Electronics

Treesearch

Ronald Sabo; Jung-Hun Seo; Zhenqiang Ma

2012-01-01

Flexible electronics have a large number of potential applications including malleable displays and wearable computers. The current research into high-speed, flexible electronic substrates employs the use of plastics for the flexible substrate, but these plastics typically have drawbacks, such as high thermal expansion coefficients. Transparent films made from...

A single-substrate model to interpret high-resolution intra-annual stable isotope signals in tree ring cellulose

NASA Astrophysics Data System (ADS)

Ogée, J.; Barbour, M. M.; Dewar, R. C.; Wingate, L.; Bert, D.; Bosc, A.; Lambrot, C.; Stievenard, M.; Bariac, T.; Berbigier, P.; Loustau, D.

2007-12-01

High-resolution measurements of the carbon and oxygen stable isotope composition of cellulose in annual tree rings (δ13Ccellulose and δ18Ocellulose, respectively) reveal well-defined seasonal patterns that could contain valuable records of past climate and tree function. Interpreting these signals is nonetheless complex because they not only record the signature of current assimilates, but also depend on carbon allocation dynamics within the trees. Here, we will present a single-substrate model for wood growth in order to interpret qualitatively and quantitatively these seasonal isotopic signals. We will also show how this model can relate to more complex models of phloem transport and cambial activity. The model will then be tested against an isotopic intra-annual chronology collected on a Pinus pinaster tree equipped with point dendrometers and growing on a Carboeurope site where climate, soil and flux variables are also monitored. The empirical δ13Ccellulose and δ18Ocellulose signals exhibit dynamic seasonal patterns with clear differences between years, which makes it suitable for model testing. We will show how our simple model of carbohydrate reserves, forced by sap flow and eddy covariance measurements, enables us to interpret these seasonal and inter-annual patterns. Finally, we will present a sensitivity analysis of the model, showing how gas-exchange parameters, carbon and water pool sizes or wood maturation times affect these isotopic signals. Acknowledgements: this study benefited from the CarboEurope-IP Bray site facilities and was funded by the French INSU programme Eclipse, with an additional support from the INRA department EFPA.

Defining Determinants and Dynamics and Cellulose Microfibril Biosynthesis, Assembly and Degredation OSP Number: 63079/A001

DOE Office of Scientific and Technical Information (OSTI.GOV)

None, None

The central paradigm for converting plant biomass into soluble sugars for subsequent conversion to transportation fuels involves the enzymatic depolymerization of lignocellulosic plant cell walls by microbial enzymes. Despite decades of intensive research, this is still a relatively inefficient process, due largely to the recalcitrance and enormous complexity of the substrate. A major obstacle is still insufficient understanding of the detailed structure and biosynthesis of major wall components, including cellulose. For example, although cellulose is generally depicted as rigid, insoluble, uniformly crystalline microfibrils that are resistant to enzymatic degradation, the in vivo structures of plant cellulose microfibrils are surprisingly complex.more » Crystallinity is frequently disrupted, for example by dislocations and areas containing chain ends, resulting in “amorphous” disordered regions. Importantly, microfibril structure and the relative proportions of crystalline and non-crystalline disordered surface regions vary substantially and yet the molecular mechanisms by which plants regulate microfibril crystallinity, and other aspects of microfibril architecture, are still entirely unknown. This obviously has a profound effect on susceptibility to enzymatic hydrolysis and so this is a critical area of research in order to characterize and optimize cellulosic biomass degradation. The entire field of cell wall assembly, as distinct from polysaccharide biosynthesis, and the degree to which they are coupled, are relatively unexplored, despite the great potential for major advances in addressing the hurdle of biomass recalcitrance. Our overarching hypothesis was that identification of the molecular machinery that determine microfibril polymerization, deposition and structure will allow the design of more effective degradative systems, and the generation of cellulosic materials with enhanced and predictable bioconversion characteristics. Our experimental framework had been based on the idea that the most effective way to address this long standing and highly complex question is to adopt a broad ‘systems approach’. Accordingly, we assembled a multi-disciplinary collaborative team with collective expertise in plant biology and molecular genetics, polymer structure and chemistry, enzyme biochemistry and biochemical engineering. We used a spectrum of cutting edge technologies, including plant functional genomics, chemical genetics, live cell imaging, advanced microscopy, high energy X-ray spectroscopy and nanotechnology, to study the molecular determinants of cellulose microfibril structure. Importantly, this research effort was closely coupled with an analytical pipeline to characterize the effects of altering microfibril architecture on bioconversion potential, with the goal of generating predictive models to help guide the identification, development and implementation of new feedstocks. This project therefore spanned core basic science and applied research, in line with the goals of the program. Over the course of the project, accomplishments included: - Establishing platforms through reverse and forward genetics to identify and manipulate candidate genes that influence cellulose microfibril synthesis and structure in a model C3 grass, Brachypodium distachyon and a model C4 grass Setaria viridis; Identifying and characterizing the effects of a number of cellulose biosynthesis inhibitors (CBIs), and particularly those that target monocots with the aim of generating resistance loci; Developing protocols for the use of high energy X-ray diffraction (XRD) to study the structure and organization of cellulose microfibrils in plant walls, notably those in Arabidopsis and Brachypodium; Using the chemical and genetic based inhibition strategies to develop new mechanistic models of cellulose microfibril crystallization, and of how altering microfibril architecture influences digestibility.« less

A Preliminary Study of the Spreading of AKD in the Presence of Capillary Structures.

PubMed

Shen, Wei; Parker, Ian H.

2001-08-01

There may be several mechanisms at work in the process of migration or redistribution of alkyl ketene dimers (AKD) on cellulose fiber surfaces during paper sizing and curing. This work is the second part of a continuing investigation of the spreading behavior of AKD on the surfaces of hydrophilic substrates. Paper sheets, single cotton, and cotton lint fibers and smooth cellulose film were used as substrates. These represent samples that have pores, V-shaped grooves, and no capillary structure at all. A very simple and effective testing method for studying the AKD migration behavior through these substrates was designed. AFM was used to study the surface capillary structures of cotton and cotton lint fibers. The results of this study provide hard evidence supporting our finding that capillary structures in the form of either interfiber pores in a paper sheet or V-shaped grooves on the surface of single fibers are essential in order for the spreading of molten AKD on a cellulose substrate to occur. Some preliminary results on the existence and the surface diffusion of an autophobic precursor of AKD are also presented. The results support the conclusion we reached in the first part of this investigation; i.e., the molten AKD wets but does not spread on smooth, capillary-free hydrophilic surfaces such as glass and cellulose. The driving force from interfacial energy alone does not cause spontaneous "flow-like" spreading of molten AKD on these surfaces. This is possibly associated with the formation of an autophobic precursor in front of an AKD droplet. The results in this study do not support the perception that molten AKD forms a single molecular layer on the surface of cellulose fibers by spreading during heat treatment, although the autophobic precursor in front of an AKD droplet could theoretically be of a monolayer thickness and the surface diffusion of this precursor may contribute to the sizing development after heat treatment. Copyright 2001 Academic Press.

The influence of lignin on steam pretreatment and mechanical pulping of poplar to achieve high sugar recovery and ease of enzymatic hydrolysis.

PubMed

Chandra, Richard P; Chu, QiuLu; Hu, Jinguang; Zhong, Na; Lin, Mandy; Lee, Jin-Suk; Saddler, Jack

2016-01-01

With the goal of enhancing overall carbohydrate recovery and reducing enzyme loading refiner mechanical pulping and steam pretreatment (210°C, 5 min) were used to pretreat poplar wood chips. Neutral sulphonation post-treatment indicated that, although the lignin present in the steam pretreated substrate was less reactive, the cellulose-rich, water insoluble component was more accessible to cellulases and Simons stain. This was likely due to lignin relocation as the relative surface lignin measured by X-ray photoelectron spectroscopy increased from 0.4 to 0.8. The integration of sulphite directly into steam pretreatment resulted in the solubilisation of 60% of the lignin while more than 80% of the carbohydrate present in the original substrate was recovered in the water insoluble fraction after Na2CO3 addition. More than 80% of the sugars present in the original cellulose and xylan could be recovered after 48 h using an enzyme loading of 20 mg protein/g cellulose at a 10% substrate concentration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Aromatic amino acids in the cellulose binding domain of Penicillium crustosum endoglucanase EGL1 differentially contribute to the cellulose affinity of the enzyme

PubMed Central

Xiong, Wei; Chen, Fang-Yuan; Xu, Li; Han, Zheng-Gang

2017-01-01

The cellulose binding domain (CBD) of cellulase binding to cellulosic materials is the initiation of a synergistic action on the enzymatic hydrolysis of the most abundant renewable biomass resources in nature. The binding of the CBD domain to cellulosic substrates generally relies on the interaction between the aromatic amino acids structurally located on the flat face of the CBD domain and the glucose rings of cellulose. In this study, we found the CBD domain of a newly cloned Penicillium crustosum endoglucanase EGL1, which was phylogenetically related to Aspergillus, Fusarium and Rhizopus, and divergent from the well-characterized Trichoderma reeseis cellulase CBD domain, contain two conserved aromatic amino acid-rich regions, Y451-Y452 and Y477-Y478-Y479, among which three amino acids Y451, Y477, and Y478 structurally sited on a flat face of this domain. Cellulose binding assays with green fluorescence protein as the marker, adsorption isotherm assays and an isothermal titration calorimetry assays revealed that although these three amino acids participated in this process, the Y451-Y452 appears to contribute more to the cellulose binding than Y477-Y478-Y479. Further glycine scanning mutagenesis and structural modelling revealed that the binding between CBD domain and cellulosic materials might be multi-amino-acids that participated in this process. The flexible poly-glucose molecule could contact Y451, Y477, and Y478 which form the contacting flat face of CBD domain as the typical model, some other amino acids in or outside the flat face might also participate in the interaction. Thus, it is possible that the conserved Y451-Y452 of CBD might have a higher chance of contacting the cellulosic substrates, contributing more to the affinity of CBD than the other amino acids. PMID:28475645

Enhanced hydrolysis of cellulose hydrogels by morphological modification.

PubMed

Alfassi, Gilad; Rein, Dmitry M; Cohen, Yachin

2017-11-01

Cellulose is one of the most abundant bio-renewable materials on earth, yet the potential of cellulosic bio-fuels is not fully exploited, primarily due to the high costs of conversion. Hydrogel particles of regenerated cellulose constitute a useful substrate for enzymatic hydrolysis, due to their porous and amorphous structure. This article describes the influence of several structural aspects of the cellulose hydrogel on its hydrolysis. The hydrogel density was shown to be directly proportional to the cellulose concentration in the initial solution, thus affecting its hydrolysis rate. Using high-resolution scanning electron microscopy, we show that the hydrogel particles in aqueous suspension exhibit a dense external surface layer and a more porous internal network. Elimination of the external surface layer accelerated the hydrolysis rate by up to sixfold and rendered the process nearly independent of cellulose concentration. These findings may be of practical relevance to saccharification processing costs, by reducing required solvent quantities and enzyme load.

Enzymatic hydrolysis of biomimetic bacterial cellulose-hemicellulose composites.

PubMed

Penttilä, Paavo A; Imai, Tomoya; Hemming, Jarl; Willför, Stefan; Sugiyama, Junji

2018-06-15

The production of biofuels and other chemicals from lignocellulosic biomass is limited by the inefficiency of enzymatic hydrolysis. Here a biomimetic composite material consisting of bacterial cellulose and wood-based hemicelluloses was used to study the effects of hemicelluloses on the enzymatic hydrolysis with a commercial cellulase mixture. Bacterial cellulose synthesized in the presence of hemicelluloses, especially xylan, was found to be more susceptible to enzymatic hydrolysis than hemicellulose-free bacterial cellulose. The reason for the easier hydrolysis could be related to the nanoscale structure of the substrate, particularly the packing of cellulose microfibrils into ribbons or bundles. In addition, small-angle X-ray scattering was used to show that the average nanoscale morphology of bacterial cellulose remained unchanged during the enzymatic hydrolysis. The reported easier enzymatic hydrolysis of bacterial cellulose produced in the presence of wood-based xylan offers new insights to overcome biomass recalcitrance through genetic engineering. Copyright © 2018 Elsevier Ltd. All rights reserved.

The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bashline, Logan; Li, Shundai; Zhu, Xiaoyu

Here, cellulose biosynthesis is performed exclusively by plasma membrane-localized cellulose synthases (CESAs). Therefore, the trafficking of CESAs to and from the plasma membrane is an important mechanism for regulating cellulose biosynthesis. CESAs were recently identified as cargo proteins of the classic adaptor protein 2 (AP2) complex of the clathrin-mediated endocytosis (CME) pathway. The AP2 complex of the CME pathway is conserved in yeast, animals, and plants, and has been well-characterized in many systems. In contrast, the recently discovered TPLATE complex (TPC), which is proposed to function as a CME adaptor complex, is only conserved in plants and a few othermore » eukaryotes. In this study, we discovered that the TWD40-2 protein, a putative member of the TPC, is also important for the endocytosis of CESAs. Genetic analysis between TWD40-2 and AP2M of the AP2 complex revealed that the roles of TWD40-2 in CME are both distinct from and cooperative with the AP2 complex. Loss of efficient CME in twd40-2-3 resulted in the unregulated overaccumulation of CESAs at the plasma membrane. In seedlings of twd40-2-3 and other CME-deficient mutants, a direct correlation was revealed between endocytic deficiency and cellulose content deficiency, highlighting the importance of controlled CESA endocytosis in regulating cellulose biosynthesis.« less

The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis

DOE PAGES

Bashline, Logan; Li, Shundai; Zhu, Xiaoyu; ...

2015-09-28

Here, cellulose biosynthesis is performed exclusively by plasma membrane-localized cellulose synthases (CESAs). Therefore, the trafficking of CESAs to and from the plasma membrane is an important mechanism for regulating cellulose biosynthesis. CESAs were recently identified as cargo proteins of the classic adaptor protein 2 (AP2) complex of the clathrin-mediated endocytosis (CME) pathway. The AP2 complex of the CME pathway is conserved in yeast, animals, and plants, and has been well-characterized in many systems. In contrast, the recently discovered TPLATE complex (TPC), which is proposed to function as a CME adaptor complex, is only conserved in plants and a few othermore » eukaryotes. In this study, we discovered that the TWD40-2 protein, a putative member of the TPC, is also important for the endocytosis of CESAs. Genetic analysis between TWD40-2 and AP2M of the AP2 complex revealed that the roles of TWD40-2 in CME are both distinct from and cooperative with the AP2 complex. Loss of efficient CME in twd40-2-3 resulted in the unregulated overaccumulation of CESAs at the plasma membrane. In seedlings of twd40-2-3 and other CME-deficient mutants, a direct correlation was revealed between endocytic deficiency and cellulose content deficiency, highlighting the importance of controlled CESA endocytosis in regulating cellulose biosynthesis.« less

The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters.

PubMed

McDonald, James E; Houghton, James N I; Rooks, David J; Allison, Heather E; McCarthy, Alan J

2012-04-01

Cellulose is reputedly the most abundant organic polymer in the biosphere, yet despite the fundamental role of cellulolytic microorganisms in global carbon cycling and as potential sources of novel enzymes for biotechnology, their identity and ecology is not well established. Cellulose is a major component of landfill waste and its degradation is therefore a key feature of the anaerobic microbial decomposition process. Here, we targeted a number of taxa containing known cellulolytic anaerobes (members of the bacterial genus Fibrobacter, lineages of Clostridium clusters I, III, IV and XIV, and anaerobic fungi of the Neocallimastigales) in landfill leachate and colonized cellulose 'baits' via PCR and quantitative PCR (qPCR). Fibrobacter spp. and Clostridium clusters III, IV and XIV were detected in almost all leachate samples and cluster III and XIV clostridia were the most abundant (1-6% and 1-17% of total bacterial 16S rRNA gene copies respectively). Two landfill leachate microcosms were constructed to specifically assess those microbial communities that colonize and degrade cellulose substrates in situ. Scanning electron microscopy (SEM) of colonized cotton revealed extensive cellulose degradation in one microcosm, and Fibrobacter spp. and Clostridium cluster III represented 29% and 17%, respectively, of total bacterial 16S rRNA gene copies in the biofilm. Visible cellulose degradation was not observed in the second microcosm, and this correlated with negligible relative abundances of Clostridium cluster III and Fibrobacter spp. (≤ 0.1%), providing the first evidence that the novel fibrobacters recently detected in landfill sites and other non-gut environments colonize and degrade cellulose substrates in situ. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Effect of polysaccharide admixtures on expression of multiple polysaccharide-degrading enzymes in Microbulbifer strain CMC-5.

PubMed

Jonnadula, RaviChand; Imran, Md; Poduval, Preethi B; Ghadi, Sanjeev C

2018-03-01

Microbulbifer strain CMC-5 produces agarase, alginate lyase, xylanase, carboxymethyl cellulase and carrageenase. The extracellular production of the above carbohydrases was investigated by growing Microbulbifer strain CMC-5 in a sea water based medium containing homologous/heterologous polysaccharides as a single substrate or as a combination of mixed assorted substrate. Presence of singular homologous polysaccharides in the growth medium induces respective carbohydrase at high levels. Any two polysaccharides in various combinations produced high level of homologous carbohydrase and low level of other heterologous carbohydrase. All five carbohydrases were consistently produced by strain CMC-5, when carboxymethyl cellulose was included as one of the substrate in dual substrate combination, or in presence of mix blends of all five polysaccharides. Interestingly, thalli of Gracilaria sp. that contain agar and cellulose predominantly in their cell wall induces only agarase expression in strain CMC-5.

Combined enzyme mediated fermentation of cellulous and xylose to ethanol by Schizosaccharoyces pombe, cellulase, .beta.-glucosidase, and xylose isomerase

DOEpatents

Lastick, Stanley M.; Mohagheghi, Ali; Tucker, Melvin P.; Grohmann, Karel

1994-01-01

A process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol; loading the substrates with the fermentation mix composed of yeast, enzymes and substrates; fermenting the loaded substrates and enzymes under anaerobic conditions at a pH range of between about 5.0 to about 6.0 and at a temperature range of between about 35.degree. C. to about 40.degree. C. until the fermentation is completed, the xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast through means of the anaerobic fermentation; and recovering the ethanol.

Multifunctional paper strip based on GO-veiled Ag nanoparticles with highly SERS sensitive and deliverable properties for high-performance molecular detection.

PubMed

Yang, Cheng; Xu, Yuanyuan; Wang, Minghong; Li, Tianming; Huo, Yanyan; Yang, Chuanxi; Man, Baoyuan

2018-04-16

The development of paper-based SERS substrates that can allow multi-component detection in real-word scenarios is of great value for applications in molecule detection under complex conditions. Here, a multifunctional SERS-based paper sensing substrate has been developed through the uniform patterning of high-density arrays of GO-isolated Ag nanoparticles on the hydrophilic porous cellulose paper strip (GO@AgNP@paper). Wet-chemical synthesis was used to provide the cover of SERS hot spots on any part of the paper, not just limited surface deposition. In virtue of the inherent ability of paper to deliver analytes by the capillary force, the detection ability of the GO@AgNP@paper substrate was greatly promoted, allowing as low as 10 -19 M R6G detection from microliter-volume (50 μL) samples. For the components with different polarity, the paper substrate can be used as an all-in-one machine to achieve the integration of separation and high-sensitive detection for ultralow mixture components, which improves the practical application value of SERS-based paper devices.

Facile preparation of optically transparent and hydrophobic cellulose nanofibril composite films

Treesearch

Yan Qing; Zhiyong Cai; Yiqiang Wu; Chunhua Yao; Qinglin Wu; Xianjun Li

2015-01-01

Cellulose nanofibril (CNF) and epoxy nanocomposites with high visible light transmittance and low watersensitivity were manufactured by laminating thin layers of epoxy resin onto CNF films prepared through,pressurized filtration in combination with oven drying. Scanning Electron Microscopy (SEM) studiessuggest that the resin component bonded to the CNF substrate well....

Stable Low-Voltage Operation Top-Gate Organic Field-Effect Transistors on Cellulose Nanocrystal Substrates

Treesearch

Cheng-Yin Wang; Canek Fuentes-Hernandez; Jen-Chieh Liu; Amir Dindar; Sangmoo Choi; Jeffrey P. Youngblood; Robert J. Moon; Bernard Kippelen

2015-01-01

We report on the performance and the characterization of top-gate organic field-effect transistors (OFETs), comprising a bilayer gate dielectric of CYTOP/ Al2O3 and a solution-processed semiconductor layer made of a blend of TIPS-pentacene:PTAA, fabricated on recyclable cellulose nanocrystal−glycerol (CNC/glycerol...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolyticmore » activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.« less

Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae.

PubMed

Shirley, Derek; Oppert, Cris; Reynolds, Todd B; Miracle, Bethany; Oppert, Brenda; Klingeman, William E; Jurat-Fuentes, Juan Luis

2014-10-01

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

DOE PAGES

Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai; ...

2015-12-21

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolyticmore » activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.« less

Evaluation of Production Version of the NASA Improved Inorganic-Organic Separator

NASA Technical Reports Server (NTRS)

Sheibley, D.

1983-01-01

The technology of an inorganic-organic (I/O) separator, which demonstrated improved flexibility, reduced cost, production feasibility and improved cycle life was developed. Substrates to replace asbestos and waterbased separator coatings to replace the solvent based coatings were investigated. An improved fuel cell grade asbestos sheet was developed and a large scale production capability for the solvent based I/O separator was demonstrated. A cellulose based substrate and a nonwoven polypropylene fiber substrate were evaluated as replacements for the asbestos. Both the cellulose and polypropylene substrates were coated with solvent based and water based coatings to produce a modified I/O separator. The solvent based coatings were modified to produce aqueous separator coatings with acceptable separator properties. A single ply fuel cell grade asbestos with a binder (BTA) was produced. It has shown to be an acceptable substrate for the solvent and water based separator coatings, an acceptable absorber for alkaline cells, and an acceptable matrix for alkaline fuel cells. The original solvent based separator (K19W1), using asbestos as a substrate, was prepared.

Production and Characterization of Highly Thermostable β-Glucosidase during the Biodegradation of Methyl Cellulose by Fusarium oxysporum

PubMed Central

Olajuyigbe, Folasade M.; Nlekerem, Chidinma M.; Ogunyewo, Olusola A.

2016-01-01

Production of β-glucosidase from Fusarium oxysporum was investigated during degradation of some cellulosic substrates (Avicel, α-cellulose, carboxymethyl cellulose (CMC), and methylcellulose). Optimized production of β-glucosidase using the cellulosic substrate that supported highest yield of enzyme was examined over 192 h fermentation period and varied pH of 3.0–11.0. The β-glucosidase produced was characterized for its suitability for industrial application. Methyl cellulose supported the highest yield of β-glucosidase (177.5 U/mg) at pH 6.0 and 30°C at 96 h of fermentation with liberation of 2.121 μmol/mL glucose. The crude enzyme had optimum activity at pH 5.0 and 70°C. The enzyme was stable over broad pH range of 4.0–7.0 with relative residual activity above 60% after 180 min of incubation. β-glucosidase demonstrated high thermostability with 83% of its original activity retained at 70°C after 180 min of incubation. The activity of β-glucosidase was enhanced by Mn2+ and Fe2+ with relative activities of 167.67% and 205.56%, respectively, at 5 mM and 360% and 315%, respectively, at 10 mM. The properties shown by β-glucosidase suggest suitability of the enzyme for industrial applications in the improvement of hydrolysis of cellulosic compounds into fermentable sugars that can be used in energy generation and biofuel production. PMID:26977320

Preparation of cellulase concoction using differential adsorption phenomenon.

PubMed

Birhade, Sachinkumar; Pednekar, Mukesh; Sagwal, Shilpa; Odaneth, Annamma; Lali, Arvind

2017-05-28

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.

Enhanced cellulose orientation analysis in complex model plant tissues.

PubMed

Rüggeberg, Markus; Saxe, Friederike; Metzger, Till H; Sundberg, Björn; Fratzl, Peter; Burgert, Ingo

2013-09-01

The orientation distribution of cellulose microfibrils in the plant cell wall is a key parameter for understanding anisotropic plant growth and mechanical behavior. However, precisely visualizing cellulose orientation in the plant cell wall has ever been a challenge due to the small size of the cellulose microfibrils and the complex network of polymers in the plant cell wall. X-ray diffraction is one of the most frequently used methods for analyzing cellulose orientation in single cells and plant tissues, but the interpretation of the diffraction images is complex. Traditionally, circular or square cells and Gaussian orientation of the cellulose microfibrils have been assumed to elucidate cellulose orientation from the diffraction images. However, the complex tissue structures of common model plant systems such as Arabidopsis or aspen (Populus) require a more sophisticated approach. We present an evaluation procedure which takes into account the precise cell geometry and is able to deal with complex microfibril orientation distributions. The evaluation procedure reveals the entire orientation distribution of the cellulose microfibrils, reflecting different orientations within the multi-layered cell wall. By analyzing aspen wood and Arabidopsis stems we demonstrate the versatility of this method and show that simplifying assumptions on geometry and orientation distributions can lead to errors in the calculated microfibril orientation pattern. The simulation routine is intended to be used as a valuable tool for nanostructural analysis of plant cell walls and is freely available from the authors on request. Copyright © 2013 Elsevier Inc. All rights reserved.

Thermophilic degradation of cellulosic biomass

NASA Astrophysics Data System (ADS)

Ng, T.; Zeikus, J. G.

1982-12-01

The conversion of cellulosic biomass to chemical feedstocks and fuel by microbial fermentation is an important objective of developing biotechnology. Direct fermentation of cellulosic derivatives to ethanol by thermophilic bacteria offers a promising approach to this goal. Fermentations at elevated temperatures lowers the energy demand for cooling and also facilitates the recovery of volatile products. In addition, thermophilic microorganisms possess enzymes with greater stability than those from mesophilic microorganisms. Three anaerobic thermophilic cocultures that ferment cellulosic substrate mainly to ethanol have been described: Clostridium thermocellum/Clostriidium thermohydrosulfuricum, C. thermocellum/Clostridium thermosaccharolyticum, and C. thermocellum/Thermoanaerobacter ethanolicus sp. nov. The growth characteristics and metabolic features of these cocultures are reviewed.

Disposable chemical sensors and biosensors made on cellulose paper.

PubMed

Kim, Joo-Hyung; Mun, Seongcheol; Ko, Hyun-U; Yun, Gyu-Young; Kim, Jaehwan

2014-03-07

Most sensors are based on ceramic or semiconducting substrates, which have no flexibility or biocompatibility. Polymer-based sensors have been the subject of much attention due to their ability to collect molecules on their sensing surface with flexibility. Beyond polymer-based sensors, the recent discovery of cellulose as a smart material paved the way to the use of cellulose paper as a potential candidate for mechanical as well as electronic applications such as actuators and sensors. Several different paper-based sensors have been investigated and suggested. In this paper, we review the potential of cellulose materials for paper-based application devices, and suggest their feasibility for chemical and biosensor applications.

A simple, single-substrate model to interpret intra-annual stable isotope signals in tree-ring cellulose

NASA Astrophysics Data System (ADS)

Ogée, J.; Barbour, M. M.; Wingate, L.; Bert, D.; Bosc, A.; Stievenard, M.; Lambrot, C.; Pierre, M.; Bariac, T.; Dewar, R. C.

2009-04-01

High-resolution intra-annual measurements of the carbon and oxygen stable isotope composition of cellulose in annual tree rings (δ13Ccellulose and δ18Ocellulose, respectively) reveal well-defined seasonal patterns that could contain valuable records of past climate and tree function. Interpreting these signals is nonetheless complex because they not only record the signature of current assimilates, but also depend on carbon allocation dynamics within the trees. Here, we present a simple, single-substrate model for wood growth containing only 12 main parameters. The model is used to interpret an isotopic intra-annual chronology collected in an even-aged maritime pine plantation growing in the South-West of France, where climate, soil and flux variables were also monitored. The empirical δ13Ccellulose and δ18Ocellulose exhibit dynamic seasonal patterns, with clear differences between years and individuals, that are mostly captured by the model. In particular, the amplitude of both signals is reproduced satisfactorily as well as the sharp 18O enrichment at the beginning of 1997 and the less pronounced 13C and 18O depletion observed at the end of the latewood. Our results suggest that the single-substrate hypothesis is a good approximation for tree ring studies on Pinus pinaster, at least for the environmental conditions covered by this study. A sensitivity analysis revealed that, in the early wood, the model was particularly sensitive to the date when cell wall thickening begins (twt). We therefore propose to use the model to reconstruct time series of twt and explore how climate influences this key parameter of xylogenesis.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass.

PubMed

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-07-29

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

PubMed Central

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-01-01

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. PMID:19680472

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

PubMed

Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

2015-11-01

Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

Morphology and physical-chemical properties of celluloses obtained by different methods

NASA Astrophysics Data System (ADS)

Anpilova, A. Yu.; Mastalygina, E. E.; Mikhaylov, I. A.; Popov, A. A.; Kartasheva, Z. S.

2017-12-01

The morphology and structural characteristics of celluloses obtained by different methods were studied. The objects of the investigation are cellulose from pulp source, commercial celluloses produced by sodium and acid hydrolysis, laboratory produced cellulose from bleached birch kraft pulp, and cellulose obtained by thermooxidative catalytic treatment of maple leaves by peroxide. According to a complex analysis of cellulose characteristics, several types of celluloses were offered as modifying additives for polymers.

Cellulosic Substrates and Challenges Ahead

USDA-ARS?s Scientific Manuscript database

The cost of production of butanol (acetone-butanol-ethanol; or ABE) is determined by feedstock prices, fermentation, recovery, by-product credits and the waste water treatment. Along these lines, we have an intensive research program on the use of various agricultural substrates, fermentation strate...

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis.

PubMed

Sornlake, Warasirin; Rattanaphanjak, Phatcharamon; Champreda, Verawat; Eurwilaichitr, Lily; Kittisenachai, Suthathip; Roytrakul, Sittiruk; Fujii, Tatsuya; Inoue, Hiroyuki

2017-07-01

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and β-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external β-xylosidase.

Xylan hydrolysis in Populus trichocarpa × P. deltoides and model substrates during hydrothermal pretreatment.

PubMed

Trajano, Heather L; Pattathil, Sivakumar; Tomkins, Bruce A; Tschaplinski, Timothy J; Hahn, Michael G; Van Berkel, Gary J; Wyman, Charles E

2015-03-01

Previous studies defined easy and difficult to hydrolyze fractions of hemicellulose that may result from bonds among cellulose, hemicellulose, and lignin. To understand how such bonds affect hydrolysis, Populus trichocarpa × Populus deltoides, holocellulose isolated from P. trichocarpa × P. deltoides and birchwood xylan were subjected to hydrothermal flow-through pretreatment. Samples were characterized by glycome profiling, HPLC, and UPLC-MS. Glycome profiling revealed steady fragmentation and removal of glycans from solids during hydrolysis. The extent of polysaccharide fragmentation, hydrolysis rate, and total xylose yield were lowest for P. trichocarpa × P. deltoides and greatest for birchwood xylan. Comparison of results from P. trichocarpa × P. deltoides and holocellulose suggested that lignin-carbohydrate complexes reduce hydrolysis rates and limit release of large xylooligomers. Smaller differences between results with holocellulose and birchwood xylan suggest xylan-cellulose hydrogen bonds limited hydrolysis, but to a lesser extent. These findings imply cell wall structure strongly influences hydrolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Seasonal variation in functional properties of microbial communities in beech forest soil

PubMed Central

Koranda, Marianne; Kaiser, Christina; Fuchslueger, Lucia; Kitzler, Barbara; Sessitsch, Angela; Zechmeister-Boltenstern, Sophie; Richter, Andreas

2013-01-01

Substrate quality and the availability of nutrients are major factors controlling microbial decomposition processes in soils. Seasonal alteration in resource availability, which is driven by plants via belowground C allocation, nutrient uptake and litter fall, also exerts effects on soil microbial community composition. Here we investigate if seasonal and experimentally induced changes in microbial community composition lead to alterations in functional properties of microbial communities and thus microbial processes. Beech forest soils characterized by three distinct microbial communities (winter and summer community, and summer community from a tree girdling plot, in which belowground carbon allocation was interrupted) were incubated with different 13C-labeled substrates with or without inorganic N supply and analyzed for substrate use and various microbial processes. Our results clearly demonstrate that the three investigated microbial communities differed in their functional response to addition of various substrates. The winter communities revealed a higher capacity for degradation of complex C substrates (cellulose, plant cell walls) than the summer communities, indicated by enhanced cellulase activities and reduced mineralization of soil organic matter. In contrast, utilization of labile C sources (glucose) was lower in winter than in summer, demonstrating that summer and winter community were adapted to the availability of different substrates. The saprotrophic community established in girdled plots exhibited a significantly higher utilization of complex C substrates than the more plant root associated community in control plots if additional nitrogen was provided. In this study we were able to demonstrate experimentally that variation in resource availability as well as seasonality in temperate forest soils cause a seasonal variation in functional properties of soil microorganisms, which is due to shifts in community structure and physiological adaptations of microbial communities to altered resource supply. PMID:23645937

Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis.

PubMed

Kumar, Manoj; Mishra, Laxmi; Carr, Paul; Pilling, Michael; Gardner, Peter; Mansfield, Shawn D; Turner, Simon

2018-05-01

Cellulose microfibrils are the basic units of cellulose in plants. The structure of these microfibrils is at least partly determined by the structure of the cellulose synthase complex. In higher plants, this complex is composed of 18 to 24 catalytic subunits known as CELLULOSE SYNTHASE A (CESA) proteins. Three different classes of CESA proteins are required for cellulose synthesis and for secondary cell wall cellulose biosynthesis these classes are represented by CESA4, CESA7, and CESA8. To probe the relationship between CESA proteins and microfibril structure, we created mutant cesa proteins that lack catalytic activity but retain sufficient structural integrity to allow assembly of the cellulose synthase complex. Using a series of Arabidopsis ( Arabidopsis thaliana ) mutants and genetic backgrounds, we found consistent differences in the ability of these mutant cesa proteins to complement the cellulose-deficient phenotype of the cesa null mutants. The best complementation was observed with catalytically inactive cesa4, while the equivalent mutation in cesa8 exhibited significantly lower levels of complementation. Using a variety of biophysical techniques, including solid-state nuclear magnetic resonance and Fourier transform infrared microscopy, to study these mutant plants, we found evidence for changes in cellulose microfibril structure, but these changes largely correlated with cellulose content and reflected differences in the relative proportions of primary and secondary cell walls. Our results suggest that individual CESA classes have similar roles in determining cellulose microfibril structure, and it is likely that the different effects of mutating members of different CESA classes are the consequence of their different catalytic activity and their influence on the overall rate of cellulose synthesis. © 2018 American Society of Plant Biologists. All Rights Reserved.

Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis1[OPEN

PubMed Central

Mishra, Laxmi; Carr, Paul; Gardner, Peter

2018-01-01

Cellulose microfibrils are the basic units of cellulose in plants. The structure of these microfibrils is at least partly determined by the structure of the cellulose synthase complex. In higher plants, this complex is composed of 18 to 24 catalytic subunits known as CELLULOSE SYNTHASE A (CESA) proteins. Three different classes of CESA proteins are required for cellulose synthesis and for secondary cell wall cellulose biosynthesis these classes are represented by CESA4, CESA7, and CESA8. To probe the relationship between CESA proteins and microfibril structure, we created mutant cesa proteins that lack catalytic activity but retain sufficient structural integrity to allow assembly of the cellulose synthase complex. Using a series of Arabidopsis (Arabidopsis thaliana) mutants and genetic backgrounds, we found consistent differences in the ability of these mutant cesa proteins to complement the cellulose-deficient phenotype of the cesa null mutants. The best complementation was observed with catalytically inactive cesa4, while the equivalent mutation in cesa8 exhibited significantly lower levels of complementation. Using a variety of biophysical techniques, including solid-state nuclear magnetic resonance and Fourier transform infrared microscopy, to study these mutant plants, we found evidence for changes in cellulose microfibril structure, but these changes largely correlated with cellulose content and reflected differences in the relative proportions of primary and secondary cell walls. Our results suggest that individual CESA classes have similar roles in determining cellulose microfibril structure, and it is likely that the different effects of mutating members of different CESA classes are the consequence of their different catalytic activity and their influence on the overall rate of cellulose synthesis. PMID:29523715

Robust cellulosic ethanol production from SPORL-pretreated lodgepole pine using an adapted strain Saccharomyces cervisiae without detoxification

Treesearch

S. Tian; X.L. Luo; X.S. Yang; J.Y. Zhu

2010-01-01

This study reports an ethanol yield of 270 L/ton wood from lodgepole pine pretreated with sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) using an adapted strain, Saccharomyces cerevisiae Y5, without detoxification. The enzymatic hydrolysate produced from pretreated cellulosic solids substrate was combined with pretreatment hydrolysate before...

[Synthesis and properties of immobilized enzymes. X. Covalent binding of polygalacturonase to insoluble carriers].

PubMed

Flemming, C; Göbel, H; Wand, H; Gabert, A; Bock, W

1978-01-01

The pectinolytic enzymes are of practical interest for the clarification of fruit juice. In the present paper the covalent coupling of polygalacturonase (PG; E. C. 3.2.1.15) is reported. A commercially available enzyme (Rohament P; 5 U/mg) and purified Endo-PG (200 U/mg) are immobilized to the following carriers: BrCN-activated Sepharose, carbodiimide-activated CH-Sepharose, dialdehyde Sepharose, dialdehyde Sephadex, dialdehyde cellulose, CMC-azide, carbodiimide-activated CMC, macroporous glass (isothiocyanate and carbodiimide coupling) and glass beads. The implications of pore diameter (Sephadex- and Sepharose derivatives), of purity of the PG, of protein content of the PG-carrier-complexes as well as the presence of substrate during the coupling reaction, are discused in relation to the relative and specific activity of the bound protein and to the efficiency of the coupling reaction. From the carriers under study derivatives of Sepharose yield the best result (relative activity max. 88%, specific activity max. 5400 U/mg). The immobilization to isothiocyanate glass yields good results, too (relative activity 20%, specific activity 500 U/g). The mechanical instability of the PG-dialdehye Sephadex-complexes and the low relative activity of the bound enzyme are unsatisfactory. Due to their low affinity to PG, the derivatives of cellulose are also inappropriate for covalent coupling of this enzyme. All PG-carrier-complexes are largely stable both during storage at 4 degrees C and repeated activity assays.

Cellulase production from spent sulfite liquor and paper-mill waste fiber

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qu Yinbo; Zhao Xin; Gao Peiji

1991-12-31

Since a high proportion of the overall cost of the conversion of cellulosics to useful products is the expense of cellulose production (1), it is desirable to develop new processes for producing large amounts of cellulase inexpensively. So far, most of the research work on cellulose production has been carried out using milled cellulose powder and inorganic salts as substrates, which significantly increases the cost of enzyme production. In order to reduce the cost of raw materials, we tried to develop from industrial wastes a new medium for the production of cellulose. In this report, we describe a simple methodmore » by which an all-waste medium, which was composed of spent ammonium sulfite liquor and cellulosic waste of a paper mill, and a catabolite derepression mutant of Penicillium decumbens were used to produce the enzyme efficiently.« less

The plant cell-wall enzyme AtXTH3 catalyses covalent cross-linking between cellulose and cello-oligosaccharide

NASA Astrophysics Data System (ADS)

Shinohara, Naoki; Sunagawa, Naoki; Tamura, Satoru; Yokoyama, Ryusuke; Ueda, Minoru; Igarashi, Kiyohiko; Nishitani, Kazuhiko

2017-04-01

Cellulose is an economically important material, but routes of its industrial processing have not been fully explored. The plant cell wall - the major source of cellulose - harbours enzymes of the xyloglucan endotransglucosylase/hydrolase (XTH) family. This class of enzymes is unique in that it is capable of elongating polysaccharide chains without the requirement for activated nucleotide sugars (e.g., UDP-glucose) and in seamlessly splitting and reconnecting chains of xyloglucan, a naturally occurring soluble analogue of cellulose. Here, we show that a recombinant version of AtXTH3, a thus far uncharacterized member of the Arabidopsis XTH family, catalysed the transglycosylation between cellulose and cello-oligosaccharide, between cellulose and xyloglucan-oligosaccharide, and between xyloglucan and xyloglucan-oligosaccharide, with the highest reaction rate observed for the latter reaction. In addition, this enzyme formed cellulose-like insoluble material from a soluble cello-oligosaccharide in the absence of additional substrates. This newly found activity (designated “cellulose endotransglucosylase,” or CET) can potentially be involved in the formation of covalent linkages between cellulose microfibrils in the plant cell wall. It can also comprise a new route of industrial cellulose functionalization.

An Improved X-ray Diffraction Method For Cellulose Crystallinity Measurement

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Xiaohui; Bowden, Mark E.; Brown, Elvie E.

2015-06-01

We show in this work a modified X-ray diffraction method to determine cellulose crystallinity index (CrI). Nanocrystalline cellulose (NCC) dervided from bleached wood pulp was used as a model substrate. Rietveld refinement was applied with consideration of March-Dollase preferred orientation at the (001) plane. In contrast to most previous methods, three distinct amorphous peaks identified from new model samples which are used to calculate CrI. A 2 theta range from 10° to 75° was found to be more suitable to determine CrI and crystallite structural parameters such as d-spacing and crystallite size. This method enables a more reliable measurement ofmore » CrI of cellulose and may be applicable to other types of cellulose polymorphs.« less

The Effect of Alkaline Concentration on Coconut Husk Crystallinity and the Yield of Sugars Released

NASA Astrophysics Data System (ADS)

Sangian, H. F.; Widjaja, A.

2018-02-01

This work was to analyze the effect of alkaline concentration on coconut coir husk crystallinity and sugar liberated enzymatically. The data showed that the employing of alkaline on lignocellulose transformed the crystallinity. The XRD peaks increased highly which indicated that cellulose was more opened and exposed. After pretreatment, the chemical compositions (cellulose, hemicellulose, and lignin) were changed significantly. The employing 1% alkaline, the cellulosic content inclined if compared to that of non-pretreatment. When the alkaline concentration was added to 4%, the cellulose was decreased slightly which indicated that a part of cellulose and hemicellulose was dissolved into solution. It was found the alkaline pretreatment influenced by the biochemical reaction of treated substrates in producing the reducing sugars. The amounts of sugar liberated enzymatically of coconut husk treated by 1% and 4% alkaline increased to 0.26, and 0.24 g sugar/g (cellulose+hemicellulose), respectively, compared to that of native solid recorded at 0.18 g sugar/g (cellulose+hemicellulose).

Extremely thermophilic microorganisms for biomass conversion: status and prospects.

PubMed

Blumer-Schuette, Sara E; Kataeva, Irina; Westpheling, Janet; Adams, Michael Ww; Kelly, Robert M

2008-06-01

Many microorganisms that grow at elevated temperatures are able to utilize a variety of carbohydrates pertinent to the conversion of lignocellulosic biomass to bioenergy. The range of substrates utilized depends on growth temperature optimum and biotope. Hyperthermophilic marine archaea (T(opt)>or=80 degrees C) utilize alpha- and beta-linked glucans, such as starch, barley glucan, laminarin, and chitin, while hyperthermophilic marine bacteria (T(opt)>or=80 degrees C) utilize the same glucans as well as hemicellulose, such as xylans and mannans. However, none of these organisms are able to efficiently utilize crystalline cellulose. Among the thermophiles, this ability is limited to a few terrestrial bacteria with upper temperature limits for growth near 75 degrees C. Deconstruction of crystalline cellulose by these extreme thermophiles is achieved by 'free' primary cellulases, which are distinct from those typically associated with large multi-enzyme complexes known as cellulosomes. These primary cellulases also differ from the endoglucanases (referred to here as 'secondary cellulases') reported from marine hyperthermophiles that show only weak activity toward cellulose. Many extremely thermophilic enzymes implicated in the deconstruction of lignocellulose can be identified in genome sequences, and many more promising biocatalysts probably remain annotated as 'hypothetical proteins'. Characterization of these enzymes will require intensive effort but is likely to generate new opportunities for the use of renewable resources as biofuels.

Anaerobic rumen SBR for degradation of cellulosic material.

PubMed

Barnes, S P; Keller, J

2004-01-01

Hydrolysis of organic particulates under anaerobic conditions is generally regarded as the rate limiting step in solid digestion processes. Rumen-based ecosystems appear to achieve very high hydrolysis rates for cellulosic organic material. This study aimed at the development and demonstration of an anaerobic sequencing batch reactor (SBR) process operating with a rumen-based microbial inoculum. Fibrous alpha cellulose was used as sole carbon substrate and the use of an SBR operating cycle allowed the utilisation of a high liquid flow rate (hydraulic retention time of 0.67 d) while maintaining a much longer solids retention time of 7 d. Complete mass balances for carbon and nitrogen, as well as COD balancing allowed the full characterisation of the process stoichiometry and kinetics. Elemental analysis of the biomass revealed a composition of C5H4.8O2.4N0.7, which is quite different from other generic biomass compositions used in the literature. The anaerobic rumen SBR was compared with another rumen-based reactor system in the literature which used a continuous filtration process for solid/liquid separation. This comparison showed that the volatile fatty acid production rate from cellulose in the anaerobic SBR was comparable with the performance achieved in the continuous system, although loading, substrate type and media composition were quite different between these two studies. Further evaluation of the anaerobic rumen SBR is required to determine its practical application for other substrates and to demonstrate the scale-up potential of this concept.

Better together: synergy in nanocellulose blends

NASA Astrophysics Data System (ADS)

Mautner, Andreas; Mayer, Florian; Hervy, Martin; Lee, Koon-Yang; Bismarck, Alexander

2017-12-01

Cellulose nanopapers have gained significant attention in recent years as large-scale reinforcement for high-loading cellulose nanocomposites, substrates for printed electronics and filter nanopapers for water treatment. The mechanical properties of nanopapers are of fundamental importance for all these applications. Cellulose nanopapers can simply be prepared by filtering a suspension of nanocellulose, followed by heat consolidation. It was already demonstrated that the mechanical properties of cellulose nanopapers can be tailored by the fineness of the fibrils used or by modifying nanocellulose fibrils for instance by polymer adsorption, but nanocellulose blends remain underexplored. In this work, we show that the mechanical and physical properties of cellulose nanopapers can be tuned by creating nanopapers from blends of various grades of nanocellulose, i.e. (mechanically refined) bacterial cellulose or cellulose nanofibrils extracted from never-dried bleached softwood pulp by chemical and mechanical pre-treatments. We found that nanopapers made from blends of two or three nanocellulose grades show synergistic effects resulting in improved stiffness, strength, ductility, toughness and physical properties. This article is part of a discussion meeting issue `New horizons for cellulose nanotechnology'.

An Outer Membrane Protein Involved in the Uptake of Glucose Is Essential for Cytophaga hutchinsonii Cellulose Utilization

PubMed Central

Zhou, Hong; Wang, Xia; Yang, Tengteng; Zhang, Weixin; Chen, Guanjun

2016-01-01

Cytophaga hutchinsonii specializes in cellulose digestion by employing a collection of novel cell-associated proteins. Here, we identified a novel gene locus, CHU_1276, that is essential for C. hutchinsonii cellulose utilization. Disruption of CHU_1276 in C. hutchinsonii resulted in complete deficiency in cellulose degradation, as well as compromised assimilation of cellobiose or glucose at a low concentration. Further analysis showed that CHU_1276 was an outer membrane protein that could be induced by cellulose and low concentrations of glucose. Transcriptional profiling revealed that CHU_1276 exerted a profound effect on the genome-wide response to both glucose and Avicel and that the mutant lacking CHU_1276 displayed expression profiles very different from those of the wild-type strain under different culture conditions. Specifically, comparison of their transcriptional responses to cellulose led to the identification of a gene set potentially regulated by CHU_1276. These results suggest that CHU_1276 plays an essential role in cellulose utilization, probably by coordinating the extracellular hydrolysis of cellulose substrate with the intracellular uptake of the hydrolysis product in C. hutchinsonii. PMID:26773084

The identification of and relief from Fe3+ inhibition for both cellulose and cellulase in cellulose saccharification catalyzed by cellulases from Penicillium decumbens.

PubMed

Wang, Mingyu; Mu, Ziming; Wang, Junli; Hou, Shaoli; Han, Lijuan; Dong, Yanmei; Xiao, Lin; Xia, Ruirui; Fang, Xu

2013-04-01

Lignocellulosic biomass is an underutilized, renewable resource that can be converted to biofuels. The key step in this conversion is cellulose saccharification catalyzed by cellulase. In this work, the effect of metal ions on cellulose hydrolysis by cellulases from Penicillium decumbens was reported for the first time. Fe(3+) and Cu(2+) were shown to be inhibitory. Further studies on Fe(3+) inhibition showed the inhibition takes place on both enzyme and substrate levels. Fe(3+) treatment damages cellulases' capability to degrade cellulose and inhibits all major cellulase activities. Fe(3+) treatment also reduces the digestibility of cellulose, due to its oxidation. Treatment of Fe(3+)-treated cellulose with DTT and supplementation of EDTA to saccharification systems partially relieved Fe(3+) inhibition. It was concluded that Fe(3+) inhibition in cellulose degradation is a complicated process in which multiple inhibition events occur, and that relief from Fe(3+) inhibition can be achieved by the supplementation of reducing or chelating agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

Injectable TEMPO-oxidized nanofibrillated cellulose/biphasic calcium phosphate hydrogel for bone regeneration.

PubMed

Safwat, Engie; Hassan, Mohammad L; Saniour, Sayed; Zaki, Dalia Yehia; Eldeftar, Mervat; Saba, Dalia; Zazou, Mohamed

2018-05-01

Nanofibrillated cellulose, obtained from rice straw agricultural wastes was used as a substrate for the preparation of a new injectable and mineralized hydrogel for bone regeneration. Tetramethyl pyridine oxyl (TEMPO) oxidized nanofibrillated cellulose, was mineralized through the incorporation of a prepared and characterized biphasic calcium phosphate at a fixed ratio of 50 wt%. The TEMPO-oxidized rice straw nanofibrillated cellulose was characterized using transmission electron microscopy, Fourier transform infrared, and carboxylic content determination. The injectability and viscosity of the prepared hydrogel were evaluated using universal testing machine and rheometer testing, respectively. Cytotoxicity and alkaline phosphatase level tests on osteoblast like-cells for in vitro assessment of the biocompatibility were investigated. Results revealed that the isolated rice straw nanofibrillated cellulose is a nanocomposite of the cellulose nanofibers and silica nanoparticles. Rheological properties of the tested materials are suitable for use as injectable material and of nontoxic effect on osteoblast-like cells, as revealed by the positive alkaline phosphate assay. However, nanofibrillated cellulose/ biphasic calcium phosphate hydrogel showed higher cytotoxicity and lower bioactivity test results when compared to that of nanofibrillated cellulose.

Evaluation of lime and hydrothermal pretreatments for efficient enzymatic hydrolysis of raw sugarcane bagasse.

PubMed

Grimaldi, Maira Prearo; Marques, Marina Paganini; Laluce, Cecília; Cilli, Eduardo Maffud; Sponchiado, Sandra Regina Pombeiro

2015-01-01

Ethanol production from sugarcane bagasse requires a pretreatment step to disrupt the cellulose-hemicellulose-lignin complex and to increase biomass digestibility, thus allowing the obtaining of high yields of fermentable sugars for the subsequent fermentation. Hydrothermal and lime pretreatments have emerged as effective methods in preparing the lignocellulosic biomass for bioconversion. These pretreatments are advantageous because they can be performed under mild temperature and pressure conditions, resulting in less sugar degradation compared with other pretreatments, and also are cost-effective and environmentally sustainable. In this study, we evaluated the effect of these pretreatments on the efficiency of enzymatic hydrolysis of raw sugarcane bagasse obtained directly from mill without prior screening. In addition, we evaluated the structure and composition modifications of this bagasse after lime and hydrothermal pretreatments. The highest cellulose hydrolysis rate (70 % digestion) was obtained for raw sugarcane bagasse pretreated with lime [0.1 g Ca(OH)2/g raw] for 60 min at 120 °C compared with hydrothermally pretreated bagasse (21 % digestion) under the same time and temperature conditions. Chemical composition analyses showed that the lime pretreatment of bagasse promoted high solubilization of lignin (30 %) and hemicellulose (5 %) accompanied by a cellulose accumulation (11 %). Analysis of pretreated bagasse structure revealed that lime pretreatment caused considerable damage to the bagasse fibers, including rupture of the cell wall, exposing the cellulose-rich areas to enzymatic action. We showed that lime pretreatment is effective in improving enzymatic digestibility of raw sugarcane bagasse, even at low lime loading and over a short pretreatment period. It was also demonstrated that this pretreatment caused alterations in the structure and composition of raw bagasse, which had a pronounced effect on the enzymes accessibility to the substrate, resulting in an increase of cellulose hydrolysis rate. These results indicate that the use of raw sugarcane bagasse (without prior screening) pretreated with lime (cheaper and environmentally friendly reagent) may represent a cost reduction in the cellulosic ethanol production.

Effect of plasma superficial treatments on antibacterial functionalization and coloration of cellulosic fabrics

NASA Astrophysics Data System (ADS)

Ibrahim, Nabil A.; Eid, Basma M.; Abdel-Aziz, Mohamed S.

2017-01-01

Remarkable improvement in antibacterial activity and durability of different cellulosic substrates namely cotton, linen, viscose and lyocell was achieved by pre-surface modification using N2-plasma to create new active and binding sites, -NH2 groups, onto the modified fabric surfaces followed by subsequent loading of biosynthesized silver nanoparticles (Ag NPs) alone and in combination with certain antibiotics using exhaustion method. The imparted antibacterial activity against both G+ve (S. aureus) and G-ve (E. coli) pathogens was governed by type of substrate, extent of modification and subsequent loading of antibacterial agent, synergistic effect, and antibacterial activity as well as type of harmful bacteria. A remarkable antibacterial activity still retained even after 15 washings. In addition, incorporation of Ag NPs into pigment printing paste and into acid dyeing bath for combined coloration and functionalization of O2-plasma and N2-plasma pre-modified substrates respectively were successfully achieved. Moreover, both SEM images and EDS spectra of selected substrates revealed the change in surface morphology as well as the presence of the loaded Ag element onto the post-treated substrates.

Ionic Liquid Mediated Dispersion and Support of Functional Molecules on Cellulose Fibers for Stimuli-Responsive Chromic Paper Devices.

PubMed

Koga, Hirotaka; Nogi, Masaya; Isogai, Akira

2017-11-22

Functional molecules play a significant role in the development of high-performance composite materials. Functional molecules should be well dispersed (ideally dissolved) and supported within an easy-to-handle substrate to take full advantage of their functionality and ensure easy handling. However, simultaneously achieving the dissolution and support of functional molecules remains a challenge. Herein, we propose the combination of a nonvolatile ionic liquid and an easy-to-handle cellulose paper substrate for achieving this goal. First, the photochromic molecule, i.e., diarylethene, was dissolved in the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([bmim]NTf 2 ). Then, diarylethene/[bmim]NTf 2 was supported on cellulose fibers within the paper, through hydrogen bonding between [bmim] cations of the ionic liquid and the abundant hydroxyl groups of cellulose. The as-prepared paper composites exhibited reversible, rapid, uniform, and vivid coloration and bleaching upon ultraviolet and visible light irradiation. The photochromic performance was superior to that of the paper prepared in the absence of [bmim]NTf 2 . This concept could be applied to other functional molecules. For example, lithium perchlorate/[bmim] tetrafluoroborate supported within cellulose paper acted as a flexible electrolyte to provide a paper-based electrochromic device. These findings are expected to further the development of composite materials with high functionality and practicality.

The effects of ethanol on hydrolysis of cellulose and pretreated barley straw by some commercial cellulolytic enzyme products

USDA-ARS?s Scientific Manuscript database

The effect of ethanol at levels ranging from 2.5% v/v to 15% v/v on the activities of two recently developed commercial cellulosic biomass hydrolytic enzyme products, Accellerase® 1500 and Accellerase® XY, was investigated. The substrates used for study of the effect of ethanol on Accellerase® 1500 ...

Determining the Elastic Modulus of Compliant Thin Films Supported on Substrates from Flat Punch Indentation Measurements

Treesearch

M.J. Wald; J.M. Considine; K.T. Turner

2013-01-01

Instrumented indentation is a technique that can be used to measure the elastic properties of soft thin films supported on stiffer substrates, including polymer films, cellulosic sheets, and thin layers of biological materials. When measuring thin film properties using indentation, the effect of the substrate must be considered. Most existing models for determining the...

Applications of bacterial cellulose and its composites in biomedicine.

PubMed

Rajwade, J M; Paknikar, K M; Kumbhar, J V

2015-03-01

Bacterial cellulose produced by few but specific microbial genera is an extremely pure natural exopolysaccharide. Besides providing adhesive properties and a competitive advantage to the cellulose over-producer, bacterial cellulose confers UV protection, ensures maintenance of an aerobic environment, retains moisture, protects against heavy metal stress, etc. This unique nanostructured matrix is being widely explored for various medical and nonmedical applications. It can be produced in various shapes and forms because of which it finds varied uses in biomedicine. The attributes of bacterial cellulose such as biocompatibility, haemocompatibility, mechanical strength, microporosity and biodegradability with its unique surface chemistry make it ideally suited for a plethora of biomedical applications. This review highlights these qualities of bacterial cellulose in detail with emphasis on reports that prove its utility in biomedicine. It also gives an in-depth account of various biomedical applications ranging from implants and scaffolds for tissue engineering, carriers for drug delivery, wound-dressing materials, etc. that are reported until date. Besides, perspectives on limitations of commercialisation of bacterial cellulose have been presented. This review is also an update on the variety of low-cost substrates used for production of bacterial cellulose and its nonmedical applications and includes patents and commercial products based on bacterial cellulose.

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers.

PubMed

Vandavasi, Venu Gopal; Putnam, Daniel K; Zhang, Qiu; Petridis, Loukas; Heller, William T; Nixon, B Tracy; Haigler, Candace H; Kalluri, Udaya; Coates, Leighton; Langan, Paul; Smith, Jeremy C; Meiler, Jens; O'Neill, Hugh

2016-01-01

A cellulose synthesis complex with a "rosette" shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the "hexamer of trimers" model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product. © 2016 American Society of Plant Biologists. All Rights Reserved.

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers1

PubMed Central

Zhang, Qiu; Petridis, Loukas; Nixon, B. Tracy; Haigler, Candace H.; Kalluri, Udaya; Coates, Leighton; Smith, Jeremy C.; Meiler, Jens

2016-01-01

A cellulose synthesis complex with a “rosette” shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the “hexamer of trimers” model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product. PMID:26556795

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers

DOE PAGES

Vandavasi, Venu Gopal; Putnam, Daniel K.; Zhang, Qiu; ...

2015-11-10

In a cellulose synthesis complex a "rosette" shape is responsible for the synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. Our work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer inmore » solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. Moreover, the conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. Finally, this study strongly supports the "hexamer of trimers" model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product.« less

Improvement of yields and rates during enzymatic hydrolysis of cellulose to glucose. Progress report, March 1, 1979-May 31, 1979

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundstrom, D W; Klei, H E; Coughlin, R W

1979-05-01

The objective of this program is to show that the conversion of cellulose to glucose can be significantly increased by enzymatically removing the inhibitory cellobiose from the reaction system using immobilized ..beta..-glucosidase (..beta..-G). An enzymatic catalyst was prepared and used in a fluidized bed with cellobiose as the substrate, only a 10% loss of activity was observed during a 500 hour period. Cellulose was hydrolyzed in two batch reactors operated side-by-side, with one reactor containing immobilized ..beta..-G and cellulose and the other reactor containing an equal amount of cellulose only. After 30 hours the reactor containing the immobilized ..beta..-G hadmore » 100% more glucose, indicating that the catalytic removal of the cellobiose had a significant effect upon the production of glucose.« less

CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lei, Lei; Singh, Abhishek; Bashline, Logan

Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasmamore » membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress.« less

Crystallographic snapshot of cellulose synthesis and membrane translocation.

PubMed

Morgan, Jacob L W; Strumillo, Joanna; Zimmer, Jochen

2013-01-10

Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

Cytoskeleton-mediated templating of complex cellulose-scaffolded extracellular structure and its association with oikosins in the urochordate Oikopleura.

PubMed

Sagane, Yoshimasa; Hosp, Julia; Zech, Karin; Thompson, Eric M

2011-05-01

Oriented cellulose deposition is critical to plant patterning and models suggest microtubules constrain cellulose synthase movements through the plasma membrane. Though widespread in plants, urochordates are the only animals that synthesize cellulose. We characterized the distinctive cellulose microfibril scaffold of the larvacean house and its interaction with house structural proteins (oikosins). Targeted disruption of cytoskeletal elements, secretory pathways, and plasma membrane organization, suggested a working model for templating extracellular cellulose microfibrils from animal cells that shows both convergence and differences to plant models. Specialized cortical F-actin arrays template microfibril orientation and glycosylphosphatidylinositol-anchored proteins in lipid rafts may act as scaffolding proteins in microfibril elongation. Microtubules deliver and maintain cellulose synthase complexes to specific cell membrane sites rather than orienting their movement through the membrane. Oikosins are incorporated into house compartments directly above their corresponding cellular field of expression and interact with the cellulose scaffold to a variable extent.

Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group.

PubMed

Sulzenbacher, G; Driguez, H; Henrissat, B; Schülein, M; Davies, G J

1996-12-03

Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall retention of configuration. The structure of the EG I from Fusarium oxysproum, complexed to a nonhydrolyzable thiooligosaccharide substrate analogue, has been determined by X-ray crystallography at a resolution of 2.7 A utilizing the 4-fold noncrystallographic symmetry present in the asymmetric unit. The electron density map clearly reveals the presence of three glucosyl units of the inhibitor, consistent with the known number of sugar-binding subsites, located at the active site of the enzyme in the -2, -1, and +1 subsites, i.e., actually spanning the point of enzymatic cleavage. The pyranose ring at the point of potential enzymatic cleavage is clearly distorted from the standard 4C1 chair as was originally suggested for beta-retaining enzymes by Phillips [Ford, L.O., Johnson, L.N., Machin, P. A., Phillips, D.C., & Tijan, T. (1974) J. Mol. Biol, 88, 349-371]. The distortion observed goes beyond the "sofa" conformation observed in previous studies and results in a conformation whose salient feature is the resulting quasi-axial orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has recently been observed in a complex of chitobiase with its unhydrolyzed substrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The striking similarity between these two complexes extends beyond the almost identical pyranose ring distortion. The overlap of the two respective sugars places the enzymatic nucleophile of endoglucanase I in coincidence with the C2 acetamido oxygen of N-acetylglucosamine in the catalytic site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enzymes. The endoglucanase I complex with the thiosaccharide substrate analogue clearly illustrates the potential of nonhydrolyzable sulfur-linked oligosaccharides in the elucidation of substrate binding and catalysis by glycosyl hydrolases.

Formation of wood secondary cell wall may involve two type cellulose synthase complexes in Populus.

PubMed

Xi, Wang; Song, Dongliang; Sun, Jiayan; Shen, Junhui; Li, Laigeng

2017-03-01

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.

Computational Investigation of the pH Dependence of Loop Flexibility and Catalytic Function in Glycoside Hydrolases*

PubMed Central

Bu, Lintao; Crowley, Michael F.; Himmel, Michael E.; Beckham, Gregg T.

2013-01-01

Cellulase enzymes cleave glycosidic bonds in cellulose to produce cellobiose via either retaining or inverting hydrolysis mechanisms, which are significantly pH-dependent. Many fungal cellulases function optimally at pH ∼5, and their activities decrease dramatically at higher or lower pH. To understand the molecular-level implications of pH in cellulase structure, we use a hybrid, solvent-based, constant pH molecular dynamics method combined with pH-based replica exchange to determine the pKa values of titratable residues of a glycoside hydrolase (GH) family 6 cellobiohydrolase (Cel6A) and a GH family 7 cellobiohydrolase (Cel7A) from the fungus Hypocrea jecorina. For both enzymes, we demonstrate that a bound substrate significantly affects the pKa values of the acid residues at the catalytic center. The calculated pKa values of catalytic residues confirm their proposed roles from structural studies and are consistent with the experimentally measured apparent pKa values. Additionally, GHs are known to impart a strained pucker conformation in carbohydrate substrates in active sites for catalysis, and results from free energy calculations combined with constant pH molecular dynamics suggest that the correct ring pucker is stable near the optimal pH for both Cel6A and Cel7A. Much longer molecular dynamics simulations of Cel6A and Cel7A with fixed protonation states based on the calculated pKa values suggest that pH affects the flexibility of tunnel loops, which likely affects processivity and substrate complexation. Taken together, this work demonstrates several molecular-level effects of pH on GH enzymes important for cellulose turnover in the biosphere and relevant to biomass conversion processes. PMID:23504310

The use of seaweed and sugarcane bagasse for the biological treatment of metal-contaminated waters under sulfate-reducing conditions.

PubMed

Gonçalves, Márcia Monteiro Machado; de Oliveira Mello, Luiz Antonio; da Costa, Antonio Carlos Augusto

2008-03-01

When wetlands reach maximum treatment capacity to remove heavy metals, removal can still take place through precipitation as sulfide because of the biological reduction of sulfate. To achieve this goal, anaerobic conditions must be attained, a sulfate source must exist, and an adequate substrate for sulfate-reducing bacteria (SRB) is also required. In the present work, two ligneous-cellulosic materials, a brown seaweed and sugarcane bagasse, have been selected as substrates for SRB growth. Experiments were simultaneously conducted in continuous operation in two columns (0.57 L each), one containing the ligneous-cellulosic material plus inoculum and another containing only the ligneous-cellulosic material. In this work, the removal of cadmium and zinc was studied because of their presence in effluents from mining/metallurgy operations. Results obtained indicated that the inoculated reactor was able to treat the effluent more efficiently than the noninoculated reactor considering the time course of the tests.

Unraveling the effects of laccase treatment on enzymatic hydrolysis of steam-exploded wheat straw.

PubMed

Oliva-Taravilla, Alfredo; Moreno, Antonio D; Demuez, Marie; Ibarra, David; Tomás-Pejó, Elia; González-Fernández, Cristina; Ballesteros, Mercedes

2015-01-01

Laccase enzymes are promising detoxifying agents during lignocellulosic bioethanol production from wheat straw. However, they affect the enzymatic hydrolysis of this material by lowering the glucose recovery yields. This work aimed at explaining the negative effects of laccase on enzymatic hydrolysis. Relative glucose recovery in presence of laccase (10IU/g substrate) with model cellulosic substrate (Sigmacell) at 10% (w/v) was almost 10% points lower (P<0.01) than in the absence of laccase. This fact could be due to an increase in the competition of cellulose binding sites between the enzymes and a slight inhibition of β-glucosidase activity. However, enzymatic hydrolysis and infrared spectra of laccase-treated and untreated wheat straw filtered pretreated residue (WS-FPR), revealed that a grafting process of phenoxy radicals onto the lignin fiber could be the cause of diminished accessibility of cellulases to cellulose in pretreated wheat straw. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identifying and overcoming the effect of mass transfer limitation on decreased yield in enzymatic hydrolysis of lignocellulose at high solid concentrations.

PubMed

Du, Jian; Cao, Yuan; Liu, Guodong; Zhao, Jian; Li, Xuezhi; Qu, Yinbo

2017-04-01

Cellulose conversion decreases significantly with increasing solid concentrations during enzymatic hydrolysis of insoluble lignocellulosic materials. Here, mass transfer limitation was identified as a significant determining factor of this decrease by studying the hydrolysis of delignified corncob residue in shake flask, the most used reaction vessel in bench scale. Two mass transfer efficiency-related factors, mixing speed and flask filling, were shown to correlate closely with cellulose conversion at solid loadings higher than 15% DM. The role of substrate characteristics in mass transfer performance was also significant, which was revealed by the saccharification of two corn stover substrates with different pretreatment methods at the same solid loading. Several approaches including premix, fed-batch operation, and particularly the use of horizontal rotating reactor were shown to be valid in facilitating cellulose conversion via improving mass transfer efficiency at solid concentrations higher than 15% DM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nitrogen and Sulfur Requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on Cellulosic Substrates in Minimal Nutrient Media

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kridelbaugh, Donna M; Nelson, Josh C; Engle, Nancy L

2013-01-01

Growth media for cellulolytic Clostridium thermocellum and Caldicellulosiruptor bescii bacteria usually contain excess nutrients that would increase costs for consolidated bioprocessing for biofuel production and create a waste stream with nitrogen, sulfur and phosphate. C. thermocellum was grown on crystalline cellulose with varying concentrations of nitrogen and sulfur compounds, and growth rate and alcohol production response curves were determined. Both bacteria assimilated sulfate in the presence of ascorbate reductant, increasing the ratio of oxidized to reduced fermentation products. From these results, a low ionic strength, defined minimal nutrient medium with decreased nitrogen, sulfur, phosphate and vitamin supplements was developed formore » the fermentation of cellobiose, cellulose and acid-pretreated Populus. Carbon and electron balance calculations indicate the unidentified residual fermentation products must include highly reduced molecules. Both bacterial populations were maintained in co-cultures with substrates containing xylan or hemicellulose in defined medium with sulfate and basal vitamin supplements.« less

Simultaneous utilization of cellobiose, xylose, and acetic acid from lignocellulosic biomass for biofuel production by an engineered yeast platform.

PubMed

Wei, Na; Oh, Eun Joong; Million, Gyver; Cate, Jamie H D; Jin, Yong-Su

2015-06-19

The inability of fermenting microorganisms to use mixed carbon components derived from lignocellulosic biomass is a major technical barrier that hinders the development of economically viable cellulosic biofuel production. In this study, we integrated the fermentation pathways of both hexose and pentose sugars and an acetic acid reduction pathway into one Saccharomyces cerevisiae strain for the first time using synthetic biology and metabolic engineering approaches. The engineered strain coutilized cellobiose, xylose, and acetic acid to produce ethanol with a substantially higher yield and productivity than the control strains, and the results showed the unique synergistic effects of pathway coexpression. The mixed substrate coutilization strategy is important for making complete and efficient use of cellulosic carbon and will contribute to the development of consolidated bioprocessing for cellulosic biofuel. The study also presents an innovative metabolic engineering approach whereby multiple substrate consumption pathways can be integrated in a synergistic way for enhanced bioconversion.

Catalytic Fast Pyrolysis of Cellulose by Integrating Dispersed Nickel Catalyst with HZSM-5 Zeolite

NASA Astrophysics Data System (ADS)

Lei, Xiaojuan; Bi, Yadong; Zhou, Wei; Chen, Hui; Hu, Jianli

2018-01-01

The effect of integrating dispersed nickel catalyst with HZSM-5 zeolite on upgrading of vapors produced from pyrolysis of lignocellulosic biomass was investigated. The active component nickel nitrate was introduced onto the cellulose substrate by impregnation technique. Based on TGA experimental results, we discovered that nickel nitrate first released crystallization water, and then successively decomposed into nickel oxide which was reduced in-situ to metallic nickel through carbothermal reduction reaction. In-situ generated nickel nanoparticles were found highly dispersed over carbon substrate, which were responsible for catalyzing reforming and cracking of tars. In catalytic fast pyrolysis of cellulose, the addition of nickel nitrate caused more char formation at the expense of the yield of the condensable liquid products. In addition, the selectivity of linear oxygenates was increased whereas the yield of laevoglucose was reduced. Oxygen-containing compounds in pyrolysis vapors were deoxygenated into aromatics using HZSM-5. Moreover, the amount of condensable liquid products was decreased with the addition of HZSM-5.

Recyclable organic solar cells on substrates comprising cellulose nanocrystals (CNC)

DOEpatents

Kippelen, Bernard; Fuentes-Hernandez, Canek; Zhou, Yinhua; Moon, Robert; Youngblood, Jeffrey P

2015-12-01

Recyclable organic solar cells are disclosed herein. Systems and methods are further disclosed for producing, improving performance, and for recycling the solar cells. In certain example embodiments, the recyclable organic solar cells disclosed herein include: a first electrode; a second electrode; a photoactive layer disposed between the first electrode and the second electrode; an interlayer comprising a Lewis basic oligomer or polymer disposed between the photoactive layer and at least a portion of the first electrode or the second electrode; and a substrate disposed adjacent to the first electrode or the second electrode. The interlayer reduces the work function associated with the first or second electrode. In certain example embodiments, the substrate comprises cellulose nanocrystals that can be recycled. In certain example embodiments, one or more of the first electrode, the photoactive layer, and the second electrode may be applied by a film transfer lamination method.

Palladium Nanoparticle-Loaded Cellulose Paper: A Highly Efficient, Robust, and Recyclable Self-Assembled Composite Catalytic System.

PubMed

Zheng, Guangchao; Kaefer, Katharina; Mourdikoudis, Stefanos; Polavarapu, Lakshminarayana; Vaz, Belén; Cartmell, Samantha E; Bouleghlimat, Azzedine; Buurma, Niklaas J; Yate, Luis; de Lera, Ángel R; Liz-Marzán, Luis M; Pastoriza-Santos, Isabel; Pérez-Juste, Jorge

2015-01-15

We present a novel strategy based on the immobilization of palladium nanoparticles (Pd NPs) on filter paper for development of a catalytic system with high efficiency and recyclability. Oleylamine-capped Pd nanoparticles, dispersed in an organic solvent, strongly adsorb on cellulose filter paper, which shows a great ability to wick fluids due to its microfiber structure. Strong van der Waals forces and hydrophobic interactions between the particles and the substrate lead to nanoparticle immobilization, with no desorption upon further immersion in any solvent. The prepared Pd NP-loaded paper substrates were tested for several model reactions such as the oxidative homocoupling of arylboronic acids, the Suzuki cross-coupling reaction, and nitro-to-amine reduction, and they display efficient catalytic activity and excellent recyclability and reusability. This approach of using NP-loaded paper substrates as reusable catalysts is expected to open doors for new types of catalytic support for practical applications.

Effect of bovine serum albumin (BSA) on enzymatic cellulose hydrolysis.

PubMed

Wang, Hui; Mochidzuki, Kazuhiro; Kobayashi, Shinichi; Hiraide, Hatsue; Wang, Xiaofen; Cui, Zongjun

2013-06-01

Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ≥90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.

A Multispecies Fungal Biofilm Approach to Enhance the Celluloyltic Efficiency of Membrane Reactors for Consolidated Bioprocessing of Plant Biomass

PubMed Central

Xiros, Charilaos; Studer, Michael H.

2017-01-01

The constraints and advantages in cellulolytic enzymes production by fungal biofilms for a consolidated bioconversion process were investigated during this study. The biofilm cultivations were carried out in reactors designed for consolidated bioprocessing Multispecies Biofilm Membrane reactors, (MBM) where an aerobic fungal biofilm produces the lignocellulolytic enzymes while a fermenting microorganism forms the fermentation product at anaerobic conditions. It was shown that although mycelial growth was limited in the MBM reactors compared to submerged cultivations, the secretion of cellulolytic enzymes per cell dry weight was higher. When Trichoderma reesei was used as the sole enzyme producer, cellobiose accumulated in the liquid medium as the result of the deficiency of β-glucosidase in the fungal secretome. To enhance β-glucosidase activity, T. reesei was co-cultivated with A. phoenicis which is a β-glucosidase overproducer. The two fungi formed a multispecies biofilm which produced a balanced cellulolytic cocktail for the saccharification of plant biomass. The mixed biofilm reached a 2.5 fold increase in β-glucosidase production, compared to the single T. reesei biofilm. The enzymatic systems of single and mixed biofilms were evaluated regarding their efficiency on cellulosic substrates degradation. Washed solids from steam pretreated beechwood, as well as microcrystalline cellulose were used as the substrates. The enzymatic system of the multispecies biofilm released four times more glucose than the enzymatic system of T. reesei alone from both substrates and hydrolyzed 78 and 60% of the cellulose content of washed solids from beechwood and microcrystalline cellulose, respectively. PMID:29067006

Simultaneous saccharification and fermentation (SSF) using cellobiose fermenting yeast Brettanomyces custersii

DOEpatents

Spindler, Diane D.; Grohmann, Karel; Wyman, Charles E.

1992-01-01

A process for producing ethanol from plant biomass includes forming a substrate from the biomass with the substrate including hydrolysates of cellulose and hemicellulose. A species of the yeast Brettanomyces custersii (CBS 5512), which has the ability to ferment both cellobiose and glucose to ethanol, is then selected and isolated. The substrate is inoculated with this yeast, and the inoculated substrate is then fermented under conditions favorable for cell viability and conversion of hydrolysates to ethanol.

Interactions between sodium dodecyl sulphate and non-ionic cellulose derivatives studied by size exclusion chromatography with online multi-angle light scattering and refractometric detection.

PubMed

Wittgren, Bengt; Stefansson, Morgan; Porsch, Bedrich

2005-08-05

The novel approach described allows to characterise the surfactant-polymer interaction under several sodium dodecyl sulphate (SDS) concentrations (0-20 mM) using size exclusion chromatography (SEC) with online multi-angle light scattering (MALS) and refractometric (RI) detection. Three different cellulose derivatives, hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC) and hydroxyethyl cellulose (HEC), have been studied in solution containing 10 mM NaCl and various concentrations of sodium dodecyl sulphate. It is shown that this approach is well suited for successful application of both Hummel-Dreyer and multi-component light scattering principles and yields reliable molecular masses of both the polymer complex and the polymer itself within the complex, the amount of surfactant bound into the complex as well as appropriate values of the refractive index increment (dn/dc)micro, of both the complex and the polymer in question. The more hydrophobic derivatives HPC and HPMC adsorbed significantly more SDS than HEC. The inter-chain interactions close to critical aggregation concentration (cac) were clearly seen for HPC and HPMC as an almost two-fold average increase in polymer molecular mass contained in the complex.

PsB multiprotein complex of Dictyostelium discoideum. Demonstration of cellulose binding activity and order of protein subunit assembly.

PubMed

McGuire, V; Alexander, S

1996-06-14

The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.

The anisotropy1 D604N Mutation in the Arabidopsis Cellulose Synthase1 Catalytic Domain Reduces Cell Wall Crystallinity and the Velocity of Cellulose Synthase Complexes1[W][OA

PubMed Central

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T.; Galway, Moira E.; Mansfield, Shawn D.; Hocart, Charles H.; Wasteneys, Geoffrey O.

2013-01-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1’s permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature. PMID:23532584

The anisotropy1 D604N mutation in the Arabidopsis cellulose synthase1 catalytic domain reduces cell wall crystallinity and the velocity of cellulose synthase complexes.

PubMed

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T; Galway, Moira E; Mansfield, Shawn D; Hocart, Charles H; Wasteneys, Geoffrey O

2013-05-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1's permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature.

Biodegradable hydrogel derived from cellulose acetate and EDTA as a reduction substrate of leaching NPK compound fertilizer and water retention in soil.

PubMed

Senna, André M; Botaro, Vagner R

2017-08-28

To study the behavior of a biodegradable hydrogel derived from cellulose acetate and ethylenediaminetetraacetic dianhydride (EDTAD), as a reduction substrate of NPK fertilizer in soil. The biodegradable hydrogel (HEDTA) was prepared from cellulose acetate (CA) with a degree substitution (DS) 2.5 by esterification crosslinking with EDTAD catalyzed by triethylamine. We systematically investigated the performance of the HEDTA in the reducing NPK (Ammonium, phosphate and potassium) fertilizer leaching. We also compare the percentage of leaching between the HEDTA and commercial fertilizers. To characterize the esterification and crosslinking between CA and EDTAD, FTIR spectroscopy and thermogravimetric analysis (DTG) were employed. The biodegradation experiments were carried out in simulated soil (23% of sand, 23% of cattle manure, 23% of soil and 31% of water) and the HEDTA was tested in the eucalyptus planting during the dry season in the São Paulo state, Brazil. The HEDTA was able to reduce the leaching of fertilizers and improve the performance of eucalyptus seedlings and reduced the mortality of the seedlings. The HEDTA showed to be an excellent substrate for slow release and water-retention in soil, reducer of the fertilizers leaching, in addition being nontoxic, biodegradable in the soil and environmentally-friendly. Copyright © 2017 Elsevier B.V. All rights reserved.

Enzymatic saccharification and fermentation of cellulosic date palm wastes to glucose and lactic acid.

PubMed

Alrumman, Sulaiman A

2016-01-01

The bioconversion of cellulosic wastes into high-value bio-products by saccharification and fermentation processes is an important step that can reduce the environmental pollution caused by agricultural wastes. In this study, enzymatic saccharification of treated and untreated date palm cellulosic wastes by the cellulases from Geobacillus stearothermophilus was optimized. The alkaline pre-treatment of the date palm wastes was found to be effective in increasing the saccharification percentage. The maximum rate of saccharification was found at a substrate concentration of 4% and enzyme concentration of 30 FPU/g of substrate. The optimum pH and temperature for the bioconversions were 5.0 and 50°C, respectively, after 24h of incubation, with a yield of 31.56mg/mL of glucose at a saccharification degree of 71.03%. The saccharification was increased to 94.88% by removal of the hydrolysate after 24h by using a two-step hydrolysis. Significant lactic acid production (27.8mg/mL) was obtained by separate saccharification and fermentation after 72h of incubation. The results indicate that production of fermentable sugar and lactic acid is feasible and may reduce environmental pollution by using date palm wastes as a cheap substrate. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Enzymatic saccharification and fermentation of cellulosic date palm wastes to glucose and lactic acid

PubMed Central

Alrumman, Sulaiman A.

2016-01-01

The bioconversion of cellulosic wastes into high-value bio-products by saccharification and fermentation processes is an important step that can reduce the environmental pollution caused by agricultural wastes. In this study, enzymatic saccharification of treated and untreated date palm cellulosic wastes by the cellulases from Geobacillus stearothermophilus was optimized. The alkaline pre-treatment of the date palm wastes was found to be effective in increasing the saccharification percentage. The maximum rate of saccharification was found at a substrate concentration of 4% and enzyme concentration of 30 FPU/g of substrate. The optimum pH and temperature for the bioconversions were 5.0 and 50 °C, respectively, after 24 h of incubation, with a yield of 31.56 mg/mL of glucose at a saccharification degree of 71.03%. The saccharification was increased to 94.88% by removal of the hydrolysate after 24 h by using a two-step hydrolysis. Significant lactic acid production (27.8 mg/mL) was obtained by separate saccharification and fermentation after 72 h of incubation. The results indicate that production of fermentable sugar and lactic acid is feasible and may reduce environmental pollution by using date palm wastes as a cheap substrate. PMID:26887233

Microbial production of rhamnolipids using sugars as carbon sources.

PubMed

Tan, Yun Nian; Li, Qingxin

2018-06-08

Rhamnolipids are a class of biosurfactants with effective surface-active properties. The high cost of microbial production of rhamnolipids largely affects their commercial applications. To reduce the production post, research has been carried out in screening more powerful strains, engineering microbes with higher biosurfactant yields and exploring cheaper substrates to reduce the production cost. Extensive refining is required for biosurfactant production using oils and oil-containing wastes, necessitating the use of complex and expensive biosurfactant recovery methods such as extraction with solvents or acid precipitation. As raw materials normally can account for 10-30% of the overall production cost, sugars have been proven to be an alternative carbon source for microbial production of rhamnolipids due to its lower costs and straightforward processing techniques. Studies have thus been focused on using tropical agroindustrial crop residues as renewable substrates. Herein, we reviewed studies that are using sugar-containing substrates as carbon sources for producing rhamnolipids. We speculate that sugars derived from agricultural wastes rich in cellulose and sugar-containing wastes are potential carbon sources in fermentation while challenges still remain in large scales.

Synergy between cellulolytic enzymes during the biodegradation of cellulose microfibrils measured using angle-scanning surface plasmon resonance (SPR) imaging

NASA Astrophysics Data System (ADS)

Raegen, Adam; Dion, Alexander; Reiter, Kyle; Clarke, Anthony; Lipkowski, Jacek; Dutcher, John

2014-03-01

The use of cellulosic ethanol, a promising emerging energy source, is limited by the energy intensive and costly step of first converting the cellulose fibers into their constituent glucose monomers. Industrial processes mimic those that occur in nature, using mixtures or ``cocktails'' of different classes of cellulolytic enzymes derived from fungi. Despite several decades of investigation, the molecular mechanisms for enzyme synergy remain poorly understood. To gain additional insight, we have used a custom angle-scanning surface plasmon resonance (SPR) imaging apparatus to obtain a sensitive measure of enzymatic degradation. By implementing a novel SPR data analysis procedure, we have been able to track the thickness and roughness of laterally heterogeneous cellulose microfibril-coated substrates as enzymatic degradation proceeds. This has allowed us to measure the synergistic actions of the different enzymes, providing data that are directly relevant to the cellulosic ethanol industry.

Optimization of pretreatment and fermentation conditions for production of extracellular cellulase complex using sugarcane bagasse.

PubMed

Ashfaque, Mohammad; Solomon, Sushil; Pathak, Neelam

2014-01-01

Sugarcane bagasse (SCB), a lignocellulosic byproduct of juice extraction from sugarcane, is rich in cellulose (40-42%). This could be used as a substrate for the production of cellulase complex. Fermentation conditions were optimized for production of cellulase complex (CMCase, Cellulobiase and FPase) by wild type Trichoderma sp. using sugarcane bagasse as sole carbon source. Alkaline treatment (2% NaOH) of bagasse (AlSCB) was found suitable for the production of reducing sugar over the acidic pretreatment method. After 5 days of incubation period, 5% substrate concentration at pH 5.0 and 400C resulted in maximum production of CMCase (0.622 U), while maximum (3.388 U) production of cellulobiase was obtained at 300C. The CMCase was precipitated and purified to the extent of 59.06 fold by affinity chromatography with 49.09% recovery. On 12% SDS-PAGE, a single band corresponding to 33 kDa was observed. The Km and Vmax for CMCase from Trichoderma was found 507.04 mg/ml and 65.32 mM/min, respectively. The enzyme exhibited maximum activity at 300C at pH-5.0 (0.363 U) and was stable over range of 20-60°C and pH 5.0-7.5.

Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

PubMed Central

2011-01-01

Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX) and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy. PMID:21702938

Substrate-Driven Convergence of the Microbial Community in Lignocellulose-Amended Enrichments of Gut Microflora from the Canadian Beaver (Castor canadensis) and North American Moose (Alces americanus).

PubMed

Wong, Mabel T; Wang, Weijun; Lacourt, Michael; Couturier, Marie; Edwards, Elizabeth A; Master, Emma R

2016-01-01

Strategic enrichment of microcosms derived from wood foragers can facilitate the discovery of key microbes that produce enzymes for the bioconversion of plant fiber (i.e., lignocellulose) into valuable chemicals and energy. In this study, lignocellulose-degrading microorganisms from the digestive systems of Canadian beaver (Castor canadensis) and North American moose (Alces americanus) were enriched under methanogenic conditions for over 3 years using various wood-derived substrates, including (i) cellulose (C), (ii) cellulose + lignosulphonate (CL), (iii) cellulose + tannic acid (CT), and (iv) poplar hydrolysate (PH). Substantial improvement in the conversion of amended organic substrates into biogas was observed in both beaver dropping and moose rumen enrichment cultures over the enrichment phases (up to 0.36-0.68 ml biogas/mg COD added), except for enrichments amended with tannic acid where conversion was approximately 0.15 ml biogas/mg COD added. Multiplex-pyrosequencing of 16S rRNA genes revealed systematic shifts in the population of Firmicutes, Bacteroidetes, Chlorobi, Spirochaetes, Chloroflexi, and Elusimicrobia in response to the enrichment. These shifts were predominantly substrate driven, not inoculum driven, as revealed by both UPGMA clustering pattern and OTU distribution. Additionally, the relative abundance of multiple OTUs from poorly defined taxonomic lineages increased from less than 1% to 25-50% in microcosms amended with lignocellulosic substrates, including OTUs from classes SJA-28, Endomicrobia, orders Bacteroidales, OPB54, and family Lachnospiraceae. This study provides the first direct comparison of shifts in microbial communities that occurred in different environmental samples in response to multiple relevant lignocellulosic carbon sources, and demonstrates the potential of enrichment to increase the abundance of key lignocellulolytic microorganisms and encoded activities.

Substrate-Driven Convergence of the Microbial Community in Lignocellulose-Amended Enrichments of Gut Microflora from the Canadian Beaver (Castor canadensis) and North American Moose (Alces americanus)

PubMed Central

Wong, Mabel T.; Wang, Weijun; Lacourt, Michael; Couturier, Marie; Edwards, Elizabeth A.; Master, Emma R.

2016-01-01

Strategic enrichment of microcosms derived from wood foragers can facilitate the discovery of key microbes that produce enzymes for the bioconversion of plant fiber (i.e., lignocellulose) into valuable chemicals and energy. In this study, lignocellulose-degrading microorganisms from the digestive systems of Canadian beaver (Castor canadensis) and North American moose (Alces americanus) were enriched under methanogenic conditions for over 3 years using various wood-derived substrates, including (i) cellulose (C), (ii) cellulose + lignosulphonate (CL), (iii) cellulose + tannic acid (CT), and (iv) poplar hydrolysate (PH). Substantial improvement in the conversion of amended organic substrates into biogas was observed in both beaver dropping and moose rumen enrichment cultures over the enrichment phases (up to 0.36–0.68 ml biogas/mg COD added), except for enrichments amended with tannic acid where conversion was approximately 0.15 ml biogas/mg COD added. Multiplex-pyrosequencing of 16S rRNA genes revealed systematic shifts in the population of Firmicutes, Bacteroidetes, Chlorobi, Spirochaetes, Chloroflexi, and Elusimicrobia in response to the enrichment. These shifts were predominantly substrate driven, not inoculum driven, as revealed by both UPGMA clustering pattern and OTU distribution. Additionally, the relative abundance of multiple OTUs from poorly defined taxonomic lineages increased from less than 1% to 25–50% in microcosms amended with lignocellulosic substrates, including OTUs from classes SJA-28, Endomicrobia, orders Bacteroidales, OPB54, and family Lachnospiraceae. This study provides the first direct comparison of shifts in microbial communities that occurred in different environmental samples in response to multiple relevant lignocellulosic carbon sources, and demonstrates the potential of enrichment to increase the abundance of key lignocellulolytic microorganisms and encoded activities. PMID:27446004

Process for producing ethanol from plant biomass using the fungus paecilomyces sp.

DOEpatents

Wu, Jung Fu

1989-01-01

A process for producing ethanol from plant biomass is disclosed. The process in cludes forming a substrate from the biomass with the substrate including hydrolysates of cellulose and hemicellulose. A species of the fungus Paecilomyces, which has the ability to ferment both cellobiose and xylose to ethanol, is then selected and isolated. The substrate is inoculated with this fungus, and the inoculated substrate is then fermented under conditions favorable for cell viability and conversion of hydrolysates to ethanol. Finally, ethanol is recovered from the fermented substrate.

Process for producing ethanol from plant biomass using the fungus Paecilomyces sp

DOEpatents

Wu, J.F.

1985-08-08

A process for producing ethanol from plant biomass is disclosed. The process includes forming a substrate from the biomass with the substrate including hydrolysates of cellulose and hemicellulose. A species of the fungus Paecilomyces which has the ability to ferment both cellobiose and xylose to ethanol is then selected and isolated. The substrate is inoculated with this fungus, and the inoculated substrate is then fermented under conditions favorable for cell viability and conversion of hydrolysates to ethanol. Finally, ethanol is recovered from the fermented substrate. 5 figs., 3 tabs.

A survey of cellulose microfibril patterns in dividing, expanding, and differentiating cells of Arabidopsis thaliana.

PubMed

Fujita, Miki; Wasteneys, Geoffrey O

2014-05-01

Cellulose microfibrils are critical for plant cell specialization and function. Recent advances in live cell imaging of fluorescently tagged cellulose synthases to track cellulose synthesis have greatly advanced our understanding of cellulose biosynthesis. Nevertheless, cellulose deposition patterns remain poorly described in many cell types, including those in the process of division or differentiation. In this study, we used field emission scanning electron microscopy analysis of cryo-planed tissues to determine the arrangement of cellulose microfibrils in various faces of cells undergoing cytokinesis or specialized development, including cell types in which cellulose cannot be imaged by conventional approaches. In dividing cells, we detected microfibrillar meshworks in the cell plates, consistent with the concentration at the cell plate of cellulose synthase complexes, as detected by fluorescently tagged CesA6. We also observed a loss of parallel cellulose microfibril orientation in walls of the mother cell during cytokinesis, which corresponded with the loss of fluorescently tagged cellulose synthase complexes from these surfaces. In recently formed guard cells, microfibrils were randomly organized and only formed a highly ordered circumferential pattern after pore formation. In pit fields, cellulose microfibrils were arranged in circular patterns around plasmodesmata. Microfibrils were random in most cotyledon cells except the epidermis and were parallel to the growth axis in trichomes. Deposition of cellulose microfibrils was spatially delineated in metaxylem and protoxylem cells of the inflorescence stem, supporting recent studies on microtubule exclusion mechanisms.

Consolidated bioprocessing method using thermophilic microorganisms

DOEpatents

Mielenz, Jonathan Richard

2016-02-02

The present invention is directed to a method of converting biomass to biofuel, and particularly to a consolidated bioprocessing method using a co-culture of thermophilic and extremely thermophilic microorganisms which collectively can ferment the hexose and pentose sugars produced by degradation of cellulose and hemicelluloses at high substrate conversion rates. A culture medium therefor is also provided as well as use of the methods to produce and recover cellulosic ethanol.

A 24-GHz Front-End Integrated on a Multilayer Cellulose-Based Substrate for Doppler Radar Sensors.

PubMed

Alimenti, Federico; Palazzi, Valentina; Mariotti, Chiara; Virili, Marco; Orecchini, Giulia; Bonafoni, Stefania; Roselli, Luca; Mezzanotte, Paolo

2017-09-12

This paper presents a miniaturized Doppler radar that can be used as a motion sensor for low-cost Internet of things (IoT) applications. For the first time, a radar front-end and its antenna are integrated on a multilayer cellulose-based substrate, built-up by alternating paper, glue and metal layers. The circuit exploits a distributed microstrip structure that is realized using a copper adhesive laminate, so as to obtain a low-loss conductor. The radar operates at 24 GHz and transmits 5 mW of power. The antenna has a gain of 7.4 dBi and features a half power beam-width of 48 degrees. The sensor, that is just the size of a stamp, is able to detect the movement of a walking person up to 10 m in distance, while a minimum speed of 50 mm/s up to 3 m is clearly measured. Beyond this specific result, the present paper demonstrates that the attractive features of cellulose, including ultra-low cost and eco-friendliness (i.e., recyclability and biodegradability), can even be exploited for the realization of future high-frequency hardware. This opens opens the door to the implementation on cellulose of devices and systems which make up the "sensing layer" at the base of the IoT ecosystem.

Simultaneous saccharification and fermentation (SSF) using cellobiose fermenting yeast Brettanomyces custersii (CBS 5512)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spindler, D.D.; Grohmann, K.; Wyman, C.E.

1991-01-16

A process for producing ethanol from plant biomass includes forming a substrate from the biomass with the substrate including hydrolysates of cellulose and hemicellulose. A species of the yeast Brettanomyces custersii (CBS 5512), which has the ability to ferment both cellobiose and glucose to ethanol, is then selected and isolated. The substrate is inoculated with this yeast, and the inoculated substrate is then fermented under conditions favorable for cell viability and conversion of hydrolysates to ethanol.

Simultaneous saccharification and fermentation (SSF) using cellobiose fermenting yeast Brettanomyces custersii

DOEpatents

Spindler, D.D.; Grohmann, K.; Wyman, C.E.

1992-03-31

A process for producing ethanol from plant biomass includes forming a substrate from the biomass with the substrate including hydrolysates of cellulose and hemicellulose. A species of the yeast Brettanomyces custersii (CBS 5512), which has the ability to ferment both cellobiose and glucose to ethanol, is then selected and isolated. The substrate is inoculated with this yeast, and the inoculated substrate is then fermented under conditions favorable for cell viability and conversion of hydrolysates to ethanol. 2 figs.

Cellulose synthase complexes act in a concerted fashion to synthesize highly aggregated cellulose in secondary cell walls of plants

PubMed Central

Li, Shundai; Bashline, Logan; Zheng, Yunzhen; Xin, Xiaoran; Huang, Shixin; Kong, Zhaosheng; Kim, Seong H.; Cosgrove, Daniel J.; Gu, Ying

2016-01-01

Cellulose, often touted as the most abundant biopolymer on Earth, is a critical component of the plant cell wall and is synthesized by plasma membrane-spanning cellulose synthase (CESA) enzymes, which in plants are organized into rosette-like CESA complexes (CSCs). Plants construct two types of cell walls, primary cell walls (PCWs) and secondary cell walls (SCWs), which differ in composition, structure, and purpose. Cellulose in PCWs and SCWs is chemically identical but has different physical characteristics. During PCW synthesis, multiple dispersed CSCs move along a shared linear track in opposing directions while synthesizing cellulose microfibrils with low aggregation. In contrast, during SCW synthesis, we observed swaths of densely arranged CSCs that moved in the same direction along tracks while synthesizing cellulose microfibrils that became highly aggregated. Our data support a model in which distinct spatiotemporal features of active CSCs during PCW and SCW synthesis contribute to the formation of cellulose with distinct structure and organization in PCWs and SCWs of Arabidopsis thaliana. This study provides a foundation for understanding differences in the formation, structure, and organization of cellulose in PCWs and SCWs. PMID:27647923

Cellulose synthase complexes act in a concerted fashion to synthesize highly aggregated cellulose in secondary cell walls of plants.

PubMed

Li, Shundai; Bashline, Logan; Zheng, Yunzhen; Xin, Xiaoran; Huang, Shixin; Kong, Zhaosheng; Kim, Seong H; Cosgrove, Daniel J; Gu, Ying

2016-10-04

Cellulose, often touted as the most abundant biopolymer on Earth, is a critical component of the plant cell wall and is synthesized by plasma membrane-spanning cellulose synthase (CESA) enzymes, which in plants are organized into rosette-like CESA complexes (CSCs). Plants construct two types of cell walls, primary cell walls (PCWs) and secondary cell walls (SCWs), which differ in composition, structure, and purpose. Cellulose in PCWs and SCWs is chemically identical but has different physical characteristics. During PCW synthesis, multiple dispersed CSCs move along a shared linear track in opposing directions while synthesizing cellulose microfibrils with low aggregation. In contrast, during SCW synthesis, we observed swaths of densely arranged CSCs that moved in the same direction along tracks while synthesizing cellulose microfibrils that became highly aggregated. Our data support a model in which distinct spatiotemporal features of active CSCs during PCW and SCW synthesis contribute to the formation of cellulose with distinct structure and organization in PCWs and SCWs of Arabidopsis thaliana This study provides a foundation for understanding differences in the formation, structure, and organization of cellulose in PCWs and SCWs.

CBD binding domain fused γ-lactamase from Sulfolobus solfataricus is an efficient catalyst for (-) γ-lactam production.

PubMed

Wang, Jianjun; Zhu, Junge; Min, Cong; Wu, Sheng

2014-05-13

γ-lactamase is used for the resolution of γ-lactam which is utilized in the synthesizing of abacavir and peramivir. In some cases, enzymatic method is the most utilized method because of its high efficiency and productivity. The cellulose binding domain (CBD) of cellulose is often used as the bio-specific affinity matrix for enzyme immobilization. Cellulose is cheap and it has excellent chemical and physical properties. Meanwhile, binding between cellulose and CBD is tight and the desorption rarely happened. We prepared two fusion constructs of the γ-lactamase gene gla, which was from Sulfolobus solfataricus P2. These two constructs had Cbd (cellulose binding domain from Clostridium thermocellum) fused at amino or carboxyl terminus of the γ-lactamase. These two constructs were heterogeneously expressed in E. coli rosetta (DE3) as two fusion proteins. Both of them were immobilized well on Avicel (microcrystalline cellulose matrix). The apparent kinetic parameters revealed that carboxyl terminus fused protein (Gla-linker-Cbd) was a better catalyst. The V(max) and k(cat) value of Avicel immobilized Gla-linker-Cbd were 381 U mg⁻¹ and 4.7 × 10⁵ s⁻¹ respectively. And the values of the free Gla-linker-Cbd were 151 U mg⁻¹ and 1.8 × 10⁵ s⁻¹ respectively. These data indicated that the catalytic efficiency of the enzyme was upgraded after immobilization. The immobilized Gla-linker-Cbd had a 10-degree temperature optimum dropping from 80°C to 70°C but it was stable when incubated at 60°C for 48 h. It remained stable in catalyzing 20-batch reactions. After optimization, the immobilized enzyme concentration in transformation was set as 200 mg/mL. We found out that there was inhibition that occurred to the immobilized enzyme when substrate concentration exceeded 60 mM. Finally a 10 mL-volume transformation was conducted, in which 0.6 M substrate was hydrolyzed and the resolution was completed within 9 h with a 99.5% ee value. Cellulose is the most abundant and renewable material on the Earth. The absorption between Cbd domain and cellulose is a bio-green process. The cellulose immobilized fusion Gla exhibited good catalytic characters, therefore we think the cellulose immobilized Gla is a promising catalyst for the industrial preparation of (-) - γ-lactam.

Effect of D2O on growth properties and chemical structure of annual ryegrass (Lolium multiflorum)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evans, Barbara R; Bali, Garima; Reeves, David T

2014-01-01

In present paper, we report the production and detailed structural analysis of deuterium-enriched rye grass (Lolium multiflorum) for neutron scattering experiments. An efficient method to produce deuterated biomass was developed by designing hydroponic perfusion chambers. In preliminary studies, the partial deuterated rye samples were grown in increasing levels of D2O to study the seed germination and the level of deuterium incorporation as a function of D2O concentration. Solution NMR method indicated 36.9 % deuterium incorporation in 50 % D2O grown annual rye samples and further significant increase in the deuterium incorporation level was observed by germinating the rye seedlings inmore » H2O and growing in 50 % D2O inside the perfusion chambers. Moreover, in an effort to compare the substrate characteristics related to enzymatic hydrolysis on deuterated and protiated version of biomass, annual rye grown in 50 % D2O was selected for detailed biomass characterization studies. The compositional analyses, degree of polymerization and cellulose crystallinity were compared with its protiated control. The cellulose molecular weight indicated slight variation with deuteration; however, hemicellulose molecular weights and cellulose crystallinity remain unaffected with the deuteration. Besides the minor differences in biomass components, the development of deuterated biomass for neutron scattering application is essential to understand the complex biomass conversion processes.« less

Effects of wet-pressing-induced fiber hornification on enzymatic saccharification of lignocelluloses

Treesearch

X.L. Luo; Junyong Zhu; Roland Gleisner; H.Y. Zhan

2011-01-01

This article reports the effect of wet-pressing-induced fiber hornification on enzymatic saccharification of lignocelluloses. A wet cellulosic substrate of bleached kraft eucalyptus pulp and two wet sulfite-pretreated lignocellulosic substrates of aspen and lodgepole pine were pressed to various moisture (solids) contents by variation of pressing pressure and pressing...

Substrate-Related Factors Affecting Enzymatic Saccharification of Lignocelluloses: Our Recent Understanding

Treesearch

Shao-Yuan Leu; J.Y. Zhu

2013-01-01

Enzymatic saccharification of cellulose is a key step in conversion of plant biomass to advanced biofuel and chemicals. Many substrate-related factors affect saccharification. Rather than examining the role of each individual factor on overall saccharification efficiency, this study examined how each factor affects the three basic processes of a heterogeneous...

Cellulolytic enzymes production by utilizing agricultural wastes under solid state fermentation and its application for biohydrogen production.

PubMed

Saratale, Ganesh D; Kshirsagar, Siddheshwar D; Sampange, Vilas T; Saratale, Rijuta G; Oh, Sang-Eun; Govindwar, Sanjay P; Oh, Min-Kyu

2014-12-01

Phanerochaete chrysosporium was evaluated for cellulase and hemicellulase production using various agricultural wastes under solid state fermentation. Optimization of various environmental factors, type of substrate, and medium composition was systematically investigated to maximize the production of enzyme complex. Using grass powder as a carbon substrate, maximum activities of endoglucanase (188.66 U/gds), exoglucanase (24.22 U/gds), cellobiase (244.60 U/gds), filter paperase (FPU) (30.22 U/gds), glucoamylase (505.0 U/gds), and xylanase (427.0 U/gds) were produced under optimized conditions. The produced crude enzyme complex was employed for hydrolysis of untreated and mild acid pretreated rice husk. The maximum amount of reducing sugar released from enzyme treated rice husk was 485 mg/g of the substrate. Finally, the hydrolysates of rice husk were used for hydrogen production by Clostridium beijerinckii. The maximum cumulative H2 production and H2 yield were 237.97 mL and 2.93 mmoL H2/g of reducing sugar, (or 2.63 mmoL H2/g of cellulose), respectively. Biohydrogen production performance obtained from this work is better than most of the reported results from relevant studies. The present study revealed the cost-effective process combining cellulolytic enzymes production under solid state fermentation (SSF) and the conversion of agro-industrial residues into renewable energy resources.

Probing Substrate Interactions in the Active Tunnel of a Catalytically Deficient Cellobiohydrolase (Cel7)*

PubMed Central

Colussi, Francieli; Sørensen, Trine H.; Alasepp, Kadri; Kari, Jeppe; Cruys-Bagger, Nicolaj; Windahl, Michael S.; Olsen, Johan P.; Borch, Kim; Westh, Peter

2015-01-01

Cellobiohydrolases break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has ∼10 glucopyranose subsites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with a DP of 2–3 the binding constants were around 105 m−1, and for longer ligands (DP 5–8) this value was ∼107 m−1. Within each of these groups we did not find increased affinity as the ligands got longer and potentially filled more subsites. On the contrary, we found a small but consistent affinity loss as DP rose from 6 to 8, particularly at the higher investigated temperatures. Other thermodynamic functions (ΔH, ΔS, and ΔCp) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel. PMID:25477511

Nondestructive, real-time determination and visualization of cellulose, hemicellulose and lignin by luminescent oligothiophenes

NASA Astrophysics Data System (ADS)

Choong, Ferdinand X.; Bäck, Marcus; Steiner, Svava E.; Melican, Keira; Nilsson, K. Peter R.; Edlund, Ulrica; Richter-Dahlfors, Agneta

2016-10-01

Enabling technologies for efficient use of the bio-based feedstock are crucial to the replacement of oil-based products. We investigated the feasibility of luminescent conjugated oligothiophenes (LCOs) for non-destructive, rapid detection and quality assessment of lignocellulosic components in complex biomass matrices. A cationic pentameric oligothiophene denoted p-HTEA (pentamer hydrogen thiophene ethyl amine) showed unique binding affinities to cellulose, lignin, hemicelluloses, and cellulose nanofibrils in crystal, liquid and paper form. We exploited this finding using spectrofluorometric methods and fluorescence confocal laser scanning microscopy, for sensitive, simultaneous determination of the structural and compositional complexities of native lignocellulosic biomass. With exceptional photostability, p-HTEA is also demonstrated as a dynamic sensor for real-time monitoring of enzymatic cellulose degradation in cellulolysis. These results demonstrate the use of p-HTEA as a non-destructive tool for the determination of cellulose, hemicellulose and lignin in complex biomass matrices, thereby aiding in the optimization of biomass-converting technologies.

Nondestructive, real-time determination and visualization of cellulose, hemicellulose and lignin by luminescent oligothiophenes

PubMed Central

Choong, Ferdinand X.; Bäck, Marcus; Steiner, Svava E.; Melican, Keira; Nilsson, K. Peter R.; Edlund, Ulrica; Richter-Dahlfors, Agneta

2016-01-01

Enabling technologies for efficient use of the bio-based feedstock are crucial to the replacement of oil-based products. We investigated the feasibility of luminescent conjugated oligothiophenes (LCOs) for non-destructive, rapid detection and quality assessment of lignocellulosic components in complex biomass matrices. A cationic pentameric oligothiophene denoted p-HTEA (pentamer hydrogen thiophene ethyl amine) showed unique binding affinities to cellulose, lignin, hemicelluloses, and cellulose nanofibrils in crystal, liquid and paper form. We exploited this finding using spectrofluorometric methods and fluorescence confocal laser scanning microscopy, for sensitive, simultaneous determination of the structural and compositional complexities of native lignocellulosic biomass. With exceptional photostability, p-HTEA is also demonstrated as a dynamic sensor for real-time monitoring of enzymatic cellulose degradation in cellulolysis. These results demonstrate the use of p-HTEA as a non-destructive tool for the determination of cellulose, hemicellulose and lignin in complex biomass matrices, thereby aiding in the optimization of biomass-converting technologies. PMID:27759105

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

PubMed Central

Geiser, Elena; Reindl, Michèle; Blank, Lars M.; Feldbrügge, Michael

2016-01-01

ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. PMID:27316952

Effects of complex carbon addition to soil CO2 efflux and isotopic composition to soils near dead and live piñon pine trees

NASA Astrophysics Data System (ADS)

Powers, H.; McDowell, N.; Breecker, D. O.

2010-12-01

We test the hypothesis that soils collected near dead and living pinus edulous (piñon pine) trees should show a difference in their capacities to decompose complex carbon compounds. Since soils near dead trees have a large amount of cellulose and other complex carbon, the soil microbial community should be selected to metabolize cellulose. We collected soils from both live and dead piñon trees, added cellulose to half of the replicates, and placed them in microcosms for incubation. The microcosms were periodically sampled by a trace gas analyzer (TGA100, Campbell Scientific, USA) for CO2 concentration and δ13C and δ18O analysis. We found that CO2 evolution rates from live soils were significantly higher than rates from dead soils (1.1 and 0.6 ug CO2 g-1 soil s-1 respectively); soils with added cellulose displayed higher rates (1.1 and 0.8 and ug CO2 g-1 soil s-1). We did not see any significant differences in δ13C values between treatments, but there was a difference in δ18O between soils treated with cellulose and soils with no cellulose. Soils from both dead and live trees showed an increase in CO2 efflux when cellulose was added; however there was no distinguishable difference in efflux rate between live and dead soils in the cellulose added treatments.

Evaluating models of cellulose degradation by Fibrobacter succinogenes S85

DOE PAGES

Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; ...

2015-12-02

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve a combination of cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further elucidate the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding Type II and III secretion systems, fibro-slime proteins,more » and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular media, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. Furthermore, these results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.« less

Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85

PubMed Central

Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; Lipton, Mary S.; Smith, Richard D.; Suen, Garret; Callister, Stephen J.

2015-01-01

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases. PMID:26629814

Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina.

PubMed

Tangthirasunun, Narumon; Navarro, David; Garajova, Sona; Chevret, Didier; Tong, Laetitia Chan Ho; Gautier, Valérie; Hyde, Kevin D; Silar, Philippe; Berrin, Jean-Guy

2017-01-15

Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO. Copyright © 2016 American Society for Microbiology.

Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

PubMed Central

Tangthirasunun, Narumon; Navarro, David; Garajova, Sona; Chevret, Didier; Tong, Laetitia Chan Ho; Gautier, Valérie; Hyde, Kevin D.

2016-01-01

ABSTRACT Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO. PMID:27836848

Compositional and structural changes in Phoenix canariensis and Opuntia ficus-indica with pretreatment: Effects on enzymatic hydrolysis and second generation ethanol production.

PubMed

Udeh, Benard Anayo; Erkurt, Emrah Ahmet

2017-01-01

Two different plants namely Phoenix canariensis and Opuntia ficus-indica were used as substrate for reducing sugar generation and ethanol production. Dilute acid, alkaline and steam explosion were used as pretreatment methods in order to depolymerize lignin and/or hemicellulose and recover cellulose. By using alkaline pretreatment with 2.5% NaOH 71.08% for P. canariensis and 74.61% for O. ficus-indica lignin removal and 81.84% for P. canariensis and 72.66% for O. ficus-indica cellulose recovery yields were obtained. Pretreated materials were hydrolyzed by cellulase with high efficiency (87.0% and 84.5% cellulose conversion yields for P. canariensis and O. ficus-indica) and used as substrate for fermentation. Maximum ethanol production of 15.75g/L and 14.71g/L were achieved from P. canariensis and O. ficus-indica respectively. Structural differences were observed by XRD, FTIR and SEM for untreated, pretreated, hydrolyzed and fermented samples and were highly correlated with compositional analysis results. Copyright © 2016 Elsevier Ltd. All rights reserved.

Key advances in the chemical modification of nanocelluloses.

PubMed

Habibi, Youssef

2014-03-07

Nanocelluloses, including nanocrystalline cellulose, nanofibrillated cellulose and bacterial cellulose nanofibers, have become fascinating building blocks for the design of new biomaterials. Derived from the must abundant and renewable biopolymer, they are drawing a tremendous level of attention, which certainly will continue to grow in the future driven by the sustainability trend. This growing interest is related to their unsurpassed quintessential physical and chemical properties. Yet, owing to their hydrophilic nature, their utilization is restricted to applications involving hydrophilic or polar media, which limits their exploitation. With the presence of a large number of chemical functionalities within their structure, these building blocks provide a unique platform for significant surface modification through various chemistries. These chemical modifications are prerequisite, sometimes unavoidable, to adapt the interfacial properties of nanocellulose substrates or adjust their hydrophilic-hydrophobic balance. Therefore, various chemistries have been developed aiming to surface-modify these nano-sized substrates in order to confer to them specific properties, extending therefore their use to highly sophisticated applications. This review collocates current knowledge in the research and development of nanocelluloses and emphasizes more particularly on the chemical modification routes developed so far for their functionalization.

Production of humic acids from oil palm empty fruit bunch by submerged fermentation with Trichoderma viride: cellulosic substrates and nitrogen sources.

PubMed

Motta, F L; Santana, M H A

2013-01-01

The novelty of this study was to produce humic acids by submerged fermentation of empty fruit bunch (EFB) with Trichoderma viride and to investigate the effects of the cellulosic substrates and the organic sources of nitrogen on the biotechnological production of these acids. The results obtained indicate the potential application of EFB, a waste of oil palm processing, for humic acids production. Because EFB contains cellulose, hemicellulose and lignin, fermentations were also performed using these polymers as carbon sources, separately or in combination. After 120 h of fermentation, significant production of humic acids was observed only in cultures containing either EFB or a mixture of the three polymers. Use of either potato peptone or yeast extract as a nitrogen source yielded nearly identical patterns of fungal growth and production of humic acids. The data obtained from microscopic imaging of T. viride growth and sporulation in EFB, coupled with the determined rates of production of humic acids indicated that the production of these acids is related to T. viride sporulation. © 2013 American Institute of Chemical Engineers.

Influence of the crystalline structure of cellulose on the production of ethanol from lignocellulose biomass

NASA Astrophysics Data System (ADS)

Smuga-Kogut, Małgorzata; Zgórska, Kazimiera; Szymanowska-Powałowska, Daria

2016-01-01

In recent years, much attention has been devoted to the possibility of using lignocellulosic biomass for energy. Bioethanol is a promising substitute for conventional fossil fuels and can be produced from straw and wood biomass. Therefore, the aim of this paper was to investigate the effect of 1-ethyl-3-methylimidazolium pretreatment on the structure of cellulose and the acquisition of reducing sugars and bioethanol from cellulosic materials. Material used in the study was rye straw and microcrystalline cellulose subjected to ionic liquid 1-ethyl-3-methylimidazolium pretreatment. The morphology of cellulose fibres in rye straw and microcrystalline cellulose was imaged prior to and after ionic liquid pretreatment. Solutions of ionic liquid-treated and untreated cellulosic materials were subjected to enzymatic hydrolysis in order to obtain reducing sugars, which constituted a substrate for alcoholic fermentation. An influence of the ionic liquid on the cellulose structure, accumulation of reducing sugars in the process of hydrolysis of this material, and an increase in ethanol amount after fermentation was observed. The ionic liquid did not affect cellulolytic enzymes negatively and did not inhibit yeast activity. The amount of reducing sugars and ethyl alcohol was higher in samples purified with 1-ethyl-3-methy-limidazolium acetate. A change in the supramolecular structure of cellulose induced by the ionic liquid was also observed.

Discrete and structurally unique proteins (tāpirins) mediate attachment of extremely thermophilic Caldicellulosiruptor species to cellulose.

PubMed

Blumer-Schuette, Sara E; Alahuhta, Markus; Conway, Jonathan M; Lee, Laura L; Zurawski, Jeffrey V; Giannone, Richard J; Hettich, Robert L; Lunin, Vladimir V; Himmel, Michael E; Kelly, Robert M

2015-04-24

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins ("tāpirins," origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Hydrolysis rate constants at 10-25 °C can be more than doubled by a short anaerobic pre-hydrolysis at 35 °C.

PubMed

Zhang, L; Gao, R; Naka, A; Hendrickx, T L G; Rijnaarts, H H M; Zeeman, G

2016-11-01

Hydrolysis is the first step of the anaerobic digestion of complex wastewater and considered as the rate limiting step especially at low temperature. Low temperature (10-25 °C) hydrolysis was investigated with and without application of a short pre-hydrolysis at 35 °C. Batch experiments were executed using cellulose and tributyrin as model substrates for carbohydrates and lipids. The results showed that the low temperature anaerobic hydrolysis rate constants increased by a factor of 1.5-10, when the short anaerobic pre-hydrolysis at 35 °C was applied. After the pre-hydrolysis phase at 35 °C and decreasing the temperature, no lag phase was observed in any case. Without the pre-hydrolysis, the lag phase for cellulose hydrolysis at 35-10 °C was 4-30 days. Tributyrin hydrolysis showed no lag phase at any temperature. The hydrolysis efficiency of cellulose increased from 40 to 62%, and from 9.6 to 40% after 9.1 days at 15 and 10 °C, respectively, when the pre-hydrolysis at 35 °C was applied. The hydrolysis efficiency of tributyrin at low temperatures with the pre-hydrolysis at 35 °C was similar to those without the pre-hydrolysis. The hydrolytic activity of the supernatant collected from the digestate after batch digestion of cellulose and tributyrin at 35 °C was higher than that of the supernatants collected from the low temperature (≤25 °C) digestates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose*

PubMed Central

Blumer-Schuette, Sara E.; Alahuhta, Markus; Conway, Jonathan M.; Lee, Laura L.; Zurawski, Jeffrey V.; Giannone, Richard J.; Hettich, Robert L.; Lunin, Vladimir V.; Himmel, Michael E.; Kelly, Robert M.

2015-01-01

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tāpirins,” origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose. PMID:25720489

In situ, rapid, and temporally resolved measurements of cellulase adsorption onto lignocellulosic substrates by UV-vis spectrophotometry

Treesearch

Hao Liu; J. Y. Zhu; X. S. Chai

2011-01-01

This study demonstrated two in situ UV-vis spectrophotometric methods for rapid and temporally resolved measurements of cellulase adsorption onto cellulosic and lignocellulosic substrates during enzymatic hydrolysis. The cellulase protein absorption peak at 280 nm was used for quantification. The spectral interferences from light scattering by small fibers (fines) and...

Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility.

PubMed

Zhu, Zhiguang; Sathitsuksanoh, Noppadon; Vinzant, Todd; Schell, Daniel J; McMillan, James D; Zhang, Y-H Percival

2009-07-01

Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF-pretreated corn stover but reached only approximately 60% for the DA-pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were measured for the first time. The COSLIF-pretreated corn stover had a CAC of 11.57 m(2)/g, nearly twice that of the DA-pretreated biomass (5.89 m(2)/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA- and COSLIF-pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility. (c) 2009 Wiley Periodicals, Inc.

Long-Term Enrichment on Cellulose or Xylan Causes Functional and Taxonomic Convergence of Microbial Communities from Anaerobic Digesters

PubMed Central

Jia, Yangyang; Wilkins, David; Lu, Hongyuan; Cai, Mingwei

2015-01-01

Cellulose and xylan are two major components of lignocellulosic biomass, which represents a potentially important energy source, as it is abundant and can be converted to methane by microbial action. However, it is recalcitrant to hydrolysis, and the establishment of a complete anaerobic digestion system requires a specific repertoire of microbial functions. In this study, we maintained 2-year enrichment cultures of anaerobic digestion sludge amended with cellulose or xylan to investigate whether a cellulose- or xylan-digesting microbial system could be assembled from sludge previously used to treat neither of them. While efficient methane-producing communities developed under mesophilic (35°C) incubation, they did not under thermophilic (55°C) conditions. Illumina amplicon sequencing results of the archaeal and bacterial 16S rRNA genes revealed that the mature cultures were much lower in richness than the inocula and were dominated by single archaeal (genus Methanobacterium) and bacterial (order Clostridiales) groups, although at finer taxonomic levels the bacteria were differentiated by substrates. Methanogenesis was primarily via the hydrogenotrophic pathway under all conditions, although the identity and growth requirements of syntrophic acetate-oxidizing bacteria were unclear. Incubation conditions (substrate and temperature) had a much greater effect than inoculum source in shaping the mature microbial community, although analysis based on unweighted UniFrac distance found that the inoculum still determined the pool from which microbes could be enriched. Overall, this study confirmed that anaerobic digestion sludge treating nonlignocellulosic material is a potential source of microbial cellulose- and xylan-digesting functions given appropriate enrichment conditions. PMID:26712547

A novel high specific surface area conducting paper material composed of polypyrrole and Cladophora cellulose.

PubMed

Mihranyan, Albert; Nyholm, Leif; Bennett, Alfonso E Garcia; Strømme, Maria

2008-10-02

We present a novel conducting polypyrrole-based composite material, obtained by polymerization of pyrrole in the presence of iron(III) chloride on a cellulose substrate derived from the environmentally polluting Cladophora sp. algae. The material, which was doped with chloride ions, was molded into paper sheets and characterized using scanning and transmission electron microscopy, N 2 gas adsorption analysis, cyclic voltammetry, chronoamperometry and conductivity measurements at varying relative humidities. The specific surface area of the composite was found to be 57 m (2)/g and the fibrous structure of the Cladophora cellulose remained intact even after a 50 nm thick layer of polypyrrole had been coated on the cellulose fibers. The composite could be repeatedly used for electrochemically controlled extraction and desorption of chloride and an ion exchanging capacity of 370 C per g of composite was obtained as a result of the high surface area of the cellulose substrate. The influence of the oxidation and reduction potentials on the chloride ion exchange capacity and the nucleation of delocalized positive charges, forming conductive paths in the polypyrrole film, was also investigated. The creation of conductive paths during oxidation followed an effective medium rather than a percolative behavior, indicating that some conduction paths survive the polymer reduction steps. The present high surface area material should be well-suited for use in, e.g., electrochemically controlled ion exchange or separation devices, as well as sensors based on the fact that the material is compact, light, mechanically stable, and moldable into paper sheets.

Cellulose Synthesis and Its Regulation

PubMed Central

Li, Shundai; Bashline, Logan; Lei, Lei; Gu, Ying

2014-01-01

Cellulose, the most abundant biopolymer synthesized on land, is made of linear chains of ß (1–4) linked D-glucose. As a major structural component of the cell wall, cellulose is important not only for industrial use but also for plant growth and development. Cellulose microfibrils are tethered by other cell wall polysaccharides such as hemicellulose, pectin, and lignin. In higher plants, cellulose is synthesized by plasma membrane-localized rosette cellulose synthase complexes. Despite the recent advances using a combination of molecular genetics, live cell imaging, and spectroscopic tools, many aspects of the cellulose synthesis remain a mystery. In this chapter, we highlight recent research progress towards understanding the mechanism of cellulose synthesis in Arabidopsis. PMID:24465174

Production of cellulosic ethanol from cotton processing residues after pretreatment with dilute sodium hydroxide and enzymatic hydrolysis.

PubMed

Fockink, Douglas Henrique; Maceno, Marcelo Adriano Corrêa; Ramos, Luiz Pereira

2015-01-01

In this study, production of cellulosic ethanol from two cotton processing residues was investigated after pretreatment with dilute sodium hydroxide. Pretreatment performance was investigated using a 2(2) factorial design and the highest glucan conversion was achieved at the most severe alkaline conditions (0.4g NaOH g(-1) of dry biomass and 120°C), reaching 51.6% and 38.8% for cotton gin waste (CGW) and cotton gin dust (CGD), respectively. The susceptibility of pretreated substrates to enzymatic hydrolysis was also investigated and the best condition was achieved at the lowest total solids (5wt%) and the highest enzyme loading (85mg of Cellic CTec2 g(-1) of dry substrate). However, the highest concentration of fermentable sugars - 47.8 and 42.5gL(-1) for CGD and CGW, respectively - was obtained at 15wt% total solids using this same enzyme loading. Substrate hydrolysates had no inhibitory effects on the fermenting microorganism. Copyright © 2015. Published by Elsevier Ltd.

Cellulose microfibrils: visualization of biosynthetic and orienting complexes in association with the plasma membrane.

PubMed

Brown, R M; Montezinos, D

1976-01-01

Cellulose microfibril biosynthesis, assembly, and orientation in the unicellular green alga, Oocystis, is visualized in association with a linear enzyme complex embedded in the B face of the plasma membrane. Granule bands of the A face and complementary ridges of the B face are postulated to assist in the orientation of recently synthesized microfibrils. A model for microfibril synthesis and orientation is proposed and correlated with current hypotheses regarding cellulose biosynthesis in higher plants.

Microbial degradation of poly-b-esters: A mechanistic study, cellulose acetate biodegradability. Final report, 1 May 1990-31 July 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gross, R.A.

1993-08-30

In this Final Report, work carried out under ARO grant C-DAAL03-G-0111 is described. The investigations performed include the following: (1) isolation, purification and characterization of a poly(3-hydroxybutyrate) depolymerase enzyme from Penicillium funiculosum, (2) determination that the depolymerase is a serine esterase, (3) study of the effect of polymer stereochemistry and crystalline order in a semi-crystalline polymer film substrate on enzyme specificity and activity, (3) isolation, purification and characterization of cellulose acetate degrading microorganisms and (4) determination of the biodegradability of cellulose acetate with degrees of substitution up to 2.5 under aerobic thermophilic conditions. Poly(3-hydroxybutyrate) biodegradation, Poly(3-hydroxybutyrate) depolymerase enzyme, Depolymerase frommore » Penicillium funiculosum, Cellulose acetate degrading microorganisms, Composting polymer biodegradable.« less

Kinetic studies on batch cultivation of Trichoderma reesei and application to enhance cellulase production by fed-batch fermentation.

PubMed

Ma, Lijuan; Li, Chen; Yang, Zhenhua; Jia, Wendi; Zhang, Dongyuan; Chen, Shulin

2013-07-20

Reducing the production cost of cellulase as the key enzyme for cellulose hydrolysis to fermentable sugars remains a major challenge for biofuel production. Because of the complexity of cellulase production, kinetic modeling and mass balance calculation can be used as effective tools for process design and optimization. In this study, kinetic models for cell growth, substrate consumption and cellulase production in batch fermentation were developed, and then applied in fed-batch fermentation to enhance cellulase production. Inhibition effect of substrate was considered and a modified Luedeking-Piret model was developed for cellulase production and substrate consumption according to the growth characteristics of Trichoderma reesei. The model predictions fit well with the experimental data. Simulation results showed that higher initial substrate concentration led to decrease of cellulase production rate. Mass balance and kinetic simulation results were applied to determine the feeding strategy. Cellulase production and its corresponding productivity increased by 82.13% after employing the proper feeding strategy in fed-batch fermentation. This method combining mathematics and chemometrics by kinetic modeling and mass balance can not only improve cellulase fermentation process, but also help to better understand the cellulase fermentation process. The model development can also provide insight to other similar fermentation processes. Copyright © 2013 Elsevier B.V. All rights reserved.

Nanocrystalline cellulose as an eco-friendly reinforcing additive to polyurethane coating for augmented anticorrosive behavior.

PubMed

Abd El-Fattah, M; Hasan, Abdulraheim M A; Keshawy, Mohamed; El Saeed, Ashraf M; Aboelenien, Ossama M

2018-03-01

Nanocrystalline cellulose (NCC) and micro-powdered cellulose (MPC) were extracted from rice straw by mechanical and alkali treatment methods, then characterized via infrared spectroscopy and dynamic light scattering. A series of polyurethane nanocrystalline cellulose composite (PNCCC) and polyurethane micro-powdered cellulose composite (PMPCC) coatings were prepared with various loading levels of NCC and MPC from 0.5 to 2.0 wt.%, and the coatings were applied onto the pretreated mild steel substrate at room temperature. The results showed that the NCC and MPC influenced positively the studied properties of the polyurethane coating; furthermore the most pronounced anticorrosive properties were obtained at 1 wt.% NCC and MPC, as confirmed by open circuit potential (OCP) study, electrochemical impedance spectroscopy (EIS) study and salt spray test. However, the optimum enhancement of mechanical properties was found at 1.5 wt.% loading level, after which further loading of NCC and MPC led to the reduction in the mechanical properties. Copyright © 2018 Elsevier Ltd. All rights reserved.

Simultaneous catalytic conversion of cellulose and corncob xylan under temperature programming for enhanced sorbitol and xylitol production.

PubMed

Ribeiro, Lucília Sousa; Órfão, José J de Melo; Pereira, Manuel Fernando Ribeiro

2017-11-01

Sorbitol and xylitol yields can be improved by converting cellulose and xylan simultaneously, due to a synergetic effect between both substrates. Furthermore, both yields can be greatly enhanced by simply adjusting the reaction conditions regarding the optimum for the production of each product, since xylitol (from xylan) and sorbitol (from cellulose) yields are maximized when the reaction is carried out at 170 and 205°C, respectively. Therefore, the combination of a simultaneous conversion of cellulose and xylan with a two-step temperature approach, which consists in the variation of the reaction temperature from 170 to 205°C after 2h, showed to be a good strategy for maximizing the production of sorbitol and xylitol directly from mixture of cellulose and xylan. Using this new and environmentally friendly approach, yields of sorbitol and xylitol of 75 and 77%, respectively, were obtained after 6h of reaction. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electrospun ultra-fine cellulose acetate fibrous mats containing tannic acid-Fe+++ complexes

USDA-ARS?s Scientific Manuscript database

Cellulose acetate (CA) fibrous mats with improved mechanical and antioxidant properties were produced by a simple, scalable and cost-effective electrospinning method. Fibers loaded with small amounts of TA-Fe+++ complexes showed an increase in tensile strength of approximately 117% when compared to ...

Solid-state fermentation for cellulase production by Pestalotiopsis versicolor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rao, M.N.A.; Mithal, B.M.; Thakkur, R.N.

1983-03-01

Solid-state fermentation (SSF) refers to the fermentation process on solid substrate without the presence of free liquid. It is found to be ideal when the organism is a fungus and the substrate is insoluble, like cellulose. Production of cellulase by SSF has been studied in detail by Toyama and Ogawa. It has been found that more concentrated enzyme preparations can be obtained by SSF than in liquid type since the enzyme gets diluted in the whole medium in liquid culture. In the present study, a plant pathogenic fungus Pestalotiopsis versicolor has been grown on various solid cultures of cellulosic substancesmore » and production of cellulase has been studied. Earlier, we had studied the production of cellulase by P. versicolor in liquid culture. (Refs. 7).« less

Cellulosic/wool pigment prints with remarkable antibacterial functionalities.

PubMed

Ibrahim, N A; Eid, B M; Khalil, H M

2015-01-22

Several bio-active agents namely choline chloride, triclosan derivative, PEG-600 and 4-hydroxybenzophenone were successfully included into solvent-free pigment formulations, in a single-stage process, followed by screen printing and microwave-fixation to obtain antibacterial functionalized cellulosic/wool pigment prints. Results obtained signify that both the improvement in functionalization and coloration properties are governed by type of antibacterial agent, kind of substrate as well as pigment colorant. The imparted antibacterial activity of the loaded bio-active agents follows the decreasing order: G+ve (Staphylococcus aureus)>G-ve (Escherichia coli), keeping other parameters constant. The imparted functional and coloration properties showed no significant decrease even after 15 washings. Mode of interactions among the nominated substrates, the pigment paste constituents and the bioactive agents were also proposed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol.

PubMed

Ballesteros, I; Ballesteros, M; Cabañas, A; Carrasco, J; Martín, C; Negro, M J; Saez, F; Saez, R

1991-01-01

A total of 27 yeast strains belonging to the groups Candida, Saccharomyces, and Kluyveromyces were screened for their ability to grow and ferment glucose at temperatures ranging 32-45 degrees C. K. marxianus and K. fragilis were found to be the best ethanol producing organisms at the higher temperature tested and, so, were selected for subsequent simultaneous saccharification and fermentation (SSF) studies. SSF experiments were performed at 42 and 45 degrees C, utilizing Solkafloc (10%) as cellulose substrate and a cellulase loading of 15 FPU/g substrate. Best results were achieved at 42 degrees C with K. marxianus L. G. and K. fragilis L. G., both of which produced close to 38 g/L ethanol and 0.5 ethanol yield, in 78 h.

Custom fabrication of biomass containment devices using 3-D printing enables bacterial growth analyses with complex insoluble substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, Cassandra E.; Beri, Nina R.; Gardner, Jeffrey G.

Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interactionmore » between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. Furthermore, we applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present.« less

Custom fabrication of biomass containment devices using 3-D printing enables bacterial growth analyses with complex insoluble substrates

DOE PAGES

Nelson, Cassandra E.; Beri, Nina R.; Gardner, Jeffrey G.

2016-09-21

Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interactionmore » between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. Furthermore, we applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present.« less

Enzymatic hydrolysis of lignocellulosic biomass from Onopordum nervosum.

PubMed

Martín, C; Negro, M J; Alfonsel, M; Sáez, R

1988-07-20

Some properties of the cellulolytic complex obtained from Trichoderma reesei QM 9414 grown on Solka floc as carbon source and its ability to hydrolyze the lignocellulosic biomass of Onopordum nervosum Boiss were studied. The optimum enzyme activity was found at temperatures between 50 and 55 degrees C and pH ranging from 4.3 to 4.8. Hydrolysis of 4-nitropnenyl-beta-D-glucopyranoside (4-NPG) and cellobiose by the beta-glucosidase of the complex, showed competitive inhibition by glucose with a K(i) value of 0.8 mM for 4-NPG and 2. 56 mM for cellobiose. Enzymatic hydrolysis yield of Onopordum nervosum, evaluated as glucose production after 48 h, showed a threefold increase by pretreating the lignocellulosic substrate with alkali. When the loss of glucose incurred by de pretreatment was taken into account, a 160% increase in the final cellulose to glucose conversion was found to be due to the pretreatment.

A 24-GHz Front-End Integrated on a Multilayer Cellulose-Based Substrate for Doppler Radar Sensors †

PubMed Central

Mariotti, Chiara; Virili, Marco; Orecchini, Giulia; Roselli, Luca; Mezzanotte, Paolo

2017-01-01

This paper presents a miniaturized Doppler radar that can be used as a motion sensor for low-cost Internet of things (IoT) applications. For the first time, a radar front-end and its antenna are integrated on a multilayer cellulose-based substrate, built-up by alternating paper, glue and metal layers. The circuit exploits a distributed microstrip structure that is realized using a copper adhesive laminate, so as to obtain a low-loss conductor. The radar operates at 24 GHz and transmits 5 mW of power. The antenna has a gain of 7.4 dBi and features a half power beam-width of 48 degrees. The sensor, that is just the size of a stamp, is able to detect the movement of a walking person up to 10 m in distance, while a minimum speed of 50 mm/s up to 3 m is clearly measured. Beyond this specific result, the present paper demonstrates that the attractive features of cellulose, including ultra-low cost and eco-friendliness (i.e., recyclability and biodegradability), can even be exploited for the realization of future high-frequency hardware. This opens opens the door to the implementation on cellulose of devices and systems which make up the “sensing layer” at the base of the IoT ecosystem. PMID:28895914

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

PubMed

Hindié, M; Vayssade, M; Dufresne, M; Quéant, S; Warocquier-Clérout, R; Legeay, G; Vigneron, P; Olivier, V; Duval, J-L; Nagel, M-D

2006-09-01

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Differential reinforcement of enzymatic hydrolysis by adding chemicals and accessory proteins to high solid loading substrates with different pretreatments.

PubMed

Du, Jian; Song, Wenxia; Zhang, Xiu; Zhao, Jian; Liu, Guodong; Qu, Yinbo

2018-04-23

High dosage of enzyme is required to achieve effective lignocellulose hydrolysis, especially at high-solid loadings, which is a significant barrier to large-scale bioconversion of lignocellulose. Here, we screened four chemical additives and three accessory proteins for their effects on the enzymatic hydrolysis of various lignocellulosic materials. The effects were found to be highly dependent on the composition and solid loadings of substrates. For xylan-extracted lignin-rich corncob residue, the enhancing effect of PEG 6000 was most pronounced and negligibly affected by solid content, which reduced more than half of enzyme demand at 20% dry matter (DM). Lytic polysaccharide monooxygenase enhanced the hydrolysis of ammonium sulfite wheat straw pulp, and its addition reduced about half of protein demand at the solid loading of 20% DM. Supplementation of the additives in the hydrolysis of pure cellulose and complex lignocellulosic materials revealed that their effects are tightly linked to pretreatment strategies.

Modelling of different enzyme productions by solid-state fermentation on several agro-industrial residues.

PubMed

Diaz, Ana Belen; Blandino, Ana; Webb, Colin; Caro, Ildefonso

2016-11-01

A simple kinetic model, with only three fitting parameters, for several enzyme productions in Petri dishes by solid-state fermentation is proposed in this paper, which may be a valuable tool for simulation of this type of processes. Basically, the model is able to predict temporal fungal enzyme production by solid-state fermentation on complex substrates, maximum enzyme activity expected and time at which these maxima are reached. In this work, several fermentations in solid state were performed in Petri dishes, using four filamentous fungi grown on different agro-industrial residues, measuring xylanase, exo-polygalacturonase, cellulose and laccase activities over time. Regression coefficients after fitting experimental data to the proposed model turned out to be quite high in all cases. In fact, these results are very interesting considering, on the one hand, the simplicity of the model and, on the other hand, that enzyme activities correspond to different enzymes, produced by different fungi on different substrates.

Characterization of Cellulose Synthesis in Plant Cells

PubMed Central

Maleki, Samaneh Sadat; Mohammadi, Kourosh; Ji, Kong-shu

2016-01-01

Cellulose is the most significant structural component of plant cell wall. Cellulose, polysaccharide containing repeated unbranched β (1-4) D-glucose units, is synthesized at the plasma membrane by the cellulose synthase complex (CSC) from bacteria to plants. The CSC is involved in biosynthesis of cellulose microfibrils containing 18 cellulose synthase (CesA) proteins. Macrofibrils can be formed with side by side arrangement of microfibrils. In addition, beside CesA, various proteins like the KORRIGAN, sucrose synthase, cytoskeletal components, and COBRA-like proteins have been involved in cellulose biosynthesis. Understanding the mechanisms of cellulose biosynthesis is of great importance not only for improving wood production in economically important forest trees to mankind but also for plant development. This review article covers the current knowledge about the cellulose biosynthesis-related gene family. PMID:27314060

Biomass conversion determined via fluorescent cellulose decay assay.

PubMed

Wischmann, Bente; Toft, Marianne; Malten, Marco; McFarland, K C

2012-01-01

An example of a rapid microtiter plate assay (fluorescence cellulose decay, FCD) that determines the conversion of cellulose in a washed biomass substrate is reported. The conversion, as verified by HPLC, is shown to correlate to the monitored FCD in the assay. The FCD assay activity correlates to the performance of multicomponent enzyme mixtures and is thus useful for the biomass industry. The development of an optimized setup of the 96-well microtiter plate is described, and is used to test a model that shortens the assay incubation time from 72 to 24h. A step-by-step procedure of the final assay is described. Copyright © 2012 Elsevier Inc. All rights reserved.

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes*

PubMed Central

Kuusk, Silja; Sørlie, Morten; Väljamäe, Priit

2015-01-01

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the 14C-labeled crystalline polymeric substrates 14C chitin nanowhiskers and 14C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki(obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki(obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation. PMID:25767120

The predominant molecular state of bound enzyme determines the strength and type of product inhibition in the hydrolysis of recalcitrant polysaccharides by processive enzymes.

PubMed

Kuusk, Silja; Sørlie, Morten; Väljamäe, Priit

2015-05-01

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the (14)C-labeled crystalline polymeric substrates (14)C chitin nanowhiskers and (14)C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki (obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki (obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Understanding how the complex molecular architecture of mannan-degrading hydrolases contributes to plant cell wall degradation.

PubMed

Zhang, Xiaoyang; Rogowski, Artur; Zhao, Lei; Hahn, Michael G; Avci, Utku; Knox, J Paul; Gilbert, Harry J

2014-01-24

Microbial degradation of plant cell walls is a central component of the carbon cycle and is of increasing importance in environmentally significant industries. Plant cell wall-degrading enzymes have a complex molecular architecture consisting of catalytic modules and, frequently, multiple non-catalytic carbohydrate binding modules (CBMs). It is currently unclear whether the specificities of the CBMs or the topology of the catalytic modules are the primary drivers for the specificity of these enzymes against plant cell walls. Here, we have evaluated the relationship between CBM specificity and their capacity to enhance the activity of GH5 and GH26 mannanases and CE2 esterases against intact plant cell walls. The data show that cellulose and mannan binding CBMs have the greatest impact on the removal of mannan from tobacco and Physcomitrella cell walls, respectively. Although the action of the GH5 mannanase was independent of the context of mannan in tobacco cell walls, a significant proportion of the polysaccharide was inaccessible to the GH26 enzyme. The recalcitrant mannan, however, was fully accessible to the GH26 mannanase appended to a cellulose binding CBM. Although CE2 esterases display similar specificities against acetylated substrates in vitro, only CjCE2C was active against acetylated mannan in Physcomitrella. Appending a mannan binding CBM27 to CjCE2C potentiated its activity against Physcomitrella walls, whereas a xylan binding CBM reduced the capacity of esterases to deacetylate xylan in tobacco walls. This work provides insight into the biological significance for the complex array of hydrolytic enzymes expressed by plant cell wall-degrading microorganisms.

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

PubMed Central

Raut, Mahendra P.; Karunakaran, Esther; Mukherjee, Joy; Biggs, Catherine A.; Wright, Phillip C.

2015-01-01

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane. PMID:26492413

Rheology of lyocell solutions from different cellulosic sources and development of regenerated cellulosic microfibers

NASA Astrophysics Data System (ADS)

Li, Zuopan

2003-10-01

The primary goals of the study were to develop manufactured cellulosic fibers and microfibers from wood pulps as well as from lignocellulosic agricultural by-products and to investigate alternative cellulosic sources as raw materials for lyocell solutions. A protocol was developed for the lyocell preparation from different cellulose sources. The cellulose sources included commercial dissolving pulps, commercial bleached hardwood, unbleached hardwood, bleached softwood, unbleached softwood, bleached thermomechanical pulp, unbleached thermomechanical pulp, bleached recycled newsprint, unbleached recycled newsprint, bagasse and kudzu. The rheological behavior of solutions was characterized. Complex viscosities and effective elongational viscosities were measured and the influences of parameters such as cellulose source, concentration, bleaching, and temperature were studied. One-way ANOVA post hoc tests were carried out to identify which cellulose sources have the potential to produce lyocell solutions having similar complex viscosities to those from commercial dissolving pulps. Lyocell solutions from both bleached and unbleached softwood and hardwood were classified as one homogenous subset that had the lowest complex viscosity. Kudzu solutions had the highest complex viscosity. The results showed the potential to substitute DP 1457 dissolving pulp with unbleached recycled newsprint pulps, to substitute DP 1195 dissolving pulp with bleached and unbleached thermomechanical pulps, to substitute DP 932 dissolving pulp with bleached thermomechanical pulps or bleached recycled newsprint pulps, to substitute DP 670 dissolving pulp with bagasse. Lyocell fibers were produced from selected solutions and were treated to produce microfibers. Water, sulfuric acid solutions and sodium hydroxide solutions were used. The treatment of lyocell fibers in 17.5% NaOH solutions for five minutes at 20°C successfully broke the fibers into fibrils along fiber axis. The diameters of the fibrils were generally in the range of 2 to 6 mum, and there were also finer fibrils with diameters less than 1 mum.

Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

DOE PAGES

Lewin, Gina R.; Johnson, Amanda L.; Soto, Rolando D. Moreira; ...

2016-03-21

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using materialmore » from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within general containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. Lastly, a representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.« less

Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

PubMed Central

Lewin, Gina R.; Johnson, Amanda L.; Soto, Rolando D. Moreira; Perry, Kailene; Book, Adam J.; Horn, Heidi A.; Pinto-Tomás, Adrián A.; Currie, Cameron R.

2016-01-01

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation. PMID:26999749

Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewin, Gina R.; Johnson, Amanda L.; Soto, Rolando D. Moreira

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using materialmore » from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within general containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. Lastly, a representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.« less

Understanding paper degradation: identification of products of cellulosic paper decomposition at the wet-dry "tideline" interface using GC-MS.

PubMed

Sladkevich, Sergey; Dupont, Anne-Laurence; Sablier, Michel; Seghouane, Dalila; Cole, Richard B

2016-11-01

Cellulose paper degradation products forming in the "tideline" area at the wet-dry interface of pure cellulose paper were analyzed using gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) and high-resolution electrospray ionization-mass spectrometry (ESI-MS, LTQ Orbitrap) techniques. Different extraction protocols were employed in order to solubilize the products of oxidative cellulose decomposition, i.e., a direct solvent extraction or a more laborious chromophore release and identification (CRI) technique aiming to reveal products responsible for paper discoloration in the tideline area. Several groups of low molecular weight compounds were identified, suggesting a complex pathway of cellulose decomposition in the tidelines formed at the cellulose-water-oxygen interface. Our findings, namely the appearance of a wide range of linear saturated carboxylic acids (from formic to nonanoic), support the oxidative autocatalytic mechanism of decomposition. In addition, the identification of several furanic compounds (which can be, in part, responsible for paper discoloration) plus anhydro carbohydrate derivatives sheds more light on the pathways of cellulose decomposition. Most notably, the mechanisms of tideline formation in the presence of molecular oxygen appear surprisingly similar to pathways of pyrolytic cellulose degradation. More complex chromophore compounds were not detected in this study, thereby revealing a difference between this short-term tideline experiment and longer-term cellulose aging.

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

PubMed

Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S; Mortimer, Jenny C; Brown, Steven P; Persson, Staffan; Dupree, Paul

2016-06-09

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.

Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction.

PubMed

Ghio, Silvina; Lorenzo, Gonzalo Sabarís Di; Lia, Verónica; Talia, Paola; Cataldi, Angel; Grasso, Daniel; Campos, Eleonora

2012-01-01

Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.

Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction

PubMed Central

Ghio, Silvina; Lorenzo, Gonzalo Sabarís Di; Lia, Verónica; Talia, Paola; Cataldi, Angel; Grasso, Daniel; Campos, Eleonora

2012-01-01

Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus. PMID:23301200

Distance-Based Tear Lactoferrin Assay on Microfluidic Paper Device Using Interfacial Interactions on Surface-Modified Cellulose.

PubMed

Yamada, Kentaro; Henares, Terence G; Suzuki, Koji; Citterio, Daniel

2015-11-11

"Distance-based" detection motifs on microfluidic paper-based analytical devices (μPADs) allow quantitative analysis without using signal readout instruments in a similar manner to classical analogue thermometers. To realize a cost-effective and calibration-free distance-based assay of lactoferrin in human tear fluid on a μPAD not relying on antibodies or enzymes, we investigated the fluidic mobilities of the target protein and Tb(3+) cations used as the fluorescent detection reagent on surface-modified cellulosic filter papers. Chromatographic elution experiments in a tear-like sample matrix containing electrolytes and proteins revealed a collapse of attractive electrostatic interactions between lactoferrin or Tb(3+) and the cellulosic substrate, which was overcome by the modification of the paper surface with the sulfated polysaccharide ι-carrageenan. The resulting μPAD based on the fluorescence emission distance successfully analyzed 0-4 mg mL(-1) of lactoferrin in complex human tear matrix with a lower limit of detection of 0.1 mg mL(-1) by simple visual inspection. Assay results of 18 human tear samples including ocular disease patients and healthy volunteers showed good correlation to the reference ELISA method with a slope of 0.997 and a regression coefficient of 0.948. The distance-based quantitative signal and the good batch-to-batch fabrication reproducibility relying on printing methods enable quantitative analysis by simply reading out "concentration scale marks" printed on the μPAD without performing any calibration and using any signal readout instrument.

Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.

PubMed

Wei, Hui; Wang, Wei; Yarbrough, John M; Baker, John O; Laurens, Lieve; Van Wychen, Stefanie; Chen, Xiaowen; Taylor, Larry E; Xu, Qi; Himmel, Michael E; Zhang, Min

2013-01-01

Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

Efficient Degradation of Lignocellulosic Plant Biomass, without Pretreatment, by the Thermophilic Anaerobe “Anaerocellum thermophilum” DSM 6725▿

PubMed Central

Yang, Sung-Jae; Kataeva, Irina; Hamilton-Brehm, Scott D.; Engle, Nancy L.; Tschaplinski, Timothy J.; Doeppke, Crissa; Davis, Mark; Westpheling, Janet; Adams, Michael W. W.

2009-01-01

Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that “Anaerocellum thermophilum” DSM 6725, an anaerobic bacterium that grows optimally at 75°C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75°C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70°C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms. PMID:19465524

Efficient degradation of lignocellulosic plant biomass without pretreatment by the 9 thermophilic anaerobe, Anaerocellum thermophilum DSM 6725

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Sung-Jae; Kataeva, Irina; Hamilton-Brehm, Scott

2009-01-01

Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that 'Anaerocellum thermophilum' DSM 6725, an anaerobic bacterium that grows optimally at 75 C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75 C for 18 h also served as a growthmore » substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70 C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.« less

Development of curcumin-cyclodextrin/cellulose nanocrystals complexes: New anticancer drug delivery systems.

PubMed

Ndong Ntoutoume, Gautier M A; Granet, Robert; Mbakidi, Jean Pierre; Brégier, Frédérique; Léger, David Y; Fidanzi-Dugas, Chloë; Lequart, Vincent; Joly, Nicolas; Liagre, Bertrand; Chaleix, Vincent; Sol, Vincent

2016-02-01

The synthesis of curcumin-cyclodextrin/cellulose nanocrystals (CNCx) nano complexes was performed. CNCx were functionalized by ionic association with cationic β-cyclodextrin (CD) and CD/CNCx complexes were used to encapsulate curcumin. Preliminary in vitro results showed that the resulting curcumin-CD/CNCx complexes exerted antiproliferative effect on colorectal and prostatic cancer cell lines, with IC50s lower than that of curcumin alone. Copyright © 2015 Elsevier Ltd. All rights reserved.

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously.

PubMed

Xiong, Wei; Reyes, Luis H; Michener, William E; Maness, Pin-Ching; Chou, Katherine J

2018-03-15

Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals. © 2018 Wiley Periodicals, Inc.

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

DOE PAGES

Xiong, Wei; Reyes, Luis H.; Michener, William E.; ...

2018-04-10

Here, cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration ofmore » xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.« less

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Wei; Reyes, Luis H.; Michener, William E.

Here, cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration ofmore » xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H 2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.« less

Cellulose Deficiency Is Enhanced on Hyper Accumulation of Sucrose by a H+-Coupled Sucrose Symporter1[OPEN

PubMed Central

Yeats, Trevor H.; Sorek, Hagit

2016-01-01

In order to understand factors controlling the synthesis and deposition of cellulose, we have studied the Arabidopsis (Arabidopsis thaliana) double mutant shaven3 shaven3-like1 (shv3svl1), which was shown previously to exhibit a marked cellulose deficiency. We discovered that exogenous sucrose (Suc) in growth medium greatly enhances the reduction in hypocotyl elongation and cellulose content of shv3svl1. This effect was specific to Suc and was not observed with other sugars or osmoticum. Live-cell imaging of fluorescently labeled cellulose synthase complexes revealed a slowing of cellulose synthase complexes in shv3svl1 compared with the wild type that is enhanced in a Suc-conditional manner. Solid-state nuclear magnetic resonance confirmed a cellulose deficiency of shv3svl1 but indicated that cellulose crystallinity was unaffected in the mutant. A genetic suppressor screen identified mutants of the plasma membrane Suc/H+ symporter SUC1, indicating that the accumulation of Suc underlies the Suc-dependent enhancement of shv3svl1 phenotypes. While other cellulose-deficient mutants were not specifically sensitive to exogenous Suc, the feronia (fer) receptor kinase mutant partially phenocopied shv3svl1 and exhibited a similar Suc-conditional cellulose defect. We demonstrate that shv3svl1, like fer, exhibits a hyperpolarized plasma membrane H+ gradient that likely underlies the enhanced accumulation of Suc via Suc/H+ symporters. Enhanced intracellular Suc abundance appears to favor the partitioning of carbon to starch rather than cellulose in both mutants. We conclude that SHV3-like proteins may be involved in signaling during cell expansion that coordinates proton pumping and cellulose synthesis. PMID:27013021

Bottom-up fabrication of paper-based microchips by blade coating of cellulose microfibers on a patterned surface.

PubMed

Gao, Bingbing; Liu, Hong; Gu, Zhongze

2014-12-23

We report a method for the bottom-up fabrication of paper-based capillary microchips by the blade coating of cellulose microfibers on a patterned surface. The fabrication process is similar to the paper-making process in which an aqueous suspension of cellulose microfibers is used as the starting material and is blade-coated onto a polypropylene substrate patterned using an inkjet printer. After water evaporation, the cellulose microfibers form a porous, hydrophilic, paperlike pattern that wicks aqueous solution by capillary action. This method enables simple, fast, inexpensive fabrication of paper-based capillary channels with both width and height down to about 10 μm. When this method is used, the capillary microfluidic chip for the colorimetric detection of glucose and total protein is fabricated, and the assay requires only 0.30 μL of sample, which is 240 times smaller than for paper devices fabricated using photolithography.

Mild and modular surface modification of cellulose via hetero Diels-Alder (HDA) cycloaddition.

PubMed

Goldmann, Anja S; Tischer, Thomas; Barner, Leonie; Bruns, Michael; Barner-Kowollik, Christopher

2011-04-11

A combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and hetero Diels-Alder (HDA) cycloaddition was used to effect, under mild (T ≈ 20 °C), fast, and modular conditions, the grafting of poly(isobornyl acrylate) (M(n) = 9800 g mol(-1), PDI = 1.19) onto a solid cellulose substrate. The active hydroxyl groups expressed on the cellulose fibers were converted to tosylate leaving groups, which were subsequently substituted by a highly reactive cyclopentadienyl functionality (Cp). By employing the reactive Cp-functionality as a diene, thiocarbonyl thio-capped poly(isobornyl acrylate) synthesized via RAFT polymerization (mediated by benzyl pyridine-2-yldithioformiate (BPDF)) was attached to the surface under ambient conditions by an HDA cycloaddition (reaction time: 15 h). The surface-modified cellulose samples were analyzed in-depth by X-ray photoelectron spectroscopy, scanning electron microscopy, elemental analysis, Fourier transform infrared (FT-IR) spectroscopy as well as Fourier transform infrared microscopy employing a focal plane array detector for imaging purposes. The analytical results provide strong evidence that the reaction of suitable dienophiles with Cp-functional cellulose proceeds under mild reaction conditions (T ≈ 20 °C) in an efficient fashion. In particular, the visualization of individual modified cellulose fibers via high-resolution FT-IR microscopy corroborates the homogeneous distribution of the polymer film on the cellulose fibers.

Structure of a cellulose degrading bacterial community during anaerobic digestion.

PubMed

O'Sullivan, Cathryn A; Burrell, Paul C; Clarke, William P; Blackall, Linda L

2005-12-30

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells. Copyright 2005 Wiley Periodicals, Inc

[Mode of action and inhibition of polygalacturonase covalently bound to polysaccharide and glass carriers].

PubMed

Bock, W; Krause, M; Göbel, H; Anger, H; Schawaller, H J; Flemming, C; Gabert, A

1978-01-01

Endo-polygalacturonase (EC 3.2.1.15.) from Aspergillus spec. is much changed as far as its mode of action and the interaction with vegetable inhibitors of pectinase (from green beans and cucumbers) are concerned when it is covalently bound to insoluble carriers (Sepharose, cellulose powder, macroporous glass and nonporous ballotinis). Whereas a 2% degradation of substrate by the soluble enzyme caused a 50% decrease of viscosity of citrus pectic acid, the comparable degradation of substrate was increased to a level of about 10% with the investigated polygalacturonase carrier complexes apparently independent of the properties of the carriers and the kind of binding of the enzyme. In contrast to this the higher degradation of substrate of 15 and 20% respectively which was further stated at a 50% decrease of viscosity is unambiguously connected with the carriers and is in direct correlation with the specific activity of the polygalacturonase carrier complexes. Contrary to the soluble enzyme the covalently bound enzyme produces more lower oligomerous galacturonic acids by an exo-mechanism or by multiple attack already at the beginning of the hydrolysis of pectic acid. During the final stage there is an enrichment of trigalacturonic acid besides mono- and digalacturonic acids independent of the state of solution of the enzyme. It could further be stated that the strong inhibition of the soluble endo-polygalacturonase by selected pectinase inhibitors which was described earlier is reduced by degrees with the enzyme covalently bound to the insoluble carriers.

Genome Sequence of Microbulbifer mangrovi DD-13T Reveals Its Versatility to Degrade Multiple Polysaccharides.

PubMed

Imran, Md; Pant, Poonam; Shanbhag, Yogini P; Sawant, Samir V; Ghadi, Sanjeev C

2017-02-01

Microbulbifer mangrovi strain DD-13 T is a novel-type species isolated from the mangroves of Goa, India. The draft genome sequence of strain DD-13 comprised 4,528,106 bp with G+C content of 57.15%. Out of 3479 open reading frames, functions for 3488 protein coding sequences were predicted on the basis of similarity with the cluster of orthologous groups. In addition to protein coding sequences, 34 tRNA genes and 3 rRNA genes were detected. Analysis of nucleotide sequence of predicted gene using a Carbohydrate-Active Enzymes (CAZymes) Analysis Toolkit indicates that strain DD-13 encodes a large set of CAZymes including 255 glycoside hydrolases, 76 carbohydrate esterases, 17 polysaccharide lyases, and 113 carbohydrate-binding modules (CBMs). Many genes from strain DD-13 were annotated as carbohydrases specific for degradation of agar, alginate, carrageenan, chitin, xylan, pullulan, cellulose, starch, β-glucan, pectin, etc. Some of polysaccharide-degrading genes were highly modular and were appended at least with one CBM indicating the versatility of strain DD-13 to degrade complex polysaccharides. The cell growth of strain DD-13 was validated using pure polysaccharides such as agarose or alginate as carbon source as well as by using red and brown seaweed powder as substrate. The homologous carbohydrase produced by strain DD-13 during growth degraded the polysaccharide, ensuring the production of metabolizable reducing sugars. Additionally, several other polysaccharides such as carrageenan, xylan, pullulan, pectin, starch, and carboxymethyl cellulose were also corroborated as growth substrate for strain DD-13 and were associated with concomitant production of homologous carbohydrase.

Preparation, Characterization and Activity of a Peptide-Cellulosic Aerogel Protease Sensor from Cotton.

PubMed

Edwards, J Vincent; Fontenot, Krystal R; Prevost, Nicolette T; Pircher, Nicole; Liebner, Falk; Condon, Brian D

2016-10-26

Nanocellulosic aerogels (NA) provide a lightweight biocompatible material with structural properties, like interconnected high porosity and specific surface area, suitable for biosensor design. We report here the preparation, characterization and activity of peptide-nanocellulose aerogels (PepNA) made from unprocessed cotton and designed with protease detection activity. Low-density cellulosic aerogels were prepared from greige cotton by employing calcium thiocyanate octahydrate/lithium chloride as a direct cellulose dissolving medium. Subsequent casting, coagulation, solvent exchange and supercritical carbon dioxide drying afforded homogeneous cellulose II aerogels of fibrous morphology. The cotton-based aerogel had a porosity of 99% largely dominated by mesopores (2-50 nm) and an internal surface of 163 m²·g -1 . A fluorescent tripeptide-substrate (succinyl-alanine-proline-alanine-4-amino-7-methyl-coumarin) was tethered to NA by (1) esterification of cellulose C6 surface hydroxyl groups with glycidyl-fluorenylmethyloxycarbonyl (FMOC), (2) deprotection and (3) coupling of the immobilized glycine with the tripeptide. Characterization of the NA and PepNA included techniques, such as elemental analysis, mass spectral analysis, attenuated total reflectance infrared imaging, nitrogen adsorption, scanning electron microscopy and bioactivity studies. The degree of substitution of the peptide analog attached to the anhydroglucose units of PepNA was 0.015. The findings from mass spectral analysis and attenuated total reflectance infrared imaging indicated that the peptide substrate was immobilized on to the surface of the NA. Nitrogen adsorption revealed a high specific surface area and a highly porous system, which supports the open porous structure observed from scanning electron microscopy images. Bioactivity studies of PepNA revealed a detection sensitivity of 0.13 units/milliliter for human neutrophil elastase, a diagnostic biomarker for inflammatory diseases. The physical properties of the aerogel are suitable for interfacing with an intelligent protease sequestrant wound dressing.

Heterogeneous Expression and Functional Characterization of Cellulose-Degrading Enzymes from Aspergillus niger for Enzymatic Hydrolysis of Alkali Pretreated Bamboo Biomass.

PubMed

Ali, Nasir; Ting, Zhang; Li, Hailong; Xue, Yong; Gan, Lihui; Liu, Jian; Long, Minnan

2015-09-01

Enzymatic hydrolysis of cellulosic biomass has caught much attention because of modest reaction conditions and environment friendly conditions. To reduce the cost and to achieve good quantity of cellulases, a heterologous expression system is highly favored. In this study, cellulose-degrading enzymes, GH3 family β-glucosidase (BGL), GH7 family-related cellobiohydrolases (CBHs), and endoglucanase (EG) from a newly isolated Aspergillus niger BE-2 are highly expressed in Pichia pastoris GS115. The strain produced EG, CBHs, and BGL enzymatic concentration of 0.56, 0.11, and 22 IU/mL, respectively. Mode of actions of the recombinant enzymes for substrate specificity and end product analysis are verified and found specific for cellulose degradation. Bamboo biomass saccharification with A. niger cellulase released a high level of fermentable sugars. Hydrolysis parameters are optimized to obtain reducing sugars level of 3.18 g/L. To obtain reducing sugars from a cellulosic biomass, A. niger could be a good candidate for enzymes resource of cellulase to produce reducing sugars from a cellulosic biomass. This study also facilitates the development of highly efficient enzyme cocktails for the bioconversion of lignocellulosic biomass into monosaccharides and oligosaccharides.

Binding and Movement of Individual Cel7A Cellobiohydrolases on Crystalline Cellulose Surfaces Revealed by Single-molecule Fluorescence Imaging*

PubMed Central

Jung, Jaemyeong; Sethi, Anurag; Gaiotto, Tiziano; Han, Jason J.; Jeoh, Tina; Gnanakaran, Sandrasegaram; Goodwin, Peter M.

2013-01-01

The efficient catalytic conversion of biomass to bioenergy would meet a large portion of energy requirements in the near future. A crucial step in this process is the enzyme-catalyzed hydrolysis of cellulose to glucose that is then converted into fuel such as ethanol by fermentation. Here we use single-molecule fluorescence imaging to directly monitor the movement of individual Cel7A cellobiohydrolases from Trichoderma reesei (TrCel7A) on the surface of insoluble cellulose fibrils to elucidate molecular level details of cellulase activity. The motion of multiple, individual TrCel7A cellobiohydrolases was simultaneously recorded with ∼15-nm spatial resolution. Time-resolved localization microscopy provides insights on the activity of TrCel7A on cellulose and informs on nonproductive binding and diffusion. We measured single-molecule residency time distributions of TrCel7A bound to cellulose both in the presence of and absence of cellobiose the major product and a potent inhibitor of Cel7A activity. Combining these results with a kinetic model of TrCel7A binding provides microscopic insight into interactions between TrCel7A and the cellulose substrate. PMID:23818525

Stomatal Complex Development and F-Actin Organization in Maize Leaf Epidermis Depend on Cellulose Synthesis.

PubMed

Panteris, Emmanuel; Achlati, Theonymphi; Daras, Gerasimos; Rigas, Stamatis

2018-06-06

Cellulose microfibrils reinforce the cell wall for morphogenesis in plants. Herein, we provide evidence on a series of defects regarding stomatal complex development and F-actin organization in Zea mays leaf epidermis, due to inhibition of cellulose synthesis. Formative cell divisions of stomatal complex ontogenesis were delayed or inhibited, resulting in lack of subsidiary cells and frequently in unicellular stomata, with an atypical stomatal pore. Guard cells failed to acquire a dumbbell shape, becoming rounded, while subsidiary cells, whenever present, exhibited aberrant morphogenesis. F-actin organization was also affected, since the stomatal complex-specific arrays were scarcely observed. At late developmental stages, the overall F-actin network was diminished in all epidermal cells, although thick actin bundles persisted. Taken together, stomatal complex development strongly depends on cell wall mechanical properties. Moreover, F-actin organization exhibits a tight relationship with the cell wall.

Bio-waste corn-cob cellulose supported poly(hydroxamic acid) copper complex for Huisgen reaction: Waste to wealth approach.

PubMed

Mandal, Bablu Hira; Rahman, Md Lutfor; Yusoff, Mashitah Mohd; Chong, Kwok Feng; Sarkar, Shaheen M

2017-01-20

Corn-cob cellulose supported poly(hydroxamic acid) Cu(II) complex was prepared by the surface modification of waste corn-cob cellulose through graft copolymerization and subsequent hydroximation. The complex was characterized by IR, UV, FESEM, TEM, XPS, EDX and ICP-AES analyses. The complex has been found to be an efficient catalyst for 1,3-dipolar Huisgen cycloaddition (CuAAC) of aryl/alkyl azides with a variety of alkynes as well as one-pot three-components reaction in the presence of sodium ascorbate to give the corresponding cycloaddition products in up to 96% yield and high turn over number (TON 18,600) and turn over frequency (TOF 930h -1 ) were achieved. The complex was easy to recover from the reaction mixture and reused six times without significant loss of its catalytic activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Application of Complex Fluids in Lignocellulose Processing

NASA Astrophysics Data System (ADS)

Carrillo Lugo, Carlos A.

Complex fluids such as emulsions, microemulsions and foams, have been used for different applications due to the multiplicity of properties they possess. In the present work, such fluids are introduced as effective media for processing lignocellulosic biomass. A demonstration of the generic benefits of complex fluids is presented to enhance biomass impregnation, to facilitate pretreatment for fiber deconstruction and to make compatible cellulose fibrils with hydrophobic polymers during composite manufacture. An improved impregnation of woody biomass was accomplished by application of water-continuous microemulsions. Microemulsions with high water content, > 85%, were formulated and wood samples were impregnated by wicking and capillary flooding at atmospheric pressure and temperature. Formulations were designed to effectively impregnate different wood species during shorter times and to a larger extent compared to the single components of the microemulsions (water, oil or surfactant solutions). The viscosity of the microemulsions and their interactions with cell wall constituents in fibers were critical to define the extent of impregnation and solubilization. The relation between composition and formulation variables and the extent of microemulsion penetration in different woody substrates was studied. Formulation variables such as salinity content of the aqueous phase and type of surfactant were elucidated. Likewise, composition variables such as the water-to-oil ratio and surfactant concentration were investigated. These variables affected the characteristics of the microemulsion and determined their effectiveness in wood treatment. Also, the interactions between the surfactant and the substrate had an important contribution in defining microemulsion penetration in the capillary structure of wood. Microemulsions as an alternative pretreatment for the manufacture of cellulose nanofibrils (CNFs) was also studied. Microemulsions were applied to pretreat lignin-free and lignin-containing fibers obtained from various processes. Incorporation of active agents in the microemulsion facilitated fiber pretreatment before deconstruction via grinding and microfluidization. The energy consumed during the manufacture of cellulose nanofibrils was reduced by up to 55 and 32% in the case of lignin-containing and lignin-free fibers. Moreover, such pre-treatment did not affect negatively the mechanical properties of films prepared with the produced CNF. CNF was also used to enhance the stability of normal and multiple emulsions of the water-in-oil-in-water (W/O/W) type and to prevent their creaming. This was achieved by the marked increase in viscosity of the aqueous phase in the presence CNF. Finally, water-continuous emulsions were used to prepare nanocomposite fibers containing polystyrene and CNF. The morphology of composite fibers obtained after electrospinning of emulsions incorporating polystyrene and CNF was affected by parameters such the concentration of surfactant additives present in the microemulsion and the conductivity of the aqueous phase. Overall, emulsions and microemulsions are presented as a convenient platform to improve the compatibility between polymers of different hydrophilicity, to facilitate their processing and integration in composites.

Catalytic conversion of nonfood woody biomass solids to organic liquids.

PubMed

Barta, Katalin; Ford, Peter C

2014-05-20

This Account outlines recent efforts in our laboratories addressing a fundamental challenge of sustainability chemistry, the effective utilization of biomass for production of chemicals and fuels. Efficient methods for converting renewable biomass solids to chemicals and liquid fuels would reduce society's dependence on nonrenewable petroleum resources while easing the atmospheric carbon dioxide burden. The major nonfood component of biomass is lignocellulose, a matrix of the biopolymers cellulose, hemicellulose, and lignin. New approaches are needed to effect facile conversion of lignocellulose solids to liquid fuels and to other chemical precursors without the formation of intractable side products and with sufficient specificity to give economically sustainable product streams. We have devised a novel catalytic system whereby the renewable feedstocks cellulose, organosolv lignin, and even lignocellulose composites such as sawdust are transformed into organic liquids. The reaction medium is supercritical methanol (sc-MeOH), while the catalyst is a copper-doped porous metal oxide (PMO) prepared from inexpensive, Earth-abundant starting materials. This transformation occurs in a single stage reactor operating at 300-320 °C and 160-220 bar. The reducing equivalents for these transformations are derived by the reforming of MeOH (to H2 and CO), which thereby serves as a "liquid syngas" in the present case. Water generated by deoxygenation processes is quickly removed by the water-gas shift reaction. The Cu-doped PMO serves multiple purposes, catalyzing substrate hydrogenolysis and hydrogenation as well as the methanol reforming and shift reactions. This one-pot "UCSB process" is quantitative, giving little or no biochar residual. Provided is an overview of these catalysis studies beginning with reactions of the model compound dihydrobenzofuran that help define the key processes occurring. The initial step is phenyl-ether bond hydrogenolysis, and this is followed by aromatic ring hydrogenation. The complete catalytic disassembly of the more complex organosolv lignin to monomeric units, largely propyl-cyclohexanol derivatives is then described. Operational indices based on (1)H NMR analysis are also presented that facilitate holistic evaluation of these product streams that within several hours consist largely of propyl-cyclohexanol derivatives. Lastly, we describe the application of this methodology with several types of wood (pine sawdust, etc.) and with cellulose fibers. The product distribution, albeit still complex, displays unprecedented selectivity toward the production of aliphatic alcohols and methylated derivatives thereof. These observations clearly indicate that the Cu-doped solid metal oxide catalyst combined with sc-MeOH is capable of breaking down the complex biomass derived substrates to markedly deoxygenated monomeric units with increased hydrogen content. Possible implementations of this promising system on a larger scale are discussed.

Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation.

PubMed

Watanabe, Yoichiro; Schneider, Rene; Barkwill, Sarah; Gonzales-Vigil, Eliana; Hill, Joseph L; Samuels, A Lacey; Persson, Staffan; Mansfield, Shawn D

2018-06-05

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs. Copyright © 2018 the Author(s). Published by PNAS.

Enzymatic conversion of pretreated biomass into fermentable sugars for biorefinery operation

NASA Astrophysics Data System (ADS)

Gao, Dahai

2011-12-01

Depleting petroleum reserves and potential climate change caused by fossil fuel consumption have attracted significant attention towards the use of alternative renewable resources for production of fuels and chemicals. Lignocellulosic biomass provides a plentiful resource for the sustainable production of biofuels and biochemicals and could serve as an important contributor to the world energy portfolio in the near future. Successful biological conversion of lignocellulosic biomass requires an efficient and economical pretreatment method, high glucose/xylose yields during enzymatic hydrolysis and fermentation of both hexose and pentose to ethanol. High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. Core glycosyl hydrolases were isolated and purified from various sources to help rationally optimize an enzyme cocktail to digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were endoglucanase I (EG I), cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and beta-Glucosidase (betaG). The two core hemicellulases were an endoxylanase (EX) and a beta-xylosidase (betaX). A diverse set of accessory hemicellulases from bacterial sources was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (˜20 mg protein/g glucan) using an in-house developed enzyme cocktail and this cocktail was compared to commercial enzyme. Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput Fast Protein Liquid Chromatography (HT-FPLC) based method has been developed to quantify CBH I, CBH II and EG I present in hydrolyzates of untreated, AFEX, and dilute-acid pretreated corn stover. This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall derived components. The binding characteristics of CBH I, CBH II and EG I during 48 hours hydrolysis were studied on different cellulose allomorphs: microcrystalline cellulose Avicel (cellulose Ibeta), liquid ammonia treated cellulose (cellulose III), sodium hydroxide treated cellulose (cellulose II) and phosphoric acid swollen amorphous cellulose (AC). The digestibility ranking is AC>cellulose III>cellulose II>cellulose I. However, AC has the highest initial enzyme binding capacity while cellulose III had the lowest. CBH II is less stable during hydrolysis. Time course binding studies were also performed for pretreated biomass. Ammonia Fiber Expansion (AFEX) treated corn stover (CS), dilute acid (ACID) treated CS and ionic liquid (IL) pretreated CS were compared. The results indicate that presence of lignin is responsible for significant unproductive cellulase binding. These results are critical for improving our understanding of enzyme synergism, productive/unproductive enzyme binding and the role of pretreatment on enzyme accessibility to lignocellulosic plant cell walls. The results also assist in engineering novel low unproductive binding enzyme systems and developing economic enzyme recycle options.

Enhanced Polysaccharide Binding and Activity on Linear β-Glucans through Addition of Carbohydrate-Binding Modules to Either Terminus of a Glucooligosaccharide Oxidase

PubMed Central

Foumani, Maryam; Vuong, Thu V.; MacCormick, Benjamin; Master, Emma R.

2015-01-01

The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, β-(1→3,1→4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30 % and on insoluble crystalline as well as amorphous cellulose by over 50 %. PMID:25932926

Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex

PubMed Central

Chen, Yalei; Deffenbaugh, Nathan C.; Anderson, Charles T.; Hancock, William O.

2014-01-01

The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells using variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data. The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle. These procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry. PMID:25232006

Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex

DOE PAGES

Chen, Yalei; Deffenbaugh, Nathan C.; Anderson, Charles T.; ...

2014-09-17

The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells usingmore » variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data. The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle. In conclusion, these procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry.« less

Antibacterial activity of silver nanoparticles synthesized In-situ by solution spraying onto cellulose.

PubMed

Yan, Jinhua; Abdelgawad, Abdelrahman M; El-Naggar, Mehrez E; Rojas, Orlando J

2016-08-20

Spray technique was used for the adsorption of in-situ silver nanoparticles (AgNPs) onto and inside the surface of nano- and micro- fibrillar cellulose (NFC and MFC) as well as filter paper. The abundance of hydroxyl and carboxyl groups located in NFC and MFC are used to stabilize Ag ions (Ag(+)) which were then in-situ reduced to (AgNPs) by chemical or UV reduction. The surface characteristic features, elemental analysis, particle size as well as size distribution of the obtained MFC, NFC and filter paper loaded with AgNPs were characterized via field emission scanning electron microscopy connected to energy dispersive X-ray spectroscopy (FESEM- EDX) and transmission electron microscopy (TEM). The associated chemical changes after growth of AgNPs onto the cellulose substrates were assessed by fourier transform infra-red (FT-IR) while the thermal stability of such systems were investigated by thermogravimetrical analyses (TGA). The antibacterial properties of AgNPs loaded NFC, MFC and filter paper as well was investigated against Escherichia Coli. The resulted data indicate that the particle size was found to be 11 and 26nm for AgNPs nucleated on NFC and MFC-based papers respectively. The antibacterial activity of AgNPs loaded MFC exhibited higher antibacterial activity than that of AgNPs loaded NFC. Overall, the present research demonstrates facile and fast method for in-situ antibacterial AgNPs loading on cellulose substrates. Copyright © 2016 Elsevier Ltd. All rights reserved.

A comparative study of graphene and graphite-based field effect transistor on flexible substrate

NASA Astrophysics Data System (ADS)

Bhatt, Kapil; Rani, Cheenu; Vaid, Monika; Kapoor, Ankit; Kumar, Pramod; Kumar, Sandeep; Shriwastawa, Shilpi; Sharma, Sandeep; Singh, Randhir; Tripathi, C. C.

2018-06-01

In the present era, there has been a great demand of cost-effective, biodegradable, flexible and wearable electronics which may open the gate to many applications like flexible displays, RFID tags, health monitoring devices, etc. Due to the versatile nature of plastic substrates, they have been extensively used in packaging, printing, etc. However, the fabrication of electronic devices requires specially prepared substrates with high quality surfaces, chemical compositions and solutions to the related fabrication issues along with its non-biodegradable nature. Therefore, in this report, a cost-effective, biodegradable cellulose paper as an alternative dielectric substrate material for the fabrication of flexible field effect transistor (FET) is presented. The graphite and liquid phase exfoliated graphene have been used as the material for the realisation of source, drain and channel on cellulose paper substrate for its comparative analysis. The mobility of fabricated FETs was calculated to be 83 cm2/V s (holes) and 33 cm2/V s (electrons) for graphite FET and 100 cm2/V s (holes) and 52 cm2/V s (electrons) for graphene FET, respectively. The output characteristic of the device demonstrates the linear behaviour and a comprehensive increase in conductance as a function of gate voltages. The fabricated FETs may be used for strain sensing, health care monitoring devices, human motion detection, etc.

Surface functional group dependent apatite formation on bacterial cellulose microfibrils network in a simulated body fluid.

PubMed

Nge, Thi Thi; Sugiyama, Junji

2007-04-01

The apatite forming ability of biopolymer bacterial cellulose (BC) has been investigated by soaking different BC specimens in a simulated body fluid (1.5 SBF) under physiological conditions, at 37 degrees C and pH 7.4, mimicking the natural process of apatite formation. From ATR-FTIR spectra and ICP-AES analysis, the crystalline phase nucleated on the BC microfibrils surface was calcium deficient carbonated apatite through initial formation of octacalcium phosphate (OCP) or OCP like calcium phosphate phase regardless of the substrates. Morphology of the deposits from SEM, FE-SEM, and TEM observations revealed the fine structure of thin film plates uniting together to form apatite globules of various size (from <1 mum to 3 mum) with respect to the substrates. Surface modification by TEMPO (2,2,6,6-tetramethylpyperidine-1-oxyl)-mediated oxidation, which can readily form active carboxyl functional groups upon selective oxidation of primary hydroxyl groups on the surface of BC microfibrils, enhanced the rate of apatite nucleation. Ion exchanged treatment with calcium chloride solution after TEMPO-mediated oxidation was found to be remarkably different from other BC substrates with the highest deposit weight and the smallest apatite globules size. The role of BC substrates to induce mineralization rate differs according to the nature of the BC substrates, which strongly influences the growth behavior of the apatite crystals. (c) 2006 Wiley Periodicals, Inc.

Production of bioethanol using agricultural waste: Banana pseudo stem

PubMed Central

Ingale, Snehal; Joshi, Sanket J.; Gupte, Akshaya

2014-01-01

India is amongst the largest banana (Musa acuminata) producing countries and thus banana pseudo stem is commonly available agricultural waste to be used as lignocellulosic substrate. Present study focuses on exploitation of banana pseudo stem as a source for bioethanol production from the sugars released due to different chemical and biological pretreatments. Two fungal strains Aspergillus ellipticus and Aspergillus fumigatus reported to be producing cellulolytic enzymes on sugarcane bagasse were used under co-culture fermentation on banana pseudo stem to degrade holocellulose and facilitate maximum release of reducing sugars. The hydrolysate obtained after alkali and microbial treatments was fermented by Saccharomyces cerevisiae NCIM 3570 to produce ethanol. Fermentation of cellulosic hydrolysate (4.1 g%) gave maximum ethanol (17.1 g/L) with yield (84%) and productivity (0.024 g%/h) after 72 h. Some critical aspects of fungal pretreatment for saccharification of cellulosic substrate using A. ellipticus and A. fumigatus for ethanol production by S. cerevisiae NCIM 3570 have been explored in this study. It was observed that pretreated banana pseudo stem can be economically utilized as a cheaper substrate for ethanol production. PMID:25477922

Electrospun Nanofibers Made of Silver Nanoparticles, Cellulose Nanocrystals, and Polyacrylonitrile as Substrates for Surface-Enhanced Raman Scattering

PubMed Central

Ren, Suxia; Dong, Lili; Zhang, Xiuqiang; Lei, Tingzhou; Ehrenhauser, Franz; Song, Kunlin; Li, Meichun; Sun, Xiuxuan; Wu, Qinglin

2017-01-01

Nanofibers with excellent activities in surface-enhanced Raman scattering (SERS) were developed through electrospinning precursor suspensions consisting of polyacrylonitrile (PAN), silver nanoparticles (AgNPs), silicon nanoparticles (SiNPs), and cellulose nanocrystals (CNCs). Rheology of the precursor suspensions, and morphology, thermal properties, chemical structures, and SERS sensitivity of the nanofibers were investigated. The electrospun nanofibers showed uniform diameters with a smooth surface. Hydrofluoric (HF) acid treatment of the PAN/CNC/Ag composite nanofibers (defined as p-PAN/CNC/Ag) led to rougher fiber surfaces with certain pores and increased mean fiber diameters. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) results confirmed the existence of AgNPs that were formed during heat and HF acid treatment processes. In addition, thermal stability of the electrospun nanofibers increased due to the incorporation of CNCs and AgNPs. The p-PAN/CNC/Ag nanofibers were used as a SERS substrate to detect p-aminothiophenol (p-ATP) probe molecule. The results show that this substrate exhibited high sensitivity for the p-ATP probe detection. PMID:28772428

Pretreatment of spent mushroom substrate for enhancing the conversion of fermentable sugar.

PubMed

Wu, Songqing; Lan, Yanjiao; Wu, Zhimao; Peng, Yan; Chen, Siqi; Huang, Zhipeng; Xu, Lei; Gelbič, Ivan; Guan, Xiong; Zhang, Lingling; Zou, Shuangquan

2013-11-01

To develop a cost-effective biopesticide, spent mushroom substrate (SMS) extract was studied as a potential carbon source for cultivating Bacillus thuringiensis (Bt). Several pretreatments were compared to determine the optimal method for degrading cellulose to produce reducing sugars, including dilute sulfuric acid (0.5-2.0% v/v, 50-121°C, 1h), sodium hydroxide (0.5-2% w/v, 50-121°C, 1h), calcium hydroxide (0.2-4% w/v, 50-121°C, 1h), and hot water (50-121°C, 1h). Pretreatment was followed by standard enzymatic hydrolysis and fermentation. Results showed that the highest cellulose degradation was obtained using 2% dilute sulfuric acid pretreatment at 121°C for 1h, resulting in a high yield of reducing sugar (284.24 g/kg SMS). Sporulation was also highest using the same pretreatment. Use of SMS is not only an alternative way to commercialize Bt-based biopesticide, but also a potential solution for the environmental pollution associated with accumulation of the spent substrate of the mushroom industry. Copyright © 2013 Elsevier Ltd. All rights reserved.

Advanced Cloning Tools for Construction of Designer Cellulosomes.

PubMed

Kahn, Amaranta; Bayer, Edward A; Moraïs, Sarah

2018-01-01

Cellulose deconstruction is achieved in nature through two main enzymatic paradigms, i.e., free enzymes and enzymatic complexes (called cellulosomes). Gaining insights into the mechanism of action and synergy among the different cellulases is of high interest, notably in the field of renewable energy, and specifically, for the conversion of cellulosic biomass to soluble sugars, en route to biofuels. In this context, designer cellulosomes are artificially assembled, chimaeric protein complexes that are used as a tool to comparatively study cellulose degradation by different enzymatic paradigms, and could also serve to improve cellulose deconstruction. Various molecular biology techniques are employed in order to design and engineer the various components of designer cellulosomes. In this chapter, we describe the cloning processes through which the appropriate modules are selected and assembled at the molecular level.

Interactions between Cellulolytic Enzymes with Native, Autohydrolysis, and Technical Lignins and the Effect of a Polysorbate Amphiphile in Reducing Nonproductive Binding.

PubMed

Fritz, Consuelo; Ferrer, Ana; Salas, Carlos; Jameel, Hasan; Rojas, Orlando J

2015-12-14

Understanding enzyme-substrate interactions is critical in designing strategies for bioconversion of lignocellulosic biomass. In this study we monitored molecular events, in situ and in real time, including the adsorption and desorption of cellulolytic enzymes on lignins and cellulose, by using quartz crystal microgravimetry and surface plasmon resonance. The effect of a nonionic surface active molecule was also elucidated. Three lignin substrates relevant to the sugar platform in biorefinery efforts were considered, namely, hardwood autohydrolysis cellulolytic (HWAH), hardwood native cellulolytic (MPCEL), and nonwood native cellulolytic (WSCEL) lignin. In addition, Kraft lignins derived from softwoods (SWK) and hardwoods (HWK) were used as references. The results indicated a high affinity between the lignins with both, monocomponent and multicomponent enzymes. More importantly, the addition of nonionic surfactants at concentrations above their critical micelle concentration reduced remarkably (by over 90%) the nonproductive interactions between the cellulolytic enzymes and the lignins. This effect was hypothesized to be a consequence of the balance of hydrophobic and hydrogen bonding interactions. Moreover, the reduction of surface roughness and increased wettability of lignin surfaces upon surfactant treatment contributed to a lower affinity with the enzymes. Conformational changes of cellulases were observed upon their adsorption on lignin carrying preadsorbed surfactant. Weak electrostatic interactions were determined in aqueous media at pH between 4.8 and 5.5 for the native cellulolytic lignins (MPCEL and WSCEL), whereby a ∼20% reduction in the enzyme affinity was observed. This was mainly explained by electrostatic interactions (osmotic pressure effects) between charged lignins and cellulases. Noteworthy, adsorption of nonionic surfactants onto cellulose, in the form cellulose nanofibrils, did not affect its hydrolytic conversion. Overall, our results highlight the benefit of nonionic surfactant pretreatment to reduce nonproductive enzyme binding while maintaining the reactivity of the cellulosic substrate.

Effect of bioaugmentation and biostimulation on sulfate-reducing column startup captured by functional gene profiling.

PubMed

Pereyra, Luciana P; Hiibel, Sage R; Perrault, Elizabeth M; Reardon, Kenneth F; Pruden, Amy

2012-10-01

Sulfate-reducing permeable reactive zones (SR-PRZs) depend upon a complex microbial community to utilize a lignocellulosic substrate and produce sulfides, which remediate mine drainage by binding heavy metals. To gain insight into the impact of the microbial community composition on the startup time and pseudo-steady-state performance, functional genes corresponding to cellulose-degrading (CD), fermentative, sulfate-reducing, and methanogenic microorganisms were characterized in columns simulating SR-PRZs using quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE). Duplicate columns were bioaugmented with sulfate-reducing or CD bacteria or biostimulated with ethanol or carboxymethyl cellulose and compared with baseline dairy manure inoculum and uninoculated controls. Sulfate removal began after ~ 15 days for all columns and pseudo-steady state was achieved by Day 30. Despite similar performance, DGGE profiles of 16S rRNA gene and functional genes at pseudo-steady state were distinct among the column treatments, suggesting the potential to control ultimate microbial community composition via bioaugmentation and biostimulation. qPCR revealed enrichment of functional genes in all columns between the initial and pseudo-steady-state time points. This is the first functional gene-based study of CD, fermentative and sulfate-reducing bacteria and methanogenic archaea in a lignocellulose-based environment and provides new qualitative and quantitative insight into startup of a complex microbial system. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Dramatic performance of Clostridium thermocellum explained by its wide range of cellulase modalities.

PubMed

Xu, Qi; Resch, Michael G; Podkaminer, Kara; Yang, Shihui; Baker, John O; Donohoe, Bryon S; Wilson, Charlotte; Klingeman, Dawn M; Olson, Daniel G; Decker, Stephen R; Giannone, Richard J; Hettich, Robert L; Brown, Steven D; Lynd, Lee R; Bayer, Edward A; Himmel, Michael E; Bomble, Yannick J

2016-02-01

Clostridium thermocellum is the most efficient microorganism for solubilizing lignocellulosic biomass known to date. Its high cellulose digestion capability is attributed to efficient cellulases consisting of both a free-enzyme system and a tethered cellulosomal system wherein carbohydrate active enzymes (CAZymes) are organized by primary and secondary scaffoldin proteins to generate large protein complexes attached to the bacterial cell wall. This study demonstrates that C. thermocellum also uses a type of cellulosomal system not bound to the bacterial cell wall, called the "cell-free" cellulosomal system. The cell-free cellulosome complex can be seen as a "long range cellulosome" because it can diffuse away from the cell and degrade polysaccharide substrates remotely from the bacterial cell. The contribution of these two types of cellulosomal systems in C. thermocellum was elucidated by characterization of mutants with different combinations of scaffoldin gene deletions. The primary scaffoldin, CipA, was found to play the most important role in cellulose degradation by C. thermocellum, whereas the secondary scaffoldins have less important roles. Additionally, the distinct and efficient mode of action of the C. thermocellum exoproteome, wherein the cellulosomes splay or divide biomass particles, changes when either the primary or secondary scaffolds are removed, showing that the intact wild-type cellulosomal system is necessary for this essential mode of action. This new transcriptional and proteomic evidence shows that a functional primary scaffoldin plays a more important role compared to secondary scaffoldins in the proper regulation of CAZyme genes, cellodextrin transport, and other cellular functions.

Custom fabrication of biomass containment devices using 3-D printing enables bacterial growth analyses with complex insoluble substrates.

PubMed

Nelson, Cassandra E; Beri, Nina R; Gardner, Jeffrey G

2016-11-01

Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interaction between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. We applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present. Copyright © 2016 Elsevier B.V. All rights reserved.

Influence of steam pretreatment severity on post-treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings.

PubMed

Kumar, Linoj; Chandra, Richard; Saddler, Jack

2011-10-01

It is recognized that some form of post-treatment will usually be required if reasonable hydrolysis yields (>60%) of steam pretreated softwood are to be achieved when using low enzyme loadings (5 FPU/g cellulose). In the work reported here we modified/removed lignin from steam pretreated softwood while investigating the influence that the severity of pretreatment might have on the effectiveness of subsequent post-treatments. Although treatment at a lower severity could provide better overall hemicellulose recovery, post-treatment was not as effective on the cellulosic component. Pretreatment at medium severity resulted in the best compromise, providing reasonable recovery of the water soluble hemicellulose sugars and the use of post-treatment conditions that significantly increased the enzymatic hydrolysis of the water insoluble cellulosic component. Post-treatment with alkaline hydrogen peroxide or neutral sulfonation resulted in 62% cellulose hydrolysis at an enzyme loading of 5 FPU/g cellulose, which was four times greater than was obtained when the cellulosic fraction was not post-treated. When the enzyme loading was increased to 15 FPU/g cellulose, the post-treated cellulosic fraction was almost completely hydrolyzed to glucose. Despite the higher lignin content (44%) of the sulfonated substrate, similar hydrolysis yields to those achieved after alkaline peroxide post-treatment (14% lignin content) indicated that, in addition to lignin removal, lignin modification also plays an important role in influencing the effectiveness of hydrolysis when low enzyme loadings are used. Copyright © 2011 Wiley Periodicals, Inc.

Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mayer, R.; Ross, P.; Weinhouse, H.

1991-06-15

To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either ({sup 32}P)c-di-GMP or ({alpha}-{sup 32}P)UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- andmore » 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-k-Da peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. The authors suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.« less

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

PubMed Central

Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E.; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S.; Mortimer, Jenny C.; Brown, Steven P.; Persson, Staffan; Dupree, Paul

2016-01-01

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

Katherine J. Chou | NREL

Science.gov Websites

J. Chou Photo of Katherine J. Chou Katherine Chou Microbial Physiology & Engineering , Clostridium thermocellum, through metabolic engineering. "Biological Electron Transfer and Catalysis principles governing substrate utilization. "Advance Biofuels from Cellulose via Genetic Engineering of

Role of Cellulose Nanocrystals on the Microstructure of Maleic Anhydride Plasma Polymer Thin Films.

PubMed

Brioude, Michel M; Roucoules, Vincent; Haidara, Hamidou; Vonna, Laurent; Laborie, Marie-Pierre

2015-07-01

Recently, it was shown that the microstructure of a maleic anhydride plasma polymer (MAPP) could be tailored ab initio by adjusting the plasma process parameters. In this work, we aim to investigate the ability of cellulose nanocrystals (CNCs) to induce topographical structuration. Thus, a new approach was designed based on the deposition of MAPP on CNCs model surfaces. The nanocellulosic surfaces were produced by spin-coating the CNC suspension on a silicon wafer substrate and on a hydrophobic silicon wafer substrate patterned with circular hydrophilic microsized domains (diameter of 86.9 ± 4.9 μm), resulting in different degrees of CNC aggregation. By depositing the MAPP over these surfaces, it was possible to observe that the surface fraction of nanostructures increased from 20% to 35%. This observation suggests that CNCs can act as nucleation points resulting in more structures, although a critical density of the CNCs is required.

Recent Progress in Fabrication and Applications of Superhydrophobic Coating on Cellulose-Based Substrates

PubMed Central

Liu, Hui; Gao, Shou-Wei; Cai, Jing-Sheng; He, Cheng-Lin; Mao, Jia-Jun; Zhu, Tian-Xue; Chen, Zhong; Huang, Jian-Ying; Meng, Kai; Zhang, Ke-Qin; Al-Deyab, Salem S.; Lai, Yue-Kun

2016-01-01

Multifuntional fabrics with special wettability have attracted a lot of interest in both fundamental research and industry applications over the last two decades. In this review, recent progress of various kinds of approaches and strategies to construct super-antiwetting coating on cellulose-based substrates (fabrics and paper) has been discussed in detail. We focus on the significant applications related to artificial superhydrophobic fabrics with special wettability and controllable adhesion, e.g., oil-water separation, self-cleaning, asymmetric/anisotropic wetting for microfluidic manipulation, air/liquid directional gating, and micro-template for patterning. In addition to the anti-wetting properties and promising applications, particular attention is paid to coating durability and other incorporated functionalities, e.g., air permeability, UV-shielding, photocatalytic self-cleaning, self-healing and patterned antiwetting properties. Finally, the existing difficulties and future prospects of this traditional and developing field are briefly proposed and discussed. PMID:28773253

Porous cellulose as promoter of oil production by the oleaginous yeast Lipomyces starkeyi using mixed agroindustrial wastes.

PubMed

Ganatsios, Vassilios; Koutinas, Athanasios A; Bekatorou, Argyro; Panagopoulos, Vassilios; Banat, Ibrahim M; Terpou, Antonia; Kopsahelis, Nikolaos

2017-11-01

Enhanced single cell oil (SCO) production by the oleaginous yeast Lipomyces starkeyi DSM 70296, immobilised on delignified porous cellulose, is reported. Pure glucose media were initially used. The effects of substrate pH and treatment temperature were evaluated, showing that 30°C and pH 5.0 were the optimum conditions for SCO production by the immobilised yeast. The immobilisation technique led to increased lipid accumulation and cell growth by 44% and 8%, respectively, in the glucose media, compared to free cells in suspension. This positive effect was also shown when low concentration mixed agro-industrial waste suspensions were used as substrates, leading to 85% enhanced SCO production in comparison with free cells. Higher fatty acid (HFA) analysis showed that yeast immobilisation led to increased formation of unsaturated HFAs (6%) and reduced saturated HFAs (5%) compared to free cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Elucidating the Structural Changes to Populus Lignin during Consolidated Bioprocessing with Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akinosho, Hannah O.; Yoo, Chang Geun; Dumitrache, Alexandru

During consolidated bioprocessing (CBP), Clostridium thermocellum hydrolyzes several plant cell wall components. Cellulose hydrolysis, specifically, liberates sugars for fermentation, which generates ethanol, acetate, hydrogen, and other products. While several studies indicate that C. thermocellum hydrolyzes carbohydrates in biomass, the structural changes to lignin during CBP remain unclear. In this paper, the whole plant cell walls of untreated and C. thermocellum-treated Populus trichocarpa were characterized using NMR and FTIR. The results suggest that C. thermocellum reduces the β-O-4 linkage content and increases the lignin S/G ratio. Finally, this investigation indicates that C. thermocellum not only modifies lignin in order to accessmore » cellulose but also leaves behind a suitable lignin substrate for value-added applications in the cellulosic ethanol production scheme.« less

Elucidating the Structural Changes to Populus Lignin during Consolidated Bioprocessing with Clostridium thermocellum

DOE PAGES

Akinosho, Hannah O.; Yoo, Chang Geun; Dumitrache, Alexandru; ...

2017-07-20

During consolidated bioprocessing (CBP), Clostridium thermocellum hydrolyzes several plant cell wall components. Cellulose hydrolysis, specifically, liberates sugars for fermentation, which generates ethanol, acetate, hydrogen, and other products. While several studies indicate that C. thermocellum hydrolyzes carbohydrates in biomass, the structural changes to lignin during CBP remain unclear. In this paper, the whole plant cell walls of untreated and C. thermocellum-treated Populus trichocarpa were characterized using NMR and FTIR. The results suggest that C. thermocellum reduces the β-O-4 linkage content and increases the lignin S/G ratio. Finally, this investigation indicates that C. thermocellum not only modifies lignin in order to accessmore » cellulose but also leaves behind a suitable lignin substrate for value-added applications in the cellulosic ethanol production scheme.« less

Controlled release of chlorhexidine digluconate using β-cyclodextrin and microfibrillated cellulose.

PubMed

Lavoine, Nathalie; Tabary, Nicolas; Desloges, Isabelle; Martel, Bernard; Bras, Julien

2014-09-01

This study aims to develop a high-performance delivery system using microfibrillated cellulose (MFC)-coated papers as a controlled release system combined with the well-known drug delivery agent, β-cyclodextrin (βCD). Chlorhexidine digluconate (CHX), an antibacterial molecule, was mixed with a suspension of MFC or a βCD solution or mixed with both the substances, before coating onto a cellulosic substrate. The intermittent diffusion of CHX (i.e., diffusion interrupted by the renewal of the release medium periodically) was conducted in an aqueous medium, and the release mechanism of CHX was elucidated by field emission gun-scanning electron microscopy, SEM, NMR, and Fourier transform infrared analyses. According to the literature, both βCD and MFC are efficient controlled delivery systems. This study indicated that βCD releases CHX more gradually and over a longer period of time compared to MFC, which is mainly due to the ability of βCD to form an inclusion complex with CHX. Furthermore from the release study, a complementary action when the two compounds were combined was deduced. MFC mainly affected the burst effect, while βCD primarily controlled the amount of CHX released over time. In this paper, two different types of controlled release systems are proposed and compared. Depending on the final application, the use of βCD alone would release low amounts of active molecules over time (slow delivery), whereas the combination of β-cyclodextrin and MFC would be more suitable for the release of higher amounts of active molecules over time (rapid delivery). Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of cellulolytic microbial consortium enriched on Napier grass using metagenomic approaches.

PubMed

Kanokratana, Pattanop; Wongwilaiwalin, Sarunyou; Mhuantong, Wuttichai; Tangphatsornruang, Sithichoke; Eurwilaichitr, Lily; Champreda, Verawat

2018-04-01

Energy grass is a promising substrate for production of biogas by anaerobic digestion. However, the conversion efficiency is limited by the enzymatically recalcitrant nature of cellulosic wastes. In this study, an active, structurally stable mesophilic lignocellulolytic degrading microbial consortium (Np-LMC) was constructed from forest compost soil microbiota by successive subcultivation on Napier grass under facultative anoxic conditions. According to tagged 16S rRNA gene amplicon sequencing, increasing abundance of facultative Proteobacteria was found in the middle of batch cycle which was then subsequently replaced by the cellulose degraders Firmicutes and Bacteroidetes along with decreasing CMCase, xylanase, and β-glucanase activity profiles in the supernatant after 5 days of incubation. Anaerobic/facultative bacteria Dysgonomonas and Sedimentibacter and aerobic bacteria Comamonas were the major genera found in Np-LMC. The consortium was active on degradation of the native and delignified grass. Direct shotgun sequencing of the consortium metagenome revealed relatively high abundance of genes encoding for various lignocellulose degrading enzymes in 23 glycosyl hydrolase (GH) families compared to previously reported cellulolytic microbial communities in mammalian digestive tracts. Enzymes attacking cellulose and hemicellulose were dominated by GH2, 3, 5, 9, 10, 26, 28 and 43 in addition to a variety of carbohydrate esterases (CE) and auxiliary activities (AA), reflecting adaptation of the enzyme systems to the native herbaceous substrate. The consortium identified here represents the microcosm specifically bred on energy grass, with potential for enhancing degradation of fibrous substrates in bioenergy industry. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Enhanced cellulase recovery without β-glucosidase supplementation for cellulosic ethanol production using an engineered strain and surfactant.

PubMed

Huang, Renliang; Guo, Hong; Su, Rongxin; Qi, Wei; He, Zhimin

2017-03-01

Recycling cellulases by substrate adsorption is a promising strategy for reducing the enzyme cost of cellulosic ethanol production. However, β-glucosidase has no carbohydrate-binding module (CBM). Thus, additional enzymes are required in each cycle to achieve a high ethanol yield. In this study, we report a new method of recycling cellulases without β-glucosidase supplementation using lignocellulosic substrate, an engineered strain expressing β-glucosidase and Tween 80. The cellulases and Tween 80 were added to an aqueous suspension of diluted sulfuric acid/ammonia-treated corncobs in a simultaneous saccharification and fermentation (SSF) process for ethanol production. Subsequently, the addition of fresh pretreated corncobs to the fermentation liquor and remaining solid residue provided substrates with absorbed cellulases for the next SSF cycle. This method provided excellent ethanol production in three successive SSF cycles without requiring the addition of new cellulases. For a 10% (w/v) solid loading, a cellulase dosage of 30 filter paper units (FPU)/g cellulose, 0.5% Tween 80, and 2 g/L of the engineered strain, approximately 90% of the initial ethanol concentration from the first SSF process was obtained in the next two SSF processes, with a total ethanol production of 306.27 g/kg corncobs and an enzyme productivity of 0.044 g/FPU. Tween 80 played an important role in enhancing cellulase recovery. This new enzyme recycling method is more efficient and practical than other reported methods. Biotechnol. Bioeng. 2017;114: 543-551. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Secretion of clostridium cellulase by E. coli

DOEpatents

Yu, Ida Kuo

1998-01-01

A gene, encoding an endocellulase from a newly isolated mesophilic Clostridium strain IY-2 which can digest bamboo fibers, cellulose, rice straw, and sawdust, was isolated by shotgun cloning in an E. coli expression plasmid pLC2833. E. coli positive clones were selected based on their ability to hydrolyze milled bamboo fibers and cellulose present in agar plates. One clone contained a 2.8 kb DNA fragment that was responsible for cellulase activity. Western blot analyses indicated that the positive clone produced a secreted cellulase with a mass of about 58,000 daltons that was identical in size to the subunit of one of the three major Clostridium cellulases. The products of cellulose digestion by this cloned cellulase were cellotetraose and soluble higher polymers. The cloned DNA contained signal sequences capable of directing the secretion of heterologous proteins from an E. coli host. The invention describes a bioprocess for the treatment of cellulosic plant materials to produce cellular growth substrates and fermentation end products suitable for production of liquid fuels, solvents, and acids.

Microtubules and cellulose microfibrils: how intimate is their relationship?

PubMed

Emons, Anne Mie C; Höfte, Herman; Mulder, Bela M

2007-07-01

The recent visualization of the motion of fluorescently labeled cellulose synthase complexes by Alexander Paredez and colleagues heralds the start of a new era in the science of the plant cell wall. Upon drug-induced complete depolymerization, the movement of the complexes does not become disordered but instead establishes an apparently self-organized novel pattern. The ability to label complexes in vivo has provided us with the ideal tool for tackling the intriguing question of the underlying default mechanisms at play.

Enzymatic digestibility and ethanol fermentability of AFEX-treated starch-rich lignocellulosics such as corn silage and whole corn plant

PubMed Central

2010-01-01

Background Corn grain is an important renewable source for bioethanol production in the USA. Corn ethanol is currently produced by steam liquefaction of starch-rich grains followed by enzymatic saccharification and fermentation. Corn stover (the non-grain parts of the plant) is a potential feedstock to produce cellulosic ethanol in second-generation biorefineries. At present, corn grain is harvested by removing the grain from the living plant while leaving the stover behind on the field. Alternatively, whole corn plants can be harvested to cohydrolyze both starch and cellulose after a suitable thermochemical pretreatment to produce fermentable monomeric sugars. In this study, we used physiologically immature corn silage (CS) and matured whole corn plants (WCP) as feedstocks to produce ethanol using ammonia fiber expansion (AFEX) pretreatment followed by enzymatic hydrolysis (at low enzyme loadings) and cofermentation (for both glucose and xylose) using a cellulase-amylase-based cocktail and a recombinant Saccharomyces cerevisiae 424A (LNH-ST) strain, respectively. The effect on hydrolysis yields of AFEX pretreatment conditions and a starch/cellulose-degrading enzyme addition sequence for both substrates was also studied. Results AFEX-pretreated starch-rich substrates (for example, corn grain, soluble starch) had a 1.5-3-fold higher enzymatic hydrolysis yield compared with the untreated substrates. Sequential addition of cellulases after hydrolysis of starch within WCP resulted in 15-20% higher hydrolysis yield compared with simultaneous addition of hydrolytic enzymes. AFEX-pretreated CS gave 70% glucan conversion after 72 h of hydrolysis for 6% glucan loading (at 8 mg total enzyme loading per gram glucan). Microbial inoculation of CS before ensilation yielded a 10-15% lower glucose hydrolysis yield for the pretreated substrate, due to loss in starch content. Ethanol fermentation of AFEX-treated (at 6% w/w glucan loading) CS hydrolyzate (resulting in 28 g/L ethanol at 93% metabolic yield) and WCP (resulting in 30 g/L ethanol at 89% metabolic yield) is reported in this work. Conclusions The current results indicate the feasibility of co-utilization of whole plants (that is, starchy grains plus cellulosic residues) using an ammonia-based (AFEX) pretreatment to increase bioethanol yield and reduce overall production cost. PMID:20534126

Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass

DOE PAGES

Poudel, Suresh; Giannone, Richard J.; Rodriguez, Jr., Miguel; ...

2017-01-10

Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Though much of the work-to-date has centered on characterizing the organism s metabolism during growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand it metabolizes more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol. The most striking feature of themore » metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum s cellular membrane as the culture progresses. This is likely a response to the gradual build-up of lignocellulose-derived inhibitory compounds detected as the organism deconstructs the switchgrass to access the embedded cellulose and includes 4-hydroxybenzoic acid, vanillic acid, ferulic acid, p-coumaric acid and vanillin. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in enzymes involved in the interconversion of branched amino acids valine, leucine and isoleucine to iso- and anteiso-fatty acid precursors. Furthermore, the metabolic accumulation of hemicellulose-derived sugars and sugar-alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism / the pentose phosphate pathway, indicate that C. thermocellum either shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux or is simply a response to C5 sugar metabolite pools that build during lignocellulose deconstruction.« less

Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poudel, Suresh; Giannone, Richard J.; Rodriguez, Jr., Miguel

Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Though much of the work-to-date has centered on characterizing the organism s metabolism during growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand it metabolizes more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol. The most striking feature of themore » metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum s cellular membrane as the culture progresses. This is likely a response to the gradual build-up of lignocellulose-derived inhibitory compounds detected as the organism deconstructs the switchgrass to access the embedded cellulose and includes 4-hydroxybenzoic acid, vanillic acid, ferulic acid, p-coumaric acid and vanillin. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in enzymes involved in the interconversion of branched amino acids valine, leucine and isoleucine to iso- and anteiso-fatty acid precursors. Furthermore, the metabolic accumulation of hemicellulose-derived sugars and sugar-alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism / the pentose phosphate pathway, indicate that C. thermocellum either shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux or is simply a response to C5 sugar metabolite pools that build during lignocellulose deconstruction.« less

Designing novel cellulase systems through agent-based modeling and global sensitivity analysis.

PubMed

Apte, Advait A; Senger, Ryan S; Fong, Stephen S

2014-01-01

Experimental techniques allow engineering of biological systems to modify functionality; however, there still remains a need to develop tools to prioritize targets for modification. In this study, agent-based modeling (ABM) was used to build stochastic models of complexed and non-complexed cellulose hydrolysis, including enzymatic mechanisms for endoglucanase, exoglucanase, and β-glucosidase activity. Modeling results were consistent with experimental observations of higher efficiency in complexed systems than non-complexed systems and established relationships between specific cellulolytic mechanisms and overall efficiency. Global sensitivity analysis (GSA) of model results identified key parameters for improving overall cellulose hydrolysis efficiency including: (1) the cellulase half-life, (2) the exoglucanase activity, and (3) the cellulase composition. Overall, the following parameters were found to significantly influence cellulose consumption in a consolidated bioprocess (CBP): (1) the glucose uptake rate of the culture, (2) the bacterial cell concentration, and (3) the nature of the cellulase enzyme system (complexed or non-complexed). Broadly, these results demonstrate the utility of combining modeling and sensitivity analysis to identify key parameters and/or targets for experimental improvement.

Designing novel cellulase systems through agent-based modeling and global sensitivity analysis

PubMed Central

Apte, Advait A; Senger, Ryan S; Fong, Stephen S

2014-01-01

Experimental techniques allow engineering of biological systems to modify functionality; however, there still remains a need to develop tools to prioritize targets for modification. In this study, agent-based modeling (ABM) was used to build stochastic models of complexed and non-complexed cellulose hydrolysis, including enzymatic mechanisms for endoglucanase, exoglucanase, and β-glucosidase activity. Modeling results were consistent with experimental observations of higher efficiency in complexed systems than non-complexed systems and established relationships between specific cellulolytic mechanisms and overall efficiency. Global sensitivity analysis (GSA) of model results identified key parameters for improving overall cellulose hydrolysis efficiency including: (1) the cellulase half-life, (2) the exoglucanase activity, and (3) the cellulase composition. Overall, the following parameters were found to significantly influence cellulose consumption in a consolidated bioprocess (CBP): (1) the glucose uptake rate of the culture, (2) the bacterial cell concentration, and (3) the nature of the cellulase enzyme system (complexed or non-complexed). Broadly, these results demonstrate the utility of combining modeling and sensitivity analysis to identify key parameters and/or targets for experimental improvement. PMID:24830736

Discrete and Structurally Unique Proteins (T$$\\bar{a}$$pirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

DOE PAGES

Blumer-Schuette, S. E.; Alahuhta, M.; Conway, J. M.; ...

2015-04-24

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tmore » $$\\bar{a}$$pirins,” origin from M$$\\bar{a}$$ori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two t$$\\bar{a}$$pirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, t$$\\bar{a}$$pirins are specific to these extreme thermophiles. T$$\\bar{a}$$pirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the t$$\\bar{a}$$pirins for cellulose. Crystallization of a cellulose-binding truncation from one t$$\\bar{a}$$pirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. In addition, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ t$$\\bar{a}$$pirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.« less

Discrete and Structurally Unique Proteins (T$$\\bar{a}$$pirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blumer-Schuette, S. E.; Alahuhta, M.; Conway, J. M.

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tmore » $$\\bar{a}$$pirins,” origin from M$$\\bar{a}$$ori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two t$$\\bar{a}$$pirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, t$$\\bar{a}$$pirins are specific to these extreme thermophiles. T$$\\bar{a}$$pirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the t$$\\bar{a}$$pirins for cellulose. Crystallization of a cellulose-binding truncation from one t$$\\bar{a}$$pirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. In addition, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ t$$\\bar{a}$$pirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.« less

Cellulose microfibrils in plants: biosynthesis, deposition, and integration into the cell wall.

PubMed

Brett, C T

2000-01-01

Cellulose occurs in all higher plants and some algae, fungi, bacteria, and animals. It forms microfibrils containing the crystalline allomorphs, cellulose I alpha and I beta. Cellulose molecules are 500-15,000 glucose units long. What controls molecular size is unknown. Microfibrils are elongated by particle rosettes in the plasma membrane (cellulose synthase complexes). The precursor, UDP-glucose, may be generated from sucrose at the site of synthesis. The biosynthetic mechanism may involve lipid-linked intermediates. Cellulose synthase has been purified from bacteria, but not from plants. In plants, disrupted cellulose synthase may form callose. Cellulose synthase genes have been isolated from bacteria and plants. Cellulose-deficient mutants have been characterised. The deduced amino acid sequence suggests possible catalytic mechanisms. It is not known whether synthesis occurs at the reducing or nonreducing end. Endoglucanase may play a role in synthesis. Nascent cellulose molecules associate by Van der Waals and hydrogen bonds to form microfibrils. Cortical microtubules control microfibril orientation, thus determining the direction of cell growth. Self-assembly mechanisms may operate. Microfibril integration into the wall occurs by interactions with matrix polymers during microfibril formation.

Proteomic and Functional Analysis of the Cellulase System Expressed by Postia placenta during Brown Rot of Solid Wood

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Jae San; Shary, Semarjit; Houtman, Carl J.

2011-11-01

Abstract Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed themore » presence of 34 likely glycoside hydrolases, but only four of these-two in glycoside hydrolase family 5, one in family 10, and one in family 12-have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component.« less

Disposable diapers biodegradation by the fungus Pleurotus ostreatus.

PubMed

Espinosa-Valdemar, Rosa María; Turpin-Marion, Sylvie; Delfín-Alcalá, Irma; Vázquez-Morillas, Alethia

2011-08-01

This research assesses the feasibility of degrading used disposable diapers, an important component (5-15% in weight) of urban solid waste in Mexico, by the activity of the fungus Pleurotus ostreatus, also known as oyster mushroom. Disposable diapers contain polyethylene, polypropylene and a super absorbent polymer. Nevertheless, its main component is cellulose, which degrades slowly. P. ostreatus has been utilized extensively to degrade cellulosic materials of agroindustrial sources, using in situ techniques. The practice has been extended to the commercial farming of the mushroom. This degradation capacity was assayed to reduce mass and volume of used disposable diapers. Pilot laboratory assays were performed to estimate the usefulness of the following variables on conditioning of used diapers before they act as substrate for P. ostreatus: (1) permanence vs removal of plastic cover; (2) shredding vs grinding; (3) addition of grape wastes to improve structure, nitrogen and trace elements content. Wheat straw was used as a positive control. After 68 days, decrease of the mass of diapers and productivity of fungus was measured. Weight and volume of degradable materials was reduced up to 90%. Cellulose content was diminished in 50% and lignine content in 47%. The highest efficiency for degradation of cellulosic materials corresponded to the substrates that showed highest biological efficiency, which varied from 0% to 34%. Harvested mushrooms had good appearance and protein content and were free of human disease pathogens. This research indicates that growing P. ostreatus on disposable diapers could be a good alternative for two current problems: reduction of urban solid waste and availability of high protein food sources. Copyright © 2011 Elsevier Ltd. All rights reserved.

Tree species diversity affects decomposition through modified micro-environmental conditions across European forests.

PubMed

Joly, François-Xavier; Milcu, Alexandru; Scherer-Lorenzen, Michael; Jean, Loreline-Katia; Bussotti, Filippo; Dawud, Seid Muhie; Müller, Sandra; Pollastrini, Martina; Raulund-Rasmussen, Karsten; Vesterdal, Lars; Hättenschwiler, Stephan

2017-05-01

Different tree species influence litter decomposition directly through species-specific litter traits, and indirectly through distinct modifications of the local decomposition environment. Whether these indirect effects on decomposition are influenced by tree species diversity is presently not clear. We addressed this question by studying the decomposition of two common substrates, cellulose paper and wood sticks, in a total of 209 forest stands of varying tree species diversity across six major forest types at the scale of Europe. Tree species richness showed a weak but positive correlation with the decomposition of cellulose but not with that of wood. Surprisingly, macroclimate had only a minor effect on cellulose decomposition and no effect on wood decomposition despite the wide range in climatic conditions among sites from Mediterranean to boreal forests. Instead, forest canopy density and stand-specific litter traits affected the decomposition of both substrates, with a particularly clear negative effect of the proportion of evergreen tree litter. Our study suggests that species richness and composition of tree canopies modify decomposition indirectly through changes in microenvironmental conditions. These canopy-induced differences in the local decomposition environment control decomposition to a greater extent than continental-scale differences in macroclimatic conditions. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Hydroxypropyl cellulose methacrylate as a photo-patternable and biodegradable hybrid paper substrate for cell culture and other bioapplications.

PubMed

Qi, Aisha; Hoo, Siew Pei; Friend, James; Yeo, Leslie; Yue, Zhilian; Chan, Peggy P Y

2014-04-01

In addition to the choice of appropriate material properties of the tissue construct to be used, such as its biocompatibility, biodegradability, cytocompatibility, and mechanical rigidity, the ability to incorporate microarchitectural patterns in the construct to mimic that found in the cellular microenvironment is an important consideration in tissue engineering and regenerative medicine. Both these issues are addressed by demonstrating a method for preparing biodegradable and photo-patternable constructs, where modified cellulose is cross-linked to form an insoluble structure in an aqueous environment. Specifically, hydroxypropyl cellulose (HPC) is rendered photocrosslinkable by grafting with methylacrylic anhydride, whose linkages also render the cross-linked construct hydrolytically degradable. The HPC is then cross-linked via a photolithography-based fabrication process. The feasibility of functionalizing these HPC structures with biochemical cues is verified post-fabrication, and shown to facilitate the adhesion of mesenchymal progenitor cells. The HPC constructs are shown to be biocompatible and hydrolytically degradable, thus enabling cell proliferation and cell migration, and therefore constituting an ideal candidate for long-term cell culture and implantable tissue scaffold applications. In addition, the potential of the HPC structure is demonstrated as an alternative substrate to paper microfluidic diagnostic devices for protein and cell assays. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multiple cellobiohydrolases and cellobiose phosphorylases cooperate in the ruminal bacterium Ruminococcus albus 8 to degrade cellooligosaccharides

NASA Astrophysics Data System (ADS)

Devendran, Saravanan; Abdel-Hamid, Ahmed M.; Evans, Anton F.; Iakiviak, Michael; Kwon, In Hyuk; Mackie, Roderick I.; Cann, Isaac

2016-10-01

Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose.

Specialized activities and expression differences for Clostridium thermocellum biofilm and planktonic cells.

PubMed

Dumitrache, Alexandru; Klingeman, Dawn M; Natzke, Jace; Rodriguez, Miguel; Giannone, Richard J; Hettich, Robert L; Davison, Brian H; Brown, Steven D

2017-02-27

Clostridium (Ruminiclostridium) thermocellum is a model organism for its ability to deconstruct plant biomass and convert the cellulose into ethanol. The bacterium forms biofilms adherent to lignocellulosic feedstocks in a continuous cell-monolayer in order to efficiently break down and uptake cellulose hydrolysates. We developed a novel bioreactor design to generate separate sessile and planktonic cell populations for omics studies. Sessile cells had significantly greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division consistent with substrate replete conditions. Planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. This study demonstrates that microbial attachment to cellulose substrate produces widespread gene expression changes for critical functions of this organism and provides physiological insights for two cells populations relevant for engineering of industrially-ready phenotypes.

Mode and polarization state selected guided wave spectroscopy of orientational anisotrophy in model membrane cellulosic polymer films: relevance to lab-on-a-chip

NASA Astrophysics Data System (ADS)

Andrews, Mark P.; Kanigan, Tanya

2007-06-01

Orientation anisotropies in structural properties relevant to the use of cellulosic polymers as membranes for lab-on-chips were investigated for cellulose acetate (CA) and regenerated cellulose (RC) films deposited as slab waveguides. Anisotropy was probed with mode and polarization state selected guided wave Raman spectroscopy. CA exhibits partial chain orientation in the plane of the film, and this orientation is independent of sample substrate and film preparation conditions. RC films also show in-plane anisotropy, where the hexose sugar rings lie roughly in the plane of the film. Explanations are given of the role of artifacts in interpreting waveguide Raman spectra, including anomalous contributions to Raman spectra that arise from deviations from right angle scattering geometry, mode-dependent contributions to longitudinal electric field components and TE<-->TM mode conversion. We explore diffusion profiles of small molecules in cellulosic films by adaptations of an inverse-Wentzel-Kramers-Brillouin (iWKB) recursive, noninteger virtual mode index algorithm. Perturbations in the refractive index distribution, n(z), are recovered from the measured relative propagation constants, neffective,m, of the planar waveguide. The refractive index distribution then yields the diffusion profile.

Microorganisms applying for artificial soil regeneration technology in space greenhouses

NASA Astrophysics Data System (ADS)

Krivobok, A. S.

2012-04-01

The space greenhouse and technology for growing plants are being designed in frame of bio-technical life support systems development. During long-term space missions such greenhouse could provide the crew with vitamins and rough plant fiber. One of the important elements of the plant cultivation technology in the absence of earth gravity is organization and support the optimum root area. The capillary-porous substrate composed of anionites (FIBAN -1) and cationites (FIBAN -22-1) synthetic salt-saturated fibers is developed for plant cultivation in space and named "BIONA-V3". The BIONA main features are high productivity and usability. But the pointed features are not constant: the substrate productivity will be decreasing gradually from vegetation to vegetation course of plant residues and root secretions accumulation. Also, the basic hydro-physical characteristic of root zone will be shifted. Furthermore, saprotrophic microflora will develop and lead to increasing the level of microbial contamination of whole inhabit isolated module. Due to these changes the substrate useful life is limited and store mass is increased in long-term missions. For overhaul-period renewal it' necessary to remove the roots residues and other organic accumulation providing safety of the substrate capillary-porous structure. The basic components of 24-days old plant roots (Brassica chinensis, L) are cellulose (35 %) hemicellulose (11 %) and lignin (10 %). We see that one of the possible ways for roots residues removal from fibrous BIONA is microorganisms applying with strong cellulolytic and ligninolytic activities. The fungi Trichoderma sp., cellulolytic bacteria associations, and some genus of anaerobic thermophilic cellulolitic bacteria have been used for roots residues biodegradation. In case of applying cellulolytic fungi Trichoderma sp. considerable decrease of microcrystalline cellulose has been noted in both liquid and solid state fermentation. Cellulolytic fungi weight has been increased up to 30 % from initial roots dry weight. When the bacterial association derived from organic compost was used, the roots dry weight reduction was not exceeded 20 % in liquid state fermentation after 21 days. But the total cellulose was quietly steady, only the readily accessible soluble fractions were consumed. It was found that the most promising microorganisms for pointed task are anaerobic, thermophilic bacterium Clostridium thermocellum F9 and Caldicellulosiruptor bescii DSM 6725. It has been shown that its' in the liquid medium with the roots residuals during 10 days provides root biomass degradation up to 45 % and double decrease of crystalline cellulose. It's known that one of the possible ways to improve biodegradation process efficiency is applying of physical-chemical pretreatment for plant biomass. We used the pretreatment of BIONA substrate in microwave irradiation in 0,7 % sodium hydroxide water solution with addition of 0,5 % of hydrogen peroxide. It has allowed hydrolyzing the roots biomass partially and making the cellulose portion accessible to subsequent biodegradation. The alkaline pretreatment and the subsequent degradation by anaerobic, thermophilic bacterium Clostridium thermocellum, had lead to root biomass decrease up to 85% during 10 days. The examined procedure has allowed to restore the initial pore space volume of BIONA substrate and its' hydro-physical properties. It has made used-up BIONA suitable for the subsequent plant cultivation. The obtained results are the basis for future development of fibrous artificial soils regeneration technologies particularly for space greenhouses

S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization.

PubMed

Kumar, Manoj; Wightman, Raymond; Atanassov, Ivan; Gupta, Anjali; Hurst, Charlotte H; Hemsley, Piers A; Turner, Simon

2016-07-08

Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here, we demonstrate that all catalytic subunits of the CSC, known as cellulose synthase A (CESA) proteins, are S-acylated. Analysis of Arabidopsis CESA7 reveals four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment. Copyright © 2016, American Association for the Advancement of Science.

Photo-biohydrogen production potential of Rhodobacter capsulatus-PK from wheat straw

PubMed Central

2013-01-01

Background Biotechnological exploitation of lignocellulosic biomass is promising for sustainable and environmentally sound energy provision strategy because of the abundant availability of the renewable resources. Wheat straw (WS) comprising of 75-80% cellulose and hemicellulose is one of widely available, inexpensive and renewable lignocellulosic biomass types. The cellulosic and hemicellulose substrate can be hydrolyzed into monomeric sugars by chemical and/or biological methods. Results This study examined comparative potential of dilute acid and pre-ammonia pretreated and enzymatically hydrolyzed wheat straw (WS) for hydrogen production by purple non sulfur bacterium Rhodobacter capsulatus-PK. Gas production became noticeable after 14 h of inoculation in WS pretreated with 4% H2SO4. The detoxified liquid hydrolyzate (DLH) after overliming attained a production level of 372 mL-H2/L after 16 h under illumination of 120-150 W/m2 at 30 ± 2.0°C. Whereas the non-detoxified acid pretreated hydrolyzate (NDLH) of WS could produce only upto 254 mL-H2/L after 21 h post inoculation. Evolution of H2 became observable just after 10 ± 2.0 h of inoculation by employing 48 h age inoculum on the WS pretreated with 30% ammonia, hydrolyzed with cellulase 80 FPU/g and β-glucosidase 220 CbU/ml at 50°C. Upto 712 ml/L of culture was measured with continuous shaking for 24 h. The 47.5% and 64.2% higher hydrogen volume than the DLH and NDLH substrates, respectively appeared as a function of significantly higher monomeric sugar contents of the enzymatically hydrolyzed substrate and lesser/zero amounts of toxic derivatives including pH reducing agents. Conclusion Photofermentative hydrogen production from lignocellulosic waste is a feasible approach for eco-friendly sustainable supply of bioenergy in a cost-effective way. Results of this study provide new insight for addressing biotechnological exploitation of abundantly available and low-cost cellulosic substrates. PMID:24099439

Prospect for Developing a Consolidated Bioprocessing (CBP) Strain Using Xylan as the Substrate: the Case Study of Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Wei, Hui; Alahuhta, Markus

2016-07-08

To achieve the goal of developing a direct microbial sugar conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in thismore » study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnD worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a direct microbial conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica.« less

Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass

USDA-ARS?s Scientific Manuscript database

Critical cellulase and hemicellulase activities are identified for hydrolysis of ionic liquid (IL) pretreated poplar and switchgrass; hemicellulase rich substrates with amorphous cellulose. Enzymes from Aspergillus nidulans were expressed and purified: an endoglucanase (EG) a cellobiohydrolase (CBH)...

CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN11[OPEN

PubMed Central

Wilkop, Thomas E.; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

2015-01-01

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

CESA TRAFFICKING INHIBITOR inhibits cellulose deposition and interferes with the trafficking of cellulose synthase complexes and their associated proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.

PubMed

Worden, Natasha; Wilkop, Thomas E; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

2015-02-01

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. © 2015 American Society of Plant Biologists. All Rights Reserved.

Preparation, Characterization and Activity of a Peptide-Cellulosic Aerogel Protease Sensor from Cotton

PubMed Central

Edwards, J. Vincent; Fontenot, Krystal R.; Prevost, Nicolette T.; Pircher, Nicole; Liebner, Falk; Condon, Brian D.

2016-01-01

Nanocellulosic aerogels (NA) provide a lightweight biocompatible material with structural properties, like interconnected high porosity and specific surface area, suitable for biosensor design. We report here the preparation, characterization and activity of peptide-nanocellulose aerogels (PepNA) made from unprocessed cotton and designed with protease detection activity. Low-density cellulosic aerogels were prepared from greige cotton by employing calcium thiocyanate octahydrate/lithium chloride as a direct cellulose dissolving medium. Subsequent casting, coagulation, solvent exchange and supercritical carbon dioxide drying afforded homogeneous cellulose II aerogels of fibrous morphology. The cotton-based aerogel had a porosity of 99% largely dominated by mesopores (2–50 nm) and an internal surface of 163 m2·g−1. A fluorescent tripeptide-substrate (succinyl-alanine-proline-alanine-4-amino-7-methyl-coumarin) was tethered to NA by (1) esterification of cellulose C6 surface hydroxyl groups with glycidyl-fluorenylmethyloxycarbonyl (FMOC), (2) deprotection and (3) coupling of the immobilized glycine with the tripeptide. Characterization of the NA and PepNA included techniques, such as elemental analysis, mass spectral analysis, attenuated total reflectance infrared imaging, nitrogen adsorption, scanning electron microscopy and bioactivity studies. The degree of substitution of the peptide analog attached to the anhydroglucose units of PepNA was 0.015. The findings from mass spectral analysis and attenuated total reflectance infrared imaging indicated that the peptide substrate was immobilized on to the surface of the NA. Nitrogen adsorption revealed a high specific surface area and a highly porous system, which supports the open porous structure observed from scanning electron microscopy images. Bioactivity studies of PepNA revealed a detection sensitivity of 0.13 units/milliliter for human neutrophil elastase, a diagnostic biomarker for inflammatory diseases. The physical properties of the aerogel are suitable for interfacing with an intelligent protease sequestrant wound dressing. PMID:27792201

Effect of mechanical disruption on the effectiveness of three reactors used for dilute acid pretreatment of corn stover Part 1: chemical and physical substrate analysis

PubMed Central

2014-01-01

Background There is considerable interest in the conversion of lignocellulosic biomass to liquid fuels to provide substitutes for fossil fuels. Pretreatments, conducted to reduce biomass recalcitrance, usually remove at least some of the hemicellulose and/or lignin in cell walls. The hypothesis that led to this research was that reactor type could have a profound effect on the properties of pretreated materials and impact subsequent cellulose hydrolysis. Results Corn stover was dilute-acid pretreated using commercially relevant reactor types (ZipperClave® (ZC), Steam Gun (SG) and Horizontal Screw (HS)) under the same nominal conditions. Samples produced in the SG and HS achieved much higher cellulose digestibilities (88% and 95%, respectively), compared to the ZC sample (68%). Characterization, by chemical, physical, spectroscopic and electron microscopy methods, was used to gain an understanding of the effects causing the digestibility differences. Chemical differences were small; however, particle size differences appeared significant. Sum-frequency generation vibrational spectra indicated larger inter-fibrillar spacing or randomization of cellulose microfibrils in the HS sample. Simons’ staining indicated increased cellulose accessibility for the SG and HS samples. Electron microscopy showed that the SG and HS samples were more porous and fibrillated because of mechanical grinding and explosive depressurization occurring with these two reactors. These structural changes most likely permitted increased cellulose accessibility to enzymes, enhancing saccharification. Conclusions Dilute-acid pretreatment of corn stover using three different reactors under the same nominal conditions gave samples with very different digestibilities, although chemical differences in the pretreated substrates were small. The results of the physical and chemical analyses of the samples indicate that the explosive depressurization and mechanical grinding with these reactors increased enzyme accessibility. Pretreatment reactors using physical force to disrupt cell walls increase the effectiveness of the pretreatment process. PMID:24713111

Dissecting the molecular mechanism underlying the intimate relationship between cellulose microfibrils and cortical microtubules.

PubMed

Lei, Lei; Li, Shundai; Bashline, Logan; Gu, Ying

2014-01-01

A central question in plant cell development is how the cell wall determines directional cell expansion and therefore the final shape of the cell. As the major load-bearing component of the cell wall, cellulose microfibrils are laid down transversely to the axis of elongation, thus forming a spring-like structure that reinforces the cell laterally and while favoring longitudinal expansion in most growing cells. Mounting evidence suggests that cortical microtubules organize the deposition of cellulose microfibrils, but the precise molecular mechanisms linking microtubules to cellulose organization have remained unclear until the recent discovery of cellulose synthase interactive protein 1 , a linker protein between the cortical microtubules and the cellulose biosynthesizing machinery. In this review, we will focus on the intimate relationship between cellulose microfibrils and cortical microtubules, in particular, we will discuss microtubule arrangement and cell wall architecture, the linkage between cellulose synthase complexes and microtubules, and the feedback mechanisms between cell wall and microtubules.

Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification.

PubMed

Longoni, Paolo; Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino

2015-01-01

Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

Cellulose synthase interactive protein 1 (CSI1) mediates the intimate relationship between cellulose microfibrils and cortical microtubules.

PubMed

Lei, Lei; Li, Shundai; Gu, Ying

2012-07-01

Cellulose is synthesized at the plasma membrane by protein complexes known as cellulose synthase complexes (CSCs). The cellulose-microtubule alignment hypothesis states that there is a causal link between the orientation of cortical microtubules and orientation of nascent cellulose microfibrils. The mechanism behind the alignment hypothesis is largely unknown. CESA interactive protein 1 (CSI1) interacts with CSCs and potentially links CSCs to the cytoskeleton. CSI1 not only co-localizes with CSCs but also travels bi-directionally in a speed indistinguishable from CSCs. The linear trajectories of CSI1-RFP coincide with the underlying microtubules labeled by YFP-TUA5. In the absence of CSI1, both the distribution and the motility of CSCs are defective and the alignment of CSCs and microtubules is disrupted. These observations led to the hypothesis that CSI1 directly mediates the interaction between CSCs and microtubules. In support of this hypothesis, CSI1 binds to microtubules directly by an in vitro microtubule-binding assay. In addition to a role in serving as a messenger from microtubule to CSCs, CSI1 labels SmaCCs/MASCs, a compartment that has been proposed to be involved in CESA trafficking and/or delivery to the plasma membrane.

Cellulose synthase interactive protein 1 (CSI1) mediates the intimate relationship between cellulose microfibrils and cortical microtubules

PubMed Central

Lei, Lei; Li, Shundai; Gu, Ying

2012-01-01

Cellulose is synthesized at the plasma membrane by protein complexes known as cellulose synthase complexes (CSCs). The cellulose-microtubule alignment hypothesis states that there is a causal link between the orientation of cortical microtubules and orientation of nascent cellulose microfibrils. The mechanism behind the alignment hypothesis is largely unknown. CESA interactive protein 1 (CSI1) interacts with CSCs and potentially links CSCs to the cytoskeleton. CSI1 not only co-localizes with CSCs but also travels bi-directionally in a speed indistinguishable from CSCs. The linear trajectories of CSI1-RFP coincide with the underlying microtubules labeled by YFP-TUA5. In the absence of CSI1, both the distribution and the motility of CSCs are defective and the alignment of CSCs and microtubules is disrupted. These observations led to the hypothesis that CSI1 directly mediates the interaction between CSCs and microtubules. In support of this hypothesis, CSI1 binds to microtubules directly by an in vitro microtubule-binding assay. In addition to a role in serving as a messenger from microtubule to CSCs, CSI1 labels SmaCCs/MASCs, a compartment that has been proposed to be involved in CESA trafficking and/or delivery to the plasma membrane. PMID:22751327

Evaluation of alkali treatment for biodegradation of corn cobs by Aspergillus niger.

PubMed

Singh, A; Abidi, A B; Agrawal, A K; Darmwal, N S

1989-01-01

Effect of NaOH pretreatment on the biodegradation of corn cobs for the production of cellulase and protein was studied using Aspergillus niger. Delignification of cobs with NaOH remarkably increased the production of cellulase and protein. Treatment of cobs with 2% NaOH was found to be the best with respect to their susceptibility to biodegradation for maximum production of cellulose 1,4-beta-cellobiosidase, cellulase, beta-glucosidase soluble protein and crude protein; this also led to the highest protein recovery, maximum cellulose utilization and also for the maximum degradation of substrate.

[New strains of basidiomycetes that produce bioethanol from lignocellulose biomass].

PubMed

Kozhevnikova, E Yu; Petrova, D A; Kopitsyn, D S; Nivikov, A A; Shnyreva, A V; Barkov, A V; Vinokurov, V A

2016-01-01

Sixty six isolates were screened for ability of bioethanol production; dynamics of product accumulation and substrate utilization were investigated for two selected strains Trametes hirsuta MT-24.24 and Trametes versicolor IT-1. The strains’ efficiency was evaluated as bioethanol production by 1 g biomass. Strain T. versicolor IT-1 producing over 33 g/L of the ethanol for 9 d was selected. Direct conversion of Na-carboxymethyl cellulose, microcrystalline cellulose and straw was shown with ethanol yields of 2.1, 1.6 and 1.7 g/L, respectively, for 9 d fermentation time.

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers

DOE PAGES

Olek, Anna T.; Rayon, Catherine; Makowski, Lee; ...

2014-07-10

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice ( Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain,more » elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. As a result, the arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.« less

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers.

PubMed

Olek, Anna T; Rayon, Catherine; Makowski, Lee; Kim, Hyung Rae; Ciesielski, Peter; Badger, John; Paul, Lake N; Ghosh, Subhangi; Kihara, Daisuke; Crowley, Michael; Himmel, Michael E; Bolin, Jeffrey T; Carpita, Nicholas C

2014-07-01

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize. © 2014 American Society of Plant Biologists. All rights reserved.

Cellulose synthase (CesA) genes in the green alga Mesotaenium caldariorum.

PubMed

Roberts, Alison W; Roberts, Eric M; Delmer, Deborah P

2002-12-01

Cellulose, a microfibrillar polysaccharide consisting of bundles of beta-1,4-glucan chains, is a major component of plant and most algal cell walls and is also synthesized by some prokaryotes. Seed plants and bacteria differ in the structures of their membrane terminal complexes that make cellulose and, in turn, control the dimensions of the microfibrils produced. They also differ in the domain structures of their CesA gene products (the catalytic subunit of cellulose synthase), which have been localized to terminal complexes and appear to help maintain terminal complex structure. Terminal complex structures in algae range from rosettes (plant-like) to linear forms (bacterium-like). Thus, algal CesA genes may reveal domains that control terminal complex assembly and microfibril structure. The CesA genes from the alga Mesotaenium caldariorum, a member of the order Zygnematales, which have rosette terminal complexes, are remarkably similar to seed plant CesAs, with deduced amino acid sequence identities of up to 59%. In addition to the putative transmembrane helices and the D-D-D-QXXRW motif shared by all known CesA gene products, M. caldariorum and seed plant CesAs share a region conserved among plants, an N-terminal zinc-binding domain, and a variable or class-specific region. This indicates that the domains that characterize seed plant CesAs arose prior to the evolution of land plants and may play a role in maintaining the structures of rosette terminal complexes. The CesA genes identified in M. caldariorum are the first reported for any eukaryotic alga and will provide a basis for analyzing the CesA genes of algae with different types of terminal complexes.

Cellulase enzyme: Homology modeling, binding site identification and molecular docking

NASA Astrophysics Data System (ADS)

Selvam, K.; Senbagam, D.; Selvankumar, T.; Sudhakar, C.; Kamala-Kannan, S.; Senthilkumar, B.; Govarthanan, M.

2017-12-01

Cellulase is an enzyme that degrades the linear polysaccharide like cellulose into glucose by breaking the β-1,4- glycosidic bonds. These enzymes are the third largest enzymes with a great potential towards the ethanol production and play a vital role in degrading the biomass. The production of ethanol depends upon the ability of the cellulose to utilize the wide range of substrates. In this study, the 3D structure of cellulase from Acinetobacter sp. was modeled by using Modeler 9v9 and validated by Ramachandran plot. The accuracy of the predicted 3D structure was checked using Ramachandran plot analysis showed that 81.1% in the favored region, compatibility of an atomic model (3D) with amino acid sequence (1D) for the model was observed as 78.21% and 49.395% for Verify 3D and ERRAT at SAVES server. As the binding efficacy with the substrate might suggests the choice of the substrate as carbon and nitrogen sources, the cellobiose, cellotetraose, cellotetriose and laminaribiose were employed in the docking studies. The docking of cellobiose, cellotetraose, cellotetriose and laminaribiose with cellulase exhibited the binding energy of -6.1523 kJ/mol, -7.8759 kJ/mol,-6.1590 kJ/mol and -6.7185 kJ/mol, respectively. These docking studies revealed that cellulase has the greater potential towards the cellotetraose as a substrate for the high yield of ethanol.

Ultra-Fast Microwave Synthesis of ZnO Nanorods on Cellulose Substrates for UV Sensor Applications

PubMed Central

Pimentel, Ana; Samouco, Ana; Araújo, Andreia; Martins, Rodrigo; Fortunato, Elvira

2017-01-01

In the present work, tracing and Whatman papers were used as substrates to grow zinc oxide (ZnO) nanostructures. Cellulose-based substrates are cost-efficient, highly sensitive and environmentally friendly. ZnO nanostructures with hexagonal structure were synthesized by hydrothermal under microwave irradiation using an ultrafast approach, that is, a fixed synthesis time of 10 min. The effect of synthesis temperature on ZnO nanostructures was investigated from 70 to 130 °C. An Ultra Violet (UV)/Ozone treatment directly to the ZnO seed layer prior to microwave assisted synthesis revealed expressive differences regarding formation of the ZnO nanostructures. Structural characterization of the microwave synthesized materials was carried out by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The optical characterization has also been performed. The time resolved photocurrent of the devices in response to the UV turn on/off was investigated and it has been observed that the ZnO nanorod arrays grown on Whatman paper substrate present a responsivity 3 times superior than the ones grown on tracing paper. By using ZnO nanorods, the surface area-to-volume ratio will increase and will improve the sensor sensibility, making these types of materials good candidates for low cost and disposable UV sensors. The sensors were exposed to bending tests, proving their high stability, flexibility and adaptability to different surfaces. PMID:29140304

Human Salivary Aldehyde Dehydrogenase: Purification, Kinetic Characterization and Effect of Ethanol, Hydrogen Peroxide and Sodium Dodecyl Sulfate on the Activity of the Enzyme.

PubMed

Alam, Md Fazle; Laskar, Amaj Ahmed; Choudhary, Hadi Hasan; Younus, Hina

2016-09-01

Human salivary aldehyde dehydrogenase (hsALDH) enzyme appears to be the first line of defense in the body against exogenous toxic aldehydes. However till date much work has not been done on this important member of the ALDH family. In this study, we have purified hsALDH to homogeneity by diethylaminoethyl-cellulose (DEAE-cellulose) ion-exchange chromatography in a single step. The molecular mass of the homodimeric enzyme was determined to be approximately 108 kDa. Four aromatic substrates; benzaldehyde, cinnamaldehyde, 2-naphthaldehyde and 6-methoxy-2-naphthaldehyde were used for determining the activity of pure hsALDH. K m values for these substrates were calculated to be 147.7, 5.31, 0.71 and 3.31 μM, respectively. The best substrates were found to be cinnamaldehyde and 2-naphthaldehyde since they exhibited high V max /K m values. 6-methoxy-2-naphthaldehyde substrate was used for further kinetic characterization of pure hsALDH. The pH and temperature optima of hsALDH were measured to be pH 8 and 45 °C, respectively. The pure enzyme is highly unstable at high temperatures. Ethanol, hydrogen peroxide and SDS activate hsALDH, therefore it is safe and beneficial to include them in mouthwashes and toothpastes in low concentrations.

Complex formation of blueberry (Vaccinium angustifolium) anthocyanins during freeze-drying and its influence on their biological activity.

PubMed

Correa-Betanzo, Julieta; Padmanabhan, Priya; Corredig, Milena; Subramanian, Jayasankar; Paliyath, Gopinadhan

2015-03-25

Biological activity of polyphenols is influenced by their uptake and is highly influenced by their interactions with the food matrix. This study evaluated the complex formation of blueberry polyphenols with fruit matrixes such as pectin and cellulose and their effect on the biological and antiproliferative properties of human colon cell lines HT-29 and CRL 1790. Free or complexed polyphenols were isolated by dialyzing aqueous or methanolic blueberry homogenates. Seven phenolic compounds and thirteen anthocyanins were identified in blueberry extracts. Blueberry extracts showed varying degrees of antioxidant and antiproliferative activities, as well as α-glucosidase activity. Fruit matrix containing cellulose and pectin, or purified polygalacturonic acid and cellulose, did not retain polyphenols and showed very low antioxidant or antiproliferative activities. These findings suggest that interactions between polyphenols and the food matrix may be more complex than a simple association and may play an important role in the bioefficacy of blueberry polyphenols.

Microbial decomposition of keratin in nature-a new hypothesis of industrial relevance.

PubMed

Lange, Lene; Huang, Yuhong; Busk, Peter Kamp

2016-03-01

Discovery of keratin-degrading enzymes from fungi and bacteria has primarily focused on finding one protease with efficient keratinase activity. Recently, an investigation was conducted of all keratinases secreted from a fungus known to grow on keratinaceous materials, such as feather, horn, and hooves. The study demonstrated that a minimum of three keratinases is needed to break down keratin, an endo-acting, an exo-acting, and an oligopeptide-acting keratinase. Further, several studies have documented that disruption of sulfur bridges of the keratin structure acts synergistically with the keratinases to loosen the molecular structure, thus giving the enzymes access to their substrate, the protein structure. With such complexity, it is relevant to compare microbial keratin decomposition with the microbial decomposition of well-studied polymers such as cellulose and chitin. Interestingly, it was recently shown that the specialized enzymes, lytic polysaccharide monoxygenases (LPMOs), shown to be important for breaking the recalcitrance of cellulose and chitin, are also found in keratin-degrading fungi. A holistic view of the complex molecular self-assembling structure of keratin and knowledge about enzymatic and boosting factors needed for keratin breakdown have been used to formulate a hypothesis for mode of action of the LPMOs in keratin decomposition and for a model for degradation of keratin in nature. Testing such hypotheses and models still needs to be done. Even now, the hypothesis can serve as an inspiration for designing industrial processes for keratin decomposition for conversion of unexploited waste streams, chicken feather, and pig bristles into bioaccessible animal feed.

Dramatic performance of Clostridium thermocellum explained by its wide range of cellulase modalities

DOE PAGES

Xu, Qi; Resch, Michael G.; Podkaminer, Kara; ...

2016-02-05

Clostridium thermocellum is the most efficient microorganism for solubilizing lignocellulosic biomass known to date. Its high cellulose digestion capability is attributed to efficient cellulases consisting of both a free-enzyme system and a tethered cellulosomal system wherein carbohydrate active enzymes (CAZymes) are organized by primary and secondary scaffoldin proteins to generate large protein complexes attached to the bacterial cell wall. This study demonstrates that C. thermocellum also uses a type of cellulosomal system not bound to the bacterial cell wall, called the “cell-free” cellulosomal system. The cell-free cellulosome complex can be seen as a “long range cellulosome” because it can diffusemore » away from the cell and degrade polysaccharide substrates remotely from the bacterial cell. The contribution of these two types of cellulosomal systems in C. thermocellum was elucidated by characterization of mutants with different combinations of scaffoldin gene deletions. The primary scaffoldin, CipA, was found to play the most important role in cellulose degradation by C. thermocellum, whereas the secondary scaffoldins have less important roles. Additionally, the distinct and efficient mode of action of the C. thermocellum exoproteome, wherein the cellulosomes splay or divide biomass particles, changes when either the primary or secondary scaffolds are removed, showing that the intact wild-type cellulosomal system is necessary for this essential mode of action. As a result, this new transcriptional and proteomic evidence shows that a functional primary scaffoldin plays a more important role compared to secondary scaffoldins in the proper regulation of CAZyme genes, cellodextrin transport, and other cellular functions.« less

Dramatic performance of Clostridium thermocellum explained by its wide range of cellulase modalities

PubMed Central

Xu, Qi; Resch, Michael G.; Podkaminer, Kara; Yang, Shihui; Baker, John O.; Donohoe, Bryon S.; Wilson, Charlotte; Klingeman, Dawn M.; Olson, Daniel G.; Decker, Stephen R.; Giannone, Richard J.; Hettich, Robert L.; Brown, Steven D.; Lynd, Lee R.; Bayer, Edward A.; Himmel, Michael E.; Bomble, Yannick J.

2016-01-01

Clostridium thermocellum is the most efficient microorganism for solubilizing lignocellulosic biomass known to date. Its high cellulose digestion capability is attributed to efficient cellulases consisting of both a free-enzyme system and a tethered cellulosomal system wherein carbohydrate active enzymes (CAZymes) are organized by primary and secondary scaffoldin proteins to generate large protein complexes attached to the bacterial cell wall. This study demonstrates that C. thermocellum also uses a type of cellulosomal system not bound to the bacterial cell wall, called the “cell-free” cellulosomal system. The cell-free cellulosome complex can be seen as a “long range cellulosome” because it can diffuse away from the cell and degrade polysaccharide substrates remotely from the bacterial cell. The contribution of these two types of cellulosomal systems in C. thermocellum was elucidated by characterization of mutants with different combinations of scaffoldin gene deletions. The primary scaffoldin, CipA, was found to play the most important role in cellulose degradation by C. thermocellum, whereas the secondary scaffoldins have less important roles. Additionally, the distinct and efficient mode of action of the C. thermocellum exoproteome, wherein the cellulosomes splay or divide biomass particles, changes when either the primary or secondary scaffolds are removed, showing that the intact wild-type cellulosomal system is necessary for this essential mode of action. This new transcriptional and proteomic evidence shows that a functional primary scaffoldin plays a more important role compared to secondary scaffoldins in the proper regulation of CAZyme genes, cellodextrin transport, and other cellular functions. PMID:26989779

The Arabidopsis COBRA Protein Facilitates Cellulose Crystallization at the Plasma Membrane*

PubMed Central

Sorek, Nadav; Sorek, Hagit; Kijac, Aleksandra; Szemenyei, Heidi J.; Bauer, Stefan; Hématy, Kian; Wemmer, David E.; Somerville, Chris R.

2014-01-01

Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual β1–4-linked glucan chains with a KD of 3.2 μm. Competition assays suggests that COBRA binds individual β1–4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging β1–4-glucan chains by acting as a “polysaccharide chaperone.” PMID:25331944

Recyclable Thermoresponsive Polymer-Cellulase Bioconjugates for Biomass Depolymerization

PubMed Central

Mackenzie, Katherine J.; Francis, Matthew B.

2013-01-01

Here we report the construction and characterization of a recoverable, thermoresponsive polymer-endoglucanase bioconjugate that matches the activity of unmodified enzymes on insoluble cellulose substrates. Two copolymers exhibiting a thermoresponsive lower critical solution temperature (LCST) were created through the copolymerization of an aminooxy-bearing methacrylamide with N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMa). The aminooxy group provided a handle through which the LCST was adjusted through small-molecule quenching. This allowed materials with LCSTs ranging from 20.9 °C to 60.5 °C to be readily obtained after polymerization. The thermostable endoglucanase EGPh from the hypothermophilic Pyrococcus horikoshii was transaminated with pyridoxal-5’-phosphate to produce a ketone-bearing protein, which was then site-selectively modified through oxime linkage with benzylalkoxyamine or 5 kDa-poly(ethylene glycol)-alkoxyamine. These modified proteins showed activity comparable to the controls when assayed on an insoluble cellulosic substrate. Two polymer bioconjugates were then constructed using transaminated EGPh and the aminooxy-bearing copolymers. After twelve hours, both bioconjugates produced an equivalent amount of free reducing sugars as the unmodified control using insoluble cellulose as a substrate. The recycling ability of the NIPAm copolymer-EGPh conjugate was determined through three rounds of activity, maintaining over 60% activity after two cycles of reuse and affording significantly more soluble carbohydrates than unmodified enzyme alone. When assayed on acid-pretreated Miscanthus, this bioconjugate increased the amount of reducing sugars by 2.8-fold over three rounds of activity. The synthetic strategy of this bioconjugate allows the LCST of the material to be changed readily from a common stock of copolymer and the method of attachment is applicable to a variety of proteins, enabling the same approach to be amenable to thermophile-derived cellulases or to the separation of multiple species using polymers with different recovery temperatures. PMID:23270527

Surface modification of cellulose fibers: towards wood composites by biomimetics.

PubMed

Gradwell, Sheila E; Renneckar, Scott; Esker, Alan R; Heinze, Thomas; Gatenholm, Paul; Vaca-Garcia, Carlos; Glasser, Wolfgang

2004-01-01

A biomimetic approach was taken for studying the adsorption of a model copolymer (pullulan abietate, DS 0.027), representing the lignin-carbohydrate complex, to a model surface for cellulose fibers (Langmuir-Blodgett thin films of regenerated cellulose). Adsorption results were assayed using surface plasmon resonance spectroscopy (SPR) and atomic force microscopy (AFM). Rapid, spontaneous, and desorption-resistant surface modification resulted. This effort is viewed as a critical first step towards the permanent surface modification of cellulose fibers with a layer of molecules amenable to either enzymatic crosslinking for improved wood composites or thermoplastic consolidation.

Engineering towards a complete heterologous cellulase secretome in Yarrowia lipolytica reveals its potential for consolidated bioprocessing

DOE PAGES

Wei, Hui; Wang, Wei; Alahuhta, Markus; ...

2014-10-16

Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichodermamore » reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient degradation of cellulosic substrates. In Conclusion: Taken together, this work demonstrates the first case of successful expression of a chimeric CBHI with essentially full native activity in Y. lipolytica, and supports the notion that Y. lipolytica strains can be genetically engineered, ultimately by heterologous expression of fungal cellulases and other enzymes, to directly convert lignocellulosic substrates to biofuels.« less

Functional reconstitution of cellulose synthase in Escherichia coli.

PubMed

Imai, Tomoya; Sun, Shi-Jing; Horikawa, Yoshiki; Wada, Masahisa; Sugiyama, Junji

2014-11-10

Cellulose is a high molecular weight polysaccharide of β1 → 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials.

Dissolution mechanism of crystalline cellulose in H3PO4 as assessed by high-field NMR spectroscopy and fast field cycling NMR relaxometry.

PubMed

Conte, Pellegrino; Maccotta, Antonella; De Pasquale, Claudio; Bubici, Salvatore; Alonzo, Giuseppe

2009-10-14

Many processes have been proposed to produce glucose as a substrate for bacterial fermentation to obtain bioethanol. Among others, cellulose degradation appears as the most convenient way to achieve reliable amounts of glucose units. In fact, cellulose is the most widespread biopolymer, and it is considered also as a renewable resource. Due to extended intra- and interchain hydrogen bonds that provide a very efficient packing structure, however, cellulose is also a very stable polymer, the degradation of which is not easily achievable. In the past decade, researchers enhanced cellulose reactivity by increasing its solubility in many solvents, among which concentrated phosphoric acid (H(3)PO(4)) played the major role because of its low volatility and nontoxicity. In the present study, the solubilization mechanism of crystalline cellulose in H(3)PO(4) has been elucidated by using high- and low-field NMR spectroscopy. In particular, high-field NMR spectra showed formation of direct bonding between phosphoric acid and dissolved cellulose. On the other hand, molecular dynamics studies by low-field NMR with a fast field cycling (FFC) setup revealed two different H(3)PO(4) relaxing components. The first component, described by the fastest longitudinal relaxation rate (R(1)), was assigned to the H(3)PO(4) molecules bound to the biopolymer. Conversely, the second component, characterized by the slowest R(1), was attributed to the bulk solvent. The understanding of cellulose dissolution in H(3)PO(4) represents a very important issue because comprehension of chemical mechanisms is fundamental for process ameliorations to produce bioenergy from biomasses.

Processive Degradation of Crystalline Cellulose by a Multimodular Endoglucanase via a Wirewalking Mode.

PubMed

Zhang, Kun-Di; Li, Wen; Wang, Ye-Fei; Zheng, Yan-Lin; Tan, Fang-Cheng; Ma, Xiao-Qing; Yao, Li-Shan; Bayer, Edward A; Wang, Lu-Shan; Li, Fu-Li

2018-05-14

Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite -4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases.

The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

PubMed

Arslan, Baran; Colpan, Mert; Ju, Xiaohui; Zhang, Xiao; Kostyukova, Alla; Abu-Lail, Nehal I

2016-05-09

The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity and the surface energy of lignin that facilitates the LW forces should be a priori to avoid nonproductive binding of cellulase to kraft lignin.

Characterization of cellulose structure of Populus plants modified in candidate cellulose biosynthesis genes

DOE PAGES

Bali, Garima; Khunsupat, Ratayakorn; Akinosho, Hannah; ...

2016-09-10

Here, the recalcitrant nature of lignocellulosic biomass is a combined effect of several factors such as high crystallinity and high degree of polymerization of cellulose, lignin content and structure, and the available surface area for enzymatic degradation (i.e., accessibility). Genetic improvement of feedstock cell wall properties is a path to reducing recalcitrance of lignocellulosic biomass and improving conversion to various biofuels. An advanced understanding of the cellulose biosynthesis pathway is essential to precisely modify cellulose properties of plant cell walls. Here we report on the impact of modified expression of candidate cellulose biosynthesis pathway genes on the ultra-structure of cellulose,more » a key carbohydrate polymer of Populus cell wall using advanced nuclear magnetic resonance approaches. Noteworthy changes were observed in the cell wall characteristics of downregulated KORRIGAN 1 (KOR) and KOR 2 transgenic plants in comparison to the wild-type control. It was observed that all of the transgenic lines showed variation in cellulose ultrastructure, increase in cellulose crystallinity and decrease in the cellulose degree of polymerization. Additionally, the properties of cellulose allomorph abundance and accessibility were found to be variable. Application of such cellulose characterization techniques beyond the traditional measurement of cellulose abundance to comprehensive studies of cellulose properties in larger transgenic and naturally variable populations is expected to provide deeper insights into the complex nature of lignocellulosic material, which can significantly contribute to the development of precisely tailored plants for enhanced biofuels production.« less

Characterization of cellulose structure of Populus plants modified in candidate cellulose biosynthesis genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bali, Garima; Khunsupat, Ratayakorn; Akinosho, Hannah

Here, the recalcitrant nature of lignocellulosic biomass is a combined effect of several factors such as high crystallinity and high degree of polymerization of cellulose, lignin content and structure, and the available surface area for enzymatic degradation (i.e., accessibility). Genetic improvement of feedstock cell wall properties is a path to reducing recalcitrance of lignocellulosic biomass and improving conversion to various biofuels. An advanced understanding of the cellulose biosynthesis pathway is essential to precisely modify cellulose properties of plant cell walls. Here we report on the impact of modified expression of candidate cellulose biosynthesis pathway genes on the ultra-structure of cellulose,more » a key carbohydrate polymer of Populus cell wall using advanced nuclear magnetic resonance approaches. Noteworthy changes were observed in the cell wall characteristics of downregulated KORRIGAN 1 (KOR) and KOR 2 transgenic plants in comparison to the wild-type control. It was observed that all of the transgenic lines showed variation in cellulose ultrastructure, increase in cellulose crystallinity and decrease in the cellulose degree of polymerization. Additionally, the properties of cellulose allomorph abundance and accessibility were found to be variable. Application of such cellulose characterization techniques beyond the traditional measurement of cellulose abundance to comprehensive studies of cellulose properties in larger transgenic and naturally variable populations is expected to provide deeper insights into the complex nature of lignocellulosic material, which can significantly contribute to the development of precisely tailored plants for enhanced biofuels production.« less

Incorporation of Monolignol Conjugates into Lignin for Improved Processing

USDA-ARS?s Scientific Manuscript database

Lignin remains one of the most significant barriers to the efficient utilization of cellulosic substrates, either for pulping or for biofuels production. Now that monomer substitution in the lignification process is now well authenticated in various transgenic plants, it is opportune to begin explor...

Absorbable Antimicrobial Battlefield Hemostat

DTIC Science & Technology

2013-04-01

to graphene sheet and carbon nanotubes . Coupled with ABTS, the GQDs can be used for the detection of hydrogen peroxide with the limit of detection......dressing could be made of any material that is compatible to our coating solvent. Examples include gelatin based and cellulose based substrates. The

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol.

PubMed

Varga, Enikõ; Klinke, Helene B; Réczey, Kati; Thomsen, Anne Belinda

2004-12-05

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.

Transparent nanocellulosic multilayer thin films on polylactic acid with tunable gas barrier properties.

PubMed

Aulin, Christian; Karabulut, Erdem; Tran, Amy; Wågberg, Lars; Lindström, Tom

2013-08-14

The layer-by-layer (LbL) deposition method was used for the build-up of alternating layers of nanofibrillated cellulose (NFC) or carboxymethyl cellulose (CMC) with a branched, cationic polyelectrolyte, polyethyleneimine (PEI) on flexible poly (lactic acid) (PLA) substrates. With this procedure, optically transparent nanocellulosic films with tunable gas barrier properties were formed. 50 layer pairs of PEI/NFC and PEI/CMC deposited on PLA have oxygen permeabilities of 0.34 and 0.71 cm(3)·μm/m(2)·day·kPa at 23 °C and 50% relative humidity, respectively, which is in the same range as polyvinyl alcohol and ethylene vinyl alcohol. The oxygen permeability of these multilayer nanocomposites outperforms those of pure NFC films prepared by solvent-casting. The nanocellulosic LbL assemblies on PLA substrates was in detailed characterized using a quartz crystal microbalance with dissipation (QCM-D). Atomic force microscopy (AFM) reveals large structural differences between the PEI/NFC and the PEI/CMC assemblies, with the PEI/NFC assembly showing a highly entangled network of nanofibrils, whereas the PEI/CMC surfaces lacked structural features. Scanning electron microscopy images showed a nearly perfect uniformity of the nanocellulosic coatings on PLA, and light transmittance results revealed remarkable transparency of the LbL-coated PLA films. The present work demonstrates the first ever LbL films based on high aspect ratio, water-dispersible nanofibrillated cellulose, and water-soluble carboxymethyl cellulose polymers that can be used as multifunctional films and coatings with tailorable properties, such as gas barriers and transparency. Owing to its flexibility, transparency and high-performance gas barrier properties, these thin film assemblies are promising candidates for several large-scale applications, including flexible electronics and renewable packaging.

Adsorption and association of a symmetric PEO-PPO-PEO triblock copolymer on polypropylene, polyethylene, and cellulose surfaces.

PubMed

Li, Yan; Liu, Hongyi; Song, Junlong; Rojas, Orlando J; Hinestroza, Juan P

2011-07-01

The association of a symmetric polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO(19)-PPO(29)-PEO(19)) triblock copolymer adsorbed from aqueous solutions onto polypropylene (PP), polyethylene (PE), and cellulose surfaces was probed using Atomic Force Microscopy (AFM). Significant morphological differences between the polyolefin substrates (PP and PE) and the cellulose surfaces were observed after immersion of the films in the PEO(19)-PPO(29)-PEO(19) solutions. When the samples were scanned, while immersed in solutions of the triblock copolymer, it was revealed that the structures adsorbed on the polyolefin surfaces were smoothed by the adsorbed PEO(19)-PPO(29)-PEO(19). In contrast, those structures on the hydrophilic cellulose surfaces were sharpened. These observations were related to the roughness of the substrate and the energy of interaction between the surfaces and the PEO and PPO polymer segments. The interaction energy between each of the blocks and the surface was calculated using molecular dynamics simulations. It is speculated that the associative structures amply reported in aqueous solution at concentrations above the critical micelle concentration, CMC, are not necessarily preserved upon adsorption; instead, it appears that molecular arrangements of the anchor-buoy type and hemimicelles prevail. The reported data suggests that the roughness of the surface, as well as its degree of hydrophobicity, have a large influence on the nature of the resulting adsorbed layer. The reported observations are valuable in explaining the behavior of finishing additives and lubricants commonly used in textile and fiber processing, as well as the effect of the morphology of the boundary layers on friction and wear, especially in the case of symmetric triblock copolymers, which are commonly used as antifriction, antiwear additives.

Class III peroxidases in cellulose deficient cultured maize cells during cell wall remodelling.

PubMed

Martínez-Rubio, Romina; Acebes, José Luis; Encina, Antonio; Kärkönen, Anna

2018-02-21

Maize (Zea mays L.) suspension-cultured cells habituated to a cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a modified cell wall, in which the reduction in the cellulose content is compensated by a network of highly cross-linked feruloylated arabinoxylans and the deposition of lignin-like polymers. For both arabinoxylan cross-linking and lignin polymerization, class III peroxidases (POXs) have been demonstrated to have a prominent role. For the first time, a comparative study of POX activity and isoforms in control and cellulose-impaired cells has been addressed, also taking into account their cellular distribution in different compartments. Proteins from the spent medium (SM), soluble cellular (SC), ionically (ICW) and covalently bound cell wall protein fractions were assayed for total and specific peroxidase activity by using coniferyl and sinapyl alcohol and ferulic acid as substrates. The isoPOX profile was obtained by isoelectric focusing. POX activity was higher in DCB-habituated than in non-habituated cells in all protein fractions at all cell culture stages. For all substrates assayed, SC and ICW fractions showed higher activity at the early-log growth phase than at the late-log phase. However, the highest POX activity in the spent medium was found at the late-log phase. According to the isoPOX profiles, the highest diversity of isoPOXs was detected in the ICW and SM protein fractions. The latter fraction contained isoPOXs with higher activity in DCB-habituated cells. Some of the isoPOXs detected could be involved in cross-linking of arabinoxylans and in the lignin-like polymer formation in DCB-habituated cells. This article is protected by copyright. All rights reserved.

Microreactor-based mixing strategy suppresses product inhibition to enhance sugar yields in enzymatic hydrolysis for cellulosic biofuel production.

PubMed

Chakraborty, Saikat; Singh, Prasun Kumar; Paramashetti, Pawan

2017-08-01

A novel microreactor-based energy-efficient process of using complete convective mixing in a macroreactor till an optimal mixing time followed by no mixing in 200-400μl microreactors enhances glucose and reducing sugar yields by upto 35% and 29%, respectively, while saving 72-90% of the energy incurred on reactor mixing in the enzymatic hydrolysis of cellulose. Empirical exponential relations are provided for determining the optimal mixing time, during which convective mixing in the macroreactor promotes mass transport of the cellulase enzyme to the solid Avicel substrate, while the latter phase of no mixing in the microreactor suppresses product inhibition by preventing the inhibitors (glucose and cellobiose) from homogenizing across the reactor. Sugar yield increases linearly with liquid to solid height ratio (r h ), irrespective of substrate loading and microreactor size, since large r h allows the inhibitors to diffuse in the liquid away from the solids, thus reducing product inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Importance of cellulase cocktails favoring hydrolysis of cellulose.

PubMed

Victoria, Juliet; Odaneth, Annamma; Lali, Arvind

2017-07-03

Depolymerization of lignocellulosic biomass is catalyzed by groups of enzymes whose action is influenced by substrate features and the composition of cellulase preparation. Cellulases contain a mixture of variety of enzymes, whose proportions dictate the saccharification of biomass. In the current study, four cellulase preparation varying in their composition were used to hydrolyze two types of alkali-treated biomass (aqueous ammonia-treated rice straw and sodium hydroxide-treated rice straw) to study the effect on catalytic rate, saccharification yields, and sugar release profile. We found that substrate features affected the extent of saccharification but had minimal effect on the sugar release pattern. In addition, complete hydrolysis to glucose was observed with enzyme preparation having at least a cellobiase units (CBU)/carboxymethyl cellulose (CMC) ratio (>0.15), while a modified enzyme ratio can be used for oligosaccharide synthesis. Thus, cellulase preparation with defined ratios of the three main enzymes can improve the saccharification which is of utmost importance in defining the success of lignocellulose-based economies.

Bioconversion of sugar cane crop residues with white-rot fungiPleurotus sp.

PubMed

Ortega, G M; Martínez, E O; Betancourt, D; González, A E; Otero, M A

1992-07-01

Four mushroom strains ofPleurotus spp. were cultivated on sugar cane crop residues for 30 days at 26°C. Biochemical changes affected the substrate as a result of fungal growth, in terms of nitrogen, lignin, cellulose and hemicellulose contents. All strains showed a strong ligninolytic activity together with variable cellulolytic and xylanolytic action.Pleurotus sajor-caju attacked lignin and cellulose at the same rate, showing a degradation of 47% and 55%, respectively. A better balance was shown by theP. ostreatus-P. pulmonarius hybrid, which exhibited the poorest cellulolytic action (39%) and the highest ligninolytic activity (67%). The average composition of mushroom fruit bodies, in terms of nitrogen, carbohydrates, fats and amino acid profiles, was determined. Crude protein and total carbohydrate varied from 23% to 33% and 36% to 68% of dry matter, respectively. Fat ranged from 3.3% to 4.7% and amino acid content from 12.2% to 22.2%. Slight evidence for a nitrogen fixing capability was encountered in the substrate to fruit body balance.

A smart approach to add antibacterial functionality to cellulosic pigment prints.

PubMed

Ibrahim, N A; Eid, B M; Elmaaty, T M Abou; El-Aziz, E Abd

2013-04-15

This study was devoted to enhancing the antibacterial functionality of pigment printed cotton, linen and viscose fabrics. Ag-NP's/PVP colloid, triclosan derivatives, chitosan or choline chloride was successfully incorporated into the pigment paste followed by printing and microwave curing to impart antibacterial activity to the cellulosic prints. Results obtained demonstrate that the modified pigment prints exhibit a remarkable antibacterial activity against the G+ve (Staphylococcus aureus) and G-ve (Escherichia coli) bacteria with a noticeable durability after 20 washing cycles without adversely affecting the printing and softness properties. The extent of printability and functionality of the nominated substrates are significantly governed by the type of: bio-active ingredient, binder, pigment and substrate. TEM, SEM and EDX analysis confirmed the formation of Ag-NP's/PVP colloid, of particle size range 7-14 nm, deposition of cross-linked-binder film onto the modified pigment prints, and the existence of elementary Ag and Si loaded onto fabrics surface, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

Some factors affecting efficiency of the ultrasound-aided enzymatic hydrolysis of cotton cellulose.

PubMed

Szabo, Orsolya Erzsebet; Csiszar, Emilia

2017-01-20

The efficiency of the enzymatic hydrolysis of cellulose with low frequency ultrasound (horn-type reactor) was investigated and characterized by the concentration of reducing sugars liberated. Small squares of bleached cotton fabric were used for comparing the efficiency of different agitation methods (i.e. magnetic stirring, horizontal and vertical mechanical agitation) and ultrasound. At the same enzyme dosage and substrate level, sonication at 40, 60 and 80% amplitudes (I diss : 16.2, 32.2 and 43.4W/cm 2 , respectively) intensified the hydrolysis over the most efficient mechanical agitation (i.e. magnetic stirring) alone by 15%, 24% and 54%, respectively. For mapping the ultrasonicated field, fabric layers positioned perpendicularly to the ultrasonic probe at different distances were hydrolysed. The optimal operating conditions were reached at 60% amplitude and 9mm The yield depended mainly on important factors such as amplitude, the presence of a reflector, distance from horn and form of substrate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of CdS quantum dot-functionalized cellulose nanocrystal films for anti-counterfeiting applications.

PubMed

Chen, L; Lai, C; Marchewka, R; Berry, R M; Tam, K C

2016-07-21

Structural colors and photoluminescence have been widely used for anti-counterfeiting and security applications. We report for the first time the use of CdS quantum dot (QD)-functionalized cellulose nanocrystals (CNCs) as building blocks to fabricate nanothin films via layer-by-layer (LBL) self-assembly for anti-counterfeiting applications. Both negatively- and positively-charged CNC/QD nanohybrids with a high colloidal stability and a narrow particle size distribution were prepared. The controllable LBL coating process was characterized by scanning electron microscopy and ellipsometry. The rigid structure of CNCs leads to nanoporous structured films on poly(ethylene terephthalate) (PET) substrates with high transmittance (above 70%) over the entire range of visible light and also resulted in increased hydrophilicity (contact angles of ∼40 degrees). Nanothin films on PET substrates showed good flexibility and enhanced stability in both water and ethanol. The modified PET films with structural colors from thin-film interference and photoluminescence from QDs can be used in anti-counterfeiting applications.

Engineering yeast consortia for surface-display of complex cellulosome structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Wilfred

As our society marches toward a more technologically advanced future, energy and environmental sustainability are some of the most challenging problems we face today. Biomass is one of the most abundant renewable-feedstock for sustainable production of biofuels. However, the main technological obstacle to more widespread uses of this resource is the lack of low-cost technologies to overcome the recalcitrant nature of the cellulosic structure, especially the hydrolysis step on highly ordered celluloses. In this proposal, we successfully engineered several efficient and inexpensive whole-cell biocatalysts in an effort to produce economically compatible and sustainable biofuels, namely cellulosic ethanol. Our approach wasmore » to display of a highly efficient cellulolytic enzyme complex, named cellulosome, on the surface of a historical ethanol producer Saccharomyces cerevisiae for the simultaneous and synergistic saccharification and fermentation of cellulose to ethanol. We first demonstrated the feasibility of assembling a mini-cellulosome by incubating E. coli lysates expressing three different cellulases. Resting cells displaying mini-cellulosomes produced 4-fold more ethanol from phosphoric acid-swollen cellulose (PASC) than cultures with only added enzymes. The flexibility to assemble the mini-cellulosome structure was further demonstrated using a synthetic yeast consortium through intracellular complementation. Direct ethanol production from PASC was demonstrated with resting cell cultures. To create a microorganism suitable for a more cost-effective process, called consolidated bioprocessing (CBP), a synthetic consortium capable of displaying mini-cellulosomes on the cell surface via intercellular complementation was created. To further improve the efficiency, a new adaptive strategy of employing anchoring and adaptor scaffoldins to amplify the number of enzymatic subunits was developed, resulting in the creation of an artificial tetravalent cellulosome on the yeast surface and a significant improvement in cellulosic ethanol production. Although this adaptive strategy is ideal for assembling more complex cellulosome for large-scale production of cellulosic ethanol, a substantially larger number of enzymes (up to 10 to 12) is needed to better mimic the natural cellulosome structures for practical usage of the technology.« less

In situ imaging of single carbohydrate-binding modules on cellulose microfibrils.

PubMed

Dagel, Daryl J; Liu, Yu-San; Zhong, Lanlan; Luo, Yonghua; Himmel, Michael E; Xu, Qi; Zeng, Yining; Ding, Shi-You; Smith, Steve

2011-02-03

The low efficiency of enzymes used in the bioprocessing of biomass for biofuels is one of the primary bottlenecks that must be overcome to make lignocellulosic biofuels cost-competitive. One of the rate-limiting factors is the accessibility of the cellulase enzymes to insoluble cellulolytic substrates, facilitated by surface absorption of the carbohydrate-binding modules (CBMs), a component of most cellulase systems. Despite their importance, reports of direct observation of CBM function and activity using microscopic methods are still uncommon. Here, we examine the site-specific binding of individual CBMs to crystalline cellulose in an aqueous environment, using the single molecule fluorescence method known as Defocused Orientation and Position Imaging (DOPI). Systematic orientations were observed that are consistent with the CBMs binding to the two opposite hydrophobic faces of the cellulose microfibril, with a well-defined orientation relative to the fiber axis. The approach provides in situ physical evidence indicating the CBMs bind with a well-defined orientation on those planes, thus supporting a binding mechanism driven by chemical and structural recognition of the cellulose surface.

Pretreating wheat straw by phosphoric acid plus hydrogen peroxide for enzymatic saccharification and ethanol production at high solid loading.

PubMed

Qiu, Jingwen; Ma, Lunjie; Shen, Fei; Yang, Gang; Zhang, Yanzong; Deng, Shihuai; Zhang, Jing; Zeng, Yongmei; Hu, Yaodong

2017-08-01

Wheat straw was pretreated by phosphoric acid plus hydrogen peroxide (PHP) for enzymatic hydrolysis and ethanol fermentation at high solid loadings. Results indicated solid loading could reach 20% with 77.4% cellulose-glucose conversion and glucose concentration of 164.9g/L in hydrolysate, it even was promoted to 25% with only 3.4% decrease on cellulose-glucose conversion as the pretreated-wheat straw was dewatered by air-drying. 72.9% cellulose-glucose conversion still was achieved as the minimized enzyme input of 20mg protein/g cellulose was employed for hydrolysis at 20% solid loading. In the corresponding conditions, 100g wheat straw can yield 11.2g ethanol with concentration of 71.2g/L by simultaneous saccharification and fermentation. Thus, PHP-pretreatment benefitted the glucose or ethanol yield at high solid loadings with lower enzyme input. Additionally, decreases on the maximal cellulase adsorption and the direct-orange/direct-blue indicated drying the PHP-pretreated substrates negatively affected the hydrolysis due to the shrinkage of cellulase-size-accommodable pores. Copyright © 2017 Elsevier Ltd. All rights reserved.

A diffuse reflectance comparative study of benzil inclusion within microcrystalline cellulose and beta-cyclodextrin.

PubMed

Vieira Ferreira, Luis F; Ferreira Machado, Isabel; Da Silva, José P; Oliveira, Anabela S

2004-02-01

Diffuse reflectance and laser-induced techniques were used to study photochemical and photophysical processes of benzil adsorbed on two solid powdered supports, microcrystalline cellulose and [small beta]-cyclodextrin. In both substrates, a distribution of ground-state benzil conformers exists, largely dominated by skew conformations where the carbonyl groups are twisted one to the other. Room temperature phosphorescence was observed in air-equilibrated samples in both cases. The decay times vary greatly and the largest lifetime was obtained for benzil/[small beta]-cyclodextrin, showing that this host's cavity accommodates benzil well, enhancing its room temperature phosphorescence. Triplet-triplet absorption of benzil entrapped in cellulose was detected and benzil ketyl radical formation also occurred. With benzil included into [small beta]-cyclodextrin, and following laser excitation, benzoyl radicals were detected on the millisecond timescale. Product analysis and identification of laser-irradiated benzil samples in the two hosts clearly showed that the main degradation photoproducts were benzoic acid and benzaldehyde. The main differences were a larger benzoic acid/benzaldehyde ratio in the case of cellulose and the formation of benzyl alcohol in this support.

Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes*

PubMed Central

Nam, Young-Woo; Nihira, Takanori; Arakawa, Takatoshi; Saito, Yuka; Kitaoka, Motomitsu; Nakai, Hiroyuki; Fushinobu, Shinya

2015-01-01

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes. PMID:26041776

Chitinase-like1/pom-pom1 and its homolog CTL2 are glucan-interacting proteins important for cellulose biosynthesis in Arabidopsis.

PubMed

Sánchez-Rodríguez, Clara; Bauer, Stefan; Hématy, Kian; Saxe, Friederike; Ibáñez, Ana Belén; Vodermaier, Vera; Konlechner, Cornelia; Sampathkumar, Arun; Rüggeberg, Markus; Aichinger, Ernst; Neumetzler, Lutz; Burgert, Ingo; Somerville, Chris; Hauser, Marie-Theres; Persson, Staffan

2012-02-01

Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane-located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils.

CHITINASE-LIKE1/POM-POM1 and Its Homolog CTL2 Are Glucan-Interacting Proteins Important for Cellulose Biosynthesis in Arabidopsis[W][OA

PubMed Central

Sánchez-Rodríguez, Clara; Bauer, Stefan; Hématy, Kian; Saxe, Friederike; Ibáñez, Ana Belén; Vodermaier, Vera; Konlechner, Cornelia; Sampathkumar, Arun; Rüggeberg, Markus; Aichinger, Ernst; Neumetzler, Lutz; Burgert, Ingo; Somerville, Chris; Hauser, Marie-Theres; Persson, Staffan

2012-01-01

Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane–located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils. PMID:22327741

Cellulose synthase stoichiometry in aspen differs from Arabidopsis and Norway spruce.

PubMed

Zhang, Xueyang; Dominguez, Pia Guadalupe; Kumar, Manoj; Bygdell, Joakim; Miroshnichenko, Sergey; Sundberg, Bjorn; Wingsle, Gunnar; Niittyla, Totte

2018-05-14

Cellulose is synthesised at the plasma membrane by cellulose synthase complexes (CSCs) containing cellulose synthases (CESAs). Genetic analysis and CESA isoform quantification indicate that cellulose in the secondary cell walls of Arabidopsis (Arabidopsis thaliana) is synthesised by isoforms CESA4, CESA7 and CESA8 in equimolar amounts. Here, we used quantitative proteomics to investigate whether the CSC model based on Arabidopsis secondary cell wall CESA stoichiometry can be applied to the angiosperm tree aspen (Populus tremula) and the gymnosperm tree Norway spruce (Picea abies). In the developing xylem of aspen the secondary cell wall CESA stoichiometry was 3:2:1 for PtCESA8a/b : PtCESA4 : PtCESA7a/b, while in Norway spruce the stoichiometry was 1:1:1 as previously observed in Arabidopsis. Furthermore, in aspen tension wood the secondary cell wall CESA stoichiometry changed to 8:3:1 for PtCESA8a/b : PtCESA4 : PtCESA7a/b. PtCESA8b represented 73% of the total secondary cell wall CESA pool, and quantitative PCR analysis of CESA transcripts in cryo-sectioned tension wood revealed increased PtCESA8b expression during formation of the cellulose-enriched gelatinous layer while the transcripts of PtCESA4, PtCESA7a/b and PtCESA8a decreased. A wide-angle X-ray scattering analysis showed that the shift in CESA stoichiometry in tension wood coincided with an increase in crystalline cellulose microfibril diameter suggesting that the CSC CESA composition influences microfibril properties. The aspen CESA stoichiometry results raise the possibility of alternative CSC models, and suggest that homomeric PtCESA8b complexes are responsible for cellulose biosynthesis in the gelatinous layer in tension wood. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Morphological structure of Gluconacetobacter xylinus cellulose and cellulose-based organic-inorganic composite materials

NASA Astrophysics Data System (ADS)

Smyslov, R. Yu; Ezdakova, K. V.; Kopitsa, G. P.; Khripunov, A. K.; Bugrov, A. N.; Tkachenko, A. A.; Angelov, B.; Pipich, V.; Szekely, N. K.; Baranchikov, A. E.; Latysheva, E.; Chetverikov, Yu O.; Haramus, V.

2017-05-01

Scanning electron microscopy, ultra-small-angle neutron scattering (USANS), small-angle neutron and X-ray scattering (SANS and SAXS), as well as low-temperature nitrogen adsorption, were used in the studies of micro- and mesostructure of polymer matrix prepared from air-dry preliminarily disintegrated cellulose nano-gel film (synthesized by Gluconacetobacter xylinus) and the composites based on this bacterial cellulose. The composites included ZrO2 nanoparticles, Tb3+ in the form of low molecular weight salt and of metal-polymer complex with poly(vinylpyrrolydone)-poly(methacryloyl-o-aminobenzoic acid) copolymer. The combined analysis of the data obtained allowed revealing three levels of fractal organization in mesostructure of G. xylinus cellulose and its composites. It was shown that both the composition and an aggregation state of dopants have a significant impact on the structural characteristics of the organic-inorganic composites. The composites containing Tb3+ ions demonstrate efficient luminescence; its intensity is an order of magnitude higher in the case of the composites with the metal-polymer complex. It was found that there is the optimal content of ZrO2 nanoparticles in composites resulting in increased Tb3+ luminescence.

Synthesis and characterization of novel polymers from non-petroleum sources for use in enhanced oil recovery. Final report, July 1, 1983-June 30, 1984

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hogen-Esch, T.E.

1984-01-01

Accomplishments for the past year are discussed for grafting of acrylamide to: (1) starch and related polysaccharides; and (2) cellulose solutions. In grafting acrylamide to various polysaccharide substrates such as okra polysaccharide, yellow dextrin, waxy corn starch, potato amylose, gum arabic, the efficiency of Ce/sup 4 +/ as initiator was found to vary from 0.02 to 0.89, depending on reaction conditions. Okra polysaccharide was isolated, characterized, and evaluated for use in enhanced oil recovery. A series of experiments designed to increase the viscosifying power of certain polymers by chain extension techniques has also been conducted. Characterization of the polymers bymore » ultracentrifugation, size exclusion chromatography, membrane filtration, multi-cell equilibrium dialysis, and rheological studies has also been done. In grafting of acrylamide to cellulose solutions the following two courses were taken: (1) dissolution of cellulose in 70% aqueous zinc chloride, followed by Ce/sup 4 +/ initiated grafting of acrylamide, and (2) introduction of a 1,2-diol substituent onto the anhydroglucose units of the cellulose chain via dissolution of cellulose in concentrated aqueous NaOH, followed by treatment with glyceryl chlorohydrin. Considerable progress has been made via both approaches. 11 refs., 1 fig., 1 tab.« less

Catalytic conversion of cellulose to levulinic acid by metal chlorides.

PubMed

Peng, Lincai; Lin, Lu; Zhang, Junhua; Zhuang, Junping; Zhang, Beixiao; Gong, Yan

2010-08-02

The catalytic performance of various metal chlorides in the conversion of cellulose to levulinic acid in liquid water at high temperatures was investigated. The effects of reaction parameters on the yield of levulinic acid were also explored. The results showed that alkali and alkaline earth metal chlorides were not effective in conversion of cellulose, while transition metal chlorides, especially CrCl(3), FeCl(3) and CuCl(2) and a group IIIA metal chloride (AlCl(3)), exhibited high catalytic activity. The catalytic performance was correlated with the acidity of the reaction system due to the addition of the metal chlorides, but more dependent on the type of metal chloride. Among those metal chlorides, chromium chloride was found to be exceptionally effective for the conversion of cellulose to levulinic acid, affording an optimum yield of 67 mol % after a reaction time of 180 min, at 200 degrees C, with a catalyst dosage of 0.02 M and substrate concentration of 50 wt %. Chromium metal, most of which was present in its oxide form in the solid sample and only a small part in solution as Cr3+ ion, can be easily separated from the resulting product mixture and recycled. Finally, a plausible reaction scheme for the chromium chloride catalyzed conversion of cellulose in water was proposed.

Microbial production of biopolymers from the renewable resource wheat straw.

PubMed

Gasser, E; Ballmann, P; Dröge, S; Bohn, J; König, H

2014-10-01

Production of poly-ß-hydroxybutyrate (PHB) and the chemical basic compound lactate from the agricultural crop 'wheat straw' as a renewable carbon resource. A thermal pressure hydrolysis procedure for the breakdown of wheat straw was applied. By this means, the wheat straw was converted into a partially solubilized hemicellulosic fraction, consisting of sugar monomers, and an insoluble cellulosic fraction, containing cellulose, lignin and a small portion of hemicellulose. The insoluble cellulosic fraction was further hydrolysed by commercial enzymes in monomers. The production of PHB from the sugar monomers originating from hemicellulose or cellulose was achieved by the isolates Bacillus licheniformis IMW KHC 3 and Bacillus megaterium IMW KNaC 2. The basic chemical compound, lactate, a starting compound for the production of polylactide (PLA), was formed by some heterofermentative lactic acid bacteria (LAB) able to grow with xylose from the hemicellulosic wheat straw hydrolysate. Two strains were selected which were able to produce PHB from the sugars both from the hemicellulosic and the cellulosic fraction of the wheat straw. In addition, some of the LAB tested were capable of producing lactate from the hemicellulosic hydrolysate. The renewable resource wheat straw could serve as a substrate for microbiologically produced basic chemicals and biodegradable plastics. © 2014 The Society for Applied Microbiology.

Optimization of enzyme complexes for efficient hydrolysis of corn stover to produce glucose.

PubMed

Yu, Xiaoxiao; Liu, Yan; Meng, Jiatong; Cheng, Qiyue; Zhang, Zaixiao; Cui, Yuxiao; Liu, Jiajing; Teng, Lirong; Lu, Jiahui; Meng, Qingfan; Ren, Xiaodong

2015-05-01

Hydrolysis of cellulose to glucose is the critical step for transferring the lignocellulose to the industrial chemicals. For improving the conversion rate of cellulose of corn stover to glucose, the cocktail of celllulase with other auxiliary enzymes and chemicals was studied in this work. Single factor tests and Response Surface Methodology (RSM) were applied to optimize the enzyme mixture, targeting maximum glucose release from corn stover. The increasing rate of glucan-to-glucose conversion got the higher levels while the cellulase was added 1.7μl tween-80/g cellulose, 300μg β-glucosidase/g cellulose, 400μg pectinase/g cellulose and 0.75mg/ml sodium thiosulphate separately in single factor tests. To improve the glucan conversion, the β-glucosidase, pectinase and sodium thiosulphate were selected for next step optimization with RSM. It is showed that the maximum increasing yield was 45.8% at 377μg/g cellulose Novozyme 188, 171μg/g cellulose pectinase and 1mg/ml sodium thiosulphate.

The biofilm matrix polysaccharides cellulose and alginate both protect Pseudomonas putida mt-2 against reactive oxygen species generated under matric stress and copper exposure.

PubMed

Svenningsen, Nanna B; Martínez-García, Esteban; Nicolaisen, Mette H; de Lorenzo, Victor; Nybroe, Ole

2018-06-01

In natural environments most bacteria live in biofilms embedded in complex matrices of extracellular polymeric substances (EPS). This lifestyle is known to increase protection against environmental stress. Pseudomonas putida mt-2 harbours genes for the production of at least four different EPS polysaccharides, including alginate and cellulose. Little is known about the functional properties of cellulose, while alginate attenuates the accumulation of reactive oxygen species (ROS) caused by matric stress. By using mutants that are deficient in either alginate or cellulose production we show that even cellulose attenuates the accumulation of matric stress-induced ROS for cells in biofilms. Further, both cellulose and alginate attenuate ROS generated through exposure to copper. Interestingly, the two EPS polysaccharides protect cells in both liquid culture and in biofilms against ROS caused by matric stress, indicating that cellulose and alginate do not need to be produced as an integral part of the biofilm lifestyle to provide tolerance towards environmental stressors.

Application of X-ray and neutron small angle scattering techniques to study the hierarchical structure of plant cell walls: a review.

PubMed

Martínez-Sanz, Marta; Gidley, Michael J; Gilbert, Elliot P

2015-07-10

Plant cell walls present an extremely complex structure of hierarchically assembled cellulose microfibrils embedded in a multi-component matrix. The biosynthesis process determines the mechanism of cellulose crystallisation and assembly, as well as the interaction of cellulose with other cell wall components. Thus, a knowledge of cellulose microfibril and bundle architecture, and the structural role of matrix components, is crucial for understanding cell wall functional and technological roles. Small angle scattering techniques, combined with complementary methods, provide an efficient approach to characterise plant cell walls, covering a broad and relevant size range while minimising experimental artefacts derived from sample treatment. Given the system complexity, approaches such as component extraction and the use of plant cell wall analogues are typically employed to enable the interpretation of experimental results. This review summarises the current research status on the characterisation of the hierarchical structure of plant cell walls using small angle scattering techniques. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Isolation and characterization of two cellulose morphology mutants of Gluconacetobacter hansenii ATCC23769 producing cellulose with lower crystallinity

DOE PAGES

Deng, Ying; Nagachar, Nivedita; Fang, Lin; ...

2015-03-19

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To addressmore » this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.« less

Isolation and characterization of two cellulose morphology mutants of Gluconacetobacter hansenii ATCC23769 producing cellulose with lower crystallinity.

PubMed

Deng, Ying; Nagachar, Nivedita; Fang, Lin; Luan, Xin; Catchmark, Jeffrey M; Tien, Ming; Kao, Teh-hui

2015-01-01

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.

Isolation and Characterization of Two Cellulose Morphology Mutants of Gluconacetobacter hansenii ATCC23769 Producing Cellulose with Lower Crystallinity

PubMed Central

Deng, Ying; Nagachar, Nivedita; Fang, Lin; Luan, Xin; Catchmark, Jeffrey M.; Tien, Ming; Kao, Teh-hui

2015-01-01

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose. PMID:25790428

Effects of different fermentation methods on bacterial cellulose and acid production by Gluconacetobacter xylinus in Cantonese-style rice vinegar.

PubMed

Fu, Liang; Chen, Siqian; Yi, Jiulong; Hou, Zongxia

2014-07-01

A strain of acidogenic bacterium was isolated from the fermentation liquid of Cantonese-style rice vinegar produced by traditional surface fermentation. 16S rDNA identification confirmed the bacterium as Gluconacetobacter xylinus, which synthesizes bacterial cellulose, and the acid productivity of the strain was investigated. In the study, the effects of the membrane integrity and the comparison of the air-liquid interface membrane with immerged membrane on total acidity, cellulose production, alcohol dehydrogenase (ADH) activity and number of bacteria were investigated. The cellulose membrane and the bacteria were observed under SEM for discussing their relationship. The correlations between oxygen consumption and total acid production rate were compared in surface and shake flask fermentation. The results showed the average acid productivity of the strain was 0.02g/(100mL/h), and the integrity of cellulose membrane in surface fermentation had an important effect on total acidity and cellulose production. With a higher membrane integrity, the total acidity after 144 h of fermentation was 3.75 g/100 mL, and the cellulose production was 1.71 g/100 mL after 360 h of fermentation. However, when the membrane was crushed by mechanical force, the total acidity and the cellulose production were as low as 0.36 g/100 mL and 0.14 g/100 mL, respectively. When the cellulose membrane was forced under the surface of fermentation liquid, the total acid production rate was extremely low, but the activity of ADH in the cellulose membrane was basically the same with the one above the liquid surface. The bacteria were mainly distributed in the cellulose membrane during the fermentation. The bacterial counts in surface fermentation were more than in the shake flask fermentation and G. xylinus consumed the substrate faster, in surface fermentation than in shake flask fermentation. The oxygen consumption rate and total acid production rate of surface fermentation were respectively 26.13 times and 2.92 times that of shake flask fermentation.

Cellulose synthesizing Complexes in Vascular Plants andProcaryotes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Richard M, Jr; Saxena, Inder Mohan

2009-07-07

Continuing the work initiated under DE-FG03-94ER20145, the following major accomplishments were achieved under DE-FG02-03ER15396 from 2003-2007: (a) we purified the acsD gene product of the Acetobacter cellulose synthase operon as well as transferred the CesA cellulose gene from Gossypium into E. coli in an attempt to crystallize this protein for x-ray diffraction structural analysis; however, crystallization attempts proved unsuccessful; (b) the Acetobacter cellulose synthase operon was successfully incorporated into Synechococcus, a cyanobacterium2; (c) this operon in Synechococcus was functionally expressed; (d) we successfully immunolabeled Vigna cellulose and callose synthase components and mapped their distribution before and after wounding; (e) wemore » developed a novel method to produce replicas of cellulose synthases in tobacco BY-2 cells, and we demonstrated the cytoplasmic domain of the rosette TC; (f) from the moss Physcomitrella, we isolated two full-length cDNA sequences of cellulose synthase (PpCesA1 and PpCesA2) and attempted to obtain full genomic DNA sequences; (g) we examined the detailed molecular structure of a new form of non-crystalline cellulose known as nematic ordered cellulose (=NOC)3.« less

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

PubMed

Fujita, Miki; Lechner, Bettina; Barton, Deborah A; Overall, Robyn L; Wasteneys, Geoffrey O

2012-02-01

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule-cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.