Efficient xylose fermentation by the brown rot fungus Neolentinus lepideus.

PubMed

Okamoto, Kenji; Kanawaku, Ryuichi; Masumoto, Masaru; Yanase, Hideshi

2012-02-10

The efficient production of bioethanol on an industrial scale requires the use of renewable lignocellulosic biomass as a starting material. A limiting factor in developing efficient processes is identifying microorganisms that are able to effectively ferment xylose, the major pentose sugar found in hemicellulose, and break down carbohydrate polymers without pre-treatment steps. Here, a basidiomycete brown rot fungus was isolated as a new biocatalyst with unprecedented fermentability, as it was capable of converting not only the 6-carbon sugars constituting cellulose, but also the major 5-carbon sugar xylose in hemicelluloses, to ethanol. The fungus was identified as Neolentinus lepideus and was capable of assimilating and fermenting xylose to ethanol in yields of 0.30, 0.33, and 0.34 g of ethanol per g of xylose consumed under aerobic, oxygen-limited, and anaerobic conditions, respectively. A small amount of xylitol was detected as the major by-product of xylose metabolism. N. lepideus produced ethanol from glucose, mannose, galactose, cellobiose, maltose, and lactose with yields ranging from 0.34 to 0.38 g ethanol per g sugar consumed, and also exhibited relatively favorable conversion of non-pretreated starch, xylan, and wheat bran. These results suggest that N. lepideus is a promising candidate for cost-effective and environmentally friendly ethanol production from lignocellulosic biomass. To our knowledge, this is the first report on efficient ethanol fermentation from various carbohydrates, including xylose, by a naturally occurring brown rot fungus. Copyright © 2011 Elsevier Inc. All rights reserved.

Genetic improvement of native xylose-fermenting yeasts for ethanol production.

PubMed

Harner, Nicole K; Wen, Xin; Bajwa, Paramjit K; Austin, Glen D; Ho, Chi-Yip; Habash, Marc B; Trevors, Jack T; Lee, Hung

2015-01-01

Lignocellulosic substrates are the largest source of fermentable sugars for bioconversion to fuel ethanol and other valuable compounds. To improve the economics of biomass conversion, it is essential that all sugars in potential hydrolysates be converted efficiently into the desired product(s). While hexoses are fermented into ethanol and some high-value chemicals, the bioconversion of pentoses in hydrolysates remains inefficient. This remains one of the key challenges in lignocellulosic biomass conversion. Native pentose-fermenting yeasts can ferment both glucose and xylose in lignocellulosic biomass to ethanol. However, they perform poorly in the presence of hydrolysate inhibitors, exhibit low ethanol tolerance and glucose repression, and ferment pentoses less efficiently than the main hexoses glucose and mannose. This paper reviews classical and molecular strain improvement strategies applied to native pentose-fermenting yeasts for improved ethanol production from xylose and lignocellulosic substrates. We focus on Pachysolen tannophilus, Scheffersomyces (Candida) shehatae, Scheffersomyces (Pichia) stipitis, and Spathaspora passalidarum which are good ethanol producers among the native xylose-fermenting yeasts. Strains obtained thus far are not robust enough for efficient ethanol production from lignocellulosic hydrolysates and can benefit from further improvements.

Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation.

PubMed

Peng, Bingyin; Huang, Shuangcheng; Liu, Tingting; Geng, Anli

2015-05-17

Xylose isomerase (XI) catalyzes the conversion of xylose to xylulose, which is the key step for anaerobic ethanolic fermentation of xylose. Very few bacterial XIs can function actively in Saccharomyces cerevisiae. Here, we illustrate a group of XIs that would function for xylose fermentation in S. cerevisiae through phylogenetic analysis, recombinant yeast strain construction, and xylose fermentation. Phylogenetic analysis of deposited XI sequences showed that XI evolutionary relationship was highly consistent with the bacterial taxonomic orders and quite a few functional XIs in S. cerevisiae were clustered with XIs from mammal gut Bacteroidetes group. An XI from Bacteroides valgutus in this cluster was actively expressed in S. cerevisiae with an activity comparable to the fungal XI from Piromyces sp. Two XI genes were isolated from the environmental metagenome and they were clustered with XIs from environmental Bacteroidetes group. These two XIs could not be expressed in yeast with activity. With the XI from B. valgutus expressed in S. cerevisiae, background yeast strains were optimized by pentose metabolizing pathway enhancement and adaptive evolution in xylose medium. Afterwards, more XIs from the mammal gut Bacteroidetes group, including those from B. vulgatus, Tannerella sp. 6_1_58FAA_CT1, Paraprevotella xylaniphila and Alistipes sp. HGB5, were individually transformed into S. cerevisiae. The known functional XI from Orpinomyces sp. ukk1, a mammal gut fungus, was used as the control. All the resulting recombinant yeast strains were able to ferment xylose. The respiration-deficient strains harboring B. vulgatus and Alistipes sp. HGB5 XI genes respectively obtained specific xylose consumption rate of 0.662 and 0.704 g xylose gcdw(-1) h(-1), and ethanol specific productivity of 0.277 and 0.283 g ethanol gcdw(-1) h(-1), much comparable to those obtained by the control strain carrying Orpinomyces sp. ukk1 XI gene. This study demonstrated that XIs clustered in the

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects.

PubMed

Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

2016-02-25

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.

Co-fermentation of glucose, xylose and/or cellobiose by yeast

DOEpatents

Jeffries, Thomas W.; Willis, Laura B.; Long, Tanya M.; Su, Yi-Kai

2013-09-10

Provided herein are methods of using yeast cells to produce ethanol by contacting a mixture comprising xylose with a Spathaspora yeast cell under conditions suitable to allow the yeast to ferment at least a portion of the xylose to ethanol. The methods allow for efficient ethanol production from hydrolysates derived from lignocellulosic material and sugar mixtures including at least xylose and glucose or xylose, glucose and cellobiose.

Enhanced xylose fermentation by engineered yeast expressing NADH oxidase through high cell density inoculums.

PubMed

Zhang, Guo-Chang; Turner, Timothy L; Jin, Yong-Su

2017-03-01

Accumulation of reduced byproducts such as glycerol and xylitol during xylose fermentation by engineered Saccharomyces cerevisiae hampers the economic production of biofuels and chemicals from cellulosic hydrolysates. In particular, engineered S. cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD + -linked xylitol dehydrogenase (XDH) produces substantial amounts of the reduced byproducts under anaerobic conditions due to the cofactor difference of XR and XDH. While the additional expression of a water-forming NADH oxidase (NoxE) from Lactococcus lactis in engineered S. cerevisiae with the XR/XDH pathway led to reduced glycerol and xylitol production and increased ethanol yields from xylose, volumetric ethanol productivities by the engineered yeast decreased because of growth defects from the overexpression of noxE. In this study, we introduced noxE into an engineered yeast strain (SR8) exhibiting near-optimal xylose fermentation capacity. To overcome the growth defect caused by the overexpression of noxE, we used a high cell density inoculum for xylose fermentation by the SR8 expressing noxE. The resulting strain, SR8N, not only showed a higher ethanol yield and lower byproduct yields, but also exhibited a high ethanol productivity during xylose fermentation. As noxE overexpression elicits a negligible growth defect on glucose conditions, the beneficial effects of noxE overexpression were substantial when a mixture of glucose and xylose was used. Consumption of glucose led to rapid cell growth and therefore enhanced the subsequent xylose fermentation. As a result, the SR8N strain produced more ethanol and fewer byproducts from a mixture of glucose and xylose than the parental SR8 strain without noxE overexpression. Our results suggest that the growth defects from noxE overexpression can be overcome in the case of fermenting lignocellulose-derived sugars such as glucose and xylose.

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

PubMed Central

Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

2016-01-01

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review. PMID:26927067

Inhibitor tolerance of a recombinant flocculating industrial Saccharomyces cerevisiae strain during glucose and xylose co-fermentation.

PubMed

Li, Yun-Cheng; Gou, Zi-Xi; Zhang, Ying; Xia, Zi-Yuan; Tang, Yue-Qin; Kida, Kenji

Lignocellulose-derived inhibitors have negative effects on the ethanol fermentation capacity of Saccharomyces cerevisiae. In this study, the effects of eight typical inhibitors, including weak acids, furans, and phenols, on glucose and xylose co-fermentation of the recombinant xylose-fermenting flocculating industrial S. cerevisiae strain NAPX37 were evaluated by batch fermentation. Inhibition on glucose fermentation, not that on xylose fermentation, correlated with delayed cell growth. The weak acids and the phenols showed additive effects. The effect of inhibitors on glucose fermentation was as follows (from strongest to weakest): vanillin>phenol>syringaldehyde>5-HMF>furfural>levulinic acid>acetic acid>formic acid. The effect of inhibitors on xylose fermentation was as follows (from strongest to weakest): phenol>vanillin>syringaldehyde>furfural>5-HMF>formic acid>levulinic acid>acetic acid. The NAPX37 strain showed substantial tolerance to typical inhibitors and showed good fermentation characteristics, when a medium with inhibitor cocktail or rape straw hydrolysate was used. This research provides important clues for inhibitors tolerance of recombinant industrial xylose-fermenting S. cerevisiae. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Evaluation of fermentation kinetics of acid-treated corn cob hydrolysate for xylose fermentation in the presence of acetic acid by Pichia stipitis.

PubMed

Kashid, Mohan; Ghosalkar, Anand

2017-08-01

The efficient utilization of lignocellulosic biomass for ethanol production depends on the fermentability of the biomass hydrolysate obtained after pretreatment. In this work we evaluated the kinetics of ethanol production from xylose using Pichia stipitis in acid-treated corn cob hydrolysate. Acetic acid is one of the main inhibitors in corn cob hydrolysate that negatively impacts kinetics of xylose fermentation by P. stipitis. Unstructured kinetic model has been formulated that describes cell mass growth and ethanol production as a function of xylose, oxygen, ethanol, and acetic acid concentration. Kinetic parameters were estimated under different operating conditions affecting xylose fermentation. This is the first report on kinetics of xylose fermentation by P. stipitis which includes inhibition of acetic acid on growth and product formation. In the presence of acetic acid in the hydrolysate, the model accurately predicted reduction in maximum specific growth rate (from 0.23 to 0.15 h -1 ) and increase in ethanol yield per unit biomass (from 3 to 6.2 gg -1 ), which was also observed during experimental trials. Presence of acetic acid in the fermentation led to significant reduction in the cell growth rate, reduction in xylose consumption and ethanol production rate. The developed model accurately described physiological state of P. stipitis during corn cob hydrolysate fermentation. Proposed model can be used to predict the influence of xylose, ethanol, oxygen, and acetic acid concentration on cell growth and ethanol productivity in industrial fermentation.

Fermentation of xylose into ethanol by a new fungus strain Pestalotiopsis sp. XE-1.

PubMed

Pang, Zong-wen; Liang, Jing-juan; Huang, Ri-bo

2011-08-01

A new fungus, Pestalotiopsis sp. XE-1, which produced ethanol from xylose with yield of 0.47 g ethanol/g of consumed xylose was isolated. It also produced ethanol from arabinose, glucose, fructose, mannose, galactose, cellobiose, maltose, and sucrose with yields of 0.38, 0.47, 0.45, 0.46, 0.31, 0.25, 0.31, and 0.34 g ethanol/g of sugar consumed, respectively. It produced maximum ethanol from xylose at pH 6.5, 30°C under a semi-aerobic condition. Acetic acid produced in xylose fermenting process inhibited ethanol production of XE-1. The ethanol yield in the pH-uncontrolled batch fermentation was about 27% lower than that in the pH-controlled one. The ethanol tolerance of XE-1 was higher than most xylose-fermenting, ethanol-producing microbes, but lower than Saccharomyces cerevisiae and Hansenula polymorpha. XE-1 showed tolerance to high concentration of xylose, and was able to grow and produce ethanol even when it was cultivated in 97.71 g/l xylose.

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.

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.

Recombinant lactobacillus for fermentation of xylose to lactic acid and lactate

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Franden, Mary Ann; Mc Millan, James D.; Finkelstein, Mark

1998-01-01

A recombinant Lactobacillus MONT4 is provided which has been genetically engineered with xylose isomerase and xylulokinase genes from Lactobacillus pentosus to impart to the Lactobacillus MONT4 the ability to ferment lignocellulosic biomass containing xylose to lactic acid.

Recombinant lactobacillus for fermentation of xylose to lactic acid and lactate

DOEpatents

Picataggio, S.K.; Zhang, M.; Franden, M.A.; McMillan, J.D.; Finkelstein, M.

1998-08-25

A recombinant Lactobacillus MONT4 is provided which has been genetically engineered with xylose isomerase and xylulokinase genes from Lactobacillus pentosus to impart to the Lactobacillus MONT4 the ability to ferment lignocellulosic biomass containing xylose to lactic acid. 4 figs.

A Novel Technique that Enables Efficient Conduct of Simultaneous Isomerization and Fermentation (SIF) of Xylose

NASA Astrophysics Data System (ADS)

Rao, Kripa; Chelikani, Silpa; Relue, Patricia; Varanasi, Sasidhar

Of the sugars recovered from lignocellulose, D-glucose can be readily converted into ethanol by baker's or brewer's yeast (Saccharomyces cerevisiae). However, xylose that is obtained by the hydrolysis of the hemicellulosic portion is not fermentable by the same species of yeasts. Xylose fermentation by native yeasts can be achieved via isomerization of xylose to its ketose isomer, xylulose. Isomerization with exogenous xylose isomerase (XI) occurs optimally at a pH of 7-8, whereas subsequent fermentation of xylulose to ethanol occurs at a pH of 4-5. We present a novel scheme for efficient isomerization of xylose to xylulose at conditions suitable for the fermentation by using an immobilized enzyme system capable of sustaining two different pH microenvironments in a single vessel. The proof-of-concept of the two-enzyme pellet is presented, showing conversion of xylose to xylulose even when the immobilized enzyme pellets are suspended in a bulk solution whose pH is sub-optimal for XI activity. The co-immobilized enzyme pellets may prove extremely valuable in effectively conducting "simultaneous isomerization and fermentation" (SIF) of xylose. To help further shift the equilibrium in favor of xylulose formation, sodium tetraborate (borax) was added to the isomerization solution. Binding of tetrahydroxyborate ions to xylulose effectively reduces the concentration of xylulose and leads to increased xylose isomerization. The formation of tetrahydroxyborate ions and the enhancement in xylulose production resulting from the complexation was studied at two different bulk pH values. The addition of 0.05 M borax to the isomerization solution containing our co-immobilized enzyme pellets resulted in xylose to xylulose conversion as high as 86% under pH conditions that are suboptimal for XI activity. These initial findings, which can be optimized for industrial conditions, have significant potential for increasing the yield of ethanol from xylose in an SIF approach.

Deletion of FPS1, Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

PubMed Central

Wei, Na; Xu, Haiqing; Kim, Soo Rin

2013-01-01

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD+/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation. PMID:23475614

Simultaneous fermentation of glucose and xylose at elevated temperatures co-produces ethanol and xylitol through overexpression of a xylose-specific transporter in engineered Kluyveromyces marxianus.

PubMed

Zhang, Biao; Zhang, Jia; Wang, Dongmei; Han, Ruixiang; Ding, Rui; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

2016-09-01

Engineered Kluyveromyces marxianus strains were constructed through over-expression of various transporters for simultaneous co-fermentation of glucose and xylose. The glucose was converted into ethanol, whereas xylose was converted into xylitol which has higher value than ethanol. Over-expressing xylose-specific transporter ScGAL2-N376F mutant enabled yeast to co-ferment glucose and xylose and the co-fermentation ability was obviously improved through increasing ScGAL2-N376F expression. The production of glycerol was blocked and acetate production was reduced by disrupting gene KmGPD1. The obtained K. marxianus YZJ119 utilized 120g/L glucose and 60g/L xylose simultaneously and produced 50.10g/L ethanol and 55.88g/L xylitol at 42°C. The yield of xylitol from consumed xylose was over 98% (0.99g/g). Through simultaneous saccharification and co-fermentation at 42°C, YZJ119 produced a maximal concentration of 44.58g/L ethanol and 32.03g/L xylitol or 29.82g/L ethanol and 31.72g/L xylitol, respectively, from detoxified or non-detoxified diluted acid pretreated corncob. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae.

PubMed

Matsushika, Akinori; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki

2009-04-01

In this study, five recombinant Saccharomyces cerevisiae strains were compared for their xylose-fermenting ability. The most efficient xylose-to-ethanol fermentation was found by using the industrial strain MA-R4, in which the genes for xylose reductase and xylitol dehydrogenase from Pichia stipitis along with an endogenous xylulokinase gene were expressed by chromosomal integration of the flocculent yeast strain IR-2. The MA-R4 strain rapidly converted xylose to ethanol with a low xylitol yield. Furthermore, the MA-R4 strain had the highest ethanol production when fermenting not only a mixture of glucose and xylose, but also mixed sugars in the detoxified hydrolysate of wood chips. These results collectively suggest that MA-R4 may be a suitable recombinant strain for further study into large-scale ethanol production from mixed sugars present in lignocellulosic hydrolysates.

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

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

Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B

2011-01-01

Background: Butanol is a second generation biofuel produced by Clostridium acetobutylicum through acetonebutanol- ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production. Results: We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylosemore » substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates. Conclusion: Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.« less

Improved bioethanol production using fusants of Saccharomyces cerevisiae and xylose-fermenting yeasts.

PubMed

Kumari, Rajni; Pramanik, K

2012-06-01

The present research deals with the development of a hybrid yeast strain with the aim of converting pentose and hexose sugar components of lignocellulosic substrate to bioethanol by fermentation. Different fusant strains were obtained by fusing protoplasts of Saccharomyces cerevisiae and xylose-fermenting yeasts such as Pachysolen tannophilus, Candida shehatae and Pichia stipitis. The fusants were sorted by fluorescent-activated cell sorter and further confirmed by molecular characterization. The fusants were evaluated by fermentation of glucose-xylose mixture and the highest ethanol producing fusant was used for further study to ferment hydrolysates produced by acid pretreatment and enzymatic hydrolysis of cotton gin waste. Among the various fusant and parental strains used under present study, RPR39 was found to be stable and most efficient strain giving maximum ethanol concentration (76.8 ± 0.31 g L(-1)), ethanol productivity (1.06 g L(-1) h(-1)) and ethanol yield (0.458 g g(-1)) by fermentation of glucose-xylose mixture under test conditions. The fusant has also shown encouraging result in fermenting hydrolysates of cotton gin waste with ethanol concentration of 7.08 ± 0.142 g L(-1), ethanol yield of 0.44 g g(-1), productivity of 0.45 g L(-1) h(-1) and biomass yield of 0.40 g g(-1).

Effect of manganese ions on ethanol fermentation by xylose isomerase expressing Saccharomyces cerevisiae under acetic acid stress.

PubMed

Ko, Ja Kyong; Um, Youngsoon; Lee, Sun-Mi

2016-12-01

The efficient fermentation of lignocellulosic hydrolysates in the presence of inhibitors is highly desirable for bioethanol production. Among the inhibitors, acetic acid released during the pretreatment of lignocellulose negatively affects the fermentation performance of biofuel producing organisms. In this study, we evaluated the inhibitory effects of acetic acid on glucose and xylose fermentation by a high performance engineered strain of xylose utilizing Saccharomyces cerevisiae, SXA-R2P-E, harboring a xylose isomerase based pathway. The presence of acetic acid severely decreased the xylose fermentation performance of this strain. However, the acetic acid stress was alleviated by metal ion supplementation resulting in a 52% increased ethanol production rate under 2g/L of acetic acid stress. This study shows the inhibitory effect of acetic acid on an engineered isomerase-based xylose utilizing strain and suggests a simple but effective method to improve the co-fermentation performance under acetic acid stress for efficient bioethanol production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transcription analysis of recombinant industrial and laboratory Saccharomyces cerevisiae strains reveals the molecular basis for fermentation of glucose and xylose

PubMed Central

2014-01-01

Background There has been much research on the bioconversion of xylose found in lignocellulosic biomass to ethanol by genetically engineered Saccharomyces cerevisiae. However, the rate of ethanol production from xylose in these xylose-utilizing yeast strains is quite low compared to their glucose fermentation. In this study, two diploid xylose-utilizing S. cerevisiae strains, the industrial strain MA-R4 and the laboratory strain MA-B4, were employed to investigate the differences between anaerobic fermentation of xylose and glucose, and general differences between recombinant yeast strains, through genome-wide transcription analysis. Results In MA-R4, many genes related to ergosterol biosynthesis were expressed more highly with glucose than with xylose. Additionally, these ergosterol-related genes had higher transcript levels in MA-R4 than in MA-B4 during glucose fermentation. During xylose fermentation, several genes related to central metabolic pathways that typically increase during growth on non-fermentable carbon sources were expressed at higher levels in both strains. Xylose did not fully repress the genes encoding enzymes of the tricarboxylic acid and respiratory pathways, even under anaerobic conditions. In addition, several genes involved in spore wall metabolism and the uptake of ammonium, which are closely related to the starvation response, and many stress-responsive genes mediated by Msn2/4p, as well as trehalose synthase genes, increased in expression when fermenting with xylose, irrespective of the yeast strain. We further observed that transcript levels of genes involved in xylose metabolism, membrane transport functions, and ATP synthesis were higher in MA-R4 than in MA-B4 when strains were fermented with glucose or xylose. Conclusions Our transcriptomic approach revealed the molecular events underlying the response to xylose or glucose and differences between MA-R4 and MA-B4. Xylose-utilizing S. cerevisiae strains may recognize xylose as a non-fermentable

Separate hydrolysis and co-fermentation for improved xylose utilization in integrated ethanol production from wheat meal and wheat straw

PubMed Central

2012-01-01

Background The commercialization of second-generation bioethanol has not been realized due to several factors, including poor biomass utilization and high production cost. It is generally accepted that the most important parameters in reducing the production cost are the ethanol yield and the ethanol concentration in the fermentation broth. Agricultural residues contain large amounts of hemicellulose, and the utilization of xylose is thus a plausible way to improve the concentration and yield of ethanol during fermentation. Most naturally occurring ethanol-fermenting microorganisms do not utilize xylose, but a genetically modified yeast strain, TMB3400, has the ability to co-ferment glucose and xylose. However, the xylose uptake rate is only enhanced when the glucose concentration is low. Results Separate hydrolysis and co-fermentation of steam-pretreated wheat straw (SPWS) combined with wheat-starch hydrolysate feed was performed in two separate processes. The average yield of ethanol and the xylose consumption reached 86% and 69%, respectively, when the hydrolysate of the enzymatically hydrolyzed (18.5% WIS) unwashed SPWS solid fraction and wheat-starch hydrolysate were fed to the fermentor after 1 h of fermentation of the SPWS liquid fraction. In the other configuration, fermentation of the SPWS hydrolysate (7.0% WIS), resulted in an average ethanol yield of 93% from fermentation based on glucose and xylose and complete xylose consumption when wheat-starch hydrolysate was included in the feed. Increased initial cell density in the fermentation (from 5 to 20 g/L) did not increase the ethanol yield, but improved and accelerated xylose consumption in both cases. Conclusions Higher ethanol yield has been achieved in co-fermentation of xylose and glucose in SPWS hydrolysate when wheat-starch hydrolysate was used as feed, then in co-fermentation of the liquid fraction of SPWS fed with the mixed hydrolysates. Integration of first-generation and second

Breeding of a xylose-fermenting hybrid strain by mating genetically engineered haploid strains derived from industrial Saccharomyces cerevisiae.

PubMed

Inoue, Hiroyuki; Hashimoto, Seitaro; Matsushika, Akinori; Watanabe, Seiya; Sawayama, Shigeki

2014-12-01

The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.

A genetic overhaul of Saccharomyces cerevisiae 424A(LNH-ST) to improve xylose fermentation.

PubMed

Bera, Aloke K; Ho, Nancy W Y; Khan, Aftab; Sedlak, Miroslav

2011-05-01

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD(+)-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.

KINETICS OF GROWTH AND ETHANOL PRODUCTION ON DIFFERENT CARBON SUBSTRATES USING GENETICALLY ENGINEERED XYLOSE-FERMENTING YEAST

EPA Science Inventory

Saccharomyces cerevisiae 424A (LNH-ST) strain was used for fermentation of glucose and xylose. Growth kinetics and ethanol productivity were calculated for batch fermentation on media containing different combinations of glucose and xylose to give a final sugar concentra...

Diversity and physiological characterization of D-xylose-fermenting yeasts isolated from the Brazilian Amazonian Forest.

PubMed

Cadete, Raquel M; Melo, Monaliza A; Dussán, Kelly J; Rodrigues, Rita C L B; Silva, Silvio S; Zilli, Jerri E; Vital, Marcos J S; Gomes, Fátima C O; Lachance, Marc-André; Rosa, Carlos A

2012-01-01

This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L · h to 0.75 g/L · h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L · h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

Efficient non-sterilized fermentation of biomass-derived xylose to lactic acid by a thermotolerant Bacillus coagulans NL01.

PubMed

Ouyang, Jia; Cai, Cong; Chen, Hai; Jiang, Ting; Zheng, Zhaojuan

2012-12-01

Xylose is the major pentose and the second most abundant sugar in lignocellulosic feedstock. Its efficient utilization is regarded as a technical barrier to the commercial production of bulk chemicals from lignocellulosic biomass. This work aimed at evaluating the lactic acid production from the biomass-derived xylose using non-sterilized fermentation by Bacillus coagulans NL01. A maximum lactic acid concentration of about 75 g/L was achieved from xylose of 100 g/L after 72 h batch fermentation. Acetic acid and levulinic acid were identified as important inhibitors in xylose fermentation, which markedly reduced lactic acid productivity at 15 and 1.0 g/L, respectively. But low concentrations of formic acid (<2 g/L) exerted a stimulating effect on the lactic acid production. When prehydrolysate containing total 25.45 g/L monosaccharide was fermented with B. coagulans NL01, the same preference for glucose, xylose, and arabinose was observed and18.2 g/L lactic acid was obtained after 48 h fermentation. These results proved that B. coagulans NL01 was potentially well-suited for producing lactic acid from underutilized xylose-rich prehydrolysates.

Enhancing ethanol yields through d-xylose and l-arabinose co-fermentation after construction of a novel high efficient l-arabinose-fermenting Saccharomyces cerevisiae strain.

PubMed

Caballero, Antonio; Ramos, Juan Luis

2017-04-01

Lignocellulose contains two pentose sugars, l-arabinose and d-xylose, neither of which is naturally fermented by first generation (1G) ethanol-producing Saccharomyces cerevisiae yeast. Since these sugars are inaccessible to 1G yeast, a significant percentage of the total carbon in bioethanol production from plant residues, which are used in second generation (2G) ethanol production, remains unused. Recombinant Saccharomyces cerevisiae strains capable of fermenting d-xylose are available on the market; however, there are few examples of l-arabinose-fermenting yeasts, and commercially, there are no strains capable of fermenting both d-xylose and l-arabinose because of metabolic incompatibilities when both metabolic pathways are expressed in the same cell. To attempt to solve this problem we have tested d-xylose and l-arabinose co-fermentation. To find efficient alternative l-arabinose utilization pathways to the few existing ones, we have used stringent methodology to screen for new genes (metabolic and transporter functions) to facilitate l-arabinose fermentation in recombinant yeast. We demonstrate the feasibility of this approach in a successfully constructed yeast strain capable of using l-arabinose as the sole carbon source and capable of fully transforming it to ethanol, reaching the maximum theoretical fermentation yield (0.43 g g-1). We demonstrate that efficient co-fermentation of d-xylose and l-arabinose is feasible using two different co-cultured strains, and observed no fermentation delays, yield drops or accumulation of undesired byproducts. In this study we have identified a technically efficient strategy to enhance ethanol yields by 10 % in 2G plants in a process based on C5 sugar co-fermentation.

Genomic sequence of the xylose fermenting, insect-inhabitingyeast, Pichia stipitis

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

Jeffries, Thomas W.; Grigoriev, Igor; Grimwood, Jane

2007-06-25

Xylose is a major constituent of angiosperm lignocellulose,so its fermentation is important for bioconversion to fuels andchemicals. Pichia stipitis is the best-studied native xylose fermentingyeast. Genes from P. stipitis have been used to engineer xylosemetabolism in Saccharomycescerevisiae, and the regulation of the P.stipitis genome offers insights into the mechanisms of xylose metabolismin yeasts. We have sequenced, assembled and finished the genome ofP.stipitis. As such, it is one of only a handful of completely finishedeukaryotic organisms undergoing analysis and manual curation. Thesequence has revealed aspects of genome organization, numerous genes forbiocoversion, preliminary insights into regulation of central metabolicpathways, numerous examples ofmore » co-localized genes with related functions,and evidence of how P. stipitis manages to achieve redox balance whilegrowing on xylose under microaerobic conditions.« less

Screening and characterizing of xylanolytic and xylose-fermenting yeasts isolated from the wood-feeding termite, Reticulitermes chinensis

PubMed Central

Xie, Rongrong; Zhou, Feng; Huang, Miao

2017-01-01

The effective fermentation of xylose remains an intractable challenge in bioethanol industry. The relevant xylanase enzyme is also in a high demand from industry for several biotechnological applications that inevitably in recent times led to many efforts for screening some novel microorganisms for better xylanase production and fermentation performance. Recently, it seems that wood-feeding termites can truly be considered as highly efficient natural bioreactors. The highly specialized gut systems of such insects are not yet fully realized, particularly, in xylose fermentation and xylanase production to advance industrial bioethanol technology as well as industrial applications of xylanases. A total of 92 strains from 18 yeast species were successfully isolated and identified from the gut of wood-feeding termite, Reticulitermes chinensis. Of these yeasts and strains, seven were identified for new species: Candida gotoi, Candida pseudorhagii, Hamamotoa lignophila, Meyerozyma guilliermondii, Sugiyamaella sp.1, Sugiyamaella sp. 2, and Sugiyamaella sp.3. Based on the phylogenetic and phenotypic characterization, the type strain of C. pseudorhagii sp. nov., which was originally designated strain SSA-1542T, was the most frequently occurred yeast from termite gut samples, showed the highly xylanolytic activity as well as D-xylose fermentation. The highest xylanase activity was recorded as 1.73 and 0.98 U/mL with xylan or D-xylose substrate, respectively, from SSA-1542T. Among xylanase-producing yeasts, four novel species were identified as D-xylose-fermenting yeasts, where the yeast, C. pseudorhagii SSA-1542T, showed the highest ethanol yield (0.31 g/g), ethanol productivity (0.31 g/L·h), and its fermentation efficiency (60.7%) in 48 h. Clearly, the symbiotic yeasts isolated from termite guts have demonstrated a competitive capability to produce xylanase and ferment xylose, suggesting that the wood-feeding termite gut is a promising reservoir for novel xylanases

Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review.

PubMed

Chen, Yanli

2011-05-01

This article reviews current co-culture systems for fermenting mixtures of glucose and xylose to ethanol. Thirty-five co-culture systems that ferment either synthetic glucose and xylose mixture or various biomass hydrolysates are examined. Strain combinations, fermentation modes and conditions, and fermentation performance for these co-culture systems are compared and discussed. It is noted that the combination of Pichia stipitis with Saccharomyces cerevisiae or its respiratory-deficient mutant is most commonly used. One of the best results for fermentation of glucose and xylose mixture is achieved by using co-culture of immobilized Zymomonas mobilis and free cells of P. stipitis, giving volumetric ethanol production of 1.277 g/l/h and ethanol yield of 0.49-0.50 g/g. The review discloses that, as a strategy for efficient conversion of glucose and xylose, co-culture fermentation for ethanol production from lignocellulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature.

Xylose fermentation to ethanol by new Galactomyces geotrichum and Candida akabanensis strains.

PubMed

Valinhas, Raquel V; Pantoja, Lílian A; Maia, Ana Carolina F; Miguel, Maria Gabriela C P; Vanzela, Ana Paula F C; Nelson, David L; Santos, Alexandre S

2018-01-01

The conversion of pentoses into ethanol remains a challenge and could increase the supply of second-generation biofuels. This study sought to isolate naturally occurring yeasts from plant biomass and determine their capabilities for transforming xylose into ethanol. Three yeast strains with the ability to ferment xylose were isolated from pepper, tomato and sugarcane bagasse. The strains selected were characterized by morphological and auxanographic assays, and they were identified by homology analysis of 5.8 S and 26 S ribosomal RNA gene sequences. The identities of two lineages of microrganism were associated with Galactomyces geotrichum , and the other was associated with Candida akabanensis . Fermentative processes were conducted with liquid media containing only xylose as the carbon source. Y P/S values for the production of ethanol ranging between 0.29 and 0.35 g g -1 were observed under non-optimized conditions.

Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae.

PubMed

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2012-08-01

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

Co-fermentation of cellobiose and xylose by mixed culture of recombinant Saccharomyces cerevisiae and kinetic modeling.

PubMed

Chen, Yingying; Wu, Ying; Zhu, Baotong; Zhang, Guanyu; Wei, Na

2018-01-01

Efficient conversion of cellulosic sugars in cellulosic hydrolysates is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge. The present study reports a new approach for simultaneous fermentation of cellobiose and xylose by using the co-culture consisting of recombinant Saccharomyces cerevisiae specialist strains. The co-culture system can provide competitive advantage of modularity compared to the single culture system and can be tuned to deal with fluctuations in feedstock composition to achieve robust and cost-effective biofuel production. This study characterized fermentation kinetics of the recombinant cellobiose-consuming S. cerevisiae strain EJ2, xylose-consuming S. cerevisiae strain SR8, and their co-culture. The motivation for kinetic modeling was to provide guidance and prediction of using the co-culture system for simultaneous fermentation of mixed sugars with adjustable biomass of each specialist strain under different substrate concentrations. The kinetic model for the co-culture system was developed based on the pure culture models and incorporated the effects of product inhibition, initial substrate concentration and inoculum size. The model simulations were validated by results from independent fermentation experiments under different substrate conditions, and good agreement was found between model predictions and experimental data from batch fermentation of cellobiose, xylose and their mixtures. Additionally, with the guidance of model prediction, simultaneous co-fermentation of 60 g/L cellobiose and 20 g/L xylose was achieved with the initial cell densities of 0.45 g dry cell weight /L for EJ2 and 0.9 g dry cell weight /L SR8. The results demonstrated that the kinetic modeling could be used to guide the design and optimization of yeast co-culture conditions for achieving simultaneous fermentation of cellobiose and xylose with improved ethanol productivity, which is

Largely enhanced bioethanol production through the combined use of lignin-modified sugarcane and xylose fermenting yeast strain.

PubMed

Ko, Ja Kyong; Jung, Je Hyeong; Altpeter, Fredy; Kannan, Baskaran; Kim, Ha Eun; Kim, Kyoung Heon; Alper, Hal S; Um, Youngsoon; Lee, Sun-Mi

2018-05-01

The recalcitrant structure of lignocellulosic biomass is a major barrier in efficient biomass-to-ethanol bioconversion processes. The combination of feedstock engineering via modification in the lignin synthesis pathway of sugarcane and co-fermentation of xylose and glucose with a recombinant xylose utilizing yeast strain produced 148% more ethanol compared to that of the wild type biomass and control strain. The lignin reduced biomass led to a substantially increased release of fermentable sugars (glucose and xylose). The engineered yeast strain efficiently co-utilized glucose and xylose for fermentation, elevating ethanol yields. In this study, it was experimentally demonstrated that the combined efforts of engineering both feedstock and microorganisms largely enhances the bioconversion of lignocellulosic feedstock to bioethanol. This strategy will significantly improve the economic feasibility of lignocellulosic biofuels production. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

PubMed Central

Matsushika, Akinori; Nagashima, Atsushi; Goshima, Tetsuya; Hoshino, Tamotsu

2013-01-01

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4

Small intestinal malabsorption in chronic alcoholism: a retrospective study of alcoholic patients by the ¹⁴C-D-xylose breath test.

PubMed

Hope, Håvar; Skar, Viggo; Sandstad, Olav; Husebye, Einar; Medhus, Asle W

2012-04-01

The ¹⁴C-D-xylose breath test was used at Ullevål University Hospital in the period from 1986 TO 1995 for malabsorption testing. The objective of this retrospective study was to reveal whether patients with chronic alcoholism may have intestinal malabsorption. The consecutive ¹⁴C-D-xylose breath test database was reviewed and patients with the diagnosis of chronic alcoholism were identified. ¹⁴C-D-xylose breath test results of the alcoholic patients were compared with the results of untreated celiac patients and patient and healthy controls. In the ¹⁴C-D-xylose breath test, ¹⁴C-D-xylose was dissolved in water and given orally after overnight fast. Breath samples were taken at 30-min intervals for 210 min, and ¹⁴CO₂ : ¹²CO₂ ratios were calculated for each time point, presenting a time curve for ¹⁴C-D-xylose absorption. Urine was collected after 210 min and the fraction of the total d-xylose passed was calculated (U%). ¹⁴CO₂ in breath and ¹⁴C-D-xylose in urine were analyzed using liquid scintillation. Both breath and urine analysis revealed a pattern of malabsorption in alcoholics comparable with untreated celiac patients, with significantly reduced absorption of d-xylose compared with patient and healthy controls. Alcoholic patients have a significantly reduced ¹⁴C-D-xylose absorption, comparable with untreated celiac patients. This indicates a reduced intestinal function in chronic alcoholism.

The effect of initial cell concentration on xylose fermentation by Pichia stipitis

Treesearch

Frank K. Agbogbo; Guillermo Coward-Kelly; Mads Torry-Smith; Kevin Wenger; Thomas W. Jeffries

2007-01-01

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was...

Xylose and cellulose fractionation from corncob with three different strategies and separate fermentation of them to bioethanol.

PubMed

Chen, Yefu; Dong, Boyu; Qin, Weijun; Xiao, Dongguang

2010-09-01

To the aim of efficient utilization of both of xylose and cellulose, a laboratory xylose/cellulose fractionation and separate fermentation (XCFSF) bioethanol process was performed. Three xylose/cellulose fractionation strategies: (A) dilute sulfur acid hydrolysis and detoxification, (B) lime pretreatment and xylanase hydrolysis, (C) bio-treatment with Phanerochaete chrysosporium and xylanase hydrolysis were applied to corn cobs. As a result, the maximum xylose yields obtained from A, B and C fractionation methods were 78.47%, 57.84% and 42.54%, respectively, and 96.81%, 92.14% and 80.34% of cellulose were preserved in the corresponding solid residues. The xylose dissolved in acid and enzymatic hydrolysates was fermented to ethanol by Candida shahatae and the cellulose remaining in solid residues was converted to ethanol by simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae. Finally, for A, B, C fractionation methods, 70.40%, 52.87%, 39.22% of hemicellulose and 89.77%, 84.30%, 71.90% of cellulose in corn cobs was converted to ethanol, respectively. Copyright 2010 Elsevier Ltd. All rights reserved.

Diversity and Fermentation Products of Xylose-Utilizing Yeasts Isolated from Buffalo Feces in Thailand

PubMed Central

Lorliam, Wanlapa; Akaracharanya, Ancharida; Suzuki, Motofumi; Ohkuma, Moriya; Tanasupawat, Somboon

2013-01-01

Twenty-eight xylose-utilizing yeast strains were isolated by enrichment culture from 11 samples of feces from the rectum of Murrah buffalo and Swamp buffalo in Thailand. On the basis of their morphological and biochemical characteristics, including sequence analysis of the D1/D2 region of the large-subunit ribosomal RNA gene (LSU rDNA), they were identified as Candida tropicalis (designated as Group I, 11 isolates), Candida parasilosis (Group II, 2 isolates), Candida mengyuniae (Group III, 2 isolates), Sporopachydermia lactativora (Group IV, 2 isolates), Geotrichum sp. (Group V, 5 isolates) and Trichosporon asahii (Group VI, 6 isolates). All isolates utilized xylose as the sole carbon source but 27 isolates could ferment xylose to ethanol (0.006–0.602 g L−1) and 21 isolates could ferment xylose to xylitol (0.19–22.84 g L−1). Candida tropicalis isolates produced the highest yield of xylitol (74.80%). Their ability to convert xylose to xylitol and ethanol ranged from 15.06 g L−1 to 22.84 g L−1 xylitol and 0.110 g L−1 to 0.602 g L−1 ethanol, respectively. PMID:24005843

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...

Combining inhibitor tolerance and D-xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production.

PubMed

Demeke, Mekonnen M; Dumortier, Françoise; Li, Yingying; Broeckx, Tom; Foulquié-Moreno, María R; Thevelein, Johan M

2013-08-26

In addition to efficient pentose utilization, high inhibitor tolerance is a key trait required in any organism used for economically viable industrial bioethanol production with lignocellulose biomass. Although recent work has succeeded in establishing efficient xylose fermentation in robust industrial Saccharomyces cerevisiae strains, the resulting strains still lacked sufficient inhibitor tolerance for efficient sugar fermentation in lignocellulose hydrolysates. The aim of the present work was to combine high xylose fermentation activity and high inhibitor tolerance in a single industrial yeast strain. We have screened 580 yeast strains for high inhibitor tolerance using undetoxified acid-pretreated spruce hydrolysate and identified a triploid industrial baker's yeast strain as having the highest inhibitor tolerance. From this strain, a mating competent diploid segregant with even higher inhibitor tolerance was obtained. It was crossed with the recently developed D-xylose fermenting diploid industrial strain GS1.11-26, with the Ethanol Red genetic background. Screening of 819 diploid segregants from the tetraploid hybrid resulted in two strains, GSF335 and GSF767, combining high inhibitor tolerance and efficient xylose fermentation. In a parallel approach, meiotic recombination of GS1.11-26 with a haploid segregant of Ethanol Red and screening of 104 segregants resulted in a similar inhibitor tolerant diploid strain, GSE16. The three superior strains exhibited significantly improved tolerance to inhibitors in spruce hydrolysate, higher glucose consumption rates, higher aerobic growth rates and higher maximal ethanol accumulation capacity in very-high gravity fermentation, compared to GS1.11-26. In complex medium, the D-xylose utilization rate by the three superior strains ranged from 0.36 to 0.67 g/g DW/h, which was lower than that of GS1.11-26 (1.10 g/g DW/h). On the other hand, in batch fermentation of undetoxified acid-pretreated spruce hydrolysate, the

Combining inhibitor tolerance and D-xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production

PubMed Central

2013-01-01

Background In addition to efficient pentose utilization, high inhibitor tolerance is a key trait required in any organism used for economically viable industrial bioethanol production with lignocellulose biomass. Although recent work has succeeded in establishing efficient xylose fermentation in robust industrial Saccharomyces cerevisiae strains, the resulting strains still lacked sufficient inhibitor tolerance for efficient sugar fermentation in lignocellulose hydrolysates. The aim of the present work was to combine high xylose fermentation activity and high inhibitor tolerance in a single industrial yeast strain. Results We have screened 580 yeast strains for high inhibitor tolerance using undetoxified acid-pretreated spruce hydrolysate and identified a triploid industrial baker’s yeast strain as having the highest inhibitor tolerance. From this strain, a mating competent diploid segregant with even higher inhibitor tolerance was obtained. It was crossed with the recently developed D-xylose fermenting diploid industrial strain GS1.11-26, with the Ethanol Red genetic background. Screening of 819 diploid segregants from the tetraploid hybrid resulted in two strains, GSF335 and GSF767, combining high inhibitor tolerance and efficient xylose fermentation. In a parallel approach, meiotic recombination of GS1.11-26 with a haploid segregant of Ethanol Red and screening of 104 segregants resulted in a similar inhibitor tolerant diploid strain, GSE16. The three superior strains exhibited significantly improved tolerance to inhibitors in spruce hydrolysate, higher glucose consumption rates, higher aerobic growth rates and higher maximal ethanol accumulation capacity in very-high gravity fermentation, compared to GS1.11-26. In complex medium, the D-xylose utilization rate by the three superior strains ranged from 0.36 to 0.67 g/g DW/h, which was lower than that of GS1.11-26 (1.10 g/g DW/h). On the other hand, in batch fermentation of undetoxified acid-pretreated spruce

Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

In this study, we introduced the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) into an engineered S. cerevisiae (SR8) harboring the XR/XDH pathway and up-regulated PPP 10, to enable CO2 recycling through a synthetic rPPP during xylose fermentation (Fig. 1). ...

Recycling Carbon Dioxide during Xylose Fermentation by Engineered Saccharomyces cerevisiae.

PubMed

Xia, Peng-Fei; Zhang, Guo-Chang; Walker, Berkley; Seo, Seung-Oh; Kwak, Suryang; Liu, Jing-Jing; Kim, Heejin; Ort, Donald R; Wang, Shu-Guang; Jin, Yong-Su

2017-02-17

Global climate change caused by the emission of anthropogenic greenhouse gases (GHGs) is a grand challenge to humanity. To alleviate the trend, the consumption of fossil fuels needs to be largely reduced and alternative energy technologies capable of controlling GHG emissions are anticipated. In this study, we introduced a synthetic reductive pentose phosphate pathway (rPPP) into a xylose-fermenting Saccharomyces cerevisiae strain SR8 to achieve simultaneous lignocellulosic bioethanol production and carbon dioxide recycling. Specifically, ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and phosphoribulokinase from Spinacia oleracea were introduced into the SR8 strain. The resulting strain with the synthetic rPPP was able to exhibit a higher yield of ethanol and lower yields of byproducts (xylitol and glycerol) than a control strain. In addition, the reduced release of carbon dioxide by the engineered strain was observed during xylose fermentation, suggesting that the carbon dioxide generated by pyruvate decarboxylase was partially reassimilated through the synthetic rPPP. These results demonstrated that recycling of carbon dioxide from the ethanol fermentation pathway in yeast can be achieved during lignocellulosic bioethanol production through a synthetic carbon conservative metabolic pathway. This strategy has a great potential to alleviate GHG emissions during the production of second-generation ethanol.

Presence of glucose, xylose, and glycerol fermenting bacteria in the deep biosphere of the former Homestake gold mine, South Dakota

PubMed Central

Rastogi, Gurdeep; Gurram, Raghu N.; Bhalla, Aditya; Gonzalez, Ramon; Bischoff, Kenneth M.; Hughes, Stephen R.; Kumar, Sudhir; Sani, Rajesh K.

2012-01-01

Eight fermentative bacterial strains were isolated from mixed enrichment cultures of a composite soil sample collected at 1.34 km depth from the former Homestake gold mine in Lead, SD, USA. Phylogenetic analysis of their 16S rRNA gene sequences revealed that these isolates were affiliated with the phylum Firmicutes belonging to genera Bacillus and Clostridium. Batch fermentation studies demonstrated that isolates had the ability to ferment glucose, xylose, or glycerol to industrially valuable products such as ethanol and 1,3-propanediol (PDO). Ethanol was detected as the major fermentation end product in glucose-fermenting cultures at pH 10 with yields of 0.205–0.304 g of ethanol/g of glucose. While a xylose-fermenting strain yielded 0.189 g of ethanol/g of xylose and 0.585 g of acetic acid/g of xylose at the end of fermentation. At pH 7, glycerol-fermenting isolates produced PDO (0.323–0.458 g of PDO/g of glycerol) and ethanol (0.284–0.350 g of ethanol/g of glycerol) as major end products while acetic acid and succinic acid were identified as minor by-products in fermentation broths. These results suggest that the deep biosphere of the former Homestake gold mine harbors bacterial strains which could be used in bio-based production of ethanol and PDO. PMID:23919089

Oxidative production of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth by Gluconobacter oxydans.

PubMed

Zhang, Hongsen; Han, Xushen; Wei, Chengxiang; Bao, Jie

2017-01-01

An oxidative production process of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth was designed, experimentally investigated, and evaluated. Dry dilute acid pretreated and biodetoxified corn stover was simultaneously saccharified and fermented into 59.80g/L of ethanol (no xylose utilization). 65.39g/L of xylose was obtained in the distillation stillage without any concentrating step after ethanol was distillated. Then the xylose was completely converted into 66.42g/L of xylonic acid by Gluconobacter oxydans. The rigorous Aspen Plus modeling shows that the wastewater generation and energy consumption was significantly reduced comparing to the previous xylonic acid production process using xylose in pretreatment liquid. This study provided a practical process option for xylonic acid production from lignocellulose feedstock with significant reduction of wastewater and energy consumption. Copyright © 2016 Elsevier Ltd. All rights reserved.

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant Saccharomyces cerevisiae Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

PubMed Central

Parreiras, Lucas S.; Breuer, Rebecca J.; Avanasi Narasimhan, Ragothaman; Higbee, Alan J.; La Reau, Alex; Tremaine, Mary; Qin, Li; Willis, Laura B.; Bice, Benjamin D.; Bonfert, Brandi L.; Pinhancos, Rebeca C.; Balloon, Allison J.; Uppugundla, Nirmal; Liu, Tongjun; Li, Chenlin; Tanjore, Deepti; Ong, Irene M.; Li, Haibo; Pohlmann, Edward L.; Serate, Jose; Withers, Sydnor T.; Simmons, Blake A.; Hodge, David B.; Westphall, Michael S.; Coon, Joshua J.; Dale, Bruce E.; Balan, Venkatesh; Keating, David H.; Zhang, Yaoping; Landick, Robert; Gasch, Audrey P.; Sato, Trey K.

2014-01-01

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH. PMID:25222864

A dynamic flux balance model and bottleneck identification of glucose, xylose, xylulose co-fermentation in Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

Economically viable production of lignocellulosic ethanol requires efficient conversion of feedstock sugars to ethanol. Saccharomyces cerevisiae cannot ferment xylose, the main five-carbon sugars in biomass, but can ferment xylulose, an enzymatically derived isomer. Xylulose fermentation is slow rel...

Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain

PubMed Central

2014-01-01

Background The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized. Results In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes. Conclusions In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to

Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain.

PubMed

Signori, Lorenzo; Passolunghi, Simone; Ruohonen, Laura; Porro, Danilo; Branduardi, Paola

2014-04-08

The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized. In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes. In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to the impairment of the

Constructing xylose-assimilating pathways in Pediococcus acidilactici for high titer d-lactic acid fermentation from corn stover feedstock.

PubMed

Qiu, Zhongyang; Gao, Qiuqiang; Bao, Jie

2017-12-01

Xylose-assimilating pathway was constructed in a d-lactic acid producing Pediococcus acidilactici strain and evolutionary adapted to yield a co-fermentation strain P. acidilactici ZY15 with 97.3g/L of d-lactic acid and xylose conversion of 92.6% obtained in the high solids content simultaneous saccharification and co-fermentation (SSCF) of dry dilute acid pretreated and biodetoxified corn stover feedstock. The heterologous genes encoding xylose isomerase (xylA) and xylulokinase (xylB) were screened and integrated into the P. acidilactici chromosome. The metabolic flux to acetic acid in phosphoketolase pathway was re-directed to pentose phosphate pathway by substituting the endogenous phosphoketolase gene (pkt) with the heterologous transketolase (tkt) and transaldolase (tal) genes. The xylose-assimilating ability of the newly constructed P. acidilactici strain was significantly improved by adaptive evolution. This study provided an important strain and process prototype for high titer d-lactic acid production from lignocellulose feedstock with efficient xylose assimilation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering.

PubMed

Demeke, Mekonnen M; Dietz, Heiko; Li, Yingying; Foulquié-Moreno, María R; Mutturi, Sarma; Deprez, Sylvie; Den Abt, Tom; Bonini, Beatriz M; Liden, Gunnar; Dumortier, Françoise; Verplaetse, Alex; Boles, Eckhard; Thevelein, Johan M

2013-06-21

The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. An industrial yeast strain for bioethanol production with

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

PubMed Central

2013-01-01

Background The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. Results An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. Conclusions An industrial yeast strain for

Metabolomic and 13C-Metabolic Flux Analysis of a Xylose-Consuming Saccharomyces cerevisiae Strain Expressing Xylose Isomerase

PubMed Central

Wasylenko, Thomas M.; Stephanopoulos, Gregory

2016-01-01

Over the past two decades significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative PPP is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis. PMID:25311863

Adaptation of the xylose fermenting yeast Saccharomyces cerevisiae F12 for improving ethanol production in different fed-batch SSF processes.

PubMed

Tomás-Pejó, E; Ballesteros, M; Oliva, J M; Olsson, L

2010-11-01

An efficient fermenting microorganism for bioethanol production from lignocellulose is highly tolerant to the inhibitors released during pretreatment and is able to ferment efficiently both glucose and xylose. In this study, directed evolution was employed to improve the xylose fermenting Saccharomyces cerevisiae F12 strain for bioethanol production at high substrate loading. Adapted and parental strains were compared with respect to xylose consumption and ethanol production. Adaptation led to an evolved strain more tolerant to the toxic compounds present in the medium. When using concentrated prehydrolysate from steam-pretreated wheat straw with high inhibitor concentration, an improvement of 65 and 20% in xylose consumption and final ethanol concentration, respectively, were achieved using the adapted strain. To address the need of high substrate loadings, fed-batch SSF experiments were performed and an ethanol concentration as high as 27.4 g/l (61% of the theoretical) was obtained with 11.25% (w/w) of water insoluble solids (WIS).

Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates

USDA-ARS?s Scientific Manuscript database

An industrial ethanol-producing Saccharomyces cerevisiae strain with genes needed for xylose-fermentation integrated into its genome was used to obtain haploids and diploid isogenic strains. The isogenic strains were more effective in metabolizing xylose than their parental strain (p < 0.05) and abl...

Fermentation of xylose to ethanol by genetically modified enteric bacteria

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

Tolan, J.S.

1987-01-01

This thesis describes the fermentation of D-xylose by wild type and recombinant Klebsiella planticola ATCC 33531 and Erwinia chrysanthemi B374. The recombinant strains bear multi-copy plasmids containing the pdc gene inserted from Zymomonas mobilis. Expression of the gene in K. planticola markedly increased the yield of ethanol, up to 1.3 mole/mole xylose, or 25.1 g/L. Concurrently, there were significant decreases in the yields of formation acetate, lactate, and butanediol. Transconjugant Klebsiella grew almost as fast as the wild type and tolerated up to 4% ethanol. The plasmid was retained by the cells during at least one batch culture, even inmore » the absence of selective pressure by antibiotics to maintain the plasmid. The cells produced 31.6 g/L ethanol from 79.6 g/L of a D-glucose-D-xylose-L-arabinose mixture designed to simulate hydrolyzed hemicellulose. The physiology of the wild type K. planticola is described in more detail than in the original report of its isolation. E. chrysanthemi PDC transconjugants also produced ethanol in high yield (up to 1.45 mole/mole xylose). However, transconjugant E. chrysanthemi grew only 1/4 as rapidly as the wild type and tolerated only 2% ethanol. The plasmid PZM15 apparently exhibits pleiotropic effects when inserted into K. planticola and into E. chrysanthemi.« less

Effect of salts on the Co-fermentation of glucose and xylose by a genetically engineered strain of Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background A challenge currently facing the cellulosic biofuel industry is the efficient fermentation of both C5 and C6 sugars in the presence of inhibitors. To overcome this challenge, microorganisms that are capable of mixed-sugar fermentation need to be further developed for increased inhibitor tolerance. However, this requires an understanding of the physiological impact of inhibitors on the microorganism. This paper investigates the effect of salts on Saccharomyces cerevisiae 424A(LNH-ST), a yeast strain capable of effectively co-fermenting glucose and xylose. Results In this study, we show that salts can be significant inhibitors of S. cerevisiae. All 6 pairs of anions (chloride and sulfate) and cations (sodium, potassium, and ammonium) tested resulted in reduced cell growth rate, glucose consumption rate, and ethanol production rate. In addition, the data showed that the xylose consumption is more strongly affected by salts than glucose consumption at all concentrations. At a NaCl concentration of 0.5M, the xylose consumption rate was reduced by 64.5% compared to the control. A metabolomics study found a shift in metabolism to increased glycerol production during xylose fermentation when salt was present, which was confirmed by an increase in extracellular glycerol titers by 4 fold. There were significant differences between the different cations. The salts with potassium cations were the least inhibitory. Surprisingly, although salts of sulfate produced twice the concentration of cations as compared to salts of chloride, the degree of inhibition was the same with one exception. Potassium salts of sulfate were less inhibitory than potassium paired with chloride, suggesting that chloride is more inhibitory than sulfate. Conclusions When developing microorganisms and processes for cellulosic ethanol production, it is important to consider salt concentrations as it has a significant negative impact on yeast performance, especially with regards to xylose

Improved production of homo-D-lactic acid via xylose fermentation by introduction of xylose assimilation genes and redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-Lactate dehydrogenase gene-deficient Lactobacillus plantarum.

PubMed

Okano, Kenji; Yoshida, Shogo; Yamada, Ryosuke; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

2009-12-01

The production of optically pure d-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of d-lactic acid was produced from 50 g/liter of xylose.

Genes related to xylose fermentation and methods of using same for enhanced biofuel production

DOEpatents

Wohlbach, Dana J.; Gasch, Audrey P.

2015-09-29

The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

Genes related to xylose fermentation and methods of using same for enhanced biofuel production

DOEpatents

Wohlbach, Dana J.; Gasch, Audrey P.

2016-11-29

The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

Genes related to xylose fermentation and methods of using same for enhanced biofuel production

DOEpatents

Wohlbach, Dana J.; Gasch, Audrey P.

2014-08-05

The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

Ethanol fermentation by xylose-assimilating Saccharomyces cerevisiae using sugars in a rice straw liquid hydrolysate concentrated by nanofiltration.

PubMed

Sasaki, Kengo; Sasaki, Daisuke; Sakihama, Yuri; Teramura, Hiroshi; Yamada, Ryosuke; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

2013-11-01

Concentrating sugars using membrane separation, followed by ethanol fermentation by recombinant xylose-assimilating Saccharomyces cerevisiae, is an attractive technology. Three nanofiltration membranes (NTR-729HF, NTR-7250, and ESNA3) were effective in concentrating glucose, fructose, and sucrose from dilute molasses solution and no permeation of sucrose. The separation factors of acetate, formate, furfural, and 5-hydroxymethyl furfural, which were produced by dilute acid pretreatment of rice straw, over glucose after passage through these three membranes were 3.37-11.22, 4.71-20.27, 4.32-16.45, and 4.05-16.84, respectively, at pH 5.0, an applied pressure of 1.5 or 2.0 MPa, and 25 °C. The separation factors of these fermentation inhibitors over xylose were infinite, as there was no permeation of xylose. Ethanol production from approximately two-times concentrated liquid hydrolysate using recombinant S. cerevisiae was double (5.34-6.44 g L(-1)) that compared with fermentation of liquid hydrolysate before membrane separation (2.75 g L(-1)). Copyright © 2013 Elsevier Ltd. All rights reserved.

Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose.

PubMed

Ishchuk, Olena P; Voronovsky, Andriy Y; Stasyk, Oleh V; Gayda, Galina Z; Gonchar, Mykhailo V; Abbas, Charles A; Sibirny, Andriy A

2008-11-01

Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.

The Effect of Initial Cell Concentration on Xylose Fermentation by Pichia stipitis

NASA Astrophysics Data System (ADS)

Agbogbo, Frank K.; Coward-Kelly, Guillermo; Torry-Smith, Mads; Wenger, Kevin; Jeffries, Thomas W.

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was produced when the initial cell concentrations were high, cell density had no effect on the final ethanol yield. A two-parameter mathematical model was used to predict the cell population dynamics at the different initial cell concentrations. The model parameters, a and b correlate with the initial cell concentrations used with an R 2 of 0.99.

Genomic analysis of a xylose operon and characterization of novel xylose isomerase and xylulokinase from Bacillus coagulans NL01.

PubMed

Zheng, Zhaojuan; Lin, Xi; Jiang, Ting; Ye, Weihua; Ouyang, Jia

2016-08-01

To investigate the xylose operon and properties of xylose isomerase and xylulokinase in Bacillus coagulans that can effectively ferment xylose to lactic acid. The xylose operon is widely present in B. coagulans. It is composed of four putative ORFs. Novel xylA and xylB from B. coagulans NL01 were cloned and expressed in Escherichia coli. Sequence of xylose isomerase was more conserved than that of xylulokinase. Both the enzymes exhibited maximum activities at pH 7-8 but with a high temperature maximum of 80-85 °C, divalent metal ion was prerequisite for their activation. Xylose isomerase and xylulokinase were most effectively activated by Ni(2+) and Co(2+), respectively. Genomic analysis of xylose operon has contributed to understanding xylose metabolism in B. coagulans and the novel xylose isomerase and xylulokinase might provide new alternatives for metabolic engineering of other strains to improve their fermentation performance on xylose.

Multi-stage Continuous Culture Fermentation of Glucose-Xylose Mixtures to Fuel Ethanol using Genetically Engineered Saccharomyces cerevisiae 424A

EPA Science Inventory

Multi-stage continuous (chemostat) culture fermentation (MCCF) with variable fermentor volumes was carried out to study utilizing glucose and xylose for ethanol production by means of mixed sugar fermentation (MSF). Variable fermentor volumes were used to enable enhanced sugar u...

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase

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

Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B

2012-01-01

Economically viable production of solvents through acetone butanol ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of fivemore » proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.« less

Process intensification through microbial strain evolution: mixed glucose-xylose fermentation in wheat straw hydrolyzates by three generations of recombinant Saccharomyces cerevisiae

PubMed Central

2014-01-01

Background Lignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2. Results With all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (qGlucose ≈ 2.9 g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXylose = 0.23 g/gCDW/h) and 17.5 (IBB10B05; qXylose = 0.35 g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50

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

Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

PubMed Central

Salusjärvi, Laura; Kankainen, Matti; Soliymani, Rabah; Pitkänen, Juha-Pekka; Penttilä, Merja; Ruohonen, Laura

2008-01-01

Background Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains

Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D-xylose.

PubMed

Slininger, P J; Dien, B S; Lomont, J M; Bothast, R J; Ladisch, M R; Okos, M R

2014-08-01

Scheffersomyces (formerly Pichia) stipitis is a potential biocatalyst for converting lignocelluloses to ethanol because the yeast natively ferments xylose. An unstructured kinetic model based upon a system of linear differential equations has been formulated that describes growth and ethanol production as functions of ethanol, oxygen, and xylose concentrations for both growth and fermentation stages. The model was validated for various growth conditions including batch, cell recycle, batch with in situ ethanol removal and fed-batch. The model provides a summary of basic physiological yeast properties and is an important tool for simulating and optimizing various culture conditions and evaluating various bioreactor designs for ethanol production. © 2014 Wiley Periodicals, Inc.

Fermentative alcohol production

DOEpatents

Wilke, Charles R.; Maiorella, Brian L.; Blanch, Harvey W.; Cysewski, Gerald R.

1982-01-01

An improved fermentation process for producing alcohol which includes the combination of vacuum fermentation and vacuum distillation. Preferably, the vacuum distillation is carried out in two phases, one a fermentor proper operated at atmospheric pressure and a flash phase operated at reduced pressure with recycle of fermentation brew having a reduced alcohol content to the fermentor, using vapor recompression heating of the flash-pot recycle stream to heat the flash-pot or the distillation step, and using "water load balancing" (i.e., the molar ratio of water in the fermentor feed is the same as the molar ratio of water in the distillation overhead).

Effects of culture conditions on the fermentation of xylose to ethanol by Candida shehatae

Treesearch

T. W. Jeffries

1985-01-01

This research examined four factors on the fermentation of xylose by Candida shehatae, and the following conclusions were reached: (1) A minimal medium is effective for producing ethanol. (2) Peptone and casamino acids stimulate ethanol production. (3) Aeration is important in obtaining good ethanol production rates and yields. (4) The maximal rate of ethanol...

Pichia stipitis Genes for Alcohol Dehydrogenase with Fermentative and Respiratory Functions

PubMed Central

Cho, Jae-yong; Jeffries, Thomas W.

1998-01-01

Two genes coding for isozymes of alcohol dehydrogenase (ADH); designated PsADH1 and PsADH2, have been identified and isolated from Pichia stipitis CBS 6054 genomic DNA by Southern hybridization to Saccharomyces cerevisiae ADH genes, and their physiological roles have been characterized through disruption. The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74.7% identical to the S. cerevisiae ADH1 protein. They also show a high level identity with the group I ADH proteins from Kluyveromyces lactis. The PsADH isozymes are presumably localized in the cytoplasm, as they do not possess the amino-terminal extension of mitochondrion-targeted ADHs. Gene disruption studies suggest that PsADH1 plays a major role in xylose fermentation because PsADH1 disruption results in a lower growth rate and profoundly greater accumulation of xylitol. Disruption of PsADH2 does not significantly affect ethanol production or aerobic growth on ethanol as long as PsADH1 is present. The PsADH1 and PsADH2 isozymes appear to be equivalent in the ability to convert ethanol to acetaldehyde, and either is sufficient to allow cell growth on ethanol. However, disruption of both genes blocks growth on ethanol. P. stipitis strains disrupted in either PsADH1 or PsADH2 still accumulate ethanol, although in different amounts, when grown on xylose under oxygen-limited conditions. The PsADH double disruptant, which is unable to grow on ethanol, still produces ethanol from xylose at about 13% of the rate seen in the parental strain. Thus, deletion of both PsADH1 and PsADH2 blocks ethanol respiration but not production, implying a separate path for fermentation. PMID:9546172

Draft genome sequence of the D-Xylose-Fermenting yeast Spathaspora xylofermentans UFMG-HMD23.3

USDA-ARS?s Scientific Manuscript database

Here, we report the draft genome sequence of the yeast Spathaspora xylofermentans UFMG-HMD23.3 (CBMAI 1427=CBS 12681), a D-xylose fermenting yeast isolated from the Amazonian forest. The genome consists of 298 contigs, with a total size of 15.1 Mb, including the mitochondrial genome, and 5,948 predi...

Zymomonas with improved xylose utilization in stress conditions

DOEpatents

Caimi, Perry G; Emptage, Mark; Li, Xu; Viitanen, Paul V; Chou, Yat-Chen; Franden, Mary Ann; Zhang, Min

2013-06-18

Strains of xylose utilizing Zymomonas with improved xylose utilization and ethanol production during fermentation in stress conditions were obtained using an adaptation method. The adaptation involved continuously growing xylose utilizing Zymomonas in media containing high sugars, acetic acid, ammonia, and ethanol.

The implementation of high fermentative 2,3-butanediol production from xylose by simultaneous additions of yeast extract, Na2EDTA, and acetic acid.

PubMed

Wang, Xiao-Xiong; Hu, Hong-Ying; Liu, De-Hua; Song, Yuan-Quan

2016-01-25

The effective use of xylose may significantly enhance the feasibility of using lignocellulosic hydrolysate to produce 2,3-butanediol (2,3-BD). Previous difficulties in 2,3-BD production include that the high-concentration xylose cannot be converted completely and the fermentation rate is slow. This study investigated the effects of yeast extract, ethylenediaminetetraacetic acid disodium salt (Na2EDTA), and acetic acid on 2,3-BD production from xylose. The central composite design approach was used to optimize the concentrations of these components. It was found that simultaneous addition of yeast extract, Na2EDTA, and acetic acid could significantly improve 2,3-BD production. The optimal concentrations of yeast extract, Na2EDTA, and acetic acid were 35.2, 1.2, and 4.5 g/L, respectively. The 2,3-BD concentration in the optimized medium reached 39.7 g/L after 48 hours of shake flask fermentation, the highest value ever reported in such a short period. The xylose utilization ratio and the 2,3-BD concentration increased to 99.0% and 42.7 g/L, respectively, after 48 hours of stirred batch fermentation. Furthermore, the 2,3-BD yield was 0.475 g/g, 95.0% of the theoretical maximum value. As the major components of lignocellulosic hydrolysate are glucose, xylose, and acetic acid, the results of this study indicate the possibility of directly using the hydrolysate to effectively produce 2,3-BD. Copyright © 2015 Elsevier B.V. All rights reserved.

Fermentation Process and Metabolic Flux of Ethanol Production from the Detoxified Hydrolyzate of Cassava Residue

PubMed Central

Li, Xingjiang; Deng, Yongdong; Yang, Ying; Wei, Zhaojun; Cheng, Jieshun; Cao, Lili; Mu, Dongdong; Luo, Shuizhong; Zheng, Zhi; Jiang, Shaotong; Wu, Xuefeng

2017-01-01

With the growth of the world population, energy problems are becoming increasingly severe; therefore, sustainable energy sources have gained enormous importance. With respect to ethanol fuel production, biomass is gradually replacing grain as the main raw material. In this study, we explored the fermentation of five- and six-carbon sugars, the main biomass degradation products, into alcohol. We conducted mutagenic screening specifically for Candida tropicalis CICC1779 to obtain a strain that effectively used xylose (Candida tropicalis CICC1779-Dyd). By subsequently studying fermentation conditions under different initial liquid volume oxygen transfer coefficients (kLα), and coupling control of the aeration rate and agitation speed under optimal conditions, the optimal dissolved oxygen change curve was obtained. In addition, we constructed metabolic flow charts and equations to obtain a better understanding of the fermentation mechanism and to improve the ethanol yield. In our experiment, the ethanol production of the wild type stain was 17.58 g·L−1 at a kLα of 120. The highest ethanol yield of the mutagenic strains was 24.85 g·L−1. The ethanol yield increased to 26.56 g·L−1 when the dissolved oxygen content was optimized, and the conversion of sugar into alcohol reached 0.447 g·g−1 glucose (the theoretical titer of yeast-metabolized xylose was 0.46 g ethanol/g xylose and the glucose ethanol fermentation titer was 0.51 g ethanol/g glucose). Finally, the detected activity of xylose reductase and xylose dehydrogenase was higher in the mutant strain than in the original, which indicated that the mutant strain (CICC1779-Dyd) could effectively utilize xylose for metabolism. PMID:28878755

Harnessing genetic diversity in Saccharomyces cerevisiae for fermentation of xylose in hydrolysates of alkaline hydrogen peroxide-pretreated biomass.

PubMed

Sato, Trey K; Liu, Tongjun; Parreiras, Lucas S; Williams, Daniel L; Wohlbach, Dana J; Bice, Benjamin D; Ong, Irene M; Breuer, Rebecca J; Qin, Li; Busalacchi, Donald; Deshpande, Shweta; Daum, Chris; Gasch, Audrey P; Hodge, David B

2014-01-01

The fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeast Saccharomyces cerevisiae is known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverse S. cerevisiae strains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na(+), acetate, and p-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts of pCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose and pCA or FA with a wild S. cerevisiae strain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production.

Engineering yeasts for xylose metabolism

Treesearch

Thomas W. Jeffries

2006-01-01

Technologies for the production of alternative fuels are receiving increased attention owing to concerns over the rising cost of petrol and global warming. One such technology under development is the use of yeasts for the commercial fermentation of xylose to ethanol. Several approaches have been employed to engineer xylose metabolism. These involve modeling, flux...

Improved Ethanol Production from Xylose by Candida shehatae Induced by Dielectric Barrier Discharge Air Plasma

NASA Astrophysics Data System (ADS)

Chen, Huixia; Xiu, Zhilong; Bai, Fengwu

2014-06-01

Xylose fermentation is essential for ethanol production from lignocellulosic biomass. Exposure of the xylose-fermenting yeast Candida shehatae (C. shehatae) CICC1766 to atmospheric pressure dielectric barrier discharge (DBD) air plasma yields a clone (designated as C81015) with stability, which exhibits a higher ethanol fermentation rate from xylose, giving a maximal enhancement in ethanol production of 36.2% compared to the control (untreated). However, the biomass production of C81015 is lower than that of the control. Analysis of the NADH (nicotinamide adenine dinucleotide)- and NADPH (nicotinamide adenine dinucleotide phosphate)-linked xylose reductases and NAD+-linked xylitol dehydrogenase indicates that their activities are enhanced by 34.1%, 61.5% and 66.3%, respectively, suggesting that the activities of these three enzymes are responsible for improving ethanol fermentation in C81015 with xylose as a substrate. The results of this study show that DBD air plasma could serve as a novel and effective means of generating microbial strains that can better use xylose for ethanol fermentation.

Optimized Production of Xylitol from Xylose Using a Hyper-Acidophilic Candida tropicalis.

PubMed

Tamburini, Elena; Costa, Stefania; Marchetti, Maria Gabriella; Pedrini, Paola

2015-08-19

The yeast Candida tropicalis DSM 7524 produces xylitol, a natural, low-calorie sweetener, by fermentation of xylose. In order to increase xylitol production rate during the submerged fermentation process, some parameters-substrate (xylose) concentration, pH, aeration rate, temperature and fermentation strategy-have been optimized. The maximum xylitol yield reached at 60-80 g/L initial xylose concentration, pH 5.5 at 37 °C was 83.66% (w/w) on consumed xylose in microaerophilic conditions (kLa = 2·h(-1)). Scaling up on 3 L fermenter, with a fed-batch strategy, the best xylitol yield was 86.84% (w/w), against a 90% of theoretical yield. The hyper-acidophilic behaviour of C. tropicalis makes this strain particularly promising for industrial application, due to the possibility to work in non-sterile conditions.

Synthetic Consortium of Escherichia coli for n-Butanol Production by Fermentation of the Glucose-Xylose Mixture.

PubMed

Saini, Mukesh; Lin, Li-Jen; Chiang, Chung-Jen; Chao, Yun-Peng

2017-11-22

The microbial production of n-butanol using glucose and xylose, the major components of plant biomass, can provide a sustainable and renewable fuel as crude oil replacement. However, Escherichia coli prefers glucose to xylose as programmed by carbohydrate catabolite repression (CCR). In this study, a synthetic consortium consisting of two strains was developed by transforming the CCR-insensitive strain into a glucose-selective strain and a xylose-selective strain. Furthermore, the dual culture was reshaped by distribution of the synthetic pathway of n-butanol into two strains. Consequently, the co-culture system enabled effective co-utilization of both sugars and production of 5.2 g/L n-butanol at 30 h. The result leads to the conversion yield and productivity accounting for 63% of the theoretical yield and 0.17 g L -1 h -1 , respectively. Overall, the technology platform as proposed is useful for production of other value-added chemicals, which require complicated pathways for their synthesis by microbial fermentation of a sugar mixture.

Draft Genome Sequence of the d-Xylose-Fermenting Yeast Spathaspora arborariae UFMG-HM19.1AT

PubMed Central

Lobo, Francisco P.; Gonçalves, Davi L.; Alves, Sergio L.; Gerber, Alexandra L.; de Vasconcelos, Ana Tereza R.; Basso, Luiz C.; Franco, Glória R.; Soares, Marco A.; Cadete, Raquel M.; Rosa, Carlos A.

2014-01-01

The draft genome sequence of the yeast Spathaspora arborariae UFMG-HM19.1AT (CBS 11463 = NRRL Y-48658) is presented here. The sequenced genome size is 12.7 Mb, consisting of 41 scaffolds containing a total of 5,625 predicted open reading frames, including many genes encoding enzymes and transporters involved in d-xylose fermentation. PMID:24435867

Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae.

PubMed

Lee, Sung-Haeng; Kodaki, Tsutomu; Park, Yong-Cheol; Seo, Jin-Ho

2012-04-30

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l⁻¹ h⁻¹ xylose consumption rate, 0.25 g l⁻¹ h⁻¹ ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only. Copyright © 2011 Elsevier B.V. All rights reserved.

Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae.

PubMed

Kwak, Suryang; Kim, Soo Rin; Xu, Haiqing; Zhang, Guo-Chang; Lane, Stephan; Kim, Heejin; Jin, Yong-Su

2017-11-01

Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. Biotechnol. Bioeng. 2017;114: 2581-2591. © 2017 Wiley Periodicals, Inc. © 2017 Wiley

Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D-xylose

USDA-ARS?s Scientific Manuscript database

Scheffersomyces (formly Pichia) stipitis is a potential biocatalyst for converting lignocelluloses to ethanol because the yeast natively ferments xylose. An unstructured kinetic model based upon a system of linear differential equations has been formulated that describes growth and ethanol productio...

Optimized Production of Xylitol from Xylose Using a Hyper-Acidophilic Candida tropicalis

PubMed Central

Tamburini, Elena; Costa, Stefania; Marchetti, Maria Gabriella; Pedrini, Paola

2015-01-01

The yeast Candida tropicalis DSM 7524 produces xylitol, a natural, low-calorie sweetener, by fermentation of xylose. In order to increase xylitol production rate during the submerged fermentation process, some parameters-substrate (xylose) concentration, pH, aeration rate, temperature and fermentation strategy-have been optimized. The maximum xylitol yield reached at 60–80 g/L initial xylose concentration, pH 5.5 at 37 °C was 83.66% (w/w) on consumed xylose in microaerophilic conditions (kLa = 2·h−1). Scaling up on 3 L fermenter, with a fed-batch strategy, the best xylitol yield was 86.84% (w/w), against a 90% of theoretical yield. The hyper-acidophilic behaviour of C. tropicalis makes this strain particularly promising for industrial application, due to the possibility to work in non-sterile conditions. PMID:26295411

D-Xylose fermentation, xylitol production and xylanase activities by seven new species of Sugiyamaella.

PubMed

Sena, Letícia M F; Morais, Camila G; Lopes, Mariana R; Santos, Renata O; Uetanabaro, Ana P T; Morais, Paula B; Vital, Marcos J S; de Morais, Marcos A; Lachance, Marc-André; Rosa, Carlos A

2017-01-01

Sixteen yeast isolates identified as belonging to the genus Sugiyamaella were studied in relation to D-xylose fermentation, xylitol production, and xylanase activities. The yeasts were recovered from rotting wood and sugarcane bagasse samples in different Brazilian regions. Sequence analyses of the internal transcribed spacer (ITS) region and the D1/D2 domains of large subunit rRNA gene showed that these isolates belong to seven new species. The species are described here as Sugiyamaella ayubii f.a., sp. nov. (UFMG-CM-Y607 T  = CBS 14108 T ), Sugiyamaella bahiana f.a., sp. nov. (UFMG-CM-Y304 T  = CBS 13474 T ), Sugiyamaella bonitensis f.a., sp. nov. (UFMG-CM-Y608 T  = CBS 14270 T ), Sugiyamaella carassensis f.a., sp. nov. (UFMG-CM-Y606 T  = CBS 14107 T ), Sugiyamaella ligni f.a., sp. nov. (UFMG-CM-Y295 T  = CBS 13482 T ), Sugiyamaella valenteae f.a., sp. nov. (UFMG-CM-Y609 T  = CBS 14109 T ) and Sugiyamaella xylolytica f.a., sp. nov. (UFMG-CM-Y348 T  = CBS 13493 T ). Strains of the described species S. boreocaroliniensis, S. lignohabitans, S. novakii and S. xylanicola, isolated from rotting wood of Brazilian ecosystems, were also compared for traits relevant to xylose metabolism. S. valenteae sp. nov., S. xylolytica sp. nov., S. bahiana sp. nov., S. bonitensis sp. nov., S. boreocarolinensis, S. lignohabitans and S. xylanicola were able to ferment D-xylose to ethanol. Xylitol production was observed for all Sugiyamaella species studied, except for S. ayubii sp. nov. All species studied showed xylanolytic activity, with S. xylanicola, S. lignohabitans and S. valenteae sp. nov. having the highest values. Our results suggest these Sugiyamaella species have good potential for biotechnological applications.

Identification and characterization of D-xylulokinase from the D-xylose-fermenting fungus, Mucor circinelloides.

PubMed

Komeda, Hidenobu; Yamasaki-Yashiki, Shino; Hoshino, Kazuhiro; Asano, Yasuhisa

2014-11-01

D-Xylulokinase catalyzes the phosphorylation of D-xylulose in the final step of the pentose catabolic pathway to form d-xylulose-5-phosphate. The D-xylulokinase activity was found to be induced by both D-xylose and L-arabinose, as well as some of the other enzymes involved in the pentose catabolism, in the D-xylose-fermenting zygomycetous fungus, Mucor circinelloides NBRC 4572. The putative gene, xyl3, which may encode D-xylulokinase, was detected in the genome sequence of this strain. The amino acid sequence deduced from the gene was more similar to D-xylulokinases from an animal origin than from other fungi. The recombinant enzyme was purified from the E. coli transformant expressing xyl3 and then characterized. The ATP-dependent phosphorylative activity of the enzyme was the highest toward D-xylulose. Its kinetic parameters were determined as Km (D-xylulose) = 0.29 mM and Km (ATP) = 0.51 mM, indicating that the xyl3 gene encoded D-xylulokinase (McXK). Western blot analysis revealed that McXK was induced by L-arabinose as well as D-xylose and the induction was repressed in the presence of D-glucose, suggesting that the enzyme may be involved in the catabolism of D-xylose and L-arabinose and is subject to carbon catabolite repression in this fungus. This is the first study on D-xylulokinase from zygomycetous fungi. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Cross-reactions between engineered xylose and galactose pathways in recombinant Saccharomyces cerevisiae.

PubMed

Garcia Sanchez, Rosa; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

2010-09-01

Overexpression of the PGM2 gene encoding phosphoglucomutase (Pgm2p) has been shown to improve galactose utilization both under aerobic and under anaerobic conditions. Similarly, xylose utilization has been improved by overexpression of genes encoding xylulokinase (XK), enzymes from the non-oxidative pentose phosphate pathway (non-ox PPP) and deletion of the endogenous aldose reductase GRE3 gene in engineered Saccharomyces cerevisiae strains carrying either fungal or bacterial xylose pathways. In the present study, we investigated how the combination of these traits affect xylose and galactose utilization in the presence or absence of glucose in S. cerevisiae strains engineered with the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. In the absence of PGM2 overexpression, the combined overexpression of XK, the non-ox PPP and deletion of the GRE3 gene significantly delayed aerobic growth on galactose, whereas no difference was observed between the control strain and the xylose-engineered strain when the PGM2 gene was overexpressed. Under anaerobic conditions, the overexpression of the PGM2 gene increased the ethanol yield and the xylose consumption rate in medium containing xylose as the only carbon source. The possibility of Pgm2p acting as a xylose isomerase (XI) could be excluded by measuring the XI activity in both strains. The additional copy of the PGM2 gene also resulted in a shorter fermentation time during the co-consumption of galactose and xylose. However, the effect was lost upon addition of glucose to the growth medium. PGM2 overexpression was shown to benefit xylose and galactose fermentation, alone and in combination. In contrast, galactose fermentation was impaired in the engineered xylose-utilizing strain harbouring extra copies of the non-ox PPP genes and a deletion of the GRE3 gene, unless PGM2 was overexpressed. These cross-reactions are of particular relevance for the fermentation of mixed sugars from lignocellulosic feedstock.

Recombinant Zymomonas mobilis with improved xylose utilization

DOEpatents

Zhang, Min

2003-05-20

A strain derived from Zymomonas mobilis ATCC31821 or its derivative capable of producing ethanol upon fermentation of a carbohydrate medium containing xylose to provide enhanced xylose utilization and enhanced ethanol process yield, the strain or its derivative comprising exogenous genes encoding xylose isornerase, xylulokinase, transaldolase and transketolase, the genes are fused to at least one promotor recognized by Zymomonas which regulates the expression of at least one of the genes.

Effects of Oxygen Limitation on Xylose Fermentation, Intracellular Metabolites, and Key Enzymes of Neurospora crassa AS3.1602

NASA Astrophysics Data System (ADS)

Zhang, Zhihua; Qu, Yinbo; Zhang, Xiao; Lin, Jianqiang

The effects of oxygen limitation on xylose fermentation of Neurospora crassa AS3.1602 were studied using batch cultures. The maximum yield of ethanol was 0.34 g/g at oxygen transfer rate (OTR) of 8.4 mmol/L·h. The maximum yield of xylitol was 0.33 g/g at OTR of 5.1 mmol/L·h. Oxygen limitation greatly affected mycelia growth and xylitol and ethanol productions. The specific growth rate (μ) decreased 82% from 0.045 to 0.008 h-1 when OTR changed from 12.6 to 8.4 mmol/L·h. Intracellular metabolites of the pentose phosphate pathway, glycolysis, and tricarboxylic acid cycle were determined at various OTRs. Concentrations of most intracellular metabolites decreased with the increase in oxygen limitation. Intracellular enzyme activities of xylose reductase, xylitol dehydrogenase, and xylulokinase, the first three enzymes in xylose metabolic pathway, decreased with the increase in oxygen limitation, resulting in the decreased xylose uptake rate. Under all tested conditions, transaldolase and transketolase activities always maintained at low levels, indicating a great control on xylose metabolism. The enzyme of glucose-6-phosphate dehydrogenase played a major role in NADPH regeneration, and its activity decreased remarkably with the increase in oxygen limitation.

Metabolic characterization and transformation of the non-dairy Lactococcus lactis strain KF147, for production of ethanol from xylose.

PubMed

Petersen, Kia Vest; Liu, Jianming; Chen, Jun; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

2017-08-01

The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, the authors characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co-metabolize xylose and arginine, resulting in bi-phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, the authors eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas over-expression of phosphoketolase increased the flux through the phosphoketolase pathway. In general, significant amounts of the mixed-acid products, including lactate, formate, acetate and ethanol, were formed irrespective of xylose concentrations. To demonstrate the potential of KF147 for converting xylose into useful chemicals the authors chose to redirect metabolism towards ethanol production. A synthetic promoter library was used to drive the expression of codon-optimized versions of the Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase, and the outcome was a strain producing ethanol as the sole fermentation product with a high yield corresponding to 83% of the theoretical maximum. The results clearly indicate the great potential of using the more metabolically diverse non-dairy L. lactis strains for bio-production based on xylose containing feedstocks. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains.

PubMed

Dos Santos, Leandro Vieira; Carazzolle, Marcelo Falsarella; Nagamatsu, Sheila Tiemi; Sampaio, Nádia Maria Vieira; Almeida, Ludimila Dias; Pirolla, Renan Augusto Siqueira; Borelli, Guilherme; Corrêa, Thamy Lívia Ribeiro; Argueso, Juan Lucas; Pereira, Gonçalo Amarante Guimarães

2016-12-21

The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In this work, a robust industrial strain of Saccharomyces cerevisiae was modified by the addition of essential genes for pentose metabolism. Subsequently, taken through cycles of adaptive evolution with selection for optimal xylose utilization, strains could efficiently convert xylose to ethanol with a yield of about 0.46 g ethanol/g xylose. Though evolved independently, two strains carried shared mutations: amplification of the xylose isomerase gene and inactivation of ISU1, a gene encoding a scaffold protein involved in the assembly of iron-sulfur clusters. In addition, one of evolved strains carried a mutation in SSK2, a member of MAPKKK signaling pathway. In validation experiments, mutating ISU1 or SSK2 improved the ability to metabolize xylose of yeast cells without adaptive evolution, suggesting that these genes are key players in a regulatory network for xylose fermentation. Furthermore, addition of iron ion to the growth media improved xylose fermentation even by non-evolved cells. Our results provide promising new targets for metabolic engineering of C5-yeasts and point to iron as a potential new additive for improvement of second-generation ethanol production.

Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae

Treesearch

Yong-Su Jin; Thomas W. Jeffries

2004-01-01

Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast raditionally...

Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation

USDA-ARS?s Scientific Manuscript database

Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses nicotinamide adenine dinucl...

Impact of zinc supplementation on the improved fructose/xylose utilization and butanol production during acetone-butanol-ethanol fermentation.

PubMed

Wu, You-Duo; Xue, Chuang; Chen, Li-Jie; Bai, Feng-Wu

2016-01-01

Lignocellulosic biomass and dedicated energy crops such as Jerusalem artichoke are promising alternatives for biobutanol production by solventogenic clostridia. However, fermentable sugars such as fructose or xylose released from the hydrolysis of these feedstocks were subjected to the incomplete utilization by the strains, leading to relatively low butanol production and productivity. When 0.001 g/L ZnSO4·7H2O was supplemented into the medium containing fructose as sole carbon source, 12.8 g/L of butanol was achieved with butanol productivity of 0.089 g/L/h compared to only 4.5 g/L of butanol produced with butanol productivity of 0.028 g/L/h in the control without zinc supplementation. Micronutrient zinc also led to the improved butanol production up to 8.3 g/L derived from 45.2 g/L xylose as sole carbon source with increasing butanol productivity by 31.7%. Moreover, the decreased acids production was observed under the zinc supplementation condition, resulting in the increased butanol yields of 0.202 g/g-fructose and 0.184 g/g-xylose, respectively. Similar improvements were also observed with increasing butanol production by 130.2 % and 8.5 %, butanol productivity by 203.4% and 18.4%, respectively, in acetone-butanol-ethanol fermentations from sugar mixtures of fructose/glucose (4:1) and xylose/glucose (1:2) simulating the hydrolysates of Jerusalem artichoke tubers and corn stover. The results obtained from transcriptional analysis revealed that zinc may have regulatory mechanisms for the sugar transport and metabolism of Clostridium acetobutylicum L7. Therefore, micronutrient zinc supplementation could be an effective way for economic development of butanol production derived from these low-cost agricultural feedstocks. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains

PubMed Central

dos Santos, Leandro Vieira; Carazzolle, Marcelo Falsarella; Nagamatsu, Sheila Tiemi; Sampaio, Nádia Maria Vieira; Almeida, Ludimila Dias; Pirolla, Renan Augusto Siqueira; Borelli, Guilherme; Corrêa, Thamy Lívia Ribeiro; Argueso, Juan Lucas; Pereira, Gonçalo Amarante Guimarães

2016-01-01

The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In this work, a robust industrial strain of Saccharomyces cerevisiae was modified by the addition of essential genes for pentose metabolism. Subsequently, taken through cycles of adaptive evolution with selection for optimal xylose utilization, strains could efficiently convert xylose to ethanol with a yield of about 0.46 g ethanol/g xylose. Though evolved independently, two strains carried shared mutations: amplification of the xylose isomerase gene and inactivation of ISU1, a gene encoding a scaffold protein involved in the assembly of iron-sulfur clusters. In addition, one of evolved strains carried a mutation in SSK2, a member of MAPKKK signaling pathway. In validation experiments, mutating ISU1 or SSK2 improved the ability to metabolize xylose of yeast cells without adaptive evolution, suggesting that these genes are key players in a regulatory network for xylose fermentation. Furthermore, addition of iron ion to the growth media improved xylose fermentation even by non-evolved cells. Our results provide promising new targets for metabolic engineering of C5-yeasts and point to iron as a potential new additive for improvement of second-generation ethanol production. PMID:28000736

Cofermentation of Glucose, Xylose, and Cellobiose by the Beetle-Associated Yeast Spathaspora passalidarum

PubMed Central

Long, Tanya M.; Su, Yi-Kai; Headman, Jennifer; Higbee, Alan; Willis, Laura B.

2012-01-01

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spathaspora passalidarum, which ferments xylose and cellobiose natively, can also coferment these two sugars in the presence of 30 g/liter glucose. S. passalidarum simultaneously assimilates glucose and xylose aerobically, it simultaneously coferments glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, an adapted strain of S. passalidarum produced 39 g/liter ethanol with a yield of 0.37 g/g sugars from a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the flux of glycolytic intermediates is significantly higher on xylose than on glucose. The high affinity of its xylose reductase activities for NADH and xylose combined with allosteric activation of glycolysis probably accounts in part for its unusual capacities. These features make S. passalidarum very attractive for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts. PMID:22636012

Xylitol synthesis mutant of xylose-utilizing zymomonas for ethanol production

DOEpatents

Viitanen, Paul V.; Chou, Yat-Chen; McCutchen, Carol M.; Zhang, Min

2010-06-22

A strain of xylose-utilizing Zymomonas was engineered with a genetic modification to the glucose-fructose oxidoreductase gene resulting in reduced expression of GFOR enzyme activity. The engineered strain exhibits reduced production of xylitol, a detrimental by-product of xylose metabolism. It also consumes more xylose and produces more ethanol during mixed sugar fermentation under process-relevant conditions.

Improved fermentative alcohol production. [Patent application

DOEpatents

Wilke, C.R.; Maiorella, B.L.; Blanch, H.W.; Cysewski, G.R.

1980-11-26

An improved fermentation process is described for producing alcohol which includes the combination of vacuum fermentation and vacuum distillation. Preferably, the vacuum distillation is carried out in two phases, one a fermentor proper operated at atmospheric pressure and a flash phase operated at reduced pressure with recycle of fermentation brew having a reduced alcohol content to the fermentor, using vapor recompression heating of the flash-pot recycle stream to heat the flash-pot or the distillation step, and using water load balancing (i.e., the molar ratio of water in the fermentor feed is the same as the molar ratio of water in the distillation overhead).

The Influence of Sugar Cane Bagasse Type and Its Particle Size on Xylose Production and Xylose-to-Xylitol Bioconversion with the Yeast Debaryomyces hansenii.

PubMed

Aghcheh, Razieh Karimi; Bonakdarpour, Babak; Ashtiani, Farzin Zokaee

2016-11-01

In the present study, the effect of the type of sugar cane bagasse (non-depithed or depithed) and its particle size on the production of xylose and its subsequent fermentation to xylitol by Debaryomyces hansenii CBS767 was investigated using a full factorial experimental design. It was found that the particle size range and whether bagasse was depithed or not had a significant effect on the concentration and yield of xylose in the resulting hemicellulose hydrolysate. Depithed bagasse resulted in higher xylose concentrations compared to non-depithed bagasse. The corresponding detoxified hemicellulose hydrolysates were used as fermentation media for the production of xylitol. The hemicellulose hydrolysate prepared from depithed bagasse also yielded meaningfully higher xylitol fermentation rates compared to non-depithed bagasse. However, in the case of non-depithed bagasse, the hemicellulose hydrolysate prepared from larger particle size range resulted in higher xylitol fermentation rates, whereas the effect in the case of non-depithed bagasse was not pronounced. Therefore, depithing of bagasse is an advantageous pretreatment when it is to be employed in bioconversion processes.

Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

PubMed

Ko, Ja Kyong; Um, Youngsoon; Woo, Han Min; Kim, Kyoung Heon; Lee, Sun-Mi

2016-06-01

The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion.

PubMed

Turner, Timothy L; Zhang, Guo-Chang; Kim, Soo Rin; Subramaniam, Vijay; Steffen, David; Skory, Christopher D; Jang, Ji Yeon; Yu, Byung Jo; Jin, Yong-Su

2015-10-01

Production of lactic acid from renewable sugars has received growing attention as lactic acid can be used for making renewable and bio-based plastics. However, most prior studies have focused on production of lactic acid from glucose despite that cellulosic hydrolysates contain xylose as well as glucose. Microbial strains capable of fermenting both glucose and xylose into lactic acid are needed for sustainable and economic lactic acid production. In this study, we introduced a lactic acid-producing pathway into an engineered Saccharomyces cerevisiae capable of fermenting xylose. Specifically, ldhA from the fungi Rhizopus oryzae was overexpressed under the control of the PGK1 promoter through integration of the expression cassette in the chromosome. The resulting strain exhibited a high lactate dehydrogenase activity and produced lactic acid from glucose or xylose. Interestingly, we observed that the engineered strain exhibited substrate-dependent product formation. When the engineered yeast was cultured on glucose, the major fermentation product was ethanol while lactic acid was a minor product. In contrast, the engineered yeast produced lactic acid almost exclusively when cultured on xylose under oxygen-limited conditions. The yields of ethanol and lactic acid from glucose were 0.31 g ethanol/g glucose and 0.22 g lactic acid/g glucose, respectively. On xylose, the yields of ethanol and lactic acid were <0.01 g ethanol/g xylose and 0.69 g lactic acid/g xylose, respectively. These results demonstrate that lactic acid can be produced from xylose with a high yield by S. cerevisiae without deleting pyruvate decarboxylase, and the formation patterns of fermentations can be altered by substrates.

A Novel simultaneous-Saccharification-Fermentation Strategy for Efficient Co-fermentation of C5 and C6 Sugars Using Native, Non-GMO Yeasts

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

Varanasi, Sasidhar; Relue, Patricia

Economic bioethanol production is critically dependent upon the ability to convert both the hexose (C6) and pentose (C5) sugars resulting from cellulose and hemicellulose. C5 sugars are not readily fermentable by native Saccharomyces cerevisiae. Genetically Modified Organisms (GMOs) are designed to ferment xylose, but their stability, ethanol yield, environmental impact, and survival under conditions of industrial fermentation are unproven. In this project, we developed a novel approach for efficient fermentation of both C5 and C6 sugars using native S. Cerevisiae by exploiting its ability to produce ethanol from xylulose - the keto-isomer of xylose. While the isomerization of xylose tomore » xylulose can be accomplished via commercially (and cheaply) available Xylose Isomerase (XI) (Sweetzyme™), this conversion has an extremely unfavorable equilibrium (xylose:xylose is about 5:1). To address this, we developed two alternate strategies. In the first, the two enzymes XI and urease are coimmobilized on solid support particles to enable complete isomerization of xylose to xylulose under pH conditions suitable for fermentation, in a simultaneous-isomerization-fermentation (SIF) mode. The ability of our technology to conduct isomerization of xylose under pH conditions suitable for both saccharification and fermentation opens the possibility of SSF with native yeasts for the first time. Herein, we performed specific research tasks for implementation of our technology in several modes of operation, including simultaneous-isomerization-and-fermentation (SIF), simultaneous-saccharification-and-isomerization (SSI) followed by fermentation, and SSF mode with the biomass feedstock poplar. The projected economics of our process are very favorable in comparison to the costs associated with engineering, licensing and propagating GMOs. This novel fermentation technology is readily accessible to rural farming economies for implementation in cellulosic ethanol production

Process for producing fuel grade ethanol by continuous fermentation, solvent extraction and alcohol separation

DOEpatents

Tedder, Daniel W.

1985-05-14

Alcohol substantially free of water is prepared by continuously fermenting a fermentable biomass feedstock in a fermentation unit, thereby forming an aqueous fermentation liquor containing alcohol and microorganisms. Continuously extracting a portion of alcohol from said fermentation liquor with an organic solvent system containing an extractant for said alcohol, thereby forming an alcohol-organic solvent extract phase and an aqueous raffinate. Said alcohol is separated from said alcohol-organic solvent phase. A raffinate comprising microorganisms and unextracted alcohol is returned to the fermentation unit.

Ethanol production using xylitol synthesis mutant of xylose-utilizing zymomonas

DOEpatents

Viitanen, Paul V.; McCutchen, Carol M.; Emptage, Mark; Caimi, Perry G.; Zhang, Min; Chou, Yat-Chen

2010-06-22

Production of ethanol using a strain of xylose-utilizing Zymomonas with a genetic modification of the glucose-fructose oxidoreductase gene was found to be improved due to greatly reduced production of xylitol, a detrimental by-product of xylose metabolism synthesized during fermentation.

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis.

PubMed

Watanabe, Seiya; Abu Saleh, Ahmed; Pack, Seung Pil; Annaluru, Narayana; Kodaki, Tsutomu; Makino, Keisuke

2007-09-01

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD(+)-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k(cat)/K(m) with NADH)/(k(cat)/K(m) with NADPH)] compared with the wild-type (WT), which was due to decrease of k(cat) with NADPH in the R276H mutant and increase of K(m) with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP(+) and NADH/NAD(+) ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering. PMID:21219616

Ethanol production in fermentation of mixed sugars containing xylose

DOEpatents

Viitanen, Paul V [West Chester, PA; Mc Cutchen, Carol M [Wilmington, DE; Li,; Xu, [Newark, DE; Emptage, Mark [Wilmington, DE; Caimi, Perry G [Kennett Square, PA; Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Lakewood, CO; Franden, Mary Ann [Centennial, CO

2009-12-08

Xylose-utilizing Z. mobilis strains were found to have improved ethanol production when grown in medium containing mixed sugars including xylose if sorbitol or mannitol was included in the medium. The effect was seen in concentrations of mixed sugars where no growth lag period occurs, as well as in higher sugars concentrations.

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production.

PubMed

Yuvadetkun, Prawphan; Leksawasdi, Noppol; Boonmee, Mallika

2017-03-16

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8 g/L in xylose and 52.6 g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4 g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40 g/L of ethanol and ethanol production capacity of the yeast was 52 g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170 g/L sugar concentrations.

Genetically engineered Escherichia coli FBR5: Part II. Ethanol production from xylose and simultaneous product recovery

USDA-ARS?s Scientific Manuscript database

In these studies concentrated xylose solution was fermented to ethanol employing Escherichia coli FBR5 which can ferment both lignocellulosic sugars (hexoses and pentoses). E. coli FBR5 can produce 40-50 gL-1 ethanol from 100 gL-1 xylose in batch reactors. Increasing sugar concentration beyond this...

Homo-D-lactic acid production from mixed sugars using xylose-assimilating operon-integrated Lactobacillus plantarum.

PubMed

Yoshida, Shogo; Okano, Kenji; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

2011-10-01

In order to achieve efficient D-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (ΔldhL1-xpk1::tkt-Δxpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of ΔldhL1-xpk1::tkt-Δxpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-D-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of D-lactic acid of 99.5%. Finally, we successfully demonstrated homo-D-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-D-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.

Production of Star Fruit Alcoholic Fermented Beverage.

PubMed

Valim, Flávia de Paula; Aguiar-Oliveira, Elizama; Kamimura, Eliana Setsuko; Alves, Vanessa Dias; Maldonado, Rafael Resende

2016-12-01

Star fruit ( Averrhoa carambola ) is a nutritious tropical fruit. The aim of this study was to evaluate the production of a star fruit alcoholic fermented beverage utilizing a lyophilized commercial yeast ( Saccharomyces cerevisiae ). The study was conducted utilizing a 2 3 central composite design and the best conditions for the production were: initial soluble solids between 23.8 and 25 °Brix (g 100 g -1 ), initial pH between 4.8 and 5.0 and initial concentration of yeast between 1.6 and 2.5 g L -1 . These conditions yielded a fermented drink with an alcohol content of 11.15 °GL (L 100 L -1 ), pH of 4.13-4.22, final yeast concentration of 89 g L -1 and fermented yield from 82 to 94 %. The fermented drink also presented low levels of total and volatile acidities.

Improved ethanol production from xylose in the presence of acetic acid by the overexpression of the HAA1 gene in Saccharomyces cerevisiae.

PubMed

Sakihama, Yuri; Hasunuma, Tomohisa; Kondo, Akihiko

2015-03-01

The hydrolysis of lignocellulosic biomass liberates sugars, primarily glucose and xylose, which are subsequently converted to ethanol by microbial fermentation. The rapid and efficient fermentation of xylose by recombinant Saccharomyces cerevisiae strains is limited by weak acids generated during biomass pretreatment processes. In particular, acetic acid negatively affects cell growth, xylose fermentation rate, and ethanol production. The ability of S. cerevisiae to efficiently utilize xylose in the presence of acetic acid is an essential requirement for the cost-effective production of ethanol from lignocellulosic hydrolysates. Here, an acetic acid-responsive transcriptional activator, HAA1, was overexpressed in a recombinant xylose-fermenting S. cerevisiae strain to yield BY4741X/HAA1. This strain exhibited improved cell growth and ethanol production from xylose under aerobic and oxygen limited conditions, respectively, in the presence of acetic acid. The HAA1p regulon enhanced transcript levels in BY4741X/HAA1. The disruption of PHO13, a p-nitrophenylphosphatase gene, in BY4741X/HAA1 led to further improvement in both yeast growth and the ability to ferment xylose, indicating that HAA1 overexpression and PHO13 deletion act by different mechanisms to enhance ethanol production. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Butyric acid production from lignocellulosic biomass hydrolysates by engineered Clostridium tyrobutyricum overexpressing xylose catabolism genes for glucose and xylose co-utilization.

PubMed

Fu, Hongxin; Yang, Shang-Tian; Wang, Minqi; Wang, Jufang; Tang, I-Ching

2017-06-01

Clostridium tyrobutyricum can utilize glucose and xylose as carbon source for butyric acid production. However, xylose catabolism is inhibited by glucose, hampering butyric acid production from lignocellulosic biomass hydrolysates containing both glucose and xylose. In this study, an engineered strain of C. tyrobutyricum Ct-pTBA overexpressing heterologous xylose catabolism genes (xylT, xylA, and xylB) was investigated for co-utilizing glucose and xylose present in hydrolysates of plant biomass, including soybean hull, corn fiber, wheat straw, rice straw, and sugarcane bagasse. Compared to the wild-type strain, Ct-pTBA showed higher xylose utilization without significant glucose catabolite repression, achieving near 100% utilization of glucose and xylose present in lignocellulosic biomass hydrolysates in bioreactor at pH 6. About 42.6g/L butyrate at a productivity of 0.56g/L·h and yield of 0.36g/g was obtained in batch fermentation, demonstrating the potential of C. tyrobutyricum Ct-pTBA for butyric acid production from lignocellulosic biomass hydrolysates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mechanism of imidazolium ionic liquids toxicity in Saccharomyces cerevisiae and rational engineering of a tolerant, xylose-fermenting strain

DOE PAGES

Dickinson, Quinn; Bottoms, Scott; Hinchman, Li; ...

2016-01-20

In this study, imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae. As a result, we found that IILs likely target mitochondria as their chemical genomic profiles closely resembled that of the mitochondrial membrane disrupting agent valinomycin. Further, several deletions of genes encoding mitochondrial proteins exhibited increased sensitivity to IIL. High-throughput chemical proteomics confirmed effectsmore » of IILs on mitochondrial protein levels. IILs induced abnormal mitochondrial morphology, as well as altered polarization of mitochondrial membrane potential similar to valinomycin. Deletion of the putative serine/threonine kinase PTK2 thought to activate the plasma-membrane proton efflux pump Pma1p conferred a significant IIL-fitness advantage. Conversely, overexpression of PMA1 conferred sensitivity to IILs, suggesting that hydrogen ion efflux may be coupled to influx of the toxic imidazolium cation. PTK2 deletion conferred resistance to multiple IILs, including [EMIM]Cl, [BMIM]Cl, and [EMIM]Ac. An engineered, xylose-converting ptk2Δ S. cerevisiae (Y133-IIL) strain consumed glucose and xylose faster and produced more ethanol in the presence of 1 % [BMIM]Cl than the wild-type PTK2 strain. We propose a model of IIL toxicity and resistance. In conclusion, this work demonstrates the utility of chemical genomics-guided biodesign for development of superior microbial biocatalysts for the ever-changing landscape of fermentation inhibitors.« less

Genome sequence and physiological analysis of Yamadazyma laniorum f.a. sp. nov. and a reevaluation of the apocryphal xylose fermentation of its sister species, Candida tenuis.

PubMed

Haase, Max A B; Kominek, Jacek; Langdon, Quinn K; Kurtzman, Cletus P; Hittinger, Chris Todd

2017-05-01

Xylose fermentation is a rare trait that is immensely important to the cellulosic biofuel industry, and Candida tenuis is one of the few yeasts that has been reported with this trait. Here we report the isolation of two strains representing a candidate sister species to C. tenuis. Integrated analysis of genome sequence and physiology suggested the genetic basis of a number of traits, including variation between the novel species and C. tenuis in lactose metabolism due to the loss of genes encoding lactose permease and β-galactosidase in the former. Surprisingly, physiological characterization revealed that neither the type strain of C. tenuis nor this novel species fermented xylose in traditional assays. We reexamined three xylose-fermenting strains previously identified as C. tenuis and found that these strains belong to the genus Scheffersomyces and are not C. tenuis. We propose Yamadazyma laniorum f.a. sp. nov. to accommodate our new strains and designate its type strain as yHMH7 (=CBS 14780 = NRRL Y-63967T). Furthermore, we propose the transfer of Candida tenuis to the genus Yamadazyma as Yamadazyma tenuis comb. nov. This approach provides a roadmap for how integrated genome sequence and physiological analysis can yield insight into the mechanisms that generate yeast biodiversity. Published by Oxford University Press on behalf of FEMS 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis.

PubMed

Kim, J-H; Han, K-C; Koh, Y-H; Ryu, Y-W; Seo, J-H

2002-07-01

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l(-1)) and less than 200 g l(-1) total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l(-1) xylitol concentration, 0.75 g xylitol g xylose(-1) xylitol yield and 3.9 g xylitol l(-1) h(-1) volumetric productivity.

Spathaspora piracicabensis f. a., sp. nov., a D-xylose-fermenting yeast species isolated from rotting wood in Brazil.

PubMed

Varize, Camila S; Cadete, Raquel M; Lopes, Lucas D; Christofoleti-Furlan, Renata M; Lachance, Marc-André; Rosa, Carlos A; Basso, Luiz C

2018-04-01

Two strains of a novel yeast species were isolated from rotting wood of an ornamental tree (purple quaresmeira, Tibouchina granulosa, Melastomataceae) in an Atlantic Rainforest area in Brazil. Analysis of the sequences of the internal transcribed spacer (ITS-5.8S) region and the D1/D2 domains of the large subunit rRNA gene showed that this species belongs to the Spathaspora clade, and is phylogenetically related to Spathaspora brasiliensis, Candida materiae and Sp. girioi. The novel species ferments D-xylose, producing ethanol, with amounts between 3.37 and 3.48 g L -1 ethanol from 2% D-xylose. Ascospores were not observed from this new species. The name Spathaspora piracicabensis f. a., sp. nov. is proposed to accommodate these isolates. The type strain is UFMG-CM-Y5867 T (= CBS 15054 T  = ESALQ-I54 T ). The MycoBank number is MB 822,320.

Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.

PubMed

Waltman, Mary Jo; Yang, Zamin Koo; Langan, Paul; Graham, David E; Kovalevsky, Andrey

2014-02-01

To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

Biofuels. Engineering alcohol tolerance in yeast.

PubMed

Lam, Felix H; Ghaderi, Adel; Fink, Gerald R; Stephanopoulos, Gregory

2014-10-03

Ethanol toxicity in the yeast Saccharomyces cerevisiae limits titer and productivity in the industrial production of transportation bioethanol. We show that strengthening the opposing potassium and proton electrochemical membrane gradients is a mechanism that enhances general resistance to multiple alcohols. The elevation of extracellular potassium and pH physically bolsters these gradients, increasing tolerance to higher alcohols and ethanol fermentation in commercial and laboratory strains (including a xylose-fermenting strain) under industrial-like conditions. Production per cell remains largely unchanged, with improvements deriving from heightened population viability. Likewise, up-regulation of the potassium and proton pumps in the laboratory strain enhances performance to levels exceeding those of industrial strains. Although genetically complex, alcohol tolerance can thus be dominated by a single cellular process, one controlled by a major physicochemical component but amenable to biological augmentation. Copyright © 2014, American Association for the Advancement of Science.

Microaerobic conversion of xylose to ethanol in recombinant Saccharomyces cerevisiae SX6(MUT) expressing cofactor-balanced xylose metabolic enzymes and deficient in ALD6.

PubMed

Jo, Sung-Eun; Seong, Yeong-Je; Lee, Hyun-Soo; Lee, Soo Min; Kim, Soo-Jung; Park, Kyungmoon; Park, Yong-Cheol

2016-06-10

Xylose is a major monosugar in cellulosic biomass and should be utilized for cost-effective ethanol production. In this study, xylose-converting ability of recombinant Saccharomyces cerevisiae SX6(MUT) expressing NADH-preferring xylose reductase mutant (R276H) and other xylose-metabolic enzymes, and deficient in aldehyde dehydrogenase 6 (Ald6p) were characterized at microaerobic conditions using various sugar mixtures. The reduction of air supply from 0.5vvm to 0.1vvm increased specific ethanol production rate by 75% and did not affect specific xylose consumption rate. In batch fermentations using various concentrations of xylose (50-104g/L), higher xylose concentration enhanced xylose consumption rate and ethanol productivity but reduced ethanol yield, owing to the accumulation of xylitol and glycerol from xylose. SX6(MUT) consumed monosugars in pitch pine hydrolysates and produced 23.1g/L ethanol from 58.7g/L sugars with 0.39g/g ethanol yield, which was 14% higher than the host strain of S. cerevisiae D452-2 without the xylose assimilating enzymes. In conclusion, S. cerevisiae SX6(MUT) was characterized to possess high xylose-consuming ability in microaerobic conditions and a potential for ethanol production from cellulosic biomass. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of a recombinant flocculent Saccharomyces cerevisiae strain that co-ferments glucose and xylose: II. influence of pH and acetic acid on ethanol production.

PubMed

Matsushika, Akinori; Sawayama, Shigeki

2012-12-01

The inhibitory effects of pH and acetic acid on the co-fermentation of glucose and xylose in complex medium by recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated. In the absence of acetic acid, the fermentation performance of strain MA-R4 was similar between pH 4.0-6.0, but was negatively affected at pH 2.5. The addition of acetic acid to batch cultures resulted in negligible inhibition of several fermentation parameters at pH 6.0, whereas the interactive inhibition of pH and acetic acid on the maximum cell and ethanol concentrations, and rates of sugar consumption and ethanol production were observed at pH levels below 5.4. The inhibitory effect of acetic acid was particularly marked for the consumption rate of xylose, as compared with that of glucose. With increasing initial acetic acid concentration, the ethanol yield slightly increased at pH 5.4 and 6.0, but decreased at pH values lower than 4.7. Notably, ethanol production was nearly completely inhibited under low pH (4.0) and high acetic acid (150-200 mM) conditions. Together, these results indicate that the inhibitory effects of acetic acid and pH on ethanol fermentation by MA-R4 are highly synergistic, although the inhibition can be reduced by increasing the medium pH.

Alcoholic fermentation induces melatonin synthesis in orange juice.

PubMed

Fernández-Pachón, M S; Medina, S; Herrero-Martín, G; Cerrillo, I; Berná, G; Escudero-López, B; Ferreres, F; Martín, F; García-Parrilla, M C; Gil-Izquierdo, A

2014-01-01

Melatonin (N-acetyl-5-methoxytryptamine) is a molecule implicated in multiple biological functions. Its level decreases with age, and the intake of foods rich in melatonin has been considered an exogenous source of this important agent. Orange is a natural source of melatonin. Melatonin synthesis occurs during alcoholic fermentation of grapes, malt and pomegranate. The amino acid tryptophan is the precursor of all 5-methoxytryptamines. Indeed, melatonin appears in a shorter time in wines when tryptophan is added before fermentation. The aim of the study was to measure melatonin content during alcoholic fermentation of orange juice and to evaluate the role of the precursor tryptophan. Identification and quantification of melatonin during the alcoholic fermentation of orange juice was carried out by UHPLC-QqQ-MS/MS. Melatonin significantly increased throughout fermentation from day 0 (3.15 ng/mL) until day 15 (21.80 ng/mL) reaching larger amounts with respect to other foods. Melatonin isomer was also analysed, but its content remained stable ranging from 11.59 to 14.18 ng/mL. The enhancement of melatonin occurred mainly in the soluble fraction. Tryptophan levels significantly dropped from 13.80 mg/L (day 0) up to 3.19 mg/L (day 15) during fermentation. Melatonin was inversely and significantly correlated with tryptophan (r = 0.907). Therefore, the enhancement in melatonin could be due to both the occurrence of tryptophan and the new synthesis by yeast. In summary, the enhancement of melatonin in novel fermented orange beverage would improve the health benefits of orange juice by increasing this bioactive compound. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Polyamine Transporters and Polyamines Increase Furfural Tolerance during Xylose Fermentation with Ethanologenic Escherichia coli Strain LY180

PubMed Central

Geddes, Ryan D.; Wang, Xuan; Yomano, Lorraine P.; Miller, Elliot N.; Zheng, Huabao; Shanmugam, Keelnatham T.

2014-01-01

Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural. PMID:25063650

Electricity and H2 generation from hemicellulose by sequential fermentation and microbial fuel/electrolysis cell

NASA Astrophysics Data System (ADS)

Yan, Di; Yang, Xuewei; Yuan, Wenqiao

2015-09-01

Electricity and hydrogen generation by bacteria Geobacter sulfurreducens in a dual-chamber microbial fuel/electrolysis cell following the fermentation of hemicellulose by bacteria Moorella thermoacetica was investigated. Experimental results showed that 10 g l-1 xylose under 60 °C was appropriate for the fermentation of xylose by M. thermoacetica, yielding 0.87 g-acetic acid per gram of xylose consumed. Corncob hydrolysate could also be fermented to produce acetic acid, but with lower yield (0.74 g-acid per g-xylose). The broths of xylose and corncob hydrolysate fermented by M. thermoacetica containing acetic acid were fed to G. sulfurreducens in a dual-chamber microbial fuel/electrolysis cell for electricity and hydrogen generation. The highest open-circuit cell voltages generated were 802 and 745 mV, and hydrogen yields were 41.7 and 23.3 mmol per mol-acetate, in xylose and corncob hydrolysate fermentation broth media, respectively. The internal resistance of the microbial fuel/electrolysis cell fed with corncob hydrolysate fermentation broth (3472 Ω) was much higher than that with xylose fermentation broth (1993 Ω) or sodium acetate medium (467 Ω), which was believed to be the main cause of the variation in hydrogen yield of the three feeding media.

Time-based comparative transcriptomics in engineered xylose-utilizing Saccharomyces cerevisiae identifies temperature-responsive genes during ethanol production.

PubMed

Ismail, Ku Syahidah Ku; Sakamoto, Takatoshi; Hasunuma, Tomohisa; Kondo, Akihiko

2013-09-01

Agricultural residues comprising lignocellulosic materials are excellent sources of pentose sugar, which can be converted to ethanol as fuel. Ethanol production via consolidated bioprocessing requires a suitable microorganism to withstand the harsh fermentation environment of high temperature, high ethanol concentration, and exposure to inhibitors. We genetically enhanced an industrial Saccharomyces cerevisiae strain, sun049, enabling it to uptake xylose as the sole carbon source at high fermentation temperature. This strain was able to produce 13.9 g/l ethanol from 50 g/l xylose at 38 °C. To better understand the xylose consumption ability during long-term, high-temperature conditions, we compared by transcriptomics two fermentation conditions: high temperature (38 °C) and control temperature (30 °C) during the first 12 h of fermentation. This is the first long-term, time-based transcriptomics approach, and it allowed us to discover the role of heat-responsive genes when xylose is the sole carbon source. The results suggest that genes related to amino acid, cell wall, and ribosomal protein synthesis are down-regulated under heat stress. To allow cell stability and continuous xylose uptake in order to produce ethanol, hexose transporter HXT5, heat shock proteins, ubiquitin proteins, and proteolysis were all induced at high temperature. We also speculate that the strong relationship between high temperature and increased xylitol accumulation represents the cell's mechanism to protect itself from heat degradation.

Effect of Brönsted acidic ionic liquid 1-(1-propylsulfonic)-3-methylimidazolium chloride on growth and co-fermentation of glucose, xylose and arabinose by Zymomonas mobilis AX101.

PubMed

Gyamerah, M; Ampaw-Asiedu, M; Mackey, J; Menezes, B; Woldesenbet, S

2018-06-01

The potential of large-scale lignocellulosic biomass hydrolysis to fermentable sugars using ionic liquids has increased interest in this green chemistry route to fermentation for fuel-ethanol production. The ionic liquid 1-(1-propylsulfonic)-3-methylimidazolium chloride compared to other reported ionic liquids has the advantage of hydrolysing lignocellulosic biomass to reducing sugars at catalytic concentrations (≤0·032 mol l -1 ) in a single step. However, effects of this ionic liquid on co-fermentation of glucose, xylose and arabinose to ethanol by recombinant Zymomonas mobilisAX101 has not been studied. Authentic glucose, xylose and arabinose were used to formulate fermentation media at varying catalytic 1-(1-propylsulfonic)-3-methylimidazolium chloride concentrations for batch co-fermentation of the sugars using Z. mobilisAX101. The results showed that at 0·008, 0·016 and 0·032 mol l -1 ionic liquid in the culture medium, cell growth decreased by 10, 27 and 67% respectively compared to the control. Ethanol yields were 62·6, 61·8, 50·5 and 23·1% for the control, 0·008, 0·016 and 0·032 mol l -1 ionic liquid respectively. The results indicate that lignocellulosic biomass hydrolysed using 0·008 mol l -1 of 1-(1-propylsulfonic)-3-methylimidazolium chloride would eliminate an additional separation step and provide a ready to use fermentation substrate. This is the first reported study of the effect of the Brönsted acidic ionic liquid 1-(1-propylsulfonic)-3-methylimidazolium chloride on growth and co-fermentation of glucose, xylose and arabinose by Zymomonas mobilisAX101 in batch culture. Growth on and co-fermentation of the sugars by Z. mobilisAX 101 with no significant inhibition by the ionic liquid at the same catalytic amounts of 0·008 mol l -1 used to hydrolyse lignocellulosic biomass to reducing sugars overcome two major hurdles that adversely affect the process economics of large-scale industrial cellulosic fuel ethanol production

Co-Utilization of Glucose and Xylose for Enhanced Lignocellulosic Ethanol Production with Reverse Membrane Bioreactors

PubMed Central

Ishola, Mofoluwake M.; Ylitervo, Päivi; Taherzadeh, Mohammad J.

2015-01-01

Integrated permeate channel (IPC) flat sheet membranes were examined for use as a reverse membrane bioreactor (rMBR) for lignocellulosic ethanol production. The fermenting organism, Saccharomyces cerevisiae (T0936), a genetically-modified strain with the ability to ferment xylose, was used inside the rMBR. The rMBR was evaluated for simultaneous glucose and xylose utilization as well as in situ detoxification of furfural and hydroxylmethyl furfural (HMF). The synthetic medium was investigated, after which the pretreated wheat straw was used as a xylose-rich lignocellulosic substrate. The IPC membrane panels were successfully used as the rMBR during the batch fermentations, which lasted for up to eight days without fouling. With the rMBR, complete glucose and xylose utilization, resulting in 86% of the theoretical ethanol yield, was observed with the synthetic medium. Its application with the pretreated wheat straw resulted in complete glucose consumption and 87% xylose utilization; a final ethanol concentration of 30.3 g/L was obtained, which corresponds to 83% of the theoretical yield. Moreover, complete in situ detoxification of furfural and HMF was obtained within 36 h and 60 h, respectively, with the rMBR. The use of the rMBR is a promising technology for large-scale lignocellulosic ethanol production, since it facilitates the co-utilization of glucose and xylose; moreover, the technology would also allow the reuse of the yeast for several batches. PMID:26633530

Directed evolution reveals unexpected epistatic interactions that alter metabolic regulation and enable anaerobic xylose use by Saccharomyces cerevisiae

DOE PAGES

Sato, Trey K.; Tremaine, Mary; Parreiras, Lucas S.; ...

2016-10-14

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactionsmore » among genes encoding a xylose reductase ( GRE3), a component of MAP Kinase (MAPK) signaling ( HOG1), a regulator of Protein Kinase A (PKA) signaling ( IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis ( ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Lastly, our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.« less

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

PubMed

Sato, Trey K; Tremaine, Mary; Parreiras, Lucas S; Hebert, Alexander S; Myers, Kevin S; Higbee, Alan J; Sardi, Maria; McIlwain, Sean J; Ong, Irene M; Breuer, Rebecca J; Avanasi Narasimhan, Ragothaman; McGee, Mick A; Dickinson, Quinn; La Reau, Alex; Xie, Dan; Tian, Mingyuan; Reed, Jennifer L; Zhang, Yaoping; Coon, Joshua J; Hittinger, Chris Todd; Gasch, Audrey P; Landick, Robert

2016-10-01

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae

PubMed Central

Tremaine, Mary; Hebert, Alexander S.; Myers, Kevin S.; Sardi, Maria; Dickinson, Quinn; Reed, Jennifer L.; Zhang, Yaoping; Coon, Joshua J.; Hittinger, Chris Todd; Gasch, Audrey P.; Landick, Robert

2016-01-01

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism. PMID:27741250

Screening and evolution of a novel protist xylose isomerase from the termite Reticulitermes speratus for efficient xylose fermentation in Saccharomyces cerevisiae.

PubMed

Katahira, Satoshi; Muramoto, Nobuhiko; Moriya, Shigeharu; Nagura, Risa; Tada, Nobuki; Yasutani, Noriko; Ohkuma, Moriya; Onishi, Toru; Tokuhiro, Kenro

2017-01-01

The yeast Saccharomyces cerevisiae , a promising host for lignocellulosic bioethanol production, is unable to metabolize xylose. In attempts to confer xylose utilization ability in S. cerevisiae , a number of xylose isomerase (XI) genes have been expressed heterologously in this yeast. Although several of these XI encoding genes were functionally expressed in S. cerevisiae , the need still exists for a S. cerevisiae strain with improved xylose utilization ability for use in the commercial production of bioethanol. Although currently much effort has been devoted to achieve the objective, one of the solutions is to search for a new XI gene that would confer superior xylose utilization in S. cerevisiae . Here, we searched for novel XI genes from the protists residing in the hindgut of the termite Reticulitermes speratus . Eight novel XI genes were obtained from a cDNA library, prepared from the protists of the R. speratus hindgut, by PCR amplification using degenerated primers based on highly conserved regions of amino acid sequences of different XIs. Phylogenetic analysis classified these cloned XIs into two groups, one showed relatively high similarities to Bacteroidetes and the other was comparatively similar to Firmicutes . The growth rate and the xylose consumption rate of the S. cerevisiae strain expressing the novel XI, which exhibited highest XI activity among the eight XIs, were superior to those exhibited by the strain expressing the XI gene from Piromyces sp. E2. Substitution of the asparagine residue at position 337 of the novel XI with a cysteine further improved the xylose utilization ability of the yeast strain. Interestingly, introducing point mutations in the corresponding asparagine residues in XIs originated from other organisms, such as Piromyces sp. E2 or Clostridium phytofermentans , similarly improved xylose utilization in S. cerevisiae . A novel XI gene conferring superior xylose utilization in S. cerevisiae was successfully isolated from the

Metabolic engineering for improved fermentation of pentoses by yeasts

Treesearch

T. W. Jeffries; Jin. Y.-S.

2004-01-01

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing...

Furfural and glucose can enhance conversion of xylose to xylitol by Candida magnoliae TISTR 5663.

PubMed

Wannawilai, Siwaporn; Lee, Wen-Chien; Chisti, Yusuf; Sirisansaneeyakul, Sarote

2017-01-10

Xylitol production from xylose by the yeast Candida magnoliae TISTR 5663 was enhanced by supplementing the fermentation medium with furfural (300mg/L) and glucose (3g/L with an initial mass ratio of glucose to xylose of 1:10) together under oxygen limiting conditions. In the presence of furfural and glucose, the final concentration of xylitol was unaffected relative to control cultures but the xylitol yield on xylose increased by about 5%. Supplementation of the culture medium with glucose alone at an initial concentration of 3g/L, stimulated the volumetric and specific rates of xylose consumption and the rate of xylitol production from xylose. In a culture medium containing 30g/L xylose, 300mg/L furfural and 3g/L glucose, the volumetric production rate of xylitol was 1.04g/L h and the specific production rate was 0.169g/g h. In the absence of furfural and glucose, the volumetric production rate of xylitol was ∼35% lower and the specific production rate was nearly 30% lower. In view of these results, xylose-containing lignocellulosic hydrolysates contaminated with furfural can be effectively used for producing xylitol by fermentation so long as the glucose-to-xylose mass ratio in the hydrolysate does not exceed 1:10 and the furfural concentration is ≤300mg/L. Copyright © 2016 Elsevier B.V. All rights reserved.

Pilot-scale steam explosion for xylose production from oil palm empty fruit bunches and the use of xylose for ethanol production.

PubMed

Duangwang, Sairudee; Ruengpeerakul, Taweesak; Cheirsilp, Benjamas; Yamsaengsung, Ram; Sangwichien, Chayanoot

2016-03-01

Pilot-scale steam explosion equipments were designed and constructed, to experimentally solubilize xylose from oil palm empty fruit bunches (OPEFB) and also to enhance an enzyme accessibility of the residual cellulose pulp. The OPEFB was chemically pretreated prior to steam explosion at saturated steam (SS) and superheated steam (SHS) conditions. The acid pretreated OPEFB gave the highest xylose recovery of 87.58 ± 0.21 g/kg dried OPEFB in the liquid fraction after explosion at SHS condition. These conditions also gave the residual cellulose pulp with high enzymatic accessibility of 73.54 ± 0.41%, which is approximately threefold that of untreated OPEFB. This study has shown that the acid pretreatment prior to SHS explosion is an effective method to enhance both xylose extraction and enzyme accessibility of the exploded OPEFB. Moreover, the xylose solution obtained in this manner could directly be fermented by Candida shehatae TISTR 5843 giving high ethanol yield of 0.30 ± 0.08 g/g xylose. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genome sequence and physiological analysis of Yamadazyma laniorum f.a. sp. nov. and a reevaluation of the apocryphal xylose fermentation of its sister species, Candida tenuis

USDA-ARS?s Scientific Manuscript database

Xylose fermentation is a rare trait that is immensely important to the cellulosic biofuel industry, and Candida tenuis is one of the few yeasts that has been reported with this trait. Here we report the isolation of two strains representing a candidate sister species to C. tenuis. Integrated analysi...

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

PubMed Central

Shin, Hyun Yong; Nijland, Jeroen G.; de Waal, Paul P.

2017-01-01

ABSTRACT Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N‐terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% d‐glucose and 4% d‐xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for d‐xylose over d‐glucose with high d‐xylose transport rates. This mutant supported efficient sugar fermentation of both d‐glucose and d‐xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. Bioeng. 2017;114: 1937–1945. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:28464256

The amino-terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid.

PubMed

Shin, Hyun Yong; Nijland, Jeroen G; de Waal, Paul P; Driessen, Arnold J M

2017-09-01

Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N-terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% d-glucose and 4% d-xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for d-xylose over d-glucose with high d-xylose transport rates. This mutant supported efficient sugar fermentation of both d-glucose and d-xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. Bioeng. 2017;114: 1937-1945. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Polyamine transporters and polyamines increase furfural tolerance during xylose fermentation with ethanologenic Escherichia coli strain LY180.

PubMed

Geddes, Ryan D; Wang, Xuan; Yomano, Lorraine P; Miller, Elliot N; Zheng, Huabao; Shanmugam, Keelnatham T; Ingram, Lonnie O

2014-10-01

Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Cofermentation of Glucose, Xylose, and Cellobiose by the Beetle-Associated Yeast Spathaspora passalidarum

Treesearch

Tanya M. Long; Yi-Kai Su; Jennifer Headman; Alan Higbee; Laura B. Willis; Thomas W. Jeffries

2012-01-01

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most...

Production of Xylitol from D-Xylose by Overexpression of Xylose Reductase in Osmotolerant Yeast Candida glycerinogenes WL2002-5.

PubMed

Zhang, Cheng; Zong, Hong; Zhuge, Bin; Lu, Xinyao; Fang, Huiying; Zhuge, Jian

2015-07-01

Efficient bioconversion of D-xylose into various biochemicals is critical for the developing lignocelluloses application. In this study, we compared D-xylose utilization in Candida glycerinogenes WL2002-5 transformants expressing xylose reductase (XYL1) in D-xylose metabolism. C. glycerinogenes WL2002-5 expressing XYL1 from Schefferomyces stipitis can produce xylitol. Xylitol production by the recombinant strains was evaluated using a xylitol fermentation medium with glucose as a co-substrate. As glucose was found to be an insufficient co-substrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best co-substrate. The effects of glycerol on the xylitol production rate by a xylose reductase gene (XYL1)-overexpressed mutant of C. glycerinogenes WL2002-5 were investigated. The XYL1-overexpressed mutant produced xylitol from D-xylose using glycerol as a co-substrate for cell growth and NAD (P) H regeneration: 100 g/L D-xylose was completely converted into xylitol when at least 20 g/L glycerol was used as a co-substrate. XYL1 overexpressed mutant grown on glycerol as co-substrate accumulated 2.1-fold increased xylitol concentration over those cells grown on glucose as co-substrate. XYL1 overexpressed mutant produced xylitol with a volumetric productivity of 0.83 g/L/h, and a xylitol yield of 98 % xylose. Recombinant yeast strains obtained in this study are promising candidates for xylitol production. This is the first report of XYL1 gene overexpression of C. glycerinogenes WL2002-5 for enhancing the efficiency of xylitol production.

Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes: role of physiological fitness and microbial interactions.

PubMed

Albergaria, Helena; Arneborg, Nils

2016-03-01

Winemaking, brewing and baking are some of the oldest biotechnological processes. In all of them, alcoholic fermentation is the main biotransformation and Saccharomyces cerevisiae the primary microorganism. Although a wide variety of microbial species may participate in alcoholic fermentation and contribute to the sensory properties of end-products, the yeast S. cerevisiae invariably dominates the final stages of fermentation. The ability of S. cerevisiae to outcompete other microbial species during alcoholic fermentation processes, such as winemaking, has traditionally been ascribed to its high fermentative power and capacity to withstand the harsh environmental conditions, i.e. high levels of ethanol and organic acids, low pH values, scarce oxygen availability and depletion of certain nutrients. However, in recent years, several studies have raised evidence that S. cerevisiae, beyond its remarkable fitness for alcoholic fermentation, also uses defensive strategies mediated by different mechanisms, such as cell-to-cell contact and secretion of antimicrobial peptides, to combat other microorganisms. In this paper, we review the main physiological features underlying the special aptitude of S. cerevisiae for alcoholic fermentation and discuss the role of microbial interactions in its dominance during alcoholic fermentation, as well as its relevance for winemaking.

Production of xylitol by a Coniochaeta ligniaria strain tolerant of inhibitors and defective in growth on xylose.

PubMed

Nichols, Nancy N; Saha, Badal C

2016-05-01

In conversion of biomass to fuels or chemicals, inhibitory compounds arising from physical-chemical pretreatment of the feedstock can interfere with fermentation of the sugars to product. Fungal strain Coniochaeta ligniaria NRRL30616 metabolizes the furan aldehydes furfural and 5-hydroxymethylfurfural, as well as a number of aromatic and aliphatic acids and aldehydes. Use of NRRL30616 to condition biomass sugars by metabolizing the inhibitors improves their fermentability. Wild-type C. ligniaria has the ability to grow on xylose as sole source of carbon and energy, with no accumulation of xylitol. Mutants of C. ligniaria unable to grow on xylose were constructed. Xylose reductase and xylitol dehydrogenase activities were reduced by approximately two thirds in mutant C8100. The mutant retained ability to metabolize inhibitors in biomass hydrolysates. Although C. ligniaria C8100 did not grow on xylose, the strain converted a portion of xylose to xylitol, producing 0.59 g xylitol/g xylose in rich medium and 0.48 g xylitol/g xylose in corn stover dilute acid hydrolysate. 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016 © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:606-612, 2016. © 2016 American Institute of Chemical Engineers.

Determination of Ethyl Carbamate in Alcoholic Beverages and Fermented Foods Sold in Korea.

PubMed

Ryu, Dayeon; Choi, Bogyoung; Kim, Eunjoo; Park, Seri; Paeng, Hwijin; Kim, Cho-Il; Lee, Jee-Yeon; Yoon, Hae Jung; Koh, Eunmi

2015-09-01

Ethyl carbamate (EC) classified as a probable human carcinogen (Group 2A) is naturally formed in alcoholic beverages and fermented foods during fermentation process and/or during storage. The objective of this study was to analyze EC in 34 food items including 14 alcoholic beverages and 20 fermented foods sold in Korea. Each food was collected from 18 supermarkets in 9 metropolitan cities in Korea, and then made into composite. According to food composition and alcohol content, samples were divided into four matrices such as apple juice, milk, Soju (liquor containing about 20% alcohol), and rice porridge. The maximum EC value of 151.06 µg/kg was found in Maesilju (liquor made from Maesil and Soju). Whisky and Bokbunjaju (Korean black raspberry wine) contained 9.90 µg/kg and 6.30 µg/kg, respectively. EC was not detected in other alcoholic beverages. Of 20 fermented foods, Japanese-style soy sauce had highest level of 15.59 µg/kg and traditional one contained 4.18 µg/kg. Soybean paste had 1.18 µg/kg, however, EC was not found in other fermented foods.

Optimization of fermentation conditions for alcohol production.

PubMed

Bowman, L; Geiger, E

1984-12-01

The quantitative effects of carbohydrate levels, degree of initial saccharification, glucoamylase dosage, temperature, and fermentation time were investigated using a Box-Wilson central composite design protocol. With Saccharomyces cerevisiae ATCC 4126, it was found that the use of a partially saccharified starch substrate markedly increased yields and attainable alcohol levels. Balancing the degree of initial saccharification with the level of glucoamylase used to complete hydrolysis was found necessary to obtain optimum yields. The temperature optimum was found to be 36 degrees C. The regression equations obtained were used to model the fermentation in order to determine optimum fermentation conditions.

Engineered yeast with a CO2-fixation pathway to improve the bio-ethanol production from xylose-mixed sugars.

PubMed

Li, Yun-Jie; Wang, Miao-Miao; Chen, Ya-Wei; Wang, Meng; Fan, Li-Hai; Tan, Tian-Wei

2017-03-06

Bio-ethanol production from lignocellulosic raw materials could serve as a sustainable potential for improving the supply of liquid fuels in face of the food-to-fuel competition and the growing energy demand. Xylose is the second abundant sugar of lignocelluloses hydrolysates, but its commercial-scale conversion to ethanol by fermentation is challenged by incomplete and inefficient utilization of xylose. Here, we use a coupled strategy of simultaneous maltose utilization and in-situ carbon dioxide (CO 2 ) fixation to achieve efficient xylose fermentation by the engineered Saccharomyces cerevisiae. Our results showed that the introduction of CO 2 as electron acceptor for nicotinamide adenine dinucleotide (NADH) oxidation increased the total ethanol productivity and yield at the expense of simultaneous maltose and xylose utilization. Our achievements present an innovative strategy using CO 2 to drive and redistribute the central pathways of xylose to desirable products and demonstrate a possible breakthrough in product yield of sugars.

Determination of Ethyl Carbamate in Alcoholic Beverages and Fermented Foods Sold in Korea

PubMed Central

Ryu, Dayeon; Choi, Bogyoung; Kim, Eunjoo; Park, Seri; Paeng, Hwijin; Kim, Cho-il; Lee, Jee-yeon; Yoon, Hae Jung

2015-01-01

Ethyl carbamate (EC) classified as a probable human carcinogen (Group 2A) is naturally formed in alcoholic beverages and fermented foods during fermentation process and/or during storage. The objective of this study was to analyze EC in 34 food items including 14 alcoholic beverages and 20 fermented foods sold in Korea. Each food was collected from 18 supermarkets in 9 metropolitan cities in Korea, and then made into composite. According to food composition and alcohol content, samples were divided into four matrices such as apple juice, milk, Soju (liquor containing about 20% alcohol), and rice porridge. The maximum EC value of 151.06 µg/kg was found in Maesilju (liquor made from Maesil and Soju). Whisky and Bokbunjaju (Korean black raspberry wine) contained 9.90 µg/kg and 6.30 µg/kg, respectively. EC was not detected in other alcoholic beverages. Of 20 fermented foods, Japanese-style soy sauce had highest level of 15.59 µg/kg and traditional one contained 4.18 µg/kg. Soybean paste had 1.18 µg/kg, however, EC was not found in other fermented foods. PMID:26483888

Ethanol production from xylose with the yeast Pichia stipitis and simultaneous product recovery by gas stripping using a gas-lift loop fermentor with attached side-arm (GLSA).

PubMed

Domínguez, J M; Cao, N; Gong, C S; Tsao, G T

2000-02-05

The bioconversion of xylose into ethanol with the yeast Pichia stipitis CBS 5773 is inhibited when 20 g/L of ethanol are present in the fermentation broth. In order to avoid this limitation, the fermentation was carried out with simultaneous recovery of product by CO(2) stripping. The fermentation was also improved by attaching a side-arm to the main body of a classical gas-lift loop fermentor. This side-arm increases the liquid circulation, mass transfer, and gas distribution, reducing the amount of oxygen in the inlet gas necessary to perform the fermentation of xylose under microaerobic conditions (K(L)a approximately 16 h(-1)). The continuous stripping of ethanol from the fermentation broth in this new bioreactor system allowed the consumption of higher xylose concentrations than using Erlenmeyer shaker flasks, improved significantly the process productivity and provided a clean ethanol solution by using an ice-cooled condenser system. Finally, a fed-batch fermentation was carried out with a K(L)a = 15.8 h(-1). Starting with 248.2 g of xylose, 237.6 g of xylose was consumed to produce 88.1 g of ethanol which represents 72.6% of the theoretical yield (47.2 g/L of ethanol was recovered in the condenser, while 9.6 g/L remained in the fermentation broth). Copyright 2000 John Wiley & Sons, Inc.

Molecular simulation to investigate the cofactor specificity for pichia stipitis Xylose reductase.

PubMed

Xia, Xiao-Le; Cong, Shan; Weng, Xiao-Rong; Chen, Jin-Hua; Wang, Jing-Fang; Chou, Kuo-Chen

2013-11-01

Xylose is one of the most abundant carbohydrates in nature, and widely used to produce bioethanol via fermentation in industry. Xylulose can produce two key enzymes: xylose reductase and xylitol dehydrogenase. Owing to the disparate cofactor specificities of xylose reductase and xylitol dehydrogenase, intracellular redox imbalance is detected during the xylose fermentation, resulting in low ethanol yields. To overcome this barrier, a common strategy is applied to artificially modify the cofactor specificity of xylose reductase. In this study, we utilized molecular simulation approaches to construct a 3D (three-dimensional) structural model for the NADP-dependent Pichia stipitis xylose reductase (PsXR). Based on the 3D model, the favourable binding modes for both cofactors NAD and NADP were obtained using the flexible docking procedure and molecular dynamics simulation. Structural analysis of the favourable binding modes showed that the cofactor binding site of PsXR was composed of 3 major components: a hydrophilic pocket, a hydrophobic pocket as well as a linker channel between the aforementioned two pockets. The hydrophilic pocket could recognize the nicotinamide moiety of the cofactors by hydrogen bonding networks, while the hydrophobic pocket functioned to position the adenine moiety of the cofactors by hydrophobic and Π-Π stacking interactions. The linker channel contained some key residues for ligand-binding; their mutation could have impact to the specificity of PsXR. Finally, it was found that any of the two single mutations, K21A and K270N, might reverse the cofactor specificity of PsXR from major NADP- to NADdependent, which was further confirmed by the additional experiments. Our findings may provide useful insights into the cofactor specificity of PsXR, stimulating new strategies for better designing xylose reductase and improving ethanol production in industry.

Understanding Gut Fermentation Syndrome in the Psychiatric Evaluation of Patients with Suspected Alcohol Use Disorder

DTIC Science & Technology

2017-10-18

Fermentation Syndrome in the Psychiatric Evaluation of Patients with Suspected Alcohol Use Disorder Sb. GRANT NUMBER Sc. PROGRAM ELEMENT NUMBER 6...by ANSI Std. Z39.18 Adobe Professional 7. 0 Introduction Gut Fermentation Syndrome, also known as auto- brewery syndrome, is a phenomenon not well...patient stated abstinence from alcohol use and that Gut Fermentation Syndrome was the cause of continually elevated blood alcohol levels. We will

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

2014-01-07

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Ultrafiltration of hemicellulose hydrolysate fermentation broth

NASA Astrophysics Data System (ADS)

Kresnowati, M. T. A. P.; Desiriani, Ria; Wenten, I. G.

2017-03-01

Hemicelulosic material is often used as the main substrate to obtain high-value products such as xylose. The five carbon sugar, xylose, could be further processed by fermentation to produce xylitol. However, not only the hemicellulose hydrolysate fermentation broth contains xylitol, but also metabolite products, residual substances, biomass and mineral salts. Therefore, in order to obtain the end products, various separation processes are required to separate and purify the desired product from the fermentation broth. One of the most promising downstream processing methods of fermentation broth clarification is ultrafiltration due to its potential for energy saving and higher purity. In addition, ultrafiltration membrane has a high performance in separating inhibitory components in the fermentation broth. This paper assesses the influence of operating conditions; including trans-membrane pressure, velocity, pH of the fermentation broth solutions, and also to the xylitol concentration in the product. The challenges of the ultrafiltration process will be pointed out.

Model-based transcriptome engineering promotes a fermentative transcriptional state in yeast

PubMed Central

Michael, Drew G.; Maier, Ezekiel J.; Brown, Holly; Gish, Stacey R.; Fiore, Christopher; Brown, Randall H.; Brent, Michael R.

2016-01-01

The ability to rationally manipulate the transcriptional states of cells would be of great use in medicine and bioengineering. We have developed an algorithm, NetSurgeon, which uses genome-wide gene-regulatory networks to identify interventions that force a cell toward a desired expression state. We first validated NetSurgeon extensively on existing datasets. Next, we used NetSurgeon to select transcription factor deletions aimed at improving ethanol production in Saccharomyces cerevisiae cultures that are catabolizing xylose. We reasoned that interventions that move the transcriptional state of cells using xylose toward that of cells producing large amounts of ethanol from glucose might improve xylose fermentation. Some of the interventions selected by NetSurgeon successfully promoted a fermentative transcriptional state in the absence of glucose, resulting in strains with a 2.7-fold increase in xylose import rates, a 4-fold improvement in xylose integration into central carbon metabolism, or a 1.3-fold increase in ethanol production rate. We conclude by presenting an integrated model of transcriptional regulation and metabolic flux that will enable future efforts aimed at improving xylose fermentation to prioritize functional regulators of central carbon metabolism. PMID:27810962

Highly efficient production of L-lactic acid from xylose by newly isolated Bacillus coagulans C106.

PubMed

Ye, Lidan; Zhou, Xingding; Hudari, Mohammad Sufian Bin; Li, Zhi; Wu, Jin Chuan

2013-03-01

Cost-effective production of optically pure lactic acid from lignocellulose sugars is commercially attractive but challenging. Bacillus coagulans C106 was isolated from environment and used to produce l-lactic acid from xylose at 50°C and pH 6.0 in mineral salts medium containing 1-2% (w/v) of yeast extract without sterilizing the medium before fermentation. In batch fermentation with 85g/L of xylose, lactic acid titer and productivity reached 83.6g/L and 7.5g/Lh, respectively. When fed-batch (120+80+60g/L) fermentation was applied, they reached 215.7g/L and 4.0g/Lh, respectively. In both cases, the lactic acid yield and optical purity reached 95% and 99.6%, respectively. The lactic acid titer and productivity on xylose are the highest among those ever reported. Ca(OH)2 was found to be a better neutralizing agent than NaOH in terms of its giving higher lactic acid titer (1.2-fold) and productivity (1.8-fold) under the same conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2013-05-14

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2017-09-12

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetically modified yeast species and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

2011-05-17

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications', include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2016-08-09

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Bioethanol production from ball milled bagasse using an on-site produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis.

PubMed

Buaban, Benchaporn; Inoue, Hiroyuki; Yano, Shinichi; Tanapongpipat, Sutipa; Ruanglek, Vasimon; Champreda, Verawat; Pichyangkura, Rath; Rengpipat, Sirirat; Eurwilaichitr, Lily

2010-07-01

Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary beta-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 degrees C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 degrees C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass. 2009. Published by Elsevier B.V.

Lignocellulosic sugar management for xylitol and ethanol fermentation with multiple cell recycling by Kluyveromyces marxianus IIPE453.

PubMed

Dasgupta, Diptarka; Ghosh, Debashish; Bandhu, Sheetal; Adhikari, Dilip K

2017-07-01

Optimum utilization of fermentable sugars from lignocellulosic biomass to deliver multiple products under biorefinery concept has been reported in this work. Alcohol fermentation has been carried out with multiple cell recycling of Kluyveromyces marxianus IIPE453. The yeast utilized xylose-rich fraction from acid and steam treated biomass for cell generation and xylitol production with an average yield of 0.315±0.01g/g while the entire glucose rich saccharified fraction had been fermented to ethanol with high productivity of 0.9±0.08g/L/h. A detailed insight into its genome illustrated the strain's complete set of genes associated with sugar transport and metabolism for high-temperature fermentation. A set flocculation proteins were identified that aided in high cell recovery in successive fermentation cycles to achieve alcohols with high productivity. We have brought biomass derived sugars, yeast cell biomass generation, and ethanol and xylitol fermentation in one platform and validated the overall material balance. 2kg sugarcane bagasse yielded 193.4g yeast cell, and with multiple times cell recycling generated 125.56g xylitol and 289.2g ethanol (366mL). Copyright © 2017 Elsevier GmbH. All rights reserved.

A review on traditional Turkish fermented non-alcoholic beverages: microbiota, fermentation process and quality characteristics.

PubMed

Altay, Filiz; Karbancıoglu-Güler, Funda; Daskaya-Dikmen, Ceren; Heperkan, Dilek

2013-10-01

Shalgam juice, hardaliye, boza, ayran (yoghurt drink) and kefir are the most known traditional Turkish fermented non-alcoholic beverages. The first three are obtained from vegetables, fruits and cereals, and the last two ones are made of milk. Shalgam juice, hardaliye and ayran are produced by lactic acid fermentation. Their microbiota is mainly composed of lactic acid bacteria (LAB). Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus paracasei subsp. paracasei in shalgam fermentation and L. paracasei subsp. paracasei and Lactobacillus casei subsp. pseudoplantarum in hardaliye fermentation are predominant. Ayran is traditionally prepared by mixing yoghurt with water and salt. Yoghurt starter cultures are used in industrial ayran production. On the other hand, both alcohol and lactic acid fermentation occur in boza and kefir. Boza is prepared by using a mixture of maize, wheat and rice or their flours and water. Generally previously produced boza or sourdough/yoghurt are used as starter culture which is rich in Lactobacillus spp. and yeasts. Kefir is prepared by inoculation of raw milk with kefir grains which consists of different species of yeasts, LAB, acetic acid bacteria in a protein and polysaccharide matrix. The microbiota of boza and kefir is affected from raw materials, the origin and the production methods. In this review, physicochemical properties, manufacturing technologies, microbiota and shelf life and spoilage of traditional fermented beverages were summarized along with how fermentation conditions could affect rheological properties of end product which are important during processing and storage. Copyright © 2013. Published by Elsevier B.V.

Creation of a synthetic xylose-inducible promoter for Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

Saccharomyces cerevisiae is currently used to produce ethanol from glucose, but it cannot utilize five-carbon sugars contained in the hemicellulose component of biomass feedstocks. S. cerevisiae strains engineered for xylose fermentation have been made using constitutive promoters to express the req...

Metabolic engineering of Saccharomyces cerevisiae ethanol strains PE-2 and CAT-1 for efficient lignocellulosic fermentation.

PubMed

Romaní, Aloia; Pereira, Filipa; Johansson, Björn; Domingues, Lucília

2015-03-01

In this work, Saccharomyces cerevisiae strains PE-2 and CAT-1, commonly used in the Brazilian fuel ethanol industry, were engineered for xylose fermentation, where the first fermented xylose faster than the latter, but also produced considerable amounts of xylitol. An engineered PE-2 strain (MEC1121) efficiently consumed xylose in presence of inhibitors both in synthetic and corn-cob hydrolysates. Interestingly, the S. cerevisiae MEC1121 consumed xylose and glucose simultaneously, while a CEN.PK based strain consumed glucose and xylose sequentially. Deletion of the aldose reductase GRE3 lowered xylitol production to undetectable levels and increased xylose consumption rate which led to higher final ethanol concentrations. Fermentation of corn-cob hydrolysate using this strain, MEC1133, resulted in an ethanol yield of 0.47 g/g of total sugars which is 92% of the theoretical yield. Copyright © 2014 Elsevier Ltd. All rights reserved.

Minimum-sized ideal reactor for continuous alcohol fermentation using immobilized microorganism

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

Yamane, T.; Shimizu, S.

Recently, alcohol fermentation has gained considerable attention with the aim of lowering its production cost in the production processes of both fuel ethanol and alcoholic beverages. The over-all cost is a summation of costs of various subsystems such as raw material (sugar, starch, and cellulosic substances) treatment, fermentation process, and alcohol separation from water solutions; lowering the cost of the fermentation processes is very important in lowering the total cost. Several new techniques have been developed for economic continuous ethanol production, use of a continuous wine fermentor with no mechanical stirring, cell recycle combined with continuous removal of ethanol undermore » vaccum, a technique involving a bed of yeast admixed with an inert carrier, and use of immobilized yeast reactors in packed-bed column and in a three-stage double conical fluidized-bed bioreactor. All these techniques lead to increases more or less, in reactor productivity, which in turn result in the reduction of the reactor size for a given production rate and a particular conversion. Since an improvement in the fermentation process often leads to a reduction of fermentor size and hence, a lowering of the initial construction cost, it is important to theoretically arrive at a solution to what is the minimum-size setup of ideal reactors from the viewpoint of liquid backmixing. In this short communication, the minimum-sized ideal reactor for continuous alcohol fermentation using immobilized cells will be specifically discussed on the basis of a mathematical model. The solution will serve for designing an optimal bioreactor. (Refs. 26).« less

Accumulation of Citrulline by Microbial Arginine Metabolism during Alcoholic Fermentation of Soy Sauce.

PubMed

Fang, Fang; Zhang, Jiran; Zhou, Jingwen; Zhou, Zhaohui; Li, Tieqiao; Lu, Liling; Zeng, Weizhu; Du, Guocheng; Chen, Jian

2018-03-07

Citrulline, the major precursor of ethyl carbamate in soy sauce, is an intermediate catabolite of arginine produced by bacteria present in soy sauce moromi mash. Pediococcus acidilactici is responsible for the formation of citrulline during the lactic acid fermentation process of soy sauce. However, citrulline accumulation during the alcoholic fermentation process and the corresponding bacteria involved have not been identified. Salt-tolerant, arginine-utilizing bacteria were isolated from moromi mash during the alcoholic fermentation process. Under normal cultivation conditions, arginine utilization by these strains did not contribute to citrulline accumulation. However, the conversion of arginine to citrulline by these bacteria increased when cultivated during the alcoholic fermentation process. Additionally, the ethanol-enhanced solubility of free fatty acids in moromi mash stimulated the accumulation of citrulline. Staphylococcus exhibited the highest capability in the conversion of arginine to citrulline.

Continuous succinic acid production from xylose by Actinobacillus succinogenes.

PubMed

Bradfield, Michael F A; Nicol, Willie

2016-02-01

Continuous, anaerobic fermentations of D-xylose were performed by Actinobacillus succinogenes 130Z in a custom, biofilm reactor at dilution rates of 0.05, 0.10 and 0.30 h(-1). Succinic acid yields on xylose (0.55-0.68 g g(-1)), titres (10.9-29.4 g L(-1)) and productivities (1.5-3.4 g L(-1) h(-1)) were lower than those of a previous study on glucose, but product ratios (succinic acid/acetic acid = 3.0-5.0 g g(-1)) and carbohydrate consumption rates were similar. Also, mass balance closures on xylose were up to 18.2 % lower than those on glucose. A modified HPLC method revealed pyruvic acid excretion at appreciable concentrations (1.2-1.9 g L(-1)) which improved the mass balance closure by up to 16.8 %. Furthermore, redox balances based on the accounted xylose consumed and the excreted metabolites, indicated an overproduction of reducing power. The oxidative pentose phosphate pathway was shown to be a plausible source of the additional reducing power.

A novel method to prepare L-Arabinose from xylose mother liquor by yeast-mediated biopurification

PubMed Central

2011-01-01

Background L-arabinose is an important intermediate for anti-virus drug synthesis and has also been used in food additives for diets-controlling in recent years. Commercial production of L-arabinose is a complex progress consisting of acid hydrolysis of gum arabic, followed by multiple procedures of purification, thus making high production cost. Therefore, there is a biotechnological and commercial interest in the development of new cost-effective and high-performance methods for obtaining high purity grade L-arabinose. Results An alternative, economical method for purifying L-arabinose from xylose mother liquor was developed in this study. After screening 306 yeast strains, a strain of Pichia anomala Y161 was selected as it could effectively metabolize other sugars but not L-arabinose. Fermentation in a medium containing xylose mother liquor permitted enrichment of L-arabinose by a significant depletion of other sugars. Biochemical analysis of this yeast strain confirmed that its poor capacity for utilizing L-arabinose was due to low activities of the enzymes required for the metabolism of this sugar. Response surface methodology was employed for optimization the fermentation conditions in shake flask cultures. The optimum conditions were: 75 h fermentation time, at 32.5°C, in a medium containing 21% (v/v) xylose mother liquor. Under these conditions, the highest purity of L-arabinose reached was 86.1% of total sugar, facilitating recovery of white crystalline L-arabinose from the fermentation medium by simple methods. Conclusion Yeast-mediated biopurification provides a dynamic method to prepare high purity of L-arabinose from the feedstock solution xylose mother liqour, with cost-effective and high-performance properties. PMID:21649890

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose.

PubMed

Fu, Hongxin; Yu, Le; Lin, Meng; Wang, Jufang; Xiu, Zhilong; Yang, Shang-Tian

2017-03-01

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8g/L vs. 19.4g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28g/L·h vs. 0.16g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53g/L·h vs. 0.26g/L·h) and yield (0.32g/g vs. 0.28g/g). When the initial total sugar concentration was ~120g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4g/L, yield of 0.43g/g sugar consumed, productivity of 0.87g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

A viable method and configuration for fermenting biomass sugars to ethanol using native Saccharomyces cerevisiae.

PubMed

Yuan, Dawei; Rao, Kripa; Varanasi, Sasidhar; Relue, Patricia

2012-08-01

A system that incorporates a packed bed reactor for isomerization of xylose and a hollow fiber membrane fermentor (HFMF) for sugar fermentation by yeast was developed for facile recovery of the xylose isomerase enzyme pellets and reuse of the cartridge loaded with yeast. Fermentation of pre-isomerized poplar hydrolysate produced using ionic liquid pretreatment in HFMF resulted in ethanol yields equivalent to that of model sugar mixtures of xylose and glucose. By recirculating model sugar mixtures containing partially isomerized xylose through the packed bed and the HFMF connected in series, 39 g/l ethanol was produced within 10h with 86.4% xylose utilization. The modular nature of this configuration has the potential for easy scale-up of the simultaneous isomerization and fermentation process without significant capital costs. Copyright © 2012 Elsevier Ltd. All rights reserved.

Studying the rapid bioconversion of lignocellulosic sugars into ethanol using high cell density fermentations with cell recycle

PubMed Central

2014-01-01

Background The Rapid Bioconversion with Integrated recycle Technology (RaBIT) process reduces capital costs, processing times, and biocatalyst cost for biochemical conversion of cellulosic biomass to biofuels by reducing total bioprocessing time (enzymatic hydrolysis plus fermentation) to 48 h, increasing biofuel productivity (g/L/h) twofold, and recycling biocatalysts (enzymes and microbes) to the next cycle. To achieve these results, RaBIT utilizes 24-h high cell density fermentations along with cell recycling to solve the slow/incomplete xylose fermentation issue, which is critical for lignocellulosic biofuel fermentations. Previous studies utilizing similar fermentation conditions showed a decrease in xylose consumption when recycling cells into the next fermentation cycle. Eliminating this decrease is critical for RaBIT process effectiveness for high cycle counts. Results Nine different engineered microbial strains (including Saccharomyces cerevisiae strains, Scheffersomyces (Pichia) stipitis strains, Zymomonas mobilis 8b, and Escherichia coli KO11) were tested under RaBIT platform fermentations to determine their suitability for this platform. Fermentation conditions were then optimized for S. cerevisiae GLBRCY128. Three different nutrient sources (corn steep liquor, yeast extract, and wheat germ) were evaluated to improve xylose consumption by recycled cells. Capacitance readings were used to accurately measure viable cell mass profiles over five cycles. Conclusion The results showed that not all strains are capable of effectively performing the RaBIT process. Acceptable performance is largely correlated to the specific xylose consumption rate. Corn steep liquor was found to reduce the deleterious impacts of cell recycle and improve specific xylose consumption rates. The viable cell mass profiles indicated that reduction in specific xylose consumption rate, not a drop in viable cell mass, was the main cause for decreasing xylose consumption. PMID:24847379

Disappearance of patulin during alcoholic fermentation of apple juice.

PubMed

Stinson, E E; Osman, S F; Huhtanen, C N; Bills, D D

1978-10-01

Eight yeast strains were used in three typical American processes to ferment apple juice containing 15 mg of added patulin per liter. Patulin was reduced to less than the minimum detectable level of 50 microgram/liter in all but two cases; in all cases, the level of patulin was reduced by over 99% during alcoholic fermentation. In unfermented samples of apple juice, the concentration of added patulin declined by only 10% when the juice was held for 2 weeks, a period equivalent to the time required for fermentation.

Acetate adaptation of clostridia tyrobutyricum for improved fermentation production of butyrate.

PubMed

Jaros, Adam M; Rova, Ulrika; Berglund, Kris A

2013-12-01

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium capable of utilizing xylose for the fermentation production of butyrate. Hot water extraction of hardwood lingocellulose is an efficient method of producing xylose where autohydrolysis of xylan is catalysed by acetate originating from acetyl groups present in hemicellulose. The presence of acetic acid in the hydrolysate might have a severe impact on the subsequent fermentations. In this study the fermentation kinetics of C. tyrobutyricum cultures after being classically adapted for growth at 26.3 g/L acetate equivalents were studied. Analysis of xylose batch fermentations found that even in the presence of high levels of acetate, acetate adapted strains had similar fermentation kinetics as the parental strain cultivated without acetate. The parental strain exposed to acetate at inhibitory conditions demonstrated a pronounced lag phase (over 100 hours) in growth and butyrate production as compared to the adapted strain (25 hour lag) or non-inhibited controls (0 lag). Additional insight into the metabolic pathway of xylose consumption was gained by determining the specific activity of the acetate kinase (AK) enzyme in adapted versus control batches. AK activity was reduced by 63% in the presence of inhibitory levels of acetate, whether or not the culture had been adapted.

Improving Xylose Utilization of Saccharomyces cerevisiae by Expressing the MIG1 Mutant from the Self-Flocculating Yeast SPSC01.

PubMed

Xu, Jian-Ren; Zhao, Xin-Qing; Liu, Chen-Guang; Bai, Feng-Wu

2018-01-01

The major carbohydrate components of lignocellulosic biomass are cellulose and hemicelluloses. Saccharomyces cerevisiae cannot efficiently utilize xylose derived upon the hydrolysis of hemicelluloses. Although engineering the yeast with xylose metabolic pathway has been intensively studied, challenges are still ahead for developing robust strains for lignocellulosic bioethanol production. The main objective of this study was to reveal the role of the MIG1 mutant isolated from the self-flocculating S. cerevisiae SPSC01 in xylose utilization, glucose repression and ethanol fermentation by S. cerevisiae. The MIG1 mutant was amplified from S. cerevisiae SPSC01 by PCR and MIG1- overexpression-cassette was transformed into S. cerevisiae S288c and xylose-metabolizing strain YB-2625-T through homologous recombination. Yeast growth was measured by colony assay on plates with or without xylose supplementation. Then xylose utilization and ethanol production were further evaluated through flask fermentation when mixed sugars of glucose and xylose at 3:1 and 2:1, respectively, were supplied. Fermentation products were detected by HPLC, and activities of xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) were also measured. The transcription of genes regulated by the expression of the MIG1 mutant was analyzed by RTqPCR. Evolutionary relationship of various MIG1s was developed by gene sequencing and sequence alignment. No difference was observed for S288c growing with xylose when it was engineered with the overexpression or deletion of its native MIG1, but its growth was enhanced when overexpressing the MIG1 mutant from SPSC01. The submerged culture of YB-2625-T MIG1-SPSC engineered with xylose-metabolic pathway and the MIG1 mutant indicated that xylitol accumulation was decreased, and consequently, more biomass was accumulated. Furthermore, improved activities of the key enzymes such as XR, XDH and XK were detected in YB-2625-T MIG1-SPSC. Evolutionary

Energy Utilization for Polysaccharide Synthesis by Mixed Rumen Organisms Fermenting Soluble Carbohydrates

PubMed Central

Walker, D. J.

1968-01-01

Synthesis of reserve polysaccharide by mixed rumen organisms fermenting glucose, maltose, cellobiose, and xylose has been studied in relation to the adenosine triphosphate energy calculated to be available from substrate fermentation. About 80% of the energy available from glucose and xylose was used for polysaccharide synthesis, whereas, assuming hydrolytic cleavage of the disaccharides, more than 100% was used when cellobiose and maltose were the substrates. If, however, phosphorolytic cleavage of the disaccharides, for which there is evidence, was involved, the energy from both maltose and cellobiose fermentation was used with about the same efficiency as that from glucose and xylose fermentation. The rumen fluid used was collected 24 hr after feeding, and growth of microorganisms in such samples was sufficient to account for utilization of less than 10% of the total energy becoming available during the 40-min incubation period. PMID:16349819

Assessment of chemical and sensory quality of sugarcane alcoholic fermented beverage.

PubMed

Resende Oliveira, Érica; Caliari, Márcio; Soares Soares Júnior, Manoel; Ribeiro Oliveira, Aryane; Cristina Marques Duarte, Renata; Valério de Barros Vilas Boas, Eduardo

2018-01-01

This study aimed to verify the technological feasibility, chemical quality and sensory acceptance of alcoholic fermented beverage obtained from sugarcane juice. A completely randomized design was applied. Sugar and alcohol content, phenolic (HPLC-MS) and volatile (GS-MS) compounds, pH, density, dry matter and acidity of the fermented beverage of sugarcane were quantified, as well as the acceptance of the product was carried out. The complete fermentation of sugarcane lasted 7 days, and it was obtained an alcohol content of 8.0% v/v. Titrable acidity of the beverage was of 67.31 meq L -1 , pH 4.03, soluble solids of 5 °Brix, reducing sugar of 0.07 g glucose 100 g -1 , density of 0.991 g cm -3 , reduced dry matter of 14.15 g L -1 , sulfates lower than 0.7 g K 2 SO 4  L -1 . Various phenolic compounds, among which, gallic acid (10.97%), catechin (1.73%), chlorogenic acid (3.52%), caffeic acid (1.49%), vanillic acid (0.28%), p -coumaric acid (0.24%), ferulic acid (6.63%), m -coumaric acid (0.36%), and o -coumaric acid (0.04%). Amongst aromatic compounds, were found mainly esters with fruity aromas (ethyl ester hexanoic acid and ethyl ester octanoic acid). The sugarcane juice can be commercialized as an alternative wine, as it presented adequate features to an alcoholic fermented beverage and was sensory accepted by consumers.

Lipid Accumulation from Glucose and Xylose in an Engineered, Naturally Oleaginous Strain of Saccharomyces cerevisiae

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

Knoshaug, Eric P; Van Wychen, Stefanie R; Zhang, Min

Saccharomyces cerevisiae, a well-known industrial yeast for alcoholic fermentation, is not historically known to accumulate lipids. Four S. cerevisiae strains used in industrial applications were screened for their ability to accumulate neutral lipids. Only one, D5A, was found to accumulate up to 20% dry cell weight (dcw) lipids. This strain was further engineered by knocking out ADP-activated serine/threonine kinase (SNF1) which increased lipid accumulation to 35% dcw lipids. In addition, we engineered D5A to utilize xylose and found that D5A accumulates up to 37% dcw lipids from xylose as the sole carbon source. Further we over-expressed different diacylglycerol acyltransferase (DGA1)more » genes and boosted lipid accumulation to 50%. Fatty acid speciation showed that 94% of the extracted lipids consisted of 5 fatty acid species, C16:0 (palmitic), C16:1n7 (palmitoleic), C18:0 (stearic), C18:1n7 (vaccenic), and C18:1n9 (oleic), while the relative distributions changed depending on growth conditions. In addition, this strain accumulated lipids concurrently with ethanol production.« less

Alcoholic fermentation of raw cassava starch by Rhizopus koji without cooking

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

Fuijo, Y.; Suyanadona, P.; Attasampunna, P.

Using only wheat bran koji from the Rhizopus strain, raw cassava starch and cassava pellets converted reasonably well to alcohol (ethanol) without cooking at 35 degrees C and pH 4.5-5.0. When the initial broth contained 30 g raw cassava starch, 10 g Rhizopus species koji, and 100 mL tap water, 12.1 g of alcohol was recovered by final distillation from fermented broth. In this case, 12.1 g alcohol corresponds to an 85.5% conversion rate based on the theoretical value of the starch content. When the initial broth contained 40 g cassava starch, 14.1 g of alcohol was recovered, where 14.1more » g corresponds to a 74.5% conversion rate. The alcoholic fermentation process described in the present work is considered more effective and reasonable than the process using raw starch without cooking reported until now, since the new process makes it unnecessary to add yeast cells and glucoamylase preparation. (Refs. 15).« less

Alcoholic fermentation of raw cassava starch by Rhizopus koji without cooking

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

Fujio, Y.; Suyanadona, P.; Attasampunna, P.

Using only wheat bran koji from the Rhizopus strain, raw cassava starch and casava pellets converted reasonably well to alcohol (ethanol) without cooking at 35/sup 0/C and pH 4.5-5.0. When the initial broth contained 30 g raw cassava starch, 10 g Rhizopus sp. koji, and 100 mL tap water, 12.1 g of alcohol was recovered by final distillation from fermented broth. In this case, 12.1 g alcohol corresponds to an 85.5% conversion rate based on the theoretical value of the starch content. When the initial broth contained 40 g cassava starch, 14.1 g of alcohol was recovered, where 14.1 gmore » corresponds to a 74.5% conversion rate. The alcoholic fermentation process described in the present work is considered more effective and reasonable than the process using raw starch without cooking reported until now, since the new process makes it unnecessary to add yeast cells and glucoamylase preparation.« less

Overexpression of NADH-dependent fumarate reductase improves D-xylose fermentation in recombinant Saccharomyces cerevisiae.

PubMed

Salusjärvi, Laura; Kaunisto, Sanna; Holmström, Sami; Vehkomäki, Maija-Leena; Koivuranta, Kari; Pitkänen, Juha-Pekka; Ruohonen, Laura

2013-12-01

Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5Δ17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.

Enhanced L-lactic acid production from biomass-derived xylose by a mutant Bacillus coagulans.

PubMed

Zheng, Zhaojuan; Cai, Cong; Jiang, Ting; Zhao, Mingyue; Ouyang, Jia

2014-08-01

Xylose effective utilization is crucial for production of bulk chemicals from low-cost lignocellulosic substrates. In this study, an efficient L-lactate production process from xylose by a mutant Bacillus coagulans NL-CC-17 was demonstrated. The nutritional requirements for L-lactate production by B. coagulans NL-CC-17 were optimized statistically in shake flask fermentations. Corn steep liquor powder and yeast exact were identified as the most significant factors by the two-level Plackett-Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors, and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform batch fermentation in a 3-l bioreactor. A maximum of 90.29 g l(-1)  L-lactic acid was obtained from 100 g l(-1) xylose in 120 h. When using corn stove prehydrolysates as substrates, 23.49 g l(-1)  L-lactic acid was obtained in 36 h and the yield was 83.09 %.

D-xylose absorption

MedlinePlus

Xylose tolerance test; Diarrhea - xylose; Malnutrition - xylose; Sprue - xylose; Celiac - xylose ... test if you have: Persistent diarrhea Signs of malnutrition Unexplained weight loss This test is primarily used ...

Coproduction of xylose, lignosulfonate and ethanol from wheat straw.

PubMed

Zhu, Shengdong; Huang, Wangxiang; Huang, Wenjing; Wang, Ke; Chen, Qiming; Wu, Yuanxin

2015-06-01

A novel integrated process to coproduce xylose, lignosulfonate and ethanol from wheat straw was investigated. Firstly, wheat straw was treated by dilute sulfuric acid and xylose was recovered from its hydrolyzate. Its optimal conditions were 1.0wt% sulfuric acid, 10% (w/v) wheat straw loading, 100°C, and 2h. Then the acid treated wheat straw was treated by sulfomethylation reagent and its hydrolyzate containing lignosulfonate was directly recovered. Its optimal conditions were 150°C, 15% (w/v) acid treated wheat straw loading, and 5h. Finally, the two-step treated wheat straw was converted to ethanol through enzymatic hydrolysis and microbial fermentation. Under optimal conditions, 1kg wheat straw could produce 0.225kg xylose with 95% purity, 4.16kg hydrolyzate of sulfomethylation treatment containing 5.5% lignosulfonate, 0.183kg ethanol and 0.05kg lignin residue. Compared to present technology, this process is a potential economically profitable wheat straw biorefinery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Decreased production of higher alcohols by Saccharomyces cerevisiae for Chinese rice wine fermentation by deletion of Bat aminotransferases.

PubMed

Zhang, Cui-Ying; Qi, Ya-Nan; Ma, Hong-Xia; Li, Wei; Dai, Long-Hai; Xiao, Dong-Guang

2015-04-01

An appropriate level of higher alcohols produced by yeast during the fermentation is one of the most important factors influencing Chinese rice wine quality. In this study, BAT1 and BAT2 single- and double-gene-deletion mutant strains were constructed from an industrial yeast strain RY1 to decrease higher alcohols during Chinese rice wine fermentation. The results showed that the BAT2 single-gene-deletion mutant strain produced best improvement in the production of higher alcohols while remaining showed normal growth and fermentation characteristics. Furthermore, a BAT2 single-gene-deletion diploid engineered strain RY1-Δbat2 was constructed and produced low levels of isobutanol and isoamylol (isoamyl alcohol and active amyl alcohol) in simulated fermentation of Chinese rice wine, 92.40 and 303.31 mg/L, respectively, which were 33.00 and 14.20 % lower than those of the parental strain RY1. The differences in fermentation performance between RY1-Δbat2 and RY1 were minor. Therefore, construction of this yeast strain is important in future development in Chinese wine industry and provides insights on generating yeast strains for other fermented alcoholic beverages.

Screening wild yeast strains for alcohol fermentation from various fruits.

PubMed

Lee, Yeon-Ju; Choi, Yu-Ri; Lee, So-Young; Park, Jong-Tae; Shim, Jae-Hoon; Park, Kwan-Hwa; Kim, Jung-Wan

2011-03-01

Wild yeasts on the surface of various fruits including grapes were surveyed to obtain yeast strains suitable for fermenting a novel wine with higher alcohol content and supplemented with rice starch. We considered selected characteristics, such as tolerance to alcohol and osmotic pressure, capability of utilizing maltose, and starch hydrolysis. Among 637 putative yeast isolates, 115 strains exhibiting better growth in yeast-peptone-dextrose broth containing 30% dextrose, 7% alcohol, or 2% maltose were selected, as well as five α-amylase producers. Nucleotide sequence analysis of the 26S rDNA gene classified the strains into 13 species belonging to five genera; Pichia anomala was the most prevalent (41.7%), followed by Wickerhamomyces anomalus (19.2%), P. guilliermondii (15%), Candida spp. (5.8%), Kodamaea ohmeri (2.5%), and Metschnikowia spp. (2.5%). All of the α-amylase producers were Aureobasidium pullulans. Only one isolate (NK28) was identified as Saccharomyces cerevisiae. NK28 had all of the desired properties for the purpose of this study, except α-amylase production, and fermented alcohol better than commercial wine yeasts.

Lactic acid production from xylose by Geobacillus stearothermophilus strain 15

NASA Astrophysics Data System (ADS)

Kunasundari, B.; Naresh, S.; Chu, J. E.

2017-09-01

Lactic acid is an important compound with a wide range of industrial applications. The present study tested the efficiency of xylose, as a sole carbon source to be converted to lactic acid by Geobacillus stearothermophilus strain 15. To the best of our knowledge, limited information is available on the directed fermentation of xylose to lactic acid by this bacterium. The effects of different parameters such as temperature, pH, incubation time, agitation speed, concentrations of nitrogen and carbon sources on the lactic acid production were investigated statistically. It was found that the bacterium exhibited poor assimilation of xylose to lactic acid. Temperature, agitation rate and incubation time were determined to improve the lactic acid production slightly. The highest lactic acid yield obtained was 8.9% at 45°C, 300 RPM, 96 h, pH of 6.0 with carbon and nitrogen source concentrations were fixed at 5% w/v.

Comparative studies on the alcohol types presence in Gracilaria sp. and rice fermentation using Sasad

NASA Astrophysics Data System (ADS)

Mansa, R.; Mansuit, H.; Sipaut, C. S.; Yee, C. F.; Yasir, S. M.

2016-06-01

Alternative fuel sources such as biofuels are needed in order to overcome environmental problem caused by fossil fuel consumption. Currently, most biofuel are produced from land based crops and there is a possibility that marine biomass such as macroalgae can be an alternative source for biofuel production. The carbohydrate in macroalgae can be broken down into simple sugar through thermo-chemical hydrolysis and enzymatic hydrolysis. Dilute-acid hydrolysis was believed to be the most available and affordable method. However, the process may release inhibitors which would affect alcohol yield from fermentation. Thus, this work was aimed at investigating if it is possible to avoid this critical pre-treatment step in macroalgae fermentation process by using Sasad, a local Sabahan fermentation agent and to compare the yield with rice wine fermentation. This work hoped to determine and compare the alcohol content from Gracilaria sp. and rice fermentation with Sasad. Rice fermentation was found containing ethanol and 2 - methyl - 1 - propanol. Fermentation of Gracilaria sp. had shown the positive presence of 3 - methyl - 1 - butanol. It was found that Sasad can be used as a fermentation agent for bioalcohol production from Gracilaria sp. without the need for a pretreatment step. However further investigations are needed to determine if pre-treatment would increase the yield of alcohol.

Substituent effect of phenolic aldehyde inhibition on alcoholic fermentation by Saccharomyces cerevisiae

Treesearch

Rui Xie; Maobing Tu; Thomas Elder

2016-01-01

Phenolic compounds significantly inhibit microbial fermentation of biomass hydrolysates. To understand thequantitative structure-inhibition relationship of phenolic aldehydes on alcoholic fermentation, the effect of 11 differentsubstituted benzaldehydes on the final ethanol yield was examined. The results showed that the degree of phenolic...

Pyrazinamidase, CR-MOX agar, salicin fermentation-esculin hydrolysis, and D-xylose fermentation for identifying pathogenic serotypes of Yersinia enterocolitica.

PubMed Central

Farmer, J J; Carter, G P; Miller, V L; Falkow, S; Wachsmuth, I K

1992-01-01

We evaluated several simple laboratory tests that have been used to identify pathogenic serotypes of Yersinia enterocolitica or to indicate the pathogenic potential of individual strains. A total of 100 strains of Y. enterocolitica were studied, including 25 isolated during five outbreak investigations, 63 from sporadic cases, and 12 from stock cultures. The pyrazinamidase test, which does not depend on the Yersinia virulence plasmid, correctly identified 60 of 63 (95% sensitivity) strains of pathogenic serotypes and 34 of 37 (92% specificity) strains of nonpathogenic serotypes. Salicin fermentation-esculin hydrolysis (25 degrees C, 48 h) correctly identified all 63 (100% sensitivity) strains of the pathogenic serotypes and 34 of 37 (92% specificity) strains of the nonpathogenic serotypes. The results of the pyrazinamidase and salicin-esculin tests disagreed for only 7 of the 100 strains of Y. enterocolitica, and these would require additional testing. Congo red-magnesium oxalate (CR-MOX) agar determines Congo red dye uptake and calcium-dependent growth at 36 degrees C, and small red colonies are present only if the strain contains the Yersinia virulence plasmid. This test has proven to be extremely useful for freshly isolated cultures, but only 15 of 62 strains of pathogenic serotypes that had been stored for 1 to 10 years were CR-MOX positive. None of the 16 strains of Y. enterocolitica serotype O3 fermented D-xylose, so this test easily differentiated strains of this serotype, which now appears to be the most common in the United States. Although antisera that can actually be used to serotype strains of Y. enterocolitica are not readily available, the four simple tests described above can be used to screen for pathogenic serotypes. Images PMID:1400958

Performance testing of Zymomonas mobilis metabolically engineered for cofermentation of glucose, xylose, and arabinose.

PubMed

Lawford, Hugh G; Rousseau, Joyce D

2002-01-01

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory's metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN's feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN's biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L x h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L x h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the

Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution.

PubMed

Kim, Hee Su; Oh, Young Hoon; Jang, Young-Ah; Kang, Kyoung Hee; David, Yokimiko; Yu, Ju Hyun; Song, Bong Keun; Choi, Jong-il; Chang, Yong Keun; Joo, Jeong Chan; Park, Si Jae

2016-06-03

Lignocellulosic raw materials have extensively been examined for the production of bio-based fuels, chemicals, and polymers using microbial platforms. Since xylose is one of the major components of the hydrolyzed lignocelluloses, it is being considered a promising substrate in lignocelluloses based fermentation process. Ralstonia eutropha, one of the most powerful and natural producers of polyhydroxyalkanoates (PHAs), has extensively been examined for the production of bio-based chemicals, fuels, and polymers. However, to the best of our knowledge, lignocellulosic feedstock has not been employed for R. eutropha probably due to its narrow spectrum of substrate utilization. Thus, R. eutropha engineered to utilize xylose should be useful in the development of microbial process for bio-based products from lignocellulosic feedstock. Recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes encoding xylose isomerase and xylulokinase respectively, was constructed and examined for the synthesis of poly(3-hydroxybutyrate) [P(3HB)] using xylose as a sole carbon source. It could produce 2.31 g/L of P(3HB) with a P(3HB) content of 30.95 wt% when it was cultured in a nitrogen limited chemically defined medium containing 20.18 g/L of xylose in a batch fermentation. Also, recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes produced 5.71 g/L of P(3HB) with a P(3HB) content of 78.11 wt% from a mixture of 10.05 g/L of glucose and 10.91 g/L of xylose in the same culture condition. The P(3HB) concentration and content could be increased to 8.79 g/L and 88.69 wt%, respectively, when it was cultured in the medium containing 16.74 g/L of glucose and 6.15 g/L of xylose. Further examination of recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes by fed-batch fermentation resulted in the production of 33.70 g/L of P(3HB) in 108 h with a P(3HB) content of 79.02 wt%. The concentration of xylose could be maintained as high as 6 g/L, which is

Impact of pseudo-continuous fermentation on the ethanol tolerance of Scheffersomyces stipitis.

PubMed

Liang, Meng; Kim, Min Hea; He, Qinghua Peter; Wang, Jin

2013-09-01

In this work we conducted the pseudo-continuous fermentation, i.e., continuous fermentation with cell retention, using Scheffersomyces stipitis, and studied its effect on ethanol tolerance of the strain. During the fermentation experiments, S. stipitis was adapted to a mild concentration of ethanol (20-26 g/L) for two weeks. Two substrates (glucose and xylose) were used in different fermentation experiments. After fermentation, various experiments were performed to evaluate the ethanol tolerance of adapted cells and unadapted cells. Compared to the unadapted cells, the viability of adapted cells increased by 8 folds with glucose as the carbon source and 6 folds with xylose as the carbon source following exposure to 60 g/L ethanol for 2 h. Improved ethanol tolerance of the adapted cells was also revealed in the effects of ethanol on plasma membrane permeability, extracellular alkalization and acidification. The mathematical modeling of cell leakage, extracellular alkalization and acidification revealed that cells cultured on glucose show better ethanol tolerance than cells cultured on xylose but the differences become smaller for adapted cells. The results show that pseudo-continuous fermentation can effectively improve cell's ethanol tolerance due to the environmental pressure during the fermentation process. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Traditional low-alcoholic and non-alcoholic fermented beverages consumed in European countries: a neglected food group.

PubMed

Baschali, Aristea; Tsakalidou, Effie; Kyriacou, Adamantini; Karavasiloglou, Nena; Matalas, Antonia-Leda

2017-06-01

Fermented beverages hold a long tradition and contribution to the nutrition of many societies and cultures worldwide. Traditional fermentation has been empirically developed in ancient times as a process of raw food preservation and at the same time production of new foods with different sensorial characteristics, such as texture, flavour and aroma, as well as nutritional value. Low-alcoholic fermented beverages (LAFB) and non-alcoholic fermented beverages (NAFB) represent a subgroup of fermented beverages that have received rather little attention by consumers and scientists alike, especially with regard to their types and traditional uses in European societies. A literature review was undertaken and research articles, review papers and textbooks were searched in order to retrieve data regarding the dietary role, nutrient composition, health benefits and other relevant aspects of diverse ethnic LAFB and NAFB consumed by European populations. A variety of traditional LAFB and NAFB consumed in European regions, such as kefir, kvass, kombucha and hardaliye, are presented. Milk-based LAFB and NAFB are also available on the market, often characterised as 'functional' foods on the basis of their probiotic culture content. Future research should focus on elucidating the dietary role and nutritional value of traditional and 'functional' LAFB and NAFB, their potential health benefits and consumption trends in European countries. Such data will allow for LAFB and NAFB to be included in national food composition tables.

Promoter engineering of the Saccharomyces cerevisiae RIM15 gene for improvement of alcoholic fermentation rates under stress conditions.

PubMed

Watanabe, Daisuke; Kaneko, Akie; Sugimoto, Yukiko; Ohnuki, Shinsuke; Takagi, Hiroshi; Ohya, Yoshikazu

2017-02-01

A loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-like protein kinase, is one of the major causes of the high alcoholic fermentation rates in Saccharomyces cerevisiae sake strains closely related to Kyokai no. 7 (K7). However, impairment of Rim15p may not be beneficial under more severe fermentation conditions, such as in the late fermentation stage, as it negatively affects stress responses. To balance stress tolerance and fermentation performance, we inserted the promoter of a gluconeogenic gene, PCK1, into the 5'-untranslated region (5'-UTR) of the RIM15 gene in a laboratory strain to achieve repression of RIM15 gene expression in the glucose-rich early stage with its induction in the stressful late stage of alcoholic fermentation. The promoter-engineered strain exhibited a fermentation rate comparable to that of the RIM15-deleted strain with no decrease in cell viability. The engineered strain achieved better alcoholic fermentation performance than the RIM15-deleted strain under repetitive and high-glucose fermentation conditions. These data demonstrated the validity of promoter engineering of the RIM15 gene that governs inhibitory control of alcoholic fermentation. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Coutilization of D-Glucose, D-Xylose, and L-Arabinose in Saccharomyces cerevisiae by Coexpressing the Metabolic Pathways and Evolutionary Engineering

PubMed Central

Zhao, Jianzhi; Qiu, Chenxi; Wang, Shihao; Du, Binghai

2017-01-01

Efficient and cost-effective fuel ethanol production from lignocellulosic materials requires simultaneous cofermentation of all hydrolyzed sugars, mainly including D-glucose, D-xylose, and L-arabinose. Saccharomyces cerevisiae is a traditional D-glucose fermenting strain and could utilize D-xylose and L-arabinose after introducing the initial metabolic pathways. The efficiency and simultaneous coutilization of the two pentoses and D-glucose for ethanol production in S. cerevisiae still need to be optimized. Previously, we constructed an L-arabinose-utilizing S. cerevisiae BSW3AP. In this study, we further introduced the XI and XR-XDH metabolic pathways of D-xylose into BSW3AP to obtain D-glucose, D-xylose, and L-arabinose cofermenting strain. Benefits of evolutionary engineering: the resulting strain BSW4XA3 displayed a simultaneous coutilization of D-xylose and L-arabinose with similar consumption rates, and the D-glucose metabolic capacity was not decreased. After 120 h of fermentation on mixed D-glucose, D-xylose, and L-arabinose, BSW4XA3 consumed 24% more amounts of pentoses and the ethanol yield of mixed sugars was increased by 30% than that of BSW3AP. The resulting strain BSW4XA3 was a useful chassis for further enhancing the coutilization efficiency of mixed sugars for bioethanol production. PMID:28459063

Effect on White Grape Must of Multiflora Bee Pollen Addition during the Alcoholic Fermentation Process.

PubMed

Amores-Arrocha, Antonio; Roldán, Ana; Jiménez-Cantizano, Ana; Caro, Ildefonso; Palacios, Víctor

2018-05-31

The aim of the present study was to compare and analyze the impact of using bee pollen doses (0.1, 0.25, 1, 5, 10 and 20 g/L) as activator in the alcoholic fermentation process of Palomino fino and Riesling wines. In this regard, its influence on the musts composition, the fermentative kinetics, the evolution of the populations of Saccharomyces cerevisiae , the evolution of yeast-assimilable nitrogen and physico-chemical characteristics of final wines has been analyzed. Bee pollen addition produces significant increases in yeast-assimilable nitrogen and maximum yeasts population and exponential velocity reached during alcoholic fermentation. Bee pollen showed an important effect on yeast survival during the death phase. Final wines showed significantly increase in volatile acidity above doses higher than 10 g/L and Comisión Internacional de L'Eclairage parameters (CIELab), color intensity and Abs 420 nm, from 1 g/L. Therefore, pollen could be used as fermentative activator for the alcoholic fermentation of white wines applying doses below of 1 g/L.

New insight into microbial diversity and functions in traditional Vietnamese alcoholic fermentation.

PubMed

Thanh, Vu Nguyen; Thuy, Nguyen Thanh; Chi, Nguyen Thuy; Hien, Dinh Duc; Ha, Bui Thi Viet; Luong, Dao Thi; Ngoc, Pham Duc; Ty, Pham Van

2016-09-02

The roles of microorganisms in traditional alcoholic fermentation are often assumed based on abundance in the starter and activity in pure culture. There is a serious lack of hard evidence on the behavior and activity of individual microbial species during the actual fermentation process. In this study, microbial succession and metabolite changes during 7days of traditional Vietnamese alcoholic fermentation were monitored. Special attention was devoted to starch degradation. In total, 22 microbial species, including 6 species of filamentous fungi (Rhizopus microsporus, Rhizopus arrhizus, Mucor indicus, Mucor circinelloides, Cunninghamella elegans, Aspergillus niger), 1 yeast-like fungus (Saccharomycopsis fibuligera), 7 yeasts (Saccharomyces cerevisiae, Clavispora lusitaniae, Wickerhamomyces anomalus, Lindnera fabianii, Pichia kudriavzevii, Candida rugosa, Candida tropicalis), and 8 bacteria (Stenotrophomonas maltophilia, Lactobacillus brevis, Lactobacillus helveticus, Acinetobacter baumannii, Staphylococcus hominis, Bacillus megaterium, Enterobacter asburiae, Pediococcus pentosaceus) were identified. Despite the presence of a complex microbiota in the starter, the fermentation process is consistent and involves a limited number of functional species. Rapid change in microbial composition of fermentation mash was observed and it was correlated with ethanol content. Microbial biomass reached maximum during first 2days of solid state fermentation. Acidification of the medium took place in day 1, starch degradation in days 2, 3, 4, and alcohol accumulation from day 3. Although Sm. fibuligera dominated by cell count amongst potential starch degraders, zymography indicated that it did not produce amylase in the fermentation mash. In mixed culture with Rhizopus, amylase production by Sm. fibuligera is regulated by the moisture content of the substrate. Rhizopus was identified as the main starch degrader and S. cerevisiae as the main ethanol producer. Bacterial load was

Recombinant zymomonas for pentose fermentation

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark

1996-01-01

The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

Engineering of the redox imbalance of Fusarium oxysporum enables anaerobic growth on xylose.

PubMed

Panagiotou, Gianni; Christakopoulos, Paul; Grotkjaer, Thomas; Olsson, Lisbeth

2006-09-01

Dissimilatory nitrate reduction metabolism, of the natural xylose-fermenting fungus Fusarium oxysporum, was used as a strategy to achieve anaerobic growth and ethanol production from xylose. Beneficial alterations of the redox fluxes and thereby of the xylose metabolism were obtained by taking advantage of the regeneration of the cofactor NAD(+) during the denitrification process. In batch cultivations, nitrate sustained growth under anaerobic conditions (1.21 g L(-1) biomass) and simultaneously a maximum yield of 0.55 moles of ethanol per mole of xylose was achieved, whereas substitution of nitrate with ammonium limited the growth significantly (0.15 g L(-1) biomass). Using nitrate, the maximum acetate yield was 0.21 moles per mole of xylose and no xylitol excretion was observed. Furthermore, the network structure in the central carbon metabolism of F. oxysporum was characterized in steady state. F. oxysporum grew anaerobically on [1-(13)C] labelled glucose and unlabelled xylose in chemostat cultivation with nitrate as nitrogen source. The use of labelled substrate allowed the precise determination of the glucose and xylose contribution to the carbon fluxes in the central metabolism of this poorly described microorganism. It was demonstrated that dissimilatory nitrate reduction allows F. oxysporum to exhibit typical respiratory metabolic behaviour with a highly active TCA cycle and a large demand for NADPH.

Recombinant Zymomonas for pentose fermentation

DOEpatents

Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.

1996-05-07

The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 2 figs.

Alcoholic fermentation with flocculant Saccharomyces cerevisiae in fed-batch process.

PubMed

Guidini, Carla Zanella; Marquez, Líbia Diniz Santos; de Almeida Silva, Helisângela; de Resende, Miriam Maria; Cardoso, Vicelma Luiz; Ribeiro, Eloízio Júlio

2014-02-01

Studies have been conducted on selecting yeast strains for use in fermentation for ethanol production to improve the performance of industrial plants and decrease production costs. In this paper, we study alcoholic fermentation in a fed-batch process using a Saccharomyces cerevisiae yeast strain with flocculant characteristics. Central composite design (CCD) was used to determine the optimal combination of the variables involved, with the sucrose concentration of 170 g/L, a cellular concentration in the inoculum of 40% (v/v), and a filling time of 6 h, which resulted in a 92.20% yield relative to the theoretical maximum yield, a productivity of 6.01 g/L h and a residual sucrose concentration of 44.33 g/L. With some changes in the process such as recirculation of medium during the fermentation process and increase in cellular concentration in the inoculum after use of the CCD was possible to reduce the residual sucrose concentration to 2.8 g/L in 9 h of fermentation and increase yield and productivity for 92.75% and 9.26 g/L h, respectively. A model was developed to describe the inhibition of alcoholic fermentation kinetics by the substrate and the product. The maximum specific growth rate was 0.103 h(-1), with K(I) and K(s) values of 109.86 and 30.24 g/L, respectively. The experimental results from the fed-batch reactor show a good fit with the proposed model, resulting in a maximum growth rate of 0.080 h(-1).

A-xylosidase enhanced conversion of plant biomass into fermentable sugars

DOEpatents

Walton, Jonathan D.; Scott-Craig, John S.; Borrusch, Melissa

2016-08-02

The invention relates to increasing the availability of fermentable sugars from plant biomass, such as glucose and xylose. As described herein, .alpha.-xylosidases can be employed with cellulases to enhance biomass conversion into free, fermentable sugar residues.

Sensory profile and volatile aroma composition of reduced alcohol Merlot wines fermented with Metschnikowia pulcherrima and Saccharomyces uvarum.

PubMed

Varela, C; Barker, A; Tran, T; Borneman, A; Curtin, C

2017-07-03

Strategies for production of wines containing lower alcohol concentrations are in strong demand, for reasons of quality, health, and taxation. Development and application of wine yeasts that are less efficient at transforming grape sugars into ethanol has the potential to allow winemakers the freedom to make lower alcohol wines from grapes harvested at optimal ripeness, without the need for post-fermentation processes aimed at removing ethanol. We have recently shown that two non-conventional wine yeast species Metschnikowia pulcherrima and Saccharomyces uvarum were both able to produce wine with reduced alcohol concentration. Both species produced laboratory-scale wines with markedly different volatile aroma compound composition relative to Saccharomyces cerevisiae. This work describes the volatile composition and sensory profiles of reduced-alcohol pilot-scale Merlot wines produced with M. pulcherrima and S. uvarum. Wines fermented with M. pulcherrima contained 1.0% v/v less ethanol than S. cerevisiae fermented wines, while those fermented with S. uvarum showed a 1.7% v/v reduction in ethanol. Compared to S. cerevisiae ferments, wines produced with M. pulcherrima showed higher concentrations of ethyl acetate, total esters, total higher alcohols and total sulfur compounds, while wines fermented with S. uvarum were characterised by the highest total concentration of higher alcohols. Sensorially, M. pulcherrima wines received relatively high scores for sensory descriptors such as red fruit and fruit flavour and overall exhibited a sensory profile similar to that of wine made with S. cerevisiae, whereas the main sensory descriptors associated with wines fermented with S. uvarum were barnyard and meat. This work demonstrates the successful application of M. pulcherrima AWRI3050 for the production of pilot-scale red wines with reduced alcohol concentration and highlights the need for rigorous evaluation of non-conventional yeasts with regard to their sensory impacts

2,3-Butanediol recovery from fermentation broth by alcohol precipitation and vacuum distillation.

PubMed

Jeon, Sangjun; Kim, Duk-Ki; Song, Hyohak; Lee, Hee Jong; Park, Sunghoon; Seung, Doyoung; Chang, Yong Keun

2014-04-01

This study presents a new and effective downstream process to recover 2,3-butanediol (2,3-BD) from fermentation broth which is produced by a recombinant Klebsiella pneumoniae strain. The ldhA-deficient K. pneumoniae strain yielded about 90 g/L of 2,3-BD, along with a number of by-products, such as organic acids and alcohols, in a 65 h fed-batch fermentation. The pH-adjusted cell-free fermentation broth was firstly concentrated until 2,3-BD reached around 500 g/L by vacuum evaporation at 50°C and 50 mbar vacuum pressure. The concentrated solution was further treated using light alcohols, including methanol, ethanol, and isopropanol, for the precipitation of organic acids and inorganic salts. Isopropanol showed the highest removal efficiency, in which 92.5% and 99.8% of organic acids and inorganic salts were precipitated, respectively. At a final step, a vacuum distillation process enabled the recovery of 76.2% of the treated 2,3-BD, with 96.1% purity, indicating that fermentatively produced 2,3-BD is effectively recovered by a simple alcohol precipitation and vacuum distillation. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Recombinant Zymomonas for pentose fermentation

DOEpatents

Picataggio, S.K.; Min Zhang; Eddy, C.K.; Deanda, K.A.

1998-03-10

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

Pentose fermentation by recombinant Zymomonas

DOEpatents

Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.; Mohagheghi, A.; Newman, M.M.; McMillan, J.D.

1998-01-27

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

Pentose fermentation by recombinant zymomonas

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark; Mohagheghi, Ali; Newman, Mildred M.; McMillan, James D.

1998-01-01

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

Recombinant Zymomonas for pentose fermentation

DOEpatents

Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.

1998-01-01

The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

Procedure of brewing alcohol as a staple food: case study of the fermented cereal liquor "Parshot" as a staple food in Dirashe special woreda, southern Ethiopia.

PubMed

Sunano, Yui

2016-07-01

For most brews, alcohol fermentation and lactic fermentation take place simultaneously during the brewing process, and alcohol fermentation can progress smoothly because the propagation of various microorganisms is prevented by lactic fermentation. It is not necessary to cause lactic fermentation with a thing generated naturally and intentionally. The people living in the Dirashe area in southern Ethiopia drink three types of alcoholic beverages that are prepared from cereals. From these alcoholic beverages, parshot is prepared by the addition of plant leaves for lactic fermentation and nech chaka by adding cereal powder for lactic fermentation before alcohol fermentation. People living in the Dirashe area partake of parshot as part of their staple diet. The brewing process used for parshot and a food culture with alcoholic beverages as parts of the staple diet are rare worldwide. This article discusses the significance of using lactic fermentation before alcoholic fermentation and focuses on lactic fermentation in the brewing methods used for the three kinds of alcoholic beverages consumed in the Dirashe area. We initially observed the brewing process and obtained information about the process from the people in that area. Next, we determined the pH and analyzed the lactic acid (g/100 g) and ethanol (g/100 g) content during lactic fermentation of parshot and nech chaka; the ethyl acetate (mg/100 g) and volatile base nitrogen (mg/100 g) content during this period was also analyzed. In addition, we compared the ethanol (g/100 g) content of all three kinds of alcoholic beverages after completion of brewing. The results showed that it was possible to consume large quantities of these alcoholic beverages because of the use of lactic fermentation before alcoholic fermentation, which improved the safety and preservation characteristics of the beverages by preventing the propagation of various microorganisms, improving flavor, and controlling the alcohol level.

Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production

PubMed Central

2013-01-01

Background There has been a great deal of interest in fuel productions from lignocellulosic biomass to minimize the conflict between food and fuel use. The bioconversion of xylose, which is the second most abundant sugar present after glucose in lignocellulosic biomass, is important for the development of cost effective bioprocesses to fuels. Rhodococcus opacus PD630, an oleaginous bacterium, accumulates large amounts of triacylglycerols (TAGs), which can be processed into advanced liquid fuels. However, R. opacus PD630 does not metabolize xylose. Results We generated DNA libraries from a Streptomyces bacterium capable of utilizing xylose and introduced them into R. opacus PD630. Xsp8, one of the engineered strains, was capable of growing on up to 180 g L-1 of xylose. Xsp8 grown in batch-cultures derived from unbleached kraft hardwood pulp hydrolysate containing 70 g L-1 total sugars was able to completely and simultaneously utilize xylose and glucose present in the lignocellulosic feedstock, and yielded 11.0 g L-1 of TAGs as fatty acids, corresponding to 45.8% of the cell dry weight. The yield of total fatty acids per gram of sugars consumed was 0.178 g, which consisted primarily of palmitic acid and oleic acid. The engineered strain Xsp8 was introduced with two heterologous genes from Streptomyces: xylA, encoding xylose isomerase, and xylB, encoding xylulokinase. We further demonstrated that in addition to the introduction and the concomitant expression of heterologous xylA and xylB genes, there is another molecular target in the R. opacus genome which fully enables the functionality of xylA and xylB genes to generate the robust xylose-fermenting strain capable of efficiently producing TAGs at high xylose concentrations. Conclusion We successfully engineered a R. opacus strain that is capable of completely utilizing high concentrations of xylose or mixed xylose/glucose simultaneously, and substantiated its suitability for TAG production. This study demonstrates

Xylose Isomerase Improves Growth and Ethanol Production Rates from Biomass Sugars for Both Saccharomyces Pastorianus and Saccharomyces Cerevisiae

PubMed Central

Miller, Kristen P.; Gowtham, Yogender Kumar; Henson, J. Michael; Harcum, Sarah W.

2013-01-01

The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion. PMID:22866331

The importance of aeration strategy in fuel alcohol fermentations contaminated with Dekkera/Brettanomyces yeasts.

PubMed

Abbott, D A; Ingledew, W M

2005-11-01

Whole corn mash fermentations infected with industrially-isolated Brettanomyces yeasts were not affected even when viable Brettanomyces yeasts out-numbered Saccharomyces yeasts tenfold at the onset of fermentation. Therefore, aeration, a parameter that is pivotal to the physiology of Dekkera/Brettanomyces yeasts, was investigated in mixed culture fermentations. Results suggest that aeration strategy plays a significant role in Dekkera/Brettanomyces-mediated inhibition of fuel alcohol fermentations. Although growth of Saccharomyces cerevisiae was not impeded, mixed culture fermentations aerated at rates of > or =20 ml air l(-1) mash min(-1) showed decreased ethanol yields and an accumulation of acetic acid. The importance of aeration was examined further in combination with organic acid(s). Growth of Saccharomyces occurred more rapidly than growth of Brettanomyces yeasts in all conditions. The combination of 0.075% (w/v) acetic acid and contamination with Brettanomyces TK 1404W did not negatively impact the final ethanol yield under fermentative conditions. Aeration, however, did prove to be detrimental to final ethanol yields. With the inclusion of aeration in the control condition (no organic acid stress) and in each fermentation containing organic acid(s), the final ethanol yields were decreased. It was therefore concluded that aeration strategy is the key parameter in regards to the negative effects observed in fuel alcohol fermentations infected with Dekkera/Brettanomyces yeasts.

Methods for sequestering carbon dioxide into alcohols via gasification fermentation

DOEpatents

Gaddy, James L; Ko, Ching-Whan; Phillips, J. Randy; Slape, M. Sean

2013-11-26

The present invention is directed to improvements in gasification for use with synthesis gas fermentation. Further, the present invention is directed to improvements in gasification for the production of alcohols from a gaseous substrate containing at least one reducing gas containing at least one microorganism.

Corecovery of lipids and fermentable sugars from Rhodosporidium toruloides using ionic liquid cosolvents: application of recycle to batch fermentation.

PubMed

Severa, Godwin; Kumar, Guneet; Cooney, Michael J

2014-01-01

This work evaluates the ability of an ionic liquid-methanol cosolvent system to extract lipids and recycle fermentable sugars recovered from oil-bearing Rhodosporidium toruloides grown in batch culture on defined media using glucose and xylose as carbon sources. Growth on the recycled mixed carbon substrate was successful with glucose consumed before xylose and overall cell mass to lipid yields (YP/X ) between 57% and 61% (w/w relative to whole dried cell mass) achieved. Enzymatic hydrolysis of the delipified carbohydrate fraction recovered approximately 9%-11% (w/w) of the whole dried cell mass as fermentable sugars, which were successfully recycled as carbon sources without further purification. In total, up to 70% (w/w) of the whole dried cell mass was recovered as lipids and fermentable sugars and the substrate to lipid yields (YP/S ) was increased from 0.12 to 0.16 g lipid/g carbohydrate consumed, highlighting the promise of this approach to process lipid bearing cell biomass. © 2014 American Institute of Chemical Engineers.

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.

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae.

PubMed

Nijland, Jeroen G; Shin, Hyun Yong; Boender, Leonie G M; de Waal, Paul P; Klaassen, Paul; Driessen, Arnold J M

2017-06-01

expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast. Copyright © 2017 American Society for Microbiology.

Xylose isomerase improves growth and ethanol production rates from biomass sugars for both Saccharomyces pastorianus and Saccharomyces cerevisiae.

PubMed

Miller, Kristen P; Gowtham, Yogender Kumar; Henson, J Michael; Harcum, Sarah W

2012-01-01

The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Retooling microorganisms for the fermentative production of alcohols.

PubMed

Toogood, Helen S; Scrutton, Nigel S

2018-04-01

Bioengineering and synthetic biology approaches have revolutionised the field of biotechnology, enabling the introduction of non-native and de novo pathways for biofuels production. This 'retooling' of microorganisms is also applied to the utilisation of mixed carbon components derived from lignocellulosic biomass, a major technical barrier for the development of economically viable fermentations. This review will discuss recent advances in microorganism engineering for efficient production of alcohols from waste biomass. These advances span the introduction of new pathways to alcohols, host modifications for more cost-effective utilisation of lignocellulosic waste and modifications of existing pathways for generating new fuel additives. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

PubMed Central

2012-01-01

Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene

Melatonin is synthesised by yeast during alcoholic fermentation in wines.

PubMed

Rodriguez-Naranjo, M Isabel; Gil-Izquierdo, Angel; Troncoso, Ana M; Cantos-Villar, Emma; Garcia-Parrilla, M Carmen

2011-06-15

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone produced in the pineal gland. Its biological properties are related to the circadian rhythm. Recently, the European Food Safety Authority (EFSA) accepted the health claim related to melatonin and the alleviation of subjective feelings of jet lag. This molecule has been detected in some foods. In this work, 13 grape varieties were studied; 7 monovarietal wines were produced in an experimental winery under strictly controlled conditions and were sampled in different steps. The grape varieties used to make the wines were: Cabernet Sauvignon, Merlot, Syrah, Tempranillo, Tintilla de Rota, Palomino Fino and Alpha red. Liquid chromatography tandem mass spectrometry (LC-MS/MS) unequivocally confirmed the presence of melatonin in wines. The main contribution of this paper is the results that clearly show that melatonin is synthesised during the winemaking process, specifically after the alcoholic fermentation. Indeed, melatonin is absent in grapes and musts and is formed during alcoholic fermentation. Copyright © 2010 Elsevier Ltd. All rights reserved.

Molecular mechanism of environmental d-xylose perception by a XylFII-LytS complex in bacteria.

PubMed

Li, Jianxu; Wang, Chengyuan; Yang, Gaohua; Sun, Zhe; Guo, Hui; Shao, Kai; Gu, Yang; Jiang, Weihong; Zhang, Peng

2017-08-01

d-xylose, the main building block of plant biomass, is a pentose sugar that can be used by bacteria as a carbon source for bio-based fuel and chemical production through fermentation. In bacteria, the first step for d-xylose metabolism is signal perception at the membrane. We previously identified a three-component system in Firmicutes bacteria comprising a membrane-associated sensor protein (XylFII), a transmembrane histidine kinase (LytS) for periplasmic d-xylose sensing, and a cytoplasmic response regulator (YesN) that activates the transcription of the target ABC transporter xylFGH genes to promote the uptake of d-xylose. The molecular mechanism underlying signal perception and integration of these processes remains elusive, however. Here we purified the N-terminal periplasmic domain of LytS (LytSN) in a complex with XylFII and determined the conformational structures of the complex in its d-xylose-free and d-xylose-bound forms. LytSN contains a four-helix bundle, and XylFII contains two Rossmann fold-like globular domains with a xylose-binding cleft between them. In the absence of d-xylose, LytSN and XylFII formed a heterodimer. Specific binding of d-xylose to the cleft of XylFII induced a large conformational change that closed the cleft and brought the globular domains closer together. This conformational change led to the formation of an active XylFII-LytSN heterotetramer. Mutations at the d-xylose binding site and the heterotetramer interface diminished heterotetramer formation and impaired the d-xylose-sensing function of XylFII-LytS. Based on these data, we propose a working model of XylFII-LytS that provides a molecular basis for d-xylose utilization and metabolic modification in bacteria.

Enterococcus faecium QU 50: a novel thermophilic lactic acid bacterium for high-yield l-lactic acid production from xylose.

PubMed

Abdel-Rahman, Mohamed Ali; Tashiro, Yukihiro; Zendo, Takeshi; Sakai, Kenji; Sonomoto, Kenji

2015-01-01

Production of optically pure lactic acid from lignocellulosic material for commercial purposes is hampered by several difficulties, including heterofermentation of pentose sugars and high energy consumption by mesophilic lactic acid bacteria. Here, we report a novel lactic acid bacterium, strain QU 50, that has the potential to produce optically pure l-lactic acid (≥99.2%) in a homofermentative manner from xylose under thermophilic conditions. Strain QU 50 was isolated from Egyptian fertile soil and identified as Enterococcus faecium QU 50 by analyzing its sugar fermentation pattern and 16S rRNA gene sequence. Enterococcus faecium QU 50 fermented xylose efficiently to produce lactic acid over wide pH (6.0-10.0) and temperature ranges (30-52°C), with a pH of 6.5 and temperature of 50°C being optimal. To our knowledge, this is the first report of homofermentative lactic acid production from xylose by a thermophilic lactic acid bacterium. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Fermentation Kinetics for Xylitol Production by a Pichia stipitis d-Xylulokinase Mutant Previously Grown in Spent Sulfite Liquor

NASA Astrophysics Data System (ADS)

Rodrigues, Rita C. L. B.; Lu, Chenfeng; Lin, Bernice; Jeffries, Thomas W.

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3Δ) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h-1). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 gxylose/gcel h) and xylitol production (0.059 gxylitol/gcel h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

Xylose utilization in recombinant Zymomonas

DOEpatents

Kahsay, Robel Y; Qi, Min; Tao, Luan; Viitanen, Paul V; Yang, Jianjun

2013-01-07

Zymomonas expressing xylose isomerase from A. missouriensis was found to have improved xylose utilization, growth, and ethanol production when grown in media containing xylose. Xylose isomerases related to that of A. missouriensis were identified structurally through molecular phylogenetic and Profile Hidden Markov Model analyses, providing xylose isomerases that may be used to improve xylose utilization.

Yeast Immobilization Systems for Alcoholic Wine Fermentations: Actual Trends and Future Perspectives

PubMed Central

Moreno-García, Jaime; García-Martínez, Teresa; Mauricio, Juan C.; Moreno, Juan

2018-01-01

Yeast immobilization is defined as the physical confinement of intact cells to a region of space with conservation of biological activity. The use of these methodologies for alcoholic fermentation (AF) offers many advantages over the use of the conventional free yeast cell method and different immobilization systems have been proposed so far for different applications, like winemaking. The most studied methods for yeast immobilization include the use of natural supports (e.g., fruit pieces), organic supports (e.g., alginate), inorganic (e.g., porous ceramics), membrane systems, and multi-functional agents. Some advantages of the yeast-immobilization systems include: high cell densities, product yield improvement, lowered risk of microbial contamination, better control and reproducibility of the processes, as well as reuse of the immobilization system for batch fermentations and continuous fermentation technologies. However, these methods have some consequences on the behavior of the yeasts, affecting the final products of the fermentative metabolism. This review compiles current information about cell immobilizer requirements for winemaking purposes, the immobilization methods applied to the production of fermented beverages to date, and yeast physiological consequences of immobilization strategies. Finally, a recent inter-species immobilization methodology has been revised, where yeast cells are attached to the hyphae of a Generally Recognized As Safe fungus and remain adhered following loss of viability of the fungus. The bio-capsules formed with this method open new and promising strategies for alcoholic beverage production (wine and low ethanol content beverages). PMID:29497415

Pulque, a Traditional Mexican Alcoholic Fermented Beverage: Historical, Microbiological, and Technical Aspects.

PubMed

Escalante, Adelfo; López Soto, David R; Velázquez Gutiérrez, Judith E; Giles-Gómez, Martha; Bolívar, Francisco; López-Munguía, Agustín

2016-01-01

Pulque is a traditional Mexican alcoholic beverage produced from the fermentation of the fresh sap known as aguamiel (mead) extracted from several species of Agave (maguey) plants that grow in the Central Mexico plateau. Currently, pulque is produced, sold and consumed in popular districts of Mexico City and rural areas. The fermented product is a milky white, viscous, and slightly acidic liquid beverage with an alcohol content between 4 and 7° GL and history of consumption that dates back to pre-Hispanic times. In this contribution, we review the traditional pulque production process, including the microbiota involved in the biochemical changes that take place during aguamiel fermentation. We discuss the historical relevance and the benefits of pulque consumption, its chemical and nutritional properties, including the health benefits associated with diverse lactic acid bacteria with probiotic potential isolated from the beverage. Finally, we describe the actual status of pulque production as well as the social, scientific and technological challenges faced to preserve and improve the production of this ancestral beverage and Mexican cultural heritage.

Pulque, a Traditional Mexican Alcoholic Fermented Beverage: Historical, Microbiological, and Technical Aspects

PubMed Central

Escalante, Adelfo; López Soto, David R.; Velázquez Gutiérrez, Judith E.; Giles-Gómez, Martha; Bolívar, Francisco; López-Munguía, Agustín

2016-01-01

Pulque is a traditional Mexican alcoholic beverage produced from the fermentation of the fresh sap known as aguamiel (mead) extracted from several species of Agave (maguey) plants that grow in the Central Mexico plateau. Currently, pulque is produced, sold and consumed in popular districts of Mexico City and rural areas. The fermented product is a milky white, viscous, and slightly acidic liquid beverage with an alcohol content between 4 and 7° GL and history of consumption that dates back to pre-Hispanic times. In this contribution, we review the traditional pulque production process, including the microbiota involved in the biochemical changes that take place during aguamiel fermentation. We discuss the historical relevance and the benefits of pulque consumption, its chemical and nutritional properties, including the health benefits associated with diverse lactic acid bacteria with probiotic potential isolated from the beverage. Finally, we describe the actual status of pulque production as well as the social, scientific and technological challenges faced to preserve and improve the production of this ancestral beverage and Mexican cultural heritage. PMID:27446061

Effects of pretreatment methods for hazelnut shell hydrolysate fermentation with Pichia Stipitis to ethanol.

PubMed

Arslan, Yeşim; Eken-Saraçoğlu, Nurdan

2010-11-01

In this study, we investigated the use of hazelnut shell as a renewable and low cost lignocellulosic material for bioethanol production for the first time. High lignin content of hazelnut shell is an important obstacle for such a biotransformation. Biomass hydrolysis with acids yields reducing sugar with several inhibitors which limit the fermentability of sugars. The various conditioning methods for biomass and hydrolysate were performed to overcome the toxicity and their effects on the subsequent fermentation of hazelnut shell hydrolysate by Pichia stipitis were evaluated with shaking flasks experiments. Hazelnut shells hydrolysis with 0.7M H(2)SO(4) yielded 49 gl(-1) total reducing sugars and fermentation inhibitors in untreated hydrolysate. First, it was shown that several hydrolysate detoxification methods were solely inefficient in achieving cell growth and ethanol production in the fermentation of hazelnut shell hydrolysates derived from non-delignified biomass. Next, different pretreatments of hazelnut shells were considered for delignification and employed before hydrolysis in conjunction with hydrolysate detoxification to improve alcohol fermentation. Among six delignification methods, the most effective pretreatment regarding to ethanol concentration includes the treatment of shells with 3% (w/v) NaOH at room temperature, which was integrated with sequential hydrolysate detoxification by overliming and then treatment with charcoal twice at 60 degrees C. This treatment brought about a total reduction of 97% in furans and 88.4% in phenolics. Almost all trialed treatments caused significant sugar loss. Under the best assayed conditions, ethanol concentration of 16.79gl(-1) was reached from a hazelnut shell hyrolysate containing initial 50g total reducing sugar l(-1) after partial synthetic xylose supplementation. This value is equal to 91.25% of ethanol concentration that was obtained from synthetic d-xylose under same conditions. The present study

Production of xylitol from D-xylose by Debaryomyces hansenii

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

Dominguez, J.M.; Gong, Cheng S.; Tsao, G.T.

1997-12-31

Xylitol, a naturally occurring five-carbon sugar alcohol, can be produced from D-xylose through microbial hydrogenation. Xylitol has found increasing use in the food industries, especially in confectionary. It is the only so-called {open_quotes}second-generation polyol sweeteners{close_quotes} that is allowed to have the specific health claims in some world markets. In this study, the effect of cell density on the xylitol production by the yeast Debaryomyces hansenii NRRL Y-7426 from D-xylose under microaerobic conditions was examined. The rate of xylitol production increased with increasing yeast cell density to 3 g/L. Beyond this amount there was no increase in the xylitol production withmore » increasing cell density. The optimal pH range for xylitol production was between 4.5 and 5.5. The optimal temperature was between 28 and 37{degrees}C, and the optimal shaking speed was 300 rpm. The rate of xylitol production increased linearly with increasing initial xylose concentration. A high concentration of xylose (279 g/L) was converted rapidly and efficiently to produce xylitol with a product concentration of 221 g/L was reached after 48 h of incubation under optimum conditions. 18 refs., 5 figs.« less

Thermophilic ethanol fermentation from lignocellulose hydrolysate by genetically engineered Moorella thermoacetica.

PubMed

Rahayu, Farida; Kawai, Yuto; Iwasaki, Yuki; Yoshida, Koichiro; Kita, Akihisa; Tajima, Takahisa; Kato, Junichi; Murakami, Katsuji; Hoshino, Tamotsu; Nakashimada, Yutaka

2017-12-01

A transformant of Moorella thermoacetica was constructed for thermophilic ethanol production from lignocellulosic biomass by deleting two phosphotransacetylase genes, pdul1 and pdul2, and introducing the native aldehyde dehydrogenase gene (aldh) controlled by the promoter from glyceraldehyde-3-phosphate dehydrogenase. The transformant showed tolerance to 540mM and fermented sugars including fructose, glucose, galactose and xylose to mainly ethanol. In a mixed-sugar medium of glucose and xylose, all of the sugars were consumed to produce ethanol at the yield of 1.9mol/mol-sugar. The transformant successfully fermented sugars in hydrolysate prepared through the acid hydrolysis of lignocellulose to ethanol, suggesting that this transformant can be used to ferment the sugars in lignocellulosic biomass for ethanol production. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Characterization of Candida sp. NY7122, a novel pentose-fermenting soil yeast.

PubMed

Watanabe, Itsuki; Ando, Akira; Nakamura, Toshihide

2012-02-01

Yeasts that ferment both hexose and pentose are important for cost-effective ethanol production. We found that the soil yeast strain NY7122 isolated from a blueberry field in Tsukuba (East Japan) could ferment both hexose and pentose (D-xylose and L-arabinose). NY7122 was closely related to Candida subhashii on the basis of the results of molecular identification using the sequence in the D1/D2 domains of 26S rDNA and 5.8S-internal transcribed spacer region. NY7122 produced at least 7.40 and 3.86 g l⁻¹ ethanol from 20 g l⁻¹ D-xylose and L-arabinose within 24 h. NY7122 could produce ethanol from pentose and hexose sugars at 37°C. The highest ethanol productivity of NY7122 was achieved under a low pH condition (pH 3.5). Fermentation of mixed sugars (50 g l⁻¹ glucose, 20 g l⁻¹ D-xylose, and 10 g l⁻¹ L-arabinose) resulted in a maximum ethanol concentration of 27.3 g l⁻¹ for the NY7122 strain versus 25.1 g l⁻¹ for Scheffersomyces stipitis. This is the first study to report that Candida sp. NY7122 from a soil environment could produce ethanol from both D-xylose and L-arabinose.

Fermentation of biomass sugars to ethanol using native industrial yeast strains.

PubMed

Yuan, Dawei; Rao, Kripa; Relue, Patricia; Varanasi, Sasidhar

2011-02-01

In this paper, the feasibility of a technology for fermenting sugar mixtures representative of cellulosic biomass hydrolyzates with native industrial yeast strains is demonstrated. This paper explores the isomerization of xylose to xylulose using a bi-layered enzyme pellet system capable of sustaining a micro-environmental pH gradient. This ability allows for considerable flexibility in conducting the isomerization and fermentation steps. With this method, the isomerization and fermentation could be conducted sequentially, in fed-batch, or simultaneously to maximize utilization of both C5 and C6 sugars and ethanol yield. This system takes advantage of a pH-dependent complexation of xylulose with a supplemented additive to achieve up to 86% isomerization of xylose at fermentation conditions. Commercially-proven Saccharomyces cerevisiae strains from the corn-ethanol industry were used and shown to be very effective in implementation of the technology for ethanol production. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effect of four pretreatments on enzymatic hydrolysis and ethanol fermentation of wheat straw. Influence of inhibitors and washing.

PubMed

Toquero, Cristina; Bolado, Silvia

2014-04-01

Pretreatment is essential in the production of alcohol from lignocellulosic material. In order to increase enzymatic sugar release and bioethanol production, thermal, dilute acid, dilute basic and alkaline peroxide pretreatments were applied to wheat straw. Compositional changes in pretreated solid fractions and sugars and possible inhibitory compounds released in liquid fractions were analysed. SEM analysis showed structural changes after pretreatments. Enzymatic hydrolysis and fermentation by Pichia stipitis of unwashed and washed samples from each pretreatment were performed so as to compare sugar and ethanol yields. The effect of the main inhibitors found in hydrolysates (formic acid, acetic acid, 5-hydroxymethylfurfural and furfural) was first studied through ethanol fermentations of model media and then compared to real hydrolysates. Hydrolysates of washed alkaline peroxide pretreated biomass provided the highest sugar concentrations, 31.82g/L glucose, and 13.75g/L xylose, their fermentation yielding promising results, with ethanol concentrations reaching 17.37g/L. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fermentation of lignocellulosic sugars to acetic acid by Moorella thermoacetica.

PubMed

Ehsanipour, Mandana; Suko, Azra Vajzovic; Bura, Renata

2016-06-01

A systematic study of bioconversion of lignocellulosic sugars to acetic acid by Moorella thermoacetica (strain ATCC 39073) was conducted. Four different water-soluble fractions (hydrolysates) obtained after steam pretreatment of lignocellulosic biomass were selected and fermented to acetic acid in batch fermentations. M. thermoacetica can effectively ferment xylose and glucose in hydrolysates from wheat straw, forest residues, switchgrass, and sugarcane straw to acetic acid. Xylose and glucose were completely utilized, with xylose being consumed first. M. thermoacetica consumed up to 62 % of arabinose, 49 % galactose and 66 % of mannose within 72 h of fermentation in the mixture of lignocellulosic sugars. The highest acetic acid yield was obtained from sugarcane straw hydrolysate, with 71 % of theoretical yield based on total sugars (17 g/L acetic acid from 24 g/L total sugars). The lowest acetic acid yield was observed in forest residues hydrolysate, with 39 % of theoretical yield based on total sugars (18 g/L acetic acid from 49 g/L total sugars). Process derived compounds from steam explosion pretreatment, including 5-hydroxymethylfurfural (0.4 g/L), furfural (0.1 g/L) and total phenolics (3 g/L), did not inhibit microbial growth and acetic acid production yield. This research identified two major factors that adversely affected acetic acid yield in all hydrolysates, especially in forest residues: (i) glucose to xylose ratio and (ii) incomplete consumption of arabinose, galactose and mannose. For efficient bioconversion of lignocellulosic sugars to acetic acid, it is imperative to have an appropriate balance of sugars in a hydrolysate. Hence, the choice of lignocellulosic biomass and steam pretreatment design are fundamental steps for the industrial application of this process.

Co-expression of TAL1 and ADH1 in recombinant xylose-fermenting Saccharomyces cerevisiae improves ethanol production from lignocellulosic hydrolysates in the presence of furfural.

PubMed

Hasunuma, Tomohisa; Ismail, Ku Syahidah Ku; Nambu, Yumiko; Kondo, Akihiko

2014-02-01

Lignocellulosic biomass dedicated to bioethanol production usually contains pentoses and inhibitory compounds such as furfural that are not well tolerated by Saccharomyces cerevisiae. Thus, S. cerevisiae strains with the capability of utilizing both glucose and xylose in the presence of inhibitors such as furfural are very important in industrial ethanol production. Under the synergistic conditions of transaldolase (TAL) and alcohol dehydrogenase (ADH) overexpression, S. cerevisiae MT8-1X/TAL-ADH was able to produce 1.3-fold and 2.3-fold more ethanol in the presence of 70 mM furfural than a TAL-expressing strain and a control strain, respectively. We also tested the strains' ability by mimicking industrial ethanol production from hemicellulosic hydrolysate containing fermentation inhibitors, and ethanol production was further improved by 16% when using MT8-1X/TAL-ADH compared to the control strain. Transcript analysis further revealed that besides the pentose phosphate pathway genes TKL1 and TAL1, ADH7 was also upregulated in response to furfural stress, which resulted in higher ethanol production compared to the TAL-expressing strain. The improved capability of our modified strain was based on its capacity to more quickly reduce furfural in situ resulting in higher ethanol production. The co-expression of TAL/ADH genes is one crucial strategy to fully utilize undetoxified lignocellulosic hydrolysate, leading to cost-competitive ethanol production. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Enhanced ethanol fermentation by engineered Saccharomyces cerevisiae strains with high spermidine contents.

PubMed

Kim, Sun-Ki; Jo, Jung-Hyun; Jin, Yong-Su; Seo, Jin-Ho

2017-05-01

Construction of robust and efficient yeast strains is a prerequisite for commercializing a biofuel production process. We have demonstrated that high intracellular spermidine (SPD) contents in Saccharomyces cerevisiae can lead to improved tolerance against various fermentation inhibitors, including furan derivatives and acetic acid. In this study, we examined the potential applicability of the S. cerevisiae strains with high SPD contents under two cases of ethanol fermentation: glucose fermentation in repeated-batch fermentations and xylose fermentation in the presence of fermentation inhibitors. During the sixteen times of repeated-batch fermentations using glucose as a sole carbon source, the S. cerevisiae strains with high SPD contents maintained higher cell viability and ethanol productivities than a control strain with lower SPD contents. Specifically, at the sixteenth fermentation, the ethanol productivity of a S. cerevisiae strain with twofold higher SPD content was 31% higher than that of the control strain. When the SPD content was elevated in an engineered S. cerevisiae capable of fermenting xylose, the resulting S. cerevisiae strain exhibited much 40-50% higher ethanol productivities than the control strain during the fermentations of synthetic hydrolysate containing high concentrations of fermentation inhibitors. These results suggest that the strain engineering strategy to increase SPD content is broadly applicable for engineering yeast strains for robust and efficient production of ethanol.

Monitoring and Evaluation of Alcoholic Fermentation Processes Using a Chemocapacitor Sensor Array

PubMed Central

Oikonomou, Petros; Raptis, Ioannis; Sanopoulou, Merope

2014-01-01

The alcoholic fermentation of Savatiano must variety was initiated under laboratory conditions and monitored daily with a gas sensor array without any pre-treatment steps. The sensor array consisted of eight interdigitated chemocapacitors (IDCs) coated with specific polymers. Two batches of fermented must were tested and also subjected daily to standard chemical analysis. The chemical composition of the two fermenting musts differed from day one of laboratory monitoring (due to different storage conditions of the musts) and due to a deliberate increase of the acetic acid content of one of the musts, during the course of the process, in an effort to spoil the fermenting medium. Sensor array responses to the headspace of the fermenting medium were compared with those obtained either for pure or contaminated samples with controlled concentrations of standard ethanol solutions of impurities. Results of data processing with Principal Component Analysis (PCA), demonstrate that this sensing system could discriminate between a normal and a potential spoiled grape must fermentation process, so this gas sensing system could be potentially applied during wine production as an auxiliary qualitative control instrument. PMID:25184490

Improved efficiency of butanol production by absorbed lignocellulose fermentation.

PubMed

He, Qin; Chen, Hongzhang

2013-03-01

Alkali-treated steam-exploded corn stover (SECSAT) was used as solid substrate for acetone-butanol-ethanol (ABE) production by absorbed lignocellulose fermentation (ALF) using Clostridium acetobutylicum ATCC 824. The ABE concentration in ALF culture had increased by 47% compared with that in submerged culture. More surprisingly, the acetone production was promoted and ethanol production was lower in the presence of SECSAT than that in its absence. ALF was also successfully in cofermentation of glucose and xylose, although decreased fermentability with an increase in the proportion of xylose. An invariable chemical composition and dry weight of SECSAT was found in ALF. Partial simultaneous saccharification and fermentation of SECSAT using a certain amount of cellulase could not only enhance the ABE concentration by 71%, but also significantly increase the area proportion of fiber cells in SECSAT from 53% to 90%, which would be an excellent paper making material. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Comprehensive study of the evolution of gas-liquid partitioning of aroma compounds during wine alcoholic fermentation.

PubMed

Morakul, Sumallika; Athes, Violaine; Mouret, Jean-Roch; Sablayrolles, Jean-Marie

2010-09-22

Calculating the gas-liquid partitioning of aromatic molecules during winemaking fermentation is essential to minimize the loss of aroma and to optimize the fermentation conditions. In this study, the effect of the main fermentation parameters on the partition coefficients (ki) of higher alcohols (2-methylpropan-1-ol and 3-methyl butan-1-ol) and esters (ethyl acetate, 3-methyl-1-butyl acetate, and 2-ethyl hexanoate) was assessed. The values of ki were first determined in synthetic media simulating must and wine. They varied considerably with both the hydrophobicity of the compound and the composition of the medium. Then, the effect of temperature on ki was quantified. The absence of any effect of gas composition was also established by replacing air with CO2. Finally, the impact of CO2 stripping was assessed by running specific fermentations in which the rate of CO2 production was kept constant by perfusion with assimilable nitrogen. These fermentations showed that in contrast to temperature and must composition, CO2 stripping did not change the gas-liquid partitioning of higher alcohols and esters.

An innovative biocatalyst for production of ethanol from xylose in a continuous bioreactor.

PubMed

Silva, C R; Zangirolami, T C; Rodrigues, J P; Matugi, K; Giordano, R C; Giordano, R L C

2012-01-05

The use of the hemicellulose fraction of biomass may be important for the feasibility of the production of second generation bioethanol. Wild strains of Saccharomyces cerevisiae are widely used in industry for production of 1st generation ethanol, and the robustness of this yeast is an important advantage in large scale applications. Isomerization of xylose to xylulose is an essential step in this process. This reaction is catalyzed by glucose isomerase (GI). A new biocatalyst is presented here for the simultaneous isomerization and fermentation (SIF) of xylose. GI from Streptomyces rubiginosus was immobilized in chitosan, through crosslinking with glutaraldehyde, and the support containing the immobilized GI (IGI-Ch) was co-immobilized with S. cerevisiae, in calcium alginate gel. The immobilization experiments led to high immobilized protein loads (30-68 mg × g(support)(-1)), high yields (circa of 100%) and high recovered enzyme activity (>90%). The IGI-Ch derivative with maximum activity presented 1700 IU × g(catalyst)(-1), almost twice the activity of a commercial immobilized GI, GENSWEET(®) IGI-HF. At typical operational conditions for xylose SIF operation (pH 5, 30-35 °C, presence of nutrients and ethanol concentrations in the medium up to 70 L(-1)), both derivatives, IGI-Ch and GENSWEET(®) IGI-HF retained app. 90% of the initial activity after 120 h, while soluble GI was almost completely inactive at pH 5, 30 °C. The isomerization xylose/xylulose, catalyzed by IGI-Ch, reached the equilibrium in batch experiments after 4h, with 12,000 IU × L(-1) (7 g(der) × L(-1)), at pH 5 and 30 °C, in the presence of fermentation nutrients. After co-immobilization of IGI-Ch with yeast in alginate gel, this biocatalyst succeeded in producing 12 g × L(-1) of ethanol, 9.5 g × L(-1) of xylitol, 2.5 g × L(-1) of glycerol and 1.9 g × L(-1) of acetate after consumption of 50 g × L(-1) of xylose, in 48 h, using 32.5 × 10(3) IU × L(-1) and 20 g(yeast) × L(-1), at 35

Production of Higher Alcohols During Indonesian Tapé Ketan Fermentation †

PubMed Central

Cronk, T. C.; Mattick, L. R.; Steinkraus, K. H.; Hackler, L. R.

1979-01-01

A study was made of the higher alcohols (fusel oils) produced during the Indonesian tapé ketan fermentation using Amylomyces rouxii as the principal mold, alone or in combination with yeasts belonging to genera commonly found in the tapé ketan fermentation (Endomycopsis, Candida, and Hansenula). Total fusel oils increased with length of fermentation. Fusel oils detected in the product distillate included isobutanol and isoamyl and active amyl alcohols. No n-propanol was detected. Isobutanol and isoamyl alcohols were formed in the largest amounts. A. rouxii alone produced nearly the same quantity of fusel oils (total production, 275 mg/liter at 192 h) as it did in combination with Endomycopsis burtonii (total production, 292 mg/liter at 192 h).A. rouxii and Endomycopsis fibuliger produced fusel oils totaling 72 mg/liter at 32 h and 558 mg/liter at 192 h. A. rouxii in combination with Candida yeasts produced somewhat more fusel oils, ranging from 590 to 618 mg/liter at 192 h. A. rouxii in combination with Hansenula yeasts produced the least fusel oils, totaling 143 to 248 mg/liter at 192 h. During the first 36 h, production of fusel oils was higher at 30 and 35°C than at 25°C. At 48 h fusel oil production was slightly higher at 30°C than at 35°C. Beyond 48 h, production of fusel oils was higher at 25°C. A. rouxii in combination with Hansenula anomala and Hansenula subpelliculosa produced considerable ethyl acetate, ranging from 145 to 199 mg/liter at 36 h and 354 to 369 mg/liter at 192 h. PMID:16345385

Low Fermentation pH Is a Trigger to Alcohol Production, but a Killer to Chain Elongation.

PubMed

Ganigué, Ramon; Sánchez-Paredes, Patricia; Bañeras, Lluis; Colprim, Jesús

2016-01-01

Gasification of organic wastes coupled to syngas fermentation allows the recovery of carbon in the form of commodity chemicals, such as carboxylates and biofuels. Acetogenic bacteria ferment syngas to mainly two-carbon compounds, although a few strains can also synthesize four-, and six-carbon molecules. In general, longer carbon chain products have a higher biotechnological (and commercial) value due to their higher energy content and their lower water solubility. However, de-novo synthesis of medium-chain products from syngas is quite uncommon in acetogenic bacteria. An alternative to de-novo synthesis is bioproduction of short-chain products (C2 and C4), and their subsequent elongation to C4, C6, or C8 through reversed β-oxidation metabolism. This two-step synergistic approach has been successfully applied for the production of up to C8 compounds, although the accumulation of alcohols in these mixed cultures remained below detection limits. The present work investigates the production of higher alcohols from syngas by open mixed cultures (OMC). A syngas-fermenting community was enriched from sludge of an anaerobic digester for a period of 109 days in a lab-scale reactor. At the end of this period, stable production of ethanol and butanol was obtained. C6 compounds were only transiently produced at the beginning of the enrichment phase, during which Clostridium kluyveri, a bacterium able to carry out carbon chain elongation, was detected in the community. Further experiments showed pH as a critical parameter to maintain chain elongation activity in the co-culture. Production of C6 compounds was recovered by preventing fermentation pH to decrease below pH 4.5-5. Finally, experiments showed maximal production of C6 compounds (0.8 g/L) and alcohols (1.7 g/L of ethanol, 1.1 g/L of butanol, and 0.6 g/L of hexanol) at pH 4.8. In conclusion, low fermentation pH is critical for the production of alcohols, although detrimental to C. kluyveri. Fine control of fermentation

Phenotypic selection of a wild Saccharomyces cerevisiae strain for simultaneous saccharification and co-fermentation of AFEX pretreated corn stover

Treesearch

Mingie Jin; Cory Sarks; Christa Gunawan; Benjamin D. Bice; Shane P. Simonett; Ragothaman Avanasi Narasimhan; Laura B. Willis; Bruce E. Dale; Venkatesh Balan; Trey K. Sato

2013-01-01

Simultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Moreover,...

Effect of fermentation parameters on bio-alcohols production from glycerol using immobilized Clostridium pasteurianum: an optimization study.

PubMed

Khanna, Swati; Goyal, Arun; Moholkar, Vijayanand S

2013-01-01

This article addresses the issue of effect of fermentation parameters for conversion of glycerol (in both pure and crude form) into three value-added products, namely, ethanol, butanol, and 1,3-propanediol (1,3-PDO), by immobilized Clostridium pasteurianum and thereby addresses the statistical optimization of this process. The analysis of effect of different process parameters such as agitation rate, fermentation temperature, medium pH, and initial glycerol concentration indicated that medium pH was the most critical factor for total alcohols production in case of pure glycerol as fermentation substrate. On the other hand, initial glycerol concentration was the most significant factor for fermentation with crude glycerol. An interesting observation was that the optimized set of fermentation parameters was found to be independent of the type of glycerol (either pure or crude) used. At optimum conditions of agitation rate (200 rpm), initial glycerol concentration (25 g/L), fermentation temperature (30°C), and medium pH (7.0), the total alcohols production was almost equal in anaerobic shake flasks and 2-L bioreactor. This essentially means that at optimum process parameters, the scale of operation does not affect the output of the process. The immobilized cells could be reused for multiple cycles for both pure and crude glycerol fermentation.

Genetic improvement of Escherichia coli for ethanol production: Chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

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

Ohta, Kazuyoshi; Beall, D.S.; Mejia, J.P.

1991-04-01

Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose tomore » ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).« less

Fed-batch hydrolysate addition and cell separation by settling in high cell density lignocellulosic ethanol fermentations on AFEX™ corn stover in the Rapid Bioconversion with Integrated recycling Technology process.

PubMed

Sarks, Cory; Jin, Mingjie; Balan, Venkatesh; Dale, Bruce E

2017-09-01

The Rapid Bioconversion with Integrated recycling Technology (RaBIT) process uses enzyme and yeast recycling to improve cellulosic ethanol production economics. The previous versions of the RaBIT process exhibited decreased xylose consumption using cell recycle for a variety of different micro-organisms. Process changes were tested in an attempt to eliminate the xylose consumption decrease. Three different RaBIT process changes were evaluated in this work including (1) shortening the fermentation time, (2) fed-batch hydrolysate addition, and (3) selective cell recycling using a settling method. Shorting the RaBIT fermentation process to 11 h and introducing fed-batch hydrolysate addition eliminated any xylose consumption decrease over ten fermentation cycles; otherwise, decreased xylose consumption was apparent by the third cell recycle event. However, partial removal of yeast cells during recycle was not economical when compared to recycling all yeast cells.

Effects of Inhibitors on the Transcriptional Profiling of Gluconobater oxydans NL71 Genes after Biooxidation of Xylose into Xylonate

PubMed Central

Miao, Yuanyuan; Shen, Yi; Xu, Yong

2017-01-01

D-Xylonic acid belongs to the top 30 biomass-based platform chemicals and represents a promising application of xylose. Until today, Gluconobacter oxydans NL71 is the most efficient microbe capable of fermenting xylose into xylonate. However, its growth is seriously inhibited when concentrated lignocellulosic hydrolysates are used as substrates due to the presence of various degraded compounds formed during biomass pretreatment. Three critical lignocellulosic inhibitors were thereby identified, i.e., formic acid, furfural, and 4-hydroxybenzaldehyde. As microbe fermentation is mostly regulated at the genome level, four groups of cell transcriptomes were obtained for a comparative investigation by RNA sequencing of a control sample with samples treated separately with the above-mentioned inhibitors. The digital gene expression profiles screened 572, 714 genes, and 408 DEGs was obtained by the comparisons among four transcriptomes. A number of genes related to the different functional groups showed characteristic expression patterns induced by three inhibitors, in which 19 genes were further tested and confirmed by qRT-PCR. We extrapolated many differentially expressed genes that could explain the cellular responses to the inhibitory effects. We provide results that enable the scientific community to better define the molecular processes involved in the microbes' responses to lignocellulosic inhibitors during the cellular biooxidation of xylose into xylonic acid. PMID:28487685

Impact of assimilable nitrogen availability in glucose uptake kinetics in Saccharomyces cerevisiae during alcoholic fermentation

PubMed Central

2012-01-01

Background The expression and activity of the different Saccharomyces cerevisiae hexose uptake systems (Hxt) and the kinetics of glucose uptake are considered essential to industrial alcoholic fermentation performance. However, the dynamics of glucose uptake kinetics during the different stages of fermentation, depending on glucose and nitrogen availability, is very poorly characterized. The objective of the present work was to examine thoroughly the alterations occurring in glucose uptake kinetics during alcoholic fermentation, by the wine strain S. cerevisiae PYCC 4072, of a synthetic grape juice basal medium with either a limiting or non-limiting initial nitrogen concentration and following nitrogen supplementation of the nitrogen-depleted sluggish fermentation. Results Independently of the initial concentration of the nitrogen source, glucose transport capacity is maximal during the early stages of fermentation and presumably sustained by the low-affinity and high-capacity glucose transporter Hxt1p. During nitrogen-limited sluggish fermentation, glucose uptake capacity was reduced to approximately 20% of its initial values (Vmax = 4.9 ± 0.8 compared to 21.9 ± 1.2 μmol h-1 10-8 cells), being presumably sustained by the low-affinity glucose transporter Hxt3p (considering the calculated Km = 39.2 ± 8.6 mM). The supplementation of the sluggish fermentation broth with ammonium led to the increase of glucose transport capacity associated to the expression of different glucose uptake systems with low and high affinities for glucose (Km = 58.2 ± 9.1 and 2.7 ± 0.4 mM). A biclustering analysis carried out using microarray data, previously obtained for this yeast strain transcriptional response to equivalent fermentation conditions, indicates that the activation of the expression of genes encoding the glucose transporters Hxt2p (during the transition period to active fermentation) and Hxt3p, Hxt4p, Hxt6p and Hxt7p (during the

Influence of the presence of Zymomonas anaerobia on the conversion of cellobiose, glucose, and xylose to ethanol by Clostridium saccharolyticum

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

Asther, M.; Khan, A.W.

1984-01-01

To convert sugar mixtures containing cellobiose, glucose, and xylose to ethanol in a single step, the possibility of using a coculture consisting of Clostridium saccharolyticum and Zymomonas anaerobia was studied. In monoculture, C. saccharolyticum utilized all three sugars; however, it preferentially utilized glucose and produced acetic acid in addition to ethanol. The formation of acetic acid from the metabolism of glucose inhibited the growth of C. saccharolyticum and, consequently, the utilization of cellobiose and xylose. In monoculture, Z. anaerobia utilized glucose at a rate of 50 g/L day, but it did not ferment cellobiose or xylose. In coculture, Z. anaerobiamore » converted most of the glucose to ethanol during the lag phase of growth of C. saccharolyticum, which then converted cellobiose and xylose to ethanol. The use of this coculture increased both the rate and the efficiency of the conversion of these three sugars to ethanol, and produced relatively small amounts of acetic acid.« less

Comparing the fermentation performance of Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST) and Zymomonas mobilis AX101 for cellulosic ethanol production

PubMed Central

2010-01-01

Background Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover. Results The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation. All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested. Conclusions Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield. PMID:20507563

Xylose utilization in recombinant zymomonas

DOEpatents

Caimi, Perry G; McCole, Laura; Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V

2014-03-25

Xylose-utilizing Zymomonas strains studied were found to accumulate ribulose when grown in xylose-containing media. Engineering these strains to increase ribose-5-phosphate isomerase activity led to reduced ribulose accumulation, improved growth, improved xylose utilization, and increased ethanol production.

Structural features of soluble cereal arabinoxylan fibers associated with a slow rate of in vitro fermentation by human fecal microbiota.

PubMed

Rumpagaporn, Pinthip; Reuhs, Brad L; Kaur, Amandeep; Patterson, John A; Keshavarzian, Ali; Hamaker, Bruce R

2015-10-05

Most soluble dietary fibers ferment rapidly in the proximal colon, potentially causing discomfort and poor tolerability. Alkali-extracted arabinoxylan isolates from corn, wheat, rice and sorghum brans were prepared, through hydrolysis (except sorghum) and ethanol fractionation, to have a broad range of initial fermentation rates, and their linkage patterns were determined to understand structural aspects related to slow fermentation rate. They were all highly branched polymers with degree of substitution greater than 64%. There was no relationship of molecular mass, arabinose:xylose ratio, or degree of substitution to fermentation rate patterns. Slow fermenting wheat and corn arabinoxylans had much higher amount of terminal xylose in branches than fast fermenting rice and sorghum arabinoxylans. The slowest fermenting wheat arabinoxylan additionally contained a complex trisaccharide side chain with two arabinoses linked at the O-2 and O-3 positions of an arabinose that is O-2 linked to the xylan backbone. Structural features were proposed for tolerable slowly fermentable arabinoxylan with possible beneficial fermentation function into the distal colon. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gene expression cross-profiling in genetically modified industrial Saccharomyces cerevisiae strains during high-temperature ethanol production from xylose.

PubMed

Ismail, Ku Syahidah Ku; Sakamoto, Takatoshi; Hatanaka, Haruyo; Hasunuma, Tomohisa; Kondo, Akihiko

2013-01-10

Production of ethanol from xylose at high temperature would be an economical approach since it reduces risk of contamination and allows both the saccharification and fermentation steps in SSF to be running at elevated temperature. Eight recombinant xylose-utilizing Saccharomyces cerevisiae strains developed from industrial strains were constructed and subjected to high-temperature fermentation at 38 °C. The best performing strain was sun049T, which produced up to 15.2 g/L ethanol (63% of the theoretical production), followed by sun048T and sun588T, both with 14.1 g/L ethanol produced. Via transcriptomic analysis, expression profiling of the top three best ethanol producing strains compared to a negative control strain, sun473T, led to the discovery of genes in common that were regulated in the same direction. Identification of the 20 most highly up-regulated and the 20 most highly down-regulated genes indicated that the cells regulate their central metabolism and maintain the integrity of the cell walls in response to high temperature. We also speculate that cross-protection in the cells occurs, allowing them to maintain ethanol production at higher concentration under heat stress than the negative controls. This report provides further transcriptomics information in the interest of producing a robust microorganism for high-temperature ethanol production utilizing xylose. Copyright © 2012 Elsevier B.V. All rights reserved.

Simultaneous saccharification and co-fermentation of paper sludge to ethanol by Saccharomyces cerevisiae RWB222--Part I: kinetic modeling and parameters.

PubMed

Zhang, Jiayi; Shao, Xiongjun; Townsend, Oliver V; Lynd, Lee R

2009-12-01

A kinetic model was developed to predict batch simultaneous saccharification and co-fermentation (SSCF) of paper sludge by the xylose-utilizing yeast Saccharomyces cerevisiae RWB222 and the commercial cellulase preparation Spezyme CP. The model accounts for cellulose and xylan enzymatic hydrolysis and competitive uptake of glucose and xylose. Experimental results show that glucan and xylan enzymatic hydrolysis are highly correlated, and that the low concentrations of xylose encountered during SSCF do not have a significant inhibitory effect on enzymatic hydrolysis. Ethanol is found to not only inhibit the specific growth rate, but also to accelerate cell death. Glucose and xylose uptake rates were found to be competitively inhibitory, but this did not have a large impact during SSCF because the sugar concentrations are low. The model was used to evaluate which constants had the greatest impact on ethanol titer for a fixed substrate loading, enzyme loading, and fermentation time. The cellulose adsorption capacity and cellulose hydrolysis rate constants were found to have the greatest impact among enzymatic hydrolysis related constants, and ethanol yield and maximum ethanol tolerance had the greatest impact among fermentation related constants.

Characteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation.

PubMed

Reis, Vanda Renata; Bassi, Ana Paula Guarnieri; da Silva, Jessica Carolina Gomes; Ceccato-Antonini, Sandra Regina

2013-12-01

Among the native yeasts found in alcoholic fermentation, rough colonies associated with pseudohyphal morphology belonging to the species Saccharomyces cerevisiae are very common and undesirable during the process. The aim of this work was to perform morphological and physiological characterisations of S. cerevisiae strains that exhibited rough and smooth colonies in an attempt to identify alternatives that could contribute to the management of rough colony yeasts in alcoholic fermentation. Characterisation tests for invasiveness in Agar medium, killer activity, flocculation and fermentative capacity were performed on 22 strains (11 rough and 11 smooth colonies). The effects of acid treatment at different pH values on the growth of two strains ("52"--rough and "PE-02"--smooth) as well as batch fermentation tests with cell recycling and acid treatment of the cells were also evaluated. Invasiveness in YPD Agar medium occurred at low frequency; ten of eleven rough yeasts exhibited flocculation; none of the strains showed killer activity; and the rough strains presented lower and slower fermentative capacities compared to the smooth strains in a 48-h cycle in a batch system with sugar cane juice. The growth of the rough strain was severely affected by the acid treatment at pH values of 1.0 and 1.5; however, the growth of the smooth strain was not affected. The fermentative efficiency in mixed fermentation (smooth and rough strains in the same cell mass proportion) did not differ from the efficiency obtained with the smooth strain alone, most likely because the acid treatment was conducted at pH 1.5 in a batch cell-recycle test. A fermentative efficiency as low as 60% was observed with the rough colony alone.

Comparison of volatile and non-volatile metabolites in rice wine fermented by Koji inoculated with Saccharomycopsis fibuligera and Aspergillus oryzae.

PubMed

Son, Eun Yeong; Lee, Sang Mi; Kim, Minjoo; Seo, Jeong-Ah; Kim, Young-Suk

2018-07-01

This study investigated volatile and nonvolatile metabolite profiles of makgeolli (a traditional rice wine in Korea) fermented by koji inoculated with Saccharomycopsis fibuligera and/or Aspergillus oryzae. The enzyme activities in koji were also examined to determine their effects on the formation of metabolites. The contents of all 18 amino acids detected were the highest in makgeolli fermented by S. fibuligera CN2601-09, and increased after combining with A. oryzae CN1102-08, unlike the contents of most fatty acids. On the other hand, major volatile metabolites were fusel alcohols, acetate esters, and ethyl esters. The contents of most fusel alcohols and acetate esters were the highest in makgeolli fermented by S. fibuligera CN2601-09, for which the protease activity was the highest, leading to the largest amounts of amino acods. The makgeolli samples fermented only by koji inoculated with S. fibuligera could be discriminated on PCA plots from the makgeolli samples fermented in combination with A. oryzae. In the case of nonvolatile metabolites, all amino acids and some metabolites such as xylose, 2-methylbenzoic acid, and oxalic acid contributed mainly to the characteristics of makgeolli fermented by koji inoculated with S. fibuligera and A. oryzae. These results showed that the formations of volatile and nonvolatile metabolites in makgeolli can be significantly affected by microbial strains with different enzyme activities in koji. To our knowledge, this study is the first report on the effects of S. fibuligera strains on the formation of volatile and non-volatile metabolites in rice wine, facilitating their use in brewing rice wine. Copyright © 2018. Published by Elsevier Ltd.

Mitochondrial-morphology-targeted breeding of industrial yeast strains for alcohol fermentation.

PubMed

Kitagaki, Hiroshi

2009-05-29

Since mitochondrial genes are repressed under high glucose and low O2, and these conditions correspond to the conditions in which yeast cells are exposed during alcohol fermentation, the existence and structure of yeast mitochondria during alcohol fermentation have not been elucidated. Yeast mitochondria can be observed throughout brewing of sake (Japanese rice wine) and fragment during brewing. Furthermore, it has been revealed that Fis1 [fission 1 (mitochondrial outer membrane) homologue (Saccharomyces cerevisiae)], which is a transmembrane protein with its C-terminal anchor embedded in the outer membrane of mitochondria, is required for fragmentation of yeast mitochondria during sake brewing. By utilizing this knowledge, a fis1 disruptant of a sake yeast strain has been generated that has a networked mitochondrial structure throughout sake brewing. It transpired that this strain produces a high content of malate, which imparts a crisp acidic taste, during sake brewing. This strategy is a useful and a completely novel strategy towards developing a new yeast strain which produces a high content of malate in sake, and mitochondrial morphology has now emerged as a promising target for the breeding of practical industrial strains.

Volatile organic compounds profile during milk fermentation by Lactobacillus pentosus and correlations between volatiles flavor and carbohydrate metabolism.

PubMed

Pan, D D; Wu, Z; Peng, T; Zeng, X Q; Li, H

2014-02-01

Flavor, as one of the most important properties determining the acceptability and preference of fermented milks, is influenced by compositional and processing factors. In this study, we focused on the volatile organic compounds related to flavor during milk fermentation by Lactobacillus pentosus according to electronic nose analysis. Xylose (1% addition) metabolized by Lb. pentosus strongly affects the flavor of yogurt, with the potent volatile organic compounds of ethanol (3.08%), 2,3-butanedione (7.77%), and acetic acid (22.70%) detected using solid-phase microextraction coupled with gas chromatography-mass spectrometry analysis. Sensoryanalysis also showed skimmed yogurt fermented by Lb. pentosus with 1% xylose had the unique scores of sourness (acetic acid) and butter flavor (2,3-butanedione). Furthermore, α-acetolactate synthase and α-acetolactate decarboxylase in carbohydrate metabolism play important roles in milk fermentation. Under preferable conditions (pH 5.5, 42 °C) for α-acetolactate synthase and α-acetolactate decarboxylase, the relative content of potent flavor compound 2,3-butanedione was 10.13%, which was 2.55% higher than common culture condition (pH 4.5, 37 °C), revealing that xylose metabolized by Lb. pentosus has potential values for the milk product industry, such as the acceptability and preference of fermented milk product. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Aptitude of Saccharomyces yeasts to ferment unripe grapes harvested during cluster thinning for reducing alcohol content of wine.

PubMed

Bovo, Barbara; Nadai, Chiara; Vendramini, Chiara; Fernandes Lemos Junior, Wilson Josè; Carlot, Milena; Skelin, Andrea; Giacomini, Alessio; Corich, Viviana

2016-11-07

Among the viticultural techniques developed to obtain wine with reduced alcohol content, the use of unripe grapes with low sugar and high malic acid concentration, harvested at cluster thinning, was recently explored. So far, no studies have evaluated the fermentation performances of Saccharomyces in unripe grape musts, in terms of fermentation ability and reducing malic acid contents, to improve the quality of this low-alcohol beverage. In this work, we evaluated 24 S. cerevisiae strains isolated from Italian and Croatian vineyards with different fermentation aptitudes. Moreover, four S. paradoxus were considered, as previous works demonstrated that strains belonging to this species were able to degrade high malic acid amounts in standard musts. The industrial strain S. cerevisiae 71B was added as reference. Sugar and malic acid contents were modified in synthetic musts in order to understand the effect of their concentrations on alcoholic fermentation and malic acid degradation. S. cerevisiae fermentation performances improved when glucose concentration decreased and malic acid level increased. The conditions that simulate unripe grape must, i.e. low glucose and high malic acid content were found to enhance S. cerevisiae ability to degrade malic acid. On the contrary, S. paradoxus strains were able to degrade high amounts of malic acid only in conditions that resemble ripe grape must, i.e. high glucose and low malic acid concentration. In fermentation trials when low glucose concentrations were used, at high malic acid levels S. cerevisiae strains produced higher glycerol than at low malic acid condition. Malic acid degradation ability, tested on the best performing S. cerevisiae strains, was enhanced in fermentation trials when unripe grape must was used. Copyright © 2016 Elsevier B.V. All rights reserved.

The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations.

PubMed

Bohlscheid, J C; Fellman, J K; Wang, X D; Ansen, D; Edwards, C G

2007-02-01

To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l(-1)) and biotin (0, 1 or 10 microg l(-1)) as variables. All media contained 240 g l(-1) glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 microg l(-1) had no effect. Nitrogen x biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H(2)S). Maximum H(2)S production occurred in the medium containing 60 mg l(-1) YAN and 1 microg l(-1) biotin. Nitrogen x biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.

Sterilization of fermentation vessels by ethanol/water mixtures

DOEpatents

Wyman, Charles E.

1999-02-09

A method for sterilizing process fermentation vessels with a concentrated alcohol and water mixture integrated in a fuel alcohol or other alcohol production facility. Hot, concentrated alcohol is drawn from a distillation or other purification stage and sprayed into the empty fermentation vessels. This sterilizing alcohol/water mixture should be of a sufficient concentration, preferably higher than 12% alcohol by volume, to be toxic to undesirable microorganisms. Following sterilization, this sterilizing alcohol/water mixture can be recovered back into the same distillation or other purification stage from which it was withdrawn. The process of this invention has its best application in, but is not limited to, batch fermentation processes, wherein the fermentation vessels must be emptied, cleaned, and sterilized following completion of each batch fermentation process.

Lactic acid production from biomass-derived sugars via co-fermentation of Lactobacillus brevis and Lactobacillus plantarum.

PubMed

Zhang, Yixing; Vadlani, Praveen V

2015-06-01

Lignocellulosic biomass is an attractive alternative resource for producing chemicals and fuels. Xylose is the dominating sugar after hydrolysis of hemicellulose in the biomass, but most microorganisms either cannot ferment xylose or have a hierarchical sugar utilization pattern in which glucose is consumed first. To overcome this barrier, Lactobacillus brevis ATCC 367 was selected to produce lactic acid. This strain possesses a relaxed carbon catabolite repression mechanism that can use glucose and xylose simultaneously; however, lactic acid yield was only 0.52 g g(-1) from a mixture of glucose and xylose, and 5.1 g L(-1) of acetic acid and 8.3 g L(-1) of ethanol were also formed during production of lactic acid. The yield was significantly increased and ethanol production was significantly reduced if L. brevis was co-cultivated with Lactobacillus plantarum ATCC 21028. L. plantarum outcompeted L. brevis in glucose consumption, meaning that L. brevis was focused on converting xylose to lactic acid and the by-product, ethanol, was reduced due to less NADH generated in the fermentation system. Sequential co-fermentation of L. brevis and L. plantarum increased lactic acid yield to 0.80 g g(-1) from poplar hydrolyzate and increased yield to 0.78 g lactic acid per g of biomass from alkali-treated corn stover with minimum by-product formation. Efficient utilization of both cellulose and hemicellulose components of the biomass will improve overall lactic acid production and enable an economical process to produce biodegradable plastics. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Zymomonas with improved xylose utilization

DOEpatents

Viitanen, Paul V [West Chester, PA; Tao, Luan [Havertown, PA; Zhang, Yuying [New Hope, PA; Caimi, Perry G [Kennett Square, PA; McCutchen, Carol M [Wilmington, DE; McCole, Laura [East Fallowfield, PA; Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Lakewood, CO; Franden, Mary Ann [Centennial, CO

2011-08-16

Strains of Zymomonas were engineered by introducing a chimeric xylose isomerase gene that contains a mutant promoter of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene. The promoter directs increased expression of xylose isomerase, and when the strain is in addition engineered for expression of xylulokinase, transaldolase and transketolase, improved utilization of xylose is obtained.

Dynamic flux balance modeling of microbial co-cultures for efficient batch fermentation of glucose and xylose mixtures.

PubMed

Hanly, Timothy J; Henson, Michael A

2011-02-01

Sequential uptake of pentose and hexose sugars that compose lignocellulosic biomass limits the ability of pure microbial cultures to efficiently produce value-added bioproducts. In this work, we used dynamic flux balance modeling to examine the capability of mixed cultures of substrate-selective microbes to improve the utilization of glucose/xylose mixtures and to convert these mixed substrates into products. Co-culture simulations of Escherichia coli strains ALS1008 and ZSC113, engineered for glucose and xylose only uptake respectively, indicated that improvements in batch substrate consumption observed in previous experimental studies resulted primarily from an increase in ZSC113 xylose uptake relative to wild-type E. coli. The E. coli strain ZSC113 engineered for the elimination of glucose uptake was computationally co-cultured with wild-type Saccharomyces cerevisiae, which can only metabolize glucose, to determine if the co-culture was capable of enhanced ethanol production compared to pure cultures of wild-type E. coli and the S. cerevisiae strain RWB218 engineered for combined glucose and xylose uptake. Under the simplifying assumption that both microbes grow optimally under common environmental conditions, optimization of the strain inoculum and the aerobic to anaerobic switching time produced an almost twofold increase in ethanol productivity over the pure cultures. To examine the effect of reduced strain growth rates at non-optimal pH and temperature values, a break even analysis was performed to determine possible reductions in individual strain substrate uptake rates that resulted in the same predicted ethanol productivity as the best pure culture. © 2010 Wiley Periodicals, Inc.

Characteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation

PubMed Central

Reis, Vanda Renata; Bassi, Ana Paula Guarnieri; da Silva, Jessica Carolina Gomes; Ceccato-Antonini, Sandra Regina

2013-01-01

Among the native yeasts found in alcoholic fermentation, rough colonies associated with pseudohyphal morphology belonging to the species Saccharomyces cerevisiae are very common and undesirable during the process. The aim of this work was to perform morphological and physiological characterisations of S. cerevisiae strains that exhibited rough and smooth colonies in an attempt to identify alternatives that could contribute to the management of rough colony yeasts in alcoholic fermentation. Characterisation tests for invasiveness in Agar medium, killer activity, flocculation and fermentative capacity were performed on 22 strains (11 rough and 11 smooth colonies). The effects of acid treatment at different pH values on the growth of two strains (“52” - rough and “PE-02” - smooth) as well as batch fermentation tests with cell recycling and acid treatment of the cells were also evaluated. Invasiveness in YPD Agar medium occurred at low frequency; ten of eleven rough yeasts exhibited flocculation; none of the strains showed killer activity; and the rough strains presented lower and slower fermentative capacities compared to the smooth strains in a 48-h cycle in a batch system with sugar cane juice. The growth of the rough strain was severely affected by the acid treatment at pH values of 1.0 and 1.5; however, the growth of the smooth strain was not affected. The fermentative efficiency in mixed fermentation (smooth and rough strains in the same cell mass proportion) did not differ from the efficiency obtained with the smooth strain alone, most likely because the acid treatment was conducted at pH 1.5 in a batch cell-recycle test. A fermentative efficiency as low as 60% was observed with the rough colony alone. PMID:24688501

Sterilization of fermentation vessels by ethanol/water mixtures

DOEpatents

Wyman, C.E.

1999-02-09

A method is described for sterilizing process fermentation vessels with a concentrated alcohol and water mixture integrated in a fuel alcohol or other alcohol production facility. Hot, concentrated alcohol is drawn from a distillation or other purification stage and sprayed into the empty fermentation vessels. This sterilizing alcohol/water mixture should be of a sufficient concentration, preferably higher than 12% alcohol by volume, to be toxic to undesirable microorganisms. Following sterilization, this sterilizing alcohol/water mixture can be recovered back into the same distillation or other purification stage from which it was withdrawn. The process of this invention has its best application in, but is not limited to, batch fermentation processes, wherein the fermentation vessels must be emptied, cleaned, and sterilized following completion of each batch fermentation process. 2 figs.

Yeasts Diversity in Fermented Foods and Beverages

NASA Astrophysics Data System (ADS)

Tamang, Jyoti Prakash; Fleet, Graham H.

People across the world have learnt to culture and use the essential microorganisms for production of fermented foods and alcoholic beverages. A fermented food is produced either spontaneously or by adding mixed/pure starter culture(s). Yeasts are among the essential functional microorganisms encountered in many fermented foods, and are commercially used in production of baker's yeast, breads, wine, beer, cheese, etc. In Asia, moulds are predominant followed by amylolytic and alcohol-producing yeasts in the fermentation processes, whereas in Africa, Europe, Australia and America, fermented products are prepared exclusively using bacteria or bacteria-yeasts mixed cultures. This chapter would focus on the varieties of fermented foods and alcoholic beverages produced by yeasts, their microbiology and role in food fermentation, widely used commercial starters (pilot production, molecular aspects), production technology of some common commercial fermented foods and alcoholic beverages, toxicity and food safety using yeasts cultures and socio-economy

Burkholderia sacchari DSM 17165: A source of compositionally-tunable block-copolymeric short-chain poly(hydroxyalkanoates) from xylose and levulinic acid

USDA-ARS?s Scientific Manuscript database

Burkholderia sacchari DSM 17165 was used as a biocatalyst for the production of poly-3-hydroxybutyrate-co-3-hydroxyvalerate block copolymers (Poly-3HB-block-3HV) from xylose and levulinic acid. Among the carbon source mixtures, levulinic acid was preferred and was consumed early in the fermentations...

Optimization of CDT-1 and XYL1 Expression for Balanced Co-Production of Ethanol and Xylitol from Cellobiose and Xylose by Engineered Saccharomyces cerevisiae

PubMed Central

Zha, Jian; Li, Bing-Zhi; Shen, Ming-Hua; Hu, Meng-Long; Song, Hao; Yuan, Ying-Jin

2013-01-01

Production of ethanol and xylitol from lignocellulosic hydrolysates is an alternative to the traditional production of ethanol in utilizing biomass. However, the conversion efficiency of xylose to xylitol is restricted by glucose repression, causing a low xylitol titer. To this end, we cloned genes CDT-1 (encoding a cellodextrin transporter) and gh1-1 (encoding an intracellular β-glucosidase) from Neurospora crassa and XYL1 (encoding a xylose reductase that converts xylose into xylitol) from Scheffersomyces stipitis into Saccharomyces cerevisiae, enabling simultaneous production of ethanol and xylitol from a mixture of cellobiose and xylose (main components of lignocellulosic hydrolysates). We further optimized the expression levels of CDT-1 and XYL1 by manipulating their promoters and copy-numbers, and constructed an engineered S. cerevisiae strain (carrying one copy of PGK1p-CDT1 and two copies of TDH3p-XYL1), which showed an 85.7% increase in xylitol production from the mixture of cellobiose and xylose than that from the mixture of glucose and xylose. Thus, we achieved a balanced co-fermentation of cellobiose (0.165 g/L/h) and xylose (0.162 g/L/h) at similar rates to co-produce ethanol (0.36 g/g) and xylitol (1.00 g/g). PMID:23844185

Influence of nitrogen sources on growth and fermentation performance of different wine yeast species during alcoholic fermentation.

PubMed

Kemsawasd, Varongsiri; Viana, Tiago; Ardö, Ylva; Arneborg, Nils

2015-12-01

In this study, the influence of twenty different single (i.e. 19 amino acids and ammonium sulphate) and two multiple nitrogen sources (N-sources) on growth and fermentation (i.e. glucose consumption and ethanol production) performance of Saccharomyces cerevisiae and of four wine-related non-Saccharomyces yeast species (Lachancea thermotolerans, Metschnikowia pulcherrima, Hanseniaspora uvarum and Torulaspora delbrueckii) was investigated during alcoholic fermentation. Briefly, the N-sources with beneficial effects on all performance parameters (or for the majority of them) for each yeast species were alanine, arginine, asparagine, aspartic acid, glutamine, isoleucine, ammonium sulphate, serine, valine and mixtures of 19 amino acids and of 19 amino acids plus ammonium sulphate (for S. cerevisiae), serine (for L. thermotolerans), alanine (for H. uvarum), alanine and asparagine (for M. pulcherrima), arginine, asparagine, glutamine, isoleucine and mixture of 19 amino acids (for T. delbrueckii). Furthermore, our results showed a clear positive effect of complex mixtures of N-sources on S. cerevisiae and on T. delbrueckii (although to a lesser extent) as to all performance parameters studied, whereas for L. thermotolerans, H. uvarum and M. pulcherrima, single amino acids affected growth and fermentation performance to the same extent as the mixtures. Moreover, we found groups of N-sources with similar effects on the growth and/or fermentation performance of two or more yeast species. Finally, the influences of N-sources observed for T. delbrueckii and H. uvarum resembled those of S. cerevisiae the most and the least, respectively. Overall, this work contributes to an improved understanding of how different N-sources affect growth, glucose consumption and ethanol production of wine-related yeast species under oxygen-limited conditions, which, in turn, may be used to, e.g. optimize growth and fermentation performance of the given yeast upon N-source supplementation during

Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae

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

Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather

Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains withmore » improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. Lastly, the mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min -1•mg -1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation.« less

Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae

DOE PAGES

Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; ...

2016-01-19

Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains withmore » improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. Lastly, the mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min -1•mg -1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation.« less

The Effect of Proanthocyanidins on Growth and Alcoholic Fermentation of Wine Yeast under Copper Stress.

PubMed

Jia, Bo; Liu, Xingyan; Zhan, Jicheng; Li, Jingyuan; Huang, Weidong

2015-06-01

Proanthocyanidins (PAs) derived from the grape skin, as well as from grape seeds, grape stems, are an important group of polyphenols in wine. The aim of this study was to understand the effect of PAs (0.1, 1.0 g/L) on growth and alcoholic fermentation of 2 strains of Saccharomyces cerevisiae (commercial strain FREDDO and newly selected strain BH8) during copper-stress fermentation, using a simple model fermentation system. Our results showed that both PAs and Cu(2+) could pose significant inhibition effects on the growth of yeast cells, CO2 release, sugar consumption, and ethanol production during the initial phase of the fermentation. Compared to PAs, Cu(2+) performed more obvious inhibition on the yeast growth and fermentation. However, adding 1.0 g/L PAs increased in the vitality and metabolism activity of yeast cells at the mid-exponential phase of fermentation in the mediums with no copper and 0.1 mM Cu(2+) added, shortened the period of wine fermentation, and decreased the copper residues. It indicated that PAs could improve the ability of wine yeast to resist detrimental effects under copper-stress fermentation condition, maintaining cells metabolic activity, and fermentation could be controlled by manipulating PAs supplementation. © 2015 Institute of Food Technologists®

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

Zymomonas pentose-sugar fermenting strains and uses thereof

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Golden, CO; Howe, William [Golden, CO; Eddy, Christine [Golden, CO; Evans, Kent [Littleton, CO; Mohagheghi, Ali [Northglenn, CO

2007-05-29

Disclosed in the present invention is a Zymomonas integrant and derivatives of these integrants that posses the ability to ferment pentose into ethanol. The genetic sequences encoding for the pentose-fermenting enzymes are integrated into the Zymomonas in a two-integration event of homologous recombination and transposition. Each operon includes more than one pentose-reducing enzyme encoding sequence. The integrant in some embodiments includes enzyme sequences encoding xylose isomerase, xylulokinase, transketolase and transketolase. The Zymomonas integrants are highly stable, and retain activity for producing the pentose-fermenting enzyme for between 80 to 160 generations. The integrants are also resistant to acetate inhibition, as the integrants demonstrate efficient ethanol production even in the presence of 8 up to 16 grams acetate per liter media. These stably integrated sequences provide a unique Zymomonas that may then be used for the efficient conversion of pentose sugars (xylose, arabinose) to ethanol. Method of using the Zymomonas integrants and derivatives thereof in production of ethanol from cellulosic feedstock is also disclosed. The invention also provides a method for preparing a Zymomonas integrant as part of the present invention. The host Zymomonas strain found particularly useful in the creation of these compositions and methods is Zymomonas mobilis 31821.

Metal Dependence of the Xylose Isomerase from Piromyces sp. E2 Explored by Activity Profiling and Protein Crystallography

PubMed Central

2017-01-01

Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity. PMID:29045784

Continuous Ethanol Fermentation of Pretreated Lignocellulosic Biomasses, Waste Biomasses, Molasses and Syrup Using the Anaerobic, Thermophilic Bacterium Thermoanaerobacter italicus Pentocrobe 411

PubMed Central

Andersen, Rasmus Lund; Jensen, Karen Møller; Mikkelsen, Marie Just

2015-01-01

Lignocellosic ethanol production is now at a stage where commercial or semi-commercial plants are coming online and, provided cost effective production can be achieved, lignocellulosic ethanol will become an important part of the world bio economy. However, challenges are still to be overcome throughout the process and particularly for the fermentation of the complex sugar mixtures resulting from the hydrolysis of hemicellulose. Here we describe the continuous fermentation of glucose, xylose and arabinose from non-detoxified pretreated wheat straw, birch, corn cob, sugar cane bagasse, cardboard, mixed bio waste, oil palm empty fruit bunch and frond, sugar cane syrup and sugar cane molasses using the anaerobic, thermophilic bacterium Thermoanaerobacter Pentocrobe 411. All fermentations resulted in close to maximum theoretical ethanol yields of 0.47–0.49 g/g (based on glucose, xylose, and arabinose), volumetric ethanol productivities of 1.2–2.7 g/L/h and a total sugar conversion of 90–99% including glucose, xylose and arabinose. The results solidify the potential of Thermoanaerobacter strains as candidates for lignocellulose bioconversion. PMID:26295944

JPRS Report, Science & Technology, Europe

DTIC Science & Technology

1992-03-27

bio-engineering company, among other things, cultivates starter cultures for the production of cheese and yogurt . The customers are dairy farmers in...produces the alcohol with the help of traditional fermentation methods. This means they use ordinary baker’s yeast which fer- ments part of the sugar in...the waste liquid into ethanol. The baker’s yeast is unable to ferment the remaining sugars in the liquid, such as xylose, for example, into alcohol

Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass.

PubMed

Kim, Jae-Han; Block, David E; Mills, David A

2010-11-01

Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy.

Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass

PubMed Central

Kim, Jae-Han; Block, David E.

2010-01-01

Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy. PMID:20838789

Fermentation of sugars and sugar alcohols by plaque Lactobacillus strains.

PubMed

Almståhl, Annica; Lingström, Peter; Eliasson, Lars; Carlén, Anette

2013-07-01

The objective was to analyse the ability of Lactobacillus strains isolated from supragingival plaque of subjects with hyposalivation and from healthy controls to ferment sugars and sugar alcohols. Fifty strains isolated from interproximal plaque from subjects with radiation-induced hyposalivation (25 strains), subjects with primary Sjögren's syndrome (16 strains) and from subjects with normal salivary secretion rate (9 strains) were tested. Growth and pH were determined after 24 and 48 h of anaerobic incubation in vials containing basal media with 1 % of glucose, fructose, sucrose, mannitol, sorbitol or xylitol. No differences between strains isolated from hyposalivated subjects and controls were detected. All strains lowered the pH to <5.0 from fructose and the majority of the strains from glucose and sucrose. A pH of <5.5 was seen for 52 % of the strains using mannitol, 50 % using sorbitol and 36 % using xylitol. The ability to produce acids from sugars and sugar alcohols was highest among strains of Lactobacillus rhamnosus, Lactobacillus casei and Lactobacillus paracasei and lowest among Lactobacillus fermentum strains. A large number of Lactobacillus strains are able to ferment not only sugars but also the sugar substitutes mannitol, sorbitol and xylitol to pH levels critical for enamel demineralisation. Our findings suggest that products containing mannitol, sorbitol and/or xylitol may contribute to the acidogenic potential of the dental plaque and especially in hyposalivated subjects with high numbers of lactobacilli.

Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

DOE PAGES

Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; ...

2016-08-24

Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening,more » more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less

Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

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

Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee

Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening,more » more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

PubMed

Ribeiro, Lucas Ferreira; Tullman, Jennifer; Nicholes, Nathan; Silva, Sérgio Ruschi Bergamachi; Vieira, Davi Serradella; Ostermeier, Marc; Ward, Richard John

2016-01-01

Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose. The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA-XBP271. The XynA-XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein-protein interface with coupled movements between the XynA and XBP domains. In the XynA-XBP271 with xylose bound to the XBP domain, the mobility of a β-loop in the XynA domain results in an increased access to the

Improved fermentative production of gamma-aminobutyric acid via the putrescine route: Systems metabolic engineering for production from glucose, amino sugars, and xylose.

PubMed

Jorge, João M P; Nguyen, Anh Q D; Pérez-García, Fernando; Kind, Stefanie; Wendisch, Volker F

2017-04-01

Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of l-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of l-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory form by changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L -1 in fed-batch cultivation at maximum volumetric productivities up to 1.34 g L -1  h -1 , the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N-acetyl-glucosamine that do not have competing uses in the food or feed industries was established. Biotechnol. Bioeng. 2017;114: 862-873. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Unexpected convergence of fungal and bacterial communities during fermentation of traditional Korean alcoholic beverages inoculated with various natural starters.

PubMed

Jung, Mi-Ja; Nam, Young-Do; Roh, Seong Woon; Bae, Jin-Woo

2012-05-01

Makgeolli is a traditional Korean alcoholic beverage manufactured with a natural starter, called nuruk, and grains. Nuruk is a starchy disk or tablet formed from wheat or grist containing various fungal and bacterial strains from the surrounding environment that are allowed to incorporate naturally into the starter, each of which simultaneously participates in the makgeolli fermentation process. In the current study, changes in microbial dynamics during laboratory-scale fermentation of makgeolli inoculated with six different kinds of nuruk were evaluated by barcoded pyrosequencing using fungal- and bacterial-specific primers targeting the internal transcribed spacer 2 region and hypervariable regions V1 to V3 of the 16S rRNA gene, respectively. A total of 61,571 fungal and 68,513 bacterial sequences were used for the analysis of microbial diversity in ferment samples. During fermentation, the proportion of fungal microorganisms belonging to the family Saccharomycetaceae increased significantly, and the major bacterial phylum of the samples shifted from γ-Proteobacteria to Firmicutes. The results of quantitative PCR indicated that the bacterial content in the final ferments was higher than in commercial rice beers, while total fungi appeared similar. This is the first report of a comparative analysis of bacterial and fungal dynamics in parallel during the fermentation of Korean traditional alcoholic beverage using barcoded pyrosequencing. Copyright © 2011 Elsevier Ltd. All rights reserved.

Microbial inactivation and MLF performances of Tempranillo Rioja wines treated with PEF after alcoholic fermentation.

PubMed

González-Arenzana, Lucía; López-Alfaro, Isabel; Garde-Cerdán, Teresa; Portu, Javier; López, Rosa; Santamaría, Pilar

2018-03-23

This study was performed with the aim of reducing the microbial communities of wines after alcoholic fermentation to improve the establishment of commercial Oenococcus oeni inoculum for developing the malolactic fermentation. Microbial community reduction was accomplished by applying Pulsed Electric Field (PEF) technology to four different wines. Overall, significant reductions in yeast population were observed. To a lesser extent, lactic acid bacteria were reduced while acetic acid bacteria were completely eliminated after the PEF treatment. In three out of the four tested wines, a decrease in the competitive pressure between microorganisms due to the detected reduction led to a general but slight shortening of the malolactic fermentation duration. In the wine with the most adverse conditions to commercial starter establishment, the shortest malolactic fermentation was reached after PEF treatment. Finally, the sensorial quality of three out of the four treated wines was considered better after the PEF treatment. Therefore, PEF technology meant an important tool for improving the malolactic fermentation performance. Copyright © 2018 Elsevier B.V. All rights reserved.

Biobutanol production by Clostridium acetobutylicum using xylose recovered from birch Kraft black liquor.

PubMed

Kudahettige-Nilsson, Rasika L; Helmerius, Jonas; Nilsson, Robert T; Sjöblom, Magnus; Hodge, David B; Rova, Ulrika

2015-01-01

Acetone-butanol-ethanol (ABE) fermentation was studied using acid-hydrolyzed xylan recovered from hardwood Kraft black liquor by CO2 acidification as the only carbon source. Detoxification of hydrolyzate using activated carbon was conducted to evaluate the impact of inhibitor removal and fermentation. Xylose hydrolysis yields as high as 18.4% were demonstrated at the highest severity hydrolysis condition. Detoxification using active carbon was effective for removal of both phenolics (76-81%) and HMF (38-52%). Batch fermentation of the hydrolyzate and semi-defined P2 media resulted in a total solvent yield of 0.12-0.13g/g and 0.34g/g, corresponding to a butanol concentration of 1.8-2.1g/L and 7.3g/L respectively. This work is the first study of a process for the production of a biologically-derived biofuel from hemicelluloses solubilized during Kraft pulping and demonstrates the feasibility of utilizing xylan recovered directly from industrial Kraft pulping liquors as a feedstock for biological production of biofuels such as butanol. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Engineering a synthetic anaerobic respiration for reduction of xylose to xylitol using NADH output of glucose catabolism by Escherichia coli AI21.

PubMed

Iverson, Andrew; Garza, Erin; Manow, Ryan; Wang, Jinhua; Gao, Yuanyuan; Grayburn, Scott; Zhou, Shengde

2016-04-16

Anaerobic rather than aerobic fermentation is preferred for conversion of biomass derived sugars to high value redox-neutral and reduced commodities. This will likely result in a higher yield of substrate to product conversion and decrease production cost since substrate often accounts for a significant portion of the overall cost. To this goal, metabolic pathway engineering has been used to optimize substrate carbon flow to target products. This approach works well for the production of redox neutral products such as lactic acid from redox neutral sugars using the reducing power NADH (nicotinamide adenine dinucleotide, reduced) generated from glycolysis (2 NADH per glucose equivalent). Nevertheless, greater than two NADH per glucose catabolized is needed for the production of reduced products (such as xylitol) from redox neutral sugars by anaerobic fermentation. The Escherichia coli strain AI05 (ΔfrdBC ΔldhA ΔackA Δ(focA-pflB) ΔadhE ΔptsG ΔpdhR::pflBp 6-(aceEF-lpd)), previously engineered for reduction of xylose to xylitol using reducing power (NADH equivalent) of glucose catabolism, was further engineered by 1) deleting xylAB operon (encoding for xylose isomerase and xylulokinase) to prevent xylose from entering the pentose phosphate pathway; 2) anaerobically expressing the sdhCDAB-sucABCD operon (encoding for succinate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase) to enable an anaerobically functional tricarboxcylic acid cycle with a theoretical 10 NAD(P)H equivalent per glucose catabolized. These reducing equivalents can be oxidized by synthetic respiration via xylose reduction, producing xylitol. The resulting strain, AI21 (pAI02), achieved a 96 % xylose to xylitol conversion, with a yield of 6 xylitol per glucose catabolized (molar yield of xylitol per glucose consumed (YRPG) = 6). This represents a 33 % improvement in xylose to xylitol conversion, and a 63 % increase in xylitol yield per glucose catabolized over

27 CFR 24.212 - High fermentation wine.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., DEPARTMENT OF THE TREASURY ALCOHOL WINE Production of Other Than Standard Wine § 24.212 High fermentation wine. High fermentation wine is wine made with the addition of sugar within the limitations prescribed... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false High fermentation wine. 24...

27 CFR 24.212 - High fermentation wine.

Code of Federal Regulations, 2014 CFR

2014-04-01

..., DEPARTMENT OF THE TREASURY ALCOHOL WINE Production of Other Than Standard Wine § 24.212 High fermentation wine. High fermentation wine is wine made with the addition of sugar within the limitations prescribed... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false High fermentation wine. 24...

Alcohol

MedlinePlus

... created when grains, fruits, or vegetables are fermented . Fermentation is a process that uses yeast or bacteria ... change the sugars in the food into alcohol. Fermentation is used to produce many necessary items — everything ...

Chlorine dioxide against bacteria and yeasts from the alcoholic fermentation

PubMed Central

Meneghin, Silvana Perissatto; Reis, Fabricia Cristina; de Almeida, Paulo Garcia; Ceccato-Antonini, Sandra Regina

2008-01-01

The ethanol production in Brazil is carried out by fed-batch or continuous process with cell recycle, in such way that bacterial contaminants are also recycled and may be troublesome due to the substrate competition. Addition of sulphuric acid when inoculum cells are washed can control the bacterial growth or alternatively biocides are used. This work aimed to verify the effect of chlorine dioxide, a well-known biocide for bacterial decontamination of water and equipments, against contaminant bacteria (Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides) from alcoholic fermentation, through the method of minimum inhibitory concentration (MIC), as well as its effect on the industrial yeast inoculum. Lower MIC was found for B. subtilis (10 ppm) and Leuconostoc mesenteroides (50 ppm) than for Lactobacillus fermentum (75 ppm) and Lactobacillus plantarum (125 ppm). Additionally, these concentrations of chlorine dioxide had similar effects on bacteria as 3 ppm of Kamoran® (recommended dosage for fermentation tanks), exception for B. subtilis, which could not be controlled at this Kamoran® dosage. The growth of industrial yeasts was affected when the concentration of chlorine dioxide was higher than 50 ppm, but the effect was slightly dependent on the type of yeast strain. Smooth yeast colonies (dispersed cells) seemed to be more sensitive than wrinkled yeast colonies (clustered cells/pseudohyphal growth), both isolated from an alcohol-producing unit during the 2006/2007 sugar cane harvest. The main advantage in the usage of chlorine dioxide that it can replace antibiotics, avoiding the selection of resistant populations of microorganisms. PMID:24031227

Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2.

PubMed

Sukpipat, Wiphat; Komeda, Hidenobu; Prasertsan, Poonsuk; Asano, Yasuhisa

2017-01-01

Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.60 g/g d-xylose, and 0.61 g/g l-arabinose with 21.7 g/L ethanol, 20.2 g/L xylitol, and 30.3 g/l l-arabitol, respectively. The enzyme with l-arabitol dehydrogenase (LAD) activity was purified from the strain and found to exhibit broad specificity to polyols, such as xylitol, d-sorbitol, ribitol, and l-arabitol. Xylitol was the preferred substrate with K m =16.1 mM and k cat /K m =67.0 min -1 mM -1 , while l-arabitol was also a substrate for the enzyme with K m =31.1 mM and k cat /K m =6.5 min -1  mM -1 . Therefore, this enzyme from M. caribbica was named xylitol dehydrogenase (McXDH). McXDH had an optimum temperature and pH at 40°C and 9.5, respectively. The McXDH gene included a coding sequence of 1086 bp encoding a putative 362 amino acid protein of 39 kDa with an apparent homopentamer structure. Native McXDH and recombinant McXDH exhibited relative activities toward l-arabitol of approximately 20% that toward xylitol, suggesting the applicability of this enzyme with the functions of XDH and LAD to the development of pentose-fermenting Saccharomyces cerevisiae. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Pleiotropic functions of the yeast Greatwall-family protein kinase Rim15p: a novel target for the control of alcoholic fermentation.

PubMed

Watanabe, Daisuke; Takagi, Hiroshi

2017-06-01

Rim15p, a Greatwall-family protein kinase in yeast Saccharomyces cerevisiae, is required for cellular nutrient responses, such as the entry into quiescence and the induction of meiosis and sporulation. In higher eukaryotes, the orthologous gene products are commonly involved in the cell cycle G 2 /M transition. How are these pleiotropic functions generated from a single family of protein kinases? Recent advances in both research fields have identified the conserved Greatwall-mediated signaling pathway and a variety of downstream target molecules. In addition, our studies of S. cerevisiae sake yeast strains revealed that Rim15p also plays a significant role in the control of alcoholic fermentation. Despite an extensive history of research on glycolysis and alcoholic fermentation, there has been no critical clue to artificial modification of fermentation performance of yeast cells. Our finding of an in vivo metabolic regulatory mechanism is expected to provide a major breakthrough in yeast breeding technologies for fermentation applications.

Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation

PubMed Central

2012-01-01

Background The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic

A design-build-test cycle using modeling and experiments reveals interdependencies between upper glycolysis and xylose uptake in recombinant S. cerevisiae and improves predictive capabilities of large-scale kinetic models.

PubMed

Miskovic, Ljubisa; Alff-Tuomala, Susanne; Soh, Keng Cher; Barth, Dorothee; Salusjärvi, Laura; Pitkänen, Juha-Pekka; Ruohonen, Laura; Penttilä, Merja; Hatzimanikatis, Vassily

2017-01-01

Recent advancements in omics measurement technologies have led to an ever-increasing amount of available experimental data that necessitate systems-oriented methodologies for efficient and systematic integration of data into consistent large-scale kinetic models. These models can help us to uncover new insights into cellular physiology and also to assist in the rational design of bioreactor or fermentation processes. Optimization and Risk Analysis of Complex Living Entities (ORACLE) framework for the construction of large-scale kinetic models can be used as guidance for formulating alternative metabolic engineering strategies. We used ORACLE in a metabolic engineering problem: improvement of the xylose uptake rate during mixed glucose-xylose consumption in a recombinant Saccharomyces cerevisiae strain. Using the data from bioreactor fermentations, we characterized network flux and concentration profiles representing possible physiological states of the analyzed strain. We then identified enzymes that could lead to improved flux through xylose transporters (XTR). For some of the identified enzymes, including hexokinase (HXK), we could not deduce if their control over XTR was positive or negative. We thus performed a follow-up experiment, and we found out that HXK2 deletion improves xylose uptake rate. The data from the performed experiments were then used to prune the kinetic models, and the predictions of the pruned population of kinetic models were in agreement with the experimental data collected on the HXK2 -deficient S. cerevisiae strain. We present a design-build-test cycle composed of modeling efforts and experiments with a glucose-xylose co-utilizing recombinant S. cerevisiae and its HXK2 -deficient mutant that allowed us to uncover interdependencies between upper glycolysis and xylose uptake pathway. Through this cycle, we also obtained kinetic models with improved prediction capabilities. The present study demonstrates the potential of integrated "modeling

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

DOE PAGES

Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; ...

2015-07-22

Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

An economic comparison of different fermentation configurations to convert corn stover to ethanol using Z. mobilis and Saccharomyces.

PubMed

Dutta, Abhijit; Dowe, Nancy; Ibsen, Kelly N; Schell, Daniel J; Aden, Andy

2010-01-01

Numerous routes are being explored to lower the cost of cellulosic ethanol production and enable large-scale production. One critical area is the development of robust cofermentative organisms to convert the multiple, mixed sugars found in biomass feedstocks to ethanol at high yields and titers without the need for processing to remove inhibitors. Until such microorganisms are commercialized, the challenge is to design processes that exploit the current microorganisms' strengths. This study explored various process configurations tailored to take advantage of the specific capabilities of three microorganisms, Z. mobilis 8b, S. cerevisiae, and S. pastorianus. A technoeconomic study, based on bench-scale experimental data generated by integrated process testing, was completed to understand the resulting costs of the different process configurations. The configurations included whole slurry fermentation with a coculture, and separate cellulose simultaneous saccharification and fermentation (SSF) and xylose fermentations with none, some or all of the water to the SSF replaced with the fermented liquor from the xylose fermentation. The difference between the highest and lowest ethanol cost for the different experimental process configurations studied was $0.27 per gallon ethanol. Separate fermentation of solid and liquor streams with recycle of fermented liquor to dilute the solids gave the lowest ethanol cost, primarily because this option achieved the highest concentrations of ethanol after fermentation. Further studies, using methods similar to ones employed here, can help understand and improve the performance and hence the economics of integrated processes involving enzymes and fermentative microorganisms.

Effect of juice and fermented vinegar from Hovenia dulcis peduncles on chronically alcohol-induced liver damage in mice.

PubMed

Xiang, Jinle; Zhu, Wenxue; Li, Zhixi; Ling, Shengbao

2012-06-01

The protective effects of juice and fermented vinegar from Hovenia dulcis peduncles on chronically ethanol-induced biochemical changes in male mice were investigated. Administration of ethanol (50%, v/v, 10 mL kg⁻¹) to mice for 6 weeks induced liver damage with a significant increase (P < 0.01) of the liver index, aspartate transaminase (AST), alanine transaminase (ALT), gamma glutamyl transferase (γ-GT) in the serum and the hepatic lipid peroxidation (LPO) level. In contrast, administration of juice or fermented vinegar from Hovenia dulcis peduncles (10 mL kg⁻¹ bw) along with alcohol significantly (P < 0.05) decreased the activities of the enzymes (AST, ALT and γ-GT), liver index, concentrations of triglyceride (TG) and total cholesterol (TCH) in the serum and the hepatic TG and LPO levels. Mice treated with juice or fermented vinegar from Hovenia dulcis peduncles showed better profiles of the antioxidant systems with relatively higher glutathione (GSH) content, total superoxide dismutase (T-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. All these results were accompanied by histological observations in liver. The results demonstrate that both of the juice and fermented vinegar from Hovenia dulcis peduncles have beneficial effects in reducing the adverse effect of alcohol.

Automated Yeast Transformation Protocol to Engineer S. cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames that Express Proteins Binding to Xylose Isomerase Identified using Robotic Two-hybrid Screen

USDA-ARS?s Scientific Manuscript database

Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. Since S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI...

Cost-effective simultaneous saccharification and fermentation of l-lactic acid from bagasse sulfite pulp by Bacillus coagulans CC17.

PubMed

Zhou, Jie; Ouyang, Jia; Xu, Qianqian; Zheng, Zhaojuan

2016-12-01

The main barriers to cost-effective lactic acid production from lignocellulose are the high cost of enzymes and the ineffective utilization of the xylose within the hydrolysate. In the present study, the thermophilic Bacillus coagulans strain CC17 was used for the simultaneous saccharification and fermentation (SSF) of bagasse sulfite pulp (BSP) to produce l-lactic acid. Unexpectedly, SSF by CC17 required approximately 33.33% less fungal cellulase than did separate hydrolysis and fermentation (SHF). More interestingly, CC17 can co-ferment cellobiose and xylose without any exogenous β-glucosidase in SSF. Moreover, adding xylanase could increase the concentration of lactic acid produced via SSF. Up to 110g/L of l-lactic acid was obtained using fed-batch SSF, resulting in a lactic acid yield of 0.72g/g cellulose. These results suggest that SSF using CC17 has a remarkable advantage over SHF and that a potentially low-cost and highly-efficient fermentation process can be established using this protocol. Copyright © 2016 Elsevier Ltd. All rights reserved.

Single-cell Protein and Xylitol Production by a Novel Yeast Strain Candida intermedia FL023 from Lignocellulosic Hydrolysates and Xylose.

PubMed

Wu, Jiaqiang; Hu, Jinlong; Zhao, Shumiao; He, Mingxiong; Hu, Guoquan; Ge, Xiangyang; Peng, Nan

2018-05-01

Yeasts are good candidates to utilize the hydrolysates of lignocellulose, the most abundant bioresource, for bioproducts. This study aimed to evaluate the efficiencies of single-cell protein (SCP) and xylitol production by a novel yeast strain, Candida intermedia FL023, from lignocellulosic hydrolysates and xylose. This strain efficiently assimilated hexose, pentose, and cellubiose for cell mass production with the crude protein content of 484.2 g kg -1 dry cell mass. SCP was produced by strain FL023 using corncob hydrolysate and urea as the carbon and nitrogen sources with the dry cell mass productivity 0.86 g L -1  h -1 and the yield of 0.40 g g -1 sugar. SCP was also produced using NaOH-pretreated Miscanthus sinensis straw and corn steep liquor as the carbon and nitrogen sources through simultaneous saccharification and fermentation with the dry cell productivity of 0.23 g L -1  h -1 and yield of 0.17 g g -1 straw. C. intermedia FL023 was tolerant to 0.5 g L -1 furfural, acetic acid, and syringaldehyde in xylitol fermentation and produced 45.7 g L -1 xylitol from xylose with the productivity of 0.38 g L -1  h -1 and the yield of 0.57 g g -1 xylose. This study provides feasible methods for feed and food additive production from the abundant lignocellulosic bioresources.

Boosting dark fermentation with co-cultures of extreme thermophiles for biohythane production from garden waste.

PubMed

Abreu, Angela A; Tavares, Fábio; Alves, Maria Madalena; Pereira, Maria Alcina

2016-11-01

Proof of principle of biohythane and potential energy production from garden waste (GW) is demonstrated in this study in a two-step process coupling dark fermentation and anaerobic digestion. The synergistic effect of using co-cultures of extreme thermophiles to intensify biohydrogen dark fermentation is demonstrated using xylose, cellobiose and GW. Co-culture of Caldicellulosiruptor saccharolyticus and Thermotoga maritima showed higher hydrogen production yields from xylose (2.7±0.1molmol(-1) total sugar) and cellobiose (4.8±0.3molmol(-1) total sugar) compared to individual cultures. Co-culture of extreme thermophiles C. saccharolyticus and Caldicellulosiruptor bescii increased synergistically the hydrogen production yield from GW (98.3±6.9Lkg(-1) (VS)) compared to individual cultures and co-culture of T. maritima and C. saccharolyticus. The biochemical methane potential of the fermentation end-products was 322±10Lkg(-1) (CODt). Biohythane, a biogas enriched with 15% hydrogen could be obtained from GW, yielding a potential energy generation of 22.2MJkg(-1) (VS). Copyright © 2016 Elsevier Ltd. All rights reserved.

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae.

PubMed

Hou, Jin; Vemuri, Goutham N; Bao, Xiaoming; Olsson, Lisbeth

2009-04-01

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO(2) to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.

Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

PubMed

Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

2015-12-01

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains. © 2015 Society for Laboratory Automation and Screening.

Simultaneous Decolorization and Biohydrogen Production from Xylose by Klebsiella oxytoca GS-4-08 in the Presence of Azo Dyes with Sulfonate and Carboxyl Groups

PubMed Central

Cao, Ming-yue; Wang, Peng-tao; Wang, Shi; Yue, Ying-rong; Yuan, Wen-duo; Qiao, Wei-chuan; Wang, Fei

2017-01-01

ABSTRACT Biohydrogen production from the pulp and paper effluent containing rich lignocellulosic material could be achieved by the fermentation process. Xylose, an important hemicellulose hydrolysis product, is used less efficiently as a substrate for biohydrogen production. Moreover, azo dyes are usually added to fabricate anticounterfeiting paper, which further increases the complexity of wastewater. This study reports that xylose could serve as the sole carbon source for a pure culture of Klebsiella oxytoca GS-4-08 to achieve simultaneous decolorization and biohydrogen production. With 2 g liter−1 of xylose as the substrate, a maximum xylose utilization rate (URxyl) and a hydrogen molar yield (HMY) of 93.99% and 0.259 mol of H2 mol of xylose−1, respectively, were obtained. Biohydrogen kinetics and electron equivalent (e− equiv) balance calculations indicated that methyl red (MR) penetrates and intracellularly inhibits both the pentose phosphate pathway and pyruvate fermentation pathway, while methyl orange (MO) acted independently of the glycolysis and biohydrogen pathway. The data demonstrate that biohydrogen pathways in the presence of azo dyes with sulfonate and carboxyl groups were different, but the azo dyes could be completely reduced during the biohydrogen production period in the presence of MO or MR. The feasibility of hydrogen production from industrial pulp and paper effluent by the strain if the xylose is sufficient was also proved and was not affected by toxic substances which usually exist in such wastewater, except for chlorophenol. This study offers a promising energy-recycling strategy for treating pulp and paper wastewaters, especially for those containing azo dyes. IMPORTANCE The pulp and paper industry is a major industry in many developing countries, and the global market of pulp and paper wastewater treatment is expected to increase by 60% between 2012 and 2020. Such wastewater contains large amounts of refractory contaminants, such

Impact of nutrient imbalance on wine alcoholic fermentations: nitrogen excess enhances yeast cell death in lipid-limited must.

PubMed

Tesnière, Catherine; Delobel, Pierre; Pradal, Martine; Blondin, Bruno

2013-01-01

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations.

Impact of Nutrient Imbalance on Wine Alcoholic Fermentations: Nitrogen Excess Enhances Yeast Cell Death in Lipid-Limited Must

PubMed Central

Tesnière, Catherine; Delobel, Pierre; Pradal, Martine; Blondin, Bruno

2013-01-01

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations. PMID:23658613

Analysis of bacterial community during the fermentation of pulque, a traditional Mexican alcoholic beverage, using a polyphasic approach.

PubMed

Escalante, Adelfo; Giles-Gómez, Martha; Hernández, Georgina; Córdova-Aguilar, María Soledad; López-Munguía, Agustín; Gosset, Guillermo; Bolívar, Francisco

2008-05-31

In this study, the characterization of the bacterial community present during the fermentation of pulque, a traditional Mexican alcoholic beverage from maguey (Agave), was determined for the first time by a polyphasic approach in which both culture and non-culture dependent methods were utilized. The work included the isolation of lactic acid bacteria (LAB), aerobic mesophiles, and 16S rDNA clone libraries from total DNA extracted from the maguey sap (aguamiel) used as substrate, after inoculation with a sample of previously produced pulque and followed by 6-h fermentation. Microbiological diversity results were correlated with fermentation process parameters such as sucrose, glucose, fructose and fermentation product concentrations. In addition, medium rheological behavior analysis and scanning electron microscopy in aguamiel and during pulque fermentation were also performed. Our results showed that both culture and non-culture dependent approaches allowed the detection of several new and previously reported species within the alpha-, gamma-Proteobacteria and Firmicutes. Bacteria diversity in aguamiel was composed by the heterofermentative Leuconostoc citreum, L. mesenteroides, L. kimchi, the gamma-Proteobacteria Erwinia rhapontici, Enterobacter spp. and Acinetobacter radioresistens. Inoculation with previously fermented pulque incorporated to the system microbiota, homofermentative lactobacilli related to Lactobacillus acidophilus, several alpha-Proteobacteria such as Zymomonas mobilis and Acetobacter malorum, other gamma-Proteobacteria and an important amount of yeasts, creating a starting metabolic diversity composed by homofermentative and heterofermentative LAB, acetic and ethanol producing microorganisms. At the end of the fermentation process, the bacterial diversity was mainly composed by the homofermentative Lactobacillus acidophilus, the heterofermentative L. mesenteroides, Lactococcus lactis subsp. lactis and the alpha-Proteobacteria A. malorum. After

Comprehensive Study of the Evolution of the Gas-Liquid Partitioning of Acetaldehyde during Wine Alcoholic Fermentation.

PubMed

Aguera, Evelyne; Sire, Yannick; Mouret, Jean-Roch; Sablayrolles, Jean-Marie; Farines, Vincent

2018-06-20

Determining the gas-liquid partitioning ( K i ) of acetaldehyde during alcoholic fermentation is an important step in the optimization of fermentation control with the aim of minimizing the accumulation of this compound, which is responsible for the undesired attributes of green apples and fresh-cut grass in wines. In this work, the effects of the main fermentation parameters on the K i of acetaldehyde were assessed. K i values were found to be dependent on the temperature and composition of the medium. A nonlinear correlation between the evolution of the K i and fermentation progress was observed, attributable to the strong retention effect of ethanol at low concentrations, and it was demonstrated that the partitioning of this specific molecule was not influenced by the CO 2 production rate. A model was developed that quantifies the K i of acetaldehyde with a very accurate prediction, as the difference between the observed and predicted values did not exceed 9%.

The effect of Maillard reaction products and yeast strain on the synthesis of key higher alcohols and esters in beer fermentations.

PubMed

Dack, Rachael E; Black, Gary W; Koutsidis, Georgios; Usher, St John

2017-10-01

The effect of Maillard reaction products (MRPs), formed during the production of dark malts, on the synthesis of higher alcohols and esters in beer fermentations was investigated by headspace solid-phase microextraction GC-MS. Higher alcohol levels were significantly (p<0.05) higher in dark malt fermentations, while the synthesis of esters was inhibited, due to possible suppression of enzyme activity and/or gene expression linked to ester synthesis. Yeast strain also affected flavour synthesis with Saccharomyces cerevisiae strain A01 producing considerably lower levels of higher alcohols and esters than S288c and L04. S288c produced approximately double the higher alcohol levels and around twenty times more esters compared to L04. Further investigations into malt type-yeast strain interactions in relation to flavour development are required to gain better understanding of flavour synthesis that could assist in the development of new products and reduce R&D costs for the industry. Copyright © 2017 Elsevier Ltd. All rights reserved.

Feasibility of using a miniature NIR spectrometer to measure volumic mass during alcoholic fermentation.

PubMed

Fernández-Novales, Juan; López, María-Isabel; González-Caballero, Virginia; Ramírez, Pilar; Sánchez, María-Teresa

2011-06-01

Volumic mass-a key component of must quality control tests during alcoholic fermentation-is of great interest to the winemaking industry. Transmitance near-infrared (NIR) spectra of 124 must samples over the range of 200-1,100-nm were obtained using a miniature spectrometer. The performance of this instrument to predict volumic mass was evaluated using partial least squares (PLS) regression and multiple linear regression (MLR). The validation statistics coefficient of determination (r(2)) and the standard error of prediction (SEP) were r(2) = 0.98, n = 31 and r(2) = 0.96, n = 31, and SEP = 5.85 and 7.49 g/dm(3) for PLS and MLR equations developed to fit reference data for volumic mass and spectral data. Comparison of results from MLR and PLS demonstrates that a MLR model with six significant wavelengths (P < 0.05) fit volumic mass data to transmittance (1/T) data slightly worse than a more sophisticated PLS model using the full scanning range. The results suggest that NIR spectroscopy is a suitable technique for predicting volumic mass during alcoholic fermentation, and that a low-cost NIR instrument can be used for this purpose.

Optimization of L(+)-Lactic Acid Production from Xylose with Rhizopus Oryzae Mutant RLC41-6 Breeding by Low-Energy Ion Implantation

NASA Astrophysics Data System (ADS)

Yang, Yingge; Fan, Yonghong; Li, Wen; Wang, Dongmei; Wu, Yuejin; Zheng, Zhiming; Yu, Zengliang

2007-10-01

In order to obtain an industrial strain with a higher L(+)-lactic acid yield, the strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion beam implantation and the mutant strain Rhizopus oryzae RLC41-6 was obtained. An experimental finding was made in surprise that Rhizopus oryzae mutant RLC41-6 is not only an L(+)-lactic acid producer from corn starch but also an efficient producer of L(+)-lactic acid from xylose. Under optimal conditions, the production of L(+)-lactic acid from 100 g/L xylose reached 77.39 g/L after 144 h fed-batch fermentation. A high mutation rate and a wide mutation spectrum of low-energy ion implantation were observed in the experiment.

Novel Xylose Dehydrogenase in the Halophilic Archaeon Haloarcula marismortui†

PubMed Central

Johnsen, Ulrike; Schönheit, Peter

2004-01-01

During growth of the halophilic archaeon Haloarcula marismortui on d-xylose, a specific d-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to d-xylose, d-ribose was oxidized at similar kinetic constants, whereas d-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)—coding for a 39.9-kDA protein—was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui. PMID:15342590

Improvement on D-xylose to Xylitol Biotransformation by Candida guilliermondii Using Cells Permeabilized with Triton X-100 and Selected Process Conditions.

PubMed

Cortez, Daniela Vieira; Mussatto, Solange I; Roberto, Inês Conceição

2016-11-01

Cells of Candida guilliermondii permeabilized with Triton X-100 were able to efficiently produce xylitol from a medium composed only by D-xylose and MgCl 2 ·6H 2 O in potassium phosphate buffer, at 35 °C and pH 6.5. Under these conditions, the results were similar to those obtained when cofactor and co-substrate or nutrients were added to the medium (about 95 % D-xylose was assimilated producing 42 g/L of xylitol, corresponding to 0.80 g/g yield and 2.65 g/L h volumetric productivity). Furthermore, the permeabilized cells kept the D-xylose assimilation in about 90 % and the xylitol production in approx. 40 g/L during three bioconversion cycles of 16 h each. These values are highly relevant when compared to others reported in the literature using enzyme technology and fermentative process, thereby demonstrating the effectiveness of the proposed method. The present study reveals that the use of permeabilized cells is an interesting alternative to obtain high xylitol productivity using low cost medium formulation. This approach may allow the future development of xylitol production from xylose present in lignocellulosic biomass, with additional potential for implementation in biorefinery strategies.

Construction and Analysis of High-Ethanol-Producing Fusants with Co-Fermentation Ability through Protoplast Fusion and Double Labeling Technology

PubMed Central

Ge, Jingping; Zhao, Jingwen; Zhang, Luyan; Zhang, Mengyun; Ping, Wenxiang

2014-01-01

Double labeling of resistance markers and report genes can be used to breed engineered Saccharomyces cerevisiae strains that can assimilate xylose and glucose as a mixed carbon source for ethanol fermentation and increased ethanol production. In this study Saccharomyces cerevisiae W5 and Candida shehatae 20335 were used as parent strains to conduct protoplast fusion and the resulting fusants were screened by double labeling. High performance liquid chromatography (HPLC) was used to assess the ethanol yield following the fermentation of xylose and glucose, as both single and mixed carbon sources, by the fusants. Interestingly, one fusant (ZLYRHZ7) was demonstrated to have an excellent fermentation performance, with an ethanol yield using the mixed carbon source of 0.424 g g−1, which compares with 0.240 g g−1 (W5) and 0.353 g g−1 (20335) for the parent strains. This indicates an improvement in the ethanol yield of 43.4% and 16.7%, respectively. PMID:25268957

Construction and analysis of high-ethanol-producing fusants with co-fermentation ability through protoplast fusion and double labeling technology.

PubMed

Ge, Jingping; Zhao, Jingwen; Zhang, Luyan; Zhang, Mengyun; Ping, Wenxiang

2014-01-01

Double labeling of resistance markers and report genes can be used to breed engineered Saccharomyces cerevisiae strains that can assimilate xylose and glucose as a mixed carbon source for ethanol fermentation and increased ethanol production. In this study Saccharomyces cerevisiae W5 and Candida shehatae 20335 were used as parent strains to conduct protoplast fusion and the resulting fusants were screened by double labeling. High performance liquid chromatography (HPLC) was used to assess the ethanol yield following the fermentation of xylose and glucose, as both single and mixed carbon sources, by the fusants. Interestingly, one fusant (ZLYRHZ7) was demonstrated to have an excellent fermentation performance, with an ethanol yield using the mixed carbon source of 0.424 g g-1, which compares with 0.240 g g-1 (W5) and 0.353 g g-1 (20335) for the parent strains. This indicates an improvement in the ethanol yield of 43.4% and 16.7%, respectively.

Scale-up and integration of alkaline hydrogen peroxide pretreatment, enzymatic hydrolysis, and ethanolic fermentation.

PubMed

Banerjee, Goutami; Car, Suzana; Liu, Tongjun; Williams, Daniel L; Meza, Sarynna López; Walton, Jonathan D; Hodge, David B

2012-04-01

Alkaline hydrogen peroxide (AHP) has several attractive features as a pretreatment in the lignocellulosic biomass-to-ethanol pipeline. Here, the feasibility of scaling-up the AHP process and integrating it with enzymatic hydrolysis and fermentation was studied. Corn stover (1 kg) was subjected to AHP pretreatment, hydrolyzed enzymatically, and the resulting sugars fermented to ethanol. The AHP pretreatment was performed at 0.125 g H(2) O(2) /g biomass, 22°C, and atmospheric pressure for 48 h with periodic pH readjustment. The enzymatic hydrolysis was performed in the same reactor following pH neutralization of the biomass slurry and without washing. After 48 h, glucose and xylose yields were 75% and 71% of the theoretical maximum. Sterility was maintained during pretreatment and enzymatic hydrolysis without the use of antibiotics. During fermentation using a glucose- and xylose-utilizing strain of Saccharomyces cerevisiae, all of the Glc and 67% of the Xyl were consumed in 120 h. The final ethanol titer was 13.7 g/L. Treatment of the enzymatic hydrolysate with activated carbon prior to fermentation had little effect on Glc fermentation but markedly improved utilization of Xyl, presumably due to the removal of soluble aromatic inhibitors. The results indicate that AHP is readily scalable and can be integrated with enzyme hydrolysis and fermentation. Compared to other leading pretreatments for lignocellulosic biomass, AHP has potential advantages with regard to capital costs, process simplicity, feedstock handling, and compatibility with enzymatic deconstruction and fermentation. Biotechnol. Bioeng. 2012; 109:922-931. © 2011 Wiley Periodicals, Inc. Copyright © 2011 Wiley Periodicals, Inc.

A Feasibility Study on Monitoring Residual Sugar and Alcohol Strength in Kiwi Wine Fermentation Using a Fiber-Optic FT-NIR Spectrometry and PLS Regression.

PubMed

Wang, Bingqian; Peng, Bangzhu

2017-02-01

This work aims to investigate the potential of fiber-optic Fourier transform-near-infrared (FT-NIR) spectrometry associated with chemometric analysis, which will be applied to monitor time-related changes in residual sugar and alcohol strength during kiwi wine fermentation. NIR calibration models for residual sugar and alcohol strength during kiwi wine fermentation were established on the FT-NIR spectra of 98 samples scanned in a fiber-optic FT-NIR spectrometer, and partial least squares regression method. The results showed that R 2 and root mean square error of cross-validation could achieve 0.982 and 3.81 g/L for residual sugar, and 0.984 and 0.34% for alcohol strength, respectively. Furthermore, crucial process information on kiwi must and wine fermentations provided by fiber-optic FT-NIR spectrometry was found to agree with those obtained from traditional chemical methods, and therefore this fiber-optic FT-NIR spectrometry can be applied as an effective and suitable alternative for analyses and monitoring of those processes. The overall results suggested that fiber-optic FT-NIR spectrometry is a promising tool for monitoring and controlling the kiwi wine fermentation process. © 2017 Institute of Food Technologists®.

A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation

PubMed Central

Duc, Camille; Pradal, Martine; Sanchez, Isabelle; Noble, Jessica; Tesnière, Catherine

2017-01-01

Yeast cell death can occur during wine alcoholic fermentation. It is generally considered to result from ethanol stress that impacts membrane integrity. This cell death mainly occurs when grape musts processing reduces lipid availability, resulting in weaker membrane resistance to ethanol. However the mechanisms underlying cell death in these conditions remain unclear. We examined cell death occurrence considering yeast cells ability to elicit an appropriate response to a given nutrient limitation and thus survive starvation. We show here that a set of micronutrients (oleic acid, ergosterol, pantothenic acid and nicotinic acid) in low, growth-restricting concentrations trigger cell death in alcoholic fermentation when nitrogen level is high. We provide evidence that nitrogen signaling is involved in cell death and that either SCH9 deletion or Tor inhibition prevent cell death in several types of micronutrient limitation. Under such limitations, yeast cells fail to acquire any stress resistance and are unable to store glycogen. Unexpectedly, transcriptome analyses did not reveal any major changes in stress genes expression, suggesting that post-transcriptional events critical for stress response were not triggered by micronutrient starvation. Our data point to the fact that yeast cell death results from yeast inability to trigger an appropriate stress response under some conditions of nutrient limitations most likely not encountered by yeast in the wild. Our conclusions provide a novel frame for considering both cell death and the management of nutrients during alcoholic fermentation. PMID:28922393

Impact of available nitrogen and sugar concentration in musts on alcoholic fermentation and subsequent wine spoilage by Brettanomyces bruxellensis.

PubMed

Childs, Bradford C; Bohlscheid, Jeffri C; Edwards, Charles G

2015-04-01

The level of yeast assimilable nitrogen (YAN) supplementation required for Saccharomyces cerevisiae to complete fermentation of high sugar musts in addition to the impact of non-metabolized nitrogen on post-alcoholic spoilage by Brettanomyces bruxellensis was studied. A 2 × 3 factorial design was employed using a synthetic grape juice medium with YAN (150 or 250 mg N/L) and equal proportions of glucose/fructose (230, 250, or 270 g/L) as variables. S. cerevisiae ECA5 (low nitrogen requirement) or Uvaferm 228 (high nitrogen requirement) were inoculated at 10(5) cfu/mL while B. bruxellensis E1 or B2 were added once alcoholic fermentation ceased. Regardless of YAN concentration, musts that contained 230 or 250 g/L glucose/fructose at either nitrogen level attained dryness (mean = 0.32 g/L fructose) while those containing 270 g/L generally did not (mean = 2.5 g/L fructose). Higher concentrations of YAN present in musts yielded wines with higher amounts of α-amino acids and ammonium but very little (≤ 6 mg N/L) was needed by B. bruxellensis to attain populations ≥ 10(7) cfu/mL. While adding nitrogen to high sugar musts does not necessarily ensure completion of alcoholic fermentation, residual YAN did not affect B. bruxellensis growth as much as ethanol concentration. Copyright © 2014 Elsevier Ltd. All rights reserved.

Simultaneous co-fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol.

PubMed

Kim, Soo Rin; Ha, Suk-Jin; Wei, Na; Oh, Eun Joong; Jin, Yong-Su

2012-05-01

The lack of microbial strains capable of fermenting all sugars prevalent in plant cell wall hydrolyzates to ethanol is a major challenge. Although naturally existing or engineered microorganisms can ferment mixed sugars (glucose, xylose and galactose) in these hydrolyzates sequentially, the preferential utilization of glucose to non-glucose sugars often results in lower overall yield and productivity of ethanol. Therefore, numerous metabolic engineering approaches have been attempted to construct optimal microorganisms capable of co-fermenting mixed sugars simultaneously. Here, we present recent findings and breakthroughs in engineering yeast for improved ethanol production from mixed sugars. In particular, this review discusses new sugar transporters, various strategies for simultaneous co-fermentation of mixed sugars, and potential applications of co-fermentation for producing fuels and chemicals. Copyright © 2012 Elsevier Ltd. All rights reserved.

Candida kantuleensis sp. nov., a d-xylose-fermenting yeast species isolated from peat in a tropical peat swamp forest.

PubMed

Nitiyon, Sukanya; Khunnamwong, Pannida; Lertwattanasakul, Noppon; Limtong, Savitree

2018-05-24

Three strains (DMKU-XE11 T , DMKU-XE15 and DMKU-XE20) representing a single novel anamorphic and d-xylose-fermenting yeast species were obtained from three peat samples collected from Khan Thulee peat swamp forest in Surat Thani province, Thailand. The strains differed from each other by one to two nucleotide substitutions in the sequences of the D1/D2 region of the large subunit (LSU) rRNA gene and zero to one nucleotide substitution in the internal transcribed spacer (ITS) region. Phylogenetic analysis based on the combined sequences of the ITS and the D1/D2 regions showed that the three strains represented a single Candida species that was distinct from the other related species in the Lodderomyces/Candida albicans clade. The three strains form a subclade with the other Candida species including Candida sanyaensis, Candida tropicalis and Candida sojae. C. sanyaensis was the most closely related species, with 2.1-2.4 % nucleotide substitutions in the D1/D2 region of the LSU rRNA gene, and 3.8-4.0 % nucleotide substitutions in the ITS region. The three strains (DMKU-XE11 T , DMKU-XE15 and DMKU-XE20) were assigned as a single novel species, which was named Candida kantuleensis sp. nov. The type strain is DMKU-XE11 T (=CBS 15219 T =TBRC 7764 T ). The MycoBank number for C. kantuleensis sp. nov. is MB 824179.

27 CFR 24.197 - Production by fermentation.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Production by fermentation... fermentation. In producing special natural wine by fermentation, flavoring materials may be added before or during fermentation. Special natural wine produced by fermentation may be ameliorated in the same manner...

27 CFR 24.197 - Production by fermentation.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Production by fermentation... fermentation. In producing special natural wine by fermentation, flavoring materials may be added before or during fermentation. Special natural wine produced by fermentation may be ameliorated in the same manner...

27 CFR 24.197 - Production by fermentation.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Production by fermentation... fermentation. In producing special natural wine by fermentation, flavoring materials may be added before or during fermentation. Special natural wine produced by fermentation may be ameliorated in the same manner...

27 CFR 24.197 - Production by fermentation.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Production by fermentation... fermentation. In producing special natural wine by fermentation, flavoring materials may be added before or during fermentation. Special natural wine produced by fermentation may be ameliorated in the same manner...

27 CFR 24.197 - Production by fermentation.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Production by fermentation... fermentation. In producing special natural wine by fermentation, flavoring materials may be added before or during fermentation. Special natural wine produced by fermentation may be ameliorated in the same manner...

Improving the quality of matured coconut (Cocos nucifera Linn.) water by low alcoholic fermentation with Saccharomyces cerevisiae: antioxidant and volatile profiles.

PubMed

Zhang, Guanfei; Chen, Wenxue; Chen, Weijun; Chen, Haiming

2018-03-01

Matured coconut water (MCW) is a by-product in the coconut milk industry that is usually discarded due to its unpleasant flavor. In this study, low-alcohol coconut water (LACW) was fermented with Saccharomyces cerevisiae to improve the quality of MCW. Volatile components and nonvolatile flavor-related elements were estimated to compare the qualities of the MCW and LACW. Besides measuring the kinetic changes, the levels of fructose, glucose, sucrose and ethanol contents were also determined. The results of the organic acid assays showed that tartaric, pyruvic and succinic acids were the primary organic acids present in LACW and increased significantly with fermentation. The resulting volatile composition assay indicated that esters, alcohols and fatty acids were significantly influenced by fermentation and yeast strains. Moreover, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), cupric ion reducing antioxidant capacity and ferric reducing antioxidant power values increased significantly throughout the process, correlating with the enhancement of total phenolic content.

Reduced Production of Higher Alcohols by Saccharomyces cerevisiae in Red Wine Fermentation by Simultaneously Overexpressing BAT1 and Deleting BAT2.

PubMed

Ma, Lijuan; Huang, Shiyong; Du, Liping; Tang, Ping; Xiao, Dongguang

2017-08-16

In red wine, the contents of higher alcohols and ethyl carbamate (EC) are two significant health concerns. To reduce the production of higher alcohols by wine yeast YZ22 with low production of EC, one BAT2 was replaced by a BAT1 expression cassette first and then another BAT2 was deleted to obtain the mutant SYBB3. Real-time quantitative PCR showed that the relative expression level of BAT1 in SYBB3 improved 28 times compared with that in YZ22. The yields of isobutanol and 3-methyl-1-butanol produced by mutant SYBB3 reduced by 39.41% and 37.18% compared to those by the original strain YZ22, and the total production of higher alcohols decreased from 463.82 mg/L to 292.83 mg/L in must fermentation of Cabernet Sauvignon. Meanwhile, there were no obvious differences on fermentation characteristics of the mutant and parental strain. This research has suggested an effective strategy for decreasing production of higher alcohols in Saccharomyces cerevisiae.

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

PubMed Central

Johnsen, Ulrike; Dambeck, Michael; Zaiss, Henning; Fuhrer, Tobias; Soppa, Jörg; Sauer, Uwe; Schönheit, Peter

2009-01-01

The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following 13C-labeling patterns of proteinogenic amino acids after growth on [13C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus. PMID:19584053

Performance of several Saccharomyces strains for the alcoholic fermentation of sugar-sweetened high-strength wastewaters: Comparative analysis and kinetic modelling.

PubMed

Comelli, Raúl N; Seluy, Lisandro G; Isla, Miguel A

2016-12-25

This work focuses on the performance of ten commercial Saccharomyces yeast strains in the batch alcoholic fermentation of sugars contained in selected industrial wastewaters from the soft drink industry. Fermentation has been applied successfully to treat these effluents prior to their disposal. Although many strains were investigated, similar behaviour was observed between all of the Saccharomyces strains tested. When media were inoculated with 2gL -1 of yeast, all strains were able to completely consume the available sugars in less than 14h. Thus, any of the strains studied in this work could be used in non-conventional wastewater treatment processes based on alcoholic fermentation. However, ethanol production varied between strains, and these differences could be significant from a production point of view. Saccharomyces bayanus produced the most ethanol, with a mean yield of 0.44g ethanol g sugarconsumed -1 and an ethanol specific production rate of 5.96g ethanol (Lh) -1 . As the assayed soft drinks wastewaters contain about 105g sugar /L of fermentable sugars, the concentration of ethanol achieved after the fermentations process was 46.2g ethanol /L. A rigorous kinetic modelling methodology was used to model the Saccharomyces bayanus fermentation process. The kinetic model included coupled mass balances and a minimal number of parameters. A simple unstructured model based on the Andrews equation (substrate inhibition) was developed. This model satisfactorily described biomass growth, sugar consumption and bioethanol production. In addition to providing insights into the fermentative performance of potentially relevant strains, this work can facilitate the design of large-scale ethanol production processes that use wastewaters from the sugar-sweetened beverage industry as feedstock. Copyright © 2016 Elsevier B.V. All rights reserved.

A strain of Meyerozyma guilliermondii isolated from sugarcane juice is able to grow and ferment pentoses in synthetic and bagasse hydrolysate media.

PubMed

Martini, Cristina; Tauk-Tornisielo, Sâmia Maria; Codato, Carolina Brito; Bastos, Reinaldo Gaspar; Ceccato-Antonini, Sandra Regina

2016-05-01

The search for new microbial strains that are able to withstand inhibitors released from hemicellulosic hydrolysis and are also still able to convert sugars in ethanol/xylitol is highly desirable. A yeast strain isolated from sugarcane juice and identified as Meyerozyma guilliermondii was evaluated for the ability to grow and ferment pentoses in synthetic media and in sugarcane bagasse hydrolysate. The yeast grew in xylose, arabinose and glucose at the same rate at an initial medium pH of 5.5. At pH 4.5, the yeast grew more slowly in arabinose. There was no sugar exhaustion within 60 h. At higher xylose concentrations with a higher initial cell concentration, sugar was exhausted within 96 h at pH 4.5. An increase of 350 % in biomass was obtained in detoxified hydrolysates, whereas supplementation with 3 g/L yeast extract increased biomass production by approximately 40 %. Ethanol and xylitol were produced more significantly in supplemented hydrolysates regardless of detoxification. Xylose consumption was enhanced in supplemented hydrolysates and arabinose was consumed only when xylose and glucose were no longer available. Supplementation had a greater impact on ethanol yield and productivity than detoxification; however, the product yields obtained in the present study are still much lower when compared to other yeast species in bagasse hydrolysate. By the other hand, the fermentation of both xylose and arabinose and capability of withstanding inhibitors are important characteristics of the strain assayed.

Ethanol production from residual wood chips of cellulose industry: acid pretreatment investigation, hemicellulosic hydrolysate fermentation, and remaining solid fraction fermentation by SSF process.

PubMed

Silva, Neumara Luci Conceição; Betancur, Gabriel Jaime Vargas; Vasquez, Mariana Peñuela; Gomes, Edelvio de Barros; Pereira, Nei

2011-04-01

Current research indicates the ethanol fuel production from lignocellulosic materials, such as residual wood chips from the cellulose industry, as new emerging technology. This work aimed at evaluating the ethanol production from hemicellulose of eucalyptus chips by diluted acid pretreatment and the subsequent fermentation of the generated hydrolysate by a flocculating strain of Pichia stipitis. The remaining solid fraction generated after pretreatment was subjected to enzymatic hydrolysis, which was carried out simultaneously with glucose fermentation [saccharification and fermentation (SSF) process] using a strain of Saccharomyces cerevisiae. The acid pretreatment was evaluated using a central composite design for sulfuric acid concentration (1.0-4.0 v/v) and solid to liquid ratio (1:2-1:4, grams to milliliter) as independent variables. A maximum xylose concentration of 50 g/L was obtained in the hemicellulosic hydrolysate. The fermentation of hemicellulosic hydrolysate and the SSF process were performed in bioreactors and the final ethanol concentrations of 15.3 g/L and 28.7 g/L were obtained, respectively.

Succinic acid production by Actinobacillus succinogenes from batch fermentation of mixed sugars.

PubMed

Almqvist, Henrik; Pateraki, Chrysanthi; Alexandri, Maria; Koutinas, Apostolis; Lidén, Gunnar

2016-08-01

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8-26.8 g/L and a total yield of 0.59-0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14-0.20 g/g and formate 0.08-0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO2 sparging could replace carbonate supply in the form of MgCO3 without affecting the succinate yield.

Enzymatic and Microbial Preparation of d-Xylulose from d-Xylose

PubMed Central

Chiang, Lin-Chang; Hsiao, Humg-Yu; Ueng, Pear P.; Tsao, George T.

1981-01-01

A high-d-xylulose mixture (d-xylose-d-xylulose = 33:67) was prepared from the cold ethanol extract of preisomerized d-xylose solution (d-xylose-d-xylulose = 77:23). Fusarium oxysporum f. sp. lini and Aspergillus niger were demonstrated to preferentially utilize d-xylose in the mixture of d-xylose and d-xylulose. Chromatographically pure d-xylulose was thus obtained in 90% yield. A high-d-xylulose mixture was also incubated with Rhodotorula toruloides, Klebsiella pneumoniae, Candida utilis, or Mucor rouxii.d-Xylose and d-xylulose were simultaneously consumed. When borate was added to the mixture, a d-xylulose-borate complex was formed, and it could be used to protect d-xylulose from being utilized. PMID:16345816

Genomic diversity of Saccharomyces cerevisiae yeasts associated with alcoholic fermentation of bacanora produced by artisanal methods.

PubMed

Álvarez-Ainza, M L; Zamora-Quiñonez, K A; Moreno-Ibarra, G M; Acedo-Félix, E

2015-03-01

Bacanora is a spirituous beverage elaborated with Agave angustifolia Haw in an artisanal process. Natural fermentation is mostly performed with native yeasts and bacteria. In this study, 228 strains of yeast like Saccharomyces were isolated from the natural alcoholic fermentation on the production of bacanora. Restriction analysis of the amplified region ITS1-5.8S-ITS2 of the ribosomal DNA genes (RFLPr) were used to confirm the genus, and 182 strains were identified as Saccharomyces cerevisiae. These strains displayed high genomic variability in their chromosomes profiles by karyotyping. Electrophoretic profiles of the strains evaluated showed a large number of chromosomes the size of which ranged between 225 and 2200 kpb approximately.

Fermentation strategy for second generation ethanol production from sugarcane bagasse hydrolyzate by Spathaspora passalidarum and Scheffersomyces stipitis.

PubMed

Nakanishi, Simone C; Soares, Lauren B; Biazi, Luiz Eduardo; Nascimento, Viviane M; Costa, Aline C; Rocha, George Jackson M; Ienczak, Jaciane L

2017-10-01

Alcoholic fermentation of released sugars in pretreatment and enzymatic hydrolysis of biomass is a central feature for second generation ethanol (E2G) production. Saccharomyces cerevisiae used industrially in the production of first generation ethanol (E1G) convert sucrose, fructose, and glucose into ethanol. However, these yeasts have no ability to ferment pentose (xylose). Therefore, the present work has focused on E2G production by Scheffersomyces stipitis and Spathaspora passalidarum. The fermentation strategy with high pitch, cell recycle, fed-batch mode, and temperature decrease for each batch were performed in a hydrolyzate obtained from a pretreatment at 130°C with NaOH solution (1.5% w/v) added with 0.15% (w/w) of anthraquinone (AQ) and followed by enzymatic hydrolysis. The process strategy has increased volumetric productivity from 0.35 to 0.38 g · L -1  · h -1 (first to third batch) for S. stipitis and from 0.38 to 0.81 g · L -1  · h -1 for S. passalidarum (first to fourth batch). Mass balance for the process proposed in this work showed the production of 177.33 kg ethanol/ton of sugar cane bagasse for S. passalidarum compared to 124.13 kg ethanol/ton of sugar cane bagasse for S. stipitis fermentation. The strategy proposed in this work can be considered as a promising strategy in the production of second generation ethanol. Biotechnol. Bioeng. 2017;114: 2211-2221. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Volatile Compounds Produced by Lactobacillus paracasei During Oat Fermentation.

PubMed

Lee, Sang Mi; Oh, Jieun; Hurh, Byung-Serk; Jeong, Gwi-Hwa; Shin, Young-Keum; Kim, Young-Suk

2016-12-01

This study investigated the profiles of volatile compounds produced by Lactobacillus paracasei during oat fermentation using gas chromatography-mass spectrometry coupled with headspace solid-phase microextraction method. A total of 60 compounds, including acids, alcohols, aldehydes, esters, furan derivatives, hydrocarbons, ketones, sulfur-containing compounds, terpenes, and other compounds, were identified in fermented oat. Lipid oxidation products such as 2-pentylfuran, 1-octen-3-ol, hexanal, and nonanal were found to be the main contributors to oat samples fermented by L. paracasei with the level of 2-pentylfuran being the highest. In addition, the contents of ketones, alcohols, acids, and furan derivatives in the oat samples consistently increased with the fermentation time. On the other hand, the contents of degradation products of amino acids, such as 3-methylbutanal, benzaldehyde, acetophenone, dimethyl sulfide, and dimethyl disulfide, decreased in oat samples during fermentation. Principal component analysis (PCA) was applied to discriminate the fermented oat samples according to different fermentation times. The fermented oats were clearly differentiated on PCA plots. The initial fermentation stage was mainly affected by aldehydes, whereas the later samples of fermented oats were strongly associated with acids, alcohols, furan derivatives, and ketones. The application of PCA to data of the volatile profiles revealed that the oat samples fermented by L. paracasei could be distinguished according to fermentation time. © 2016 Institute of Food Technologists®.

Assessing the Mechanisms Responsible for Differences between Nitrogen Requirements of Saccharomyces cerevisiae Wine Yeasts in Alcoholic Fermentation

PubMed Central

Brice, Claire; Sanchez, Isabelle; Tesnière, Catherine

2014-01-01

Nitrogen is an essential nutrient for Saccharomyces cerevisiae wine yeasts during alcoholic fermentation, and its abundance determines the fermentation rate and duration. The capacity to ferment under conditions of nitrogen deficiency differs between yeasts. A characterization of the nitrogen requirements of a set of 23 strains revealed large differences in their fermentative performances under nitrogen deficiency, and these differences reflect the nitrogen requirements of the strains. We selected and compared two groups of strains, one with low nitrogen requirements (LNRs) and the other with high nitrogen requirements (HNRs). A comparison of various physiological traits indicated that the differences are not related to the ability to store nitrogen or the protein content. No differences in protein synthesis activity were detected between strains with different nitrogen requirements. Transcriptomic analysis revealed expression patterns specific to each of the two groups of strains, with an overexpression of stress genes in HNR strains and a stronger expression of biosynthetic genes in LNR strains. Our data suggest that differences in glycolytic flux may originate from variations in nitrogen sensing and signaling under conditions of starvation. PMID:24334661

A novel steam explosion sterilization improving solid-state fermentation performance.

PubMed

Zhao, Zhi-Min; Wang, Lan; Chen, Hong-Zhang

2015-09-01

Traditional sterilization of solid medium (SM) requires lengthy time, degrades nutrients, and even sterilizes inadequately compared with that of liquid medium due to its low thermal conductivity. A novel sterilization strategy, high-temperature and short-time steam explosion (SE), was exploited for SM sterilization in this study. Results showed that SE conditions for complete sterilization were 172 °C for 2 min and 128 °C for 5 min. Glucose and xylose contents in medium after SE sterilization increased by 157% and 93% respectively compared with those after conventional sterilization (121 °C, 20 min) while fermentation inhibitors were not detected. FTIR spectra revealed that the mild SE conditions helped to release monosaccharides from the polysaccharides. Bacillus subtilis fermentation productivity on medium after SE sterilization was 3.83 times of that after conventional sterilization. Therefore, SE shortened sterilization time and improved SM nutrition, which facilitated fermentability of SM and should promote economy of solid-state fermentation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Xylose induces cellulase production in Thermoascus aurantiacus.

PubMed

Schuerg, Timo; Prahl, Jan-Philip; Gabriel, Raphael; Harth, Simon; Tachea, Firehiwot; Chen, Chyi-Shin; Miller, Matthew; Masson, Fabrice; He, Qian; Brown, Sarah; Mirshiaghi, Mona; Liang, Ling; Tom, Lauren M; Tanjore, Deepti; Sun, Ning; Pray, Todd R; Singer, Steven W

2017-01-01

Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus . Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted to produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. Xylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.

Utilization of xylose for growth by the eukaryotic alga, Chlorella.

PubMed

Hawkins, R L

1999-06-01

A green alga, Chlorella, was found to be capable of utilizing xylose or other pentose sugars (xylitol, arabinose) for enhanced growth rates when grown in the light, but not when grown heterotrophically in the dark. With selection for growth in xylose-containing medium, it was possible to improve dramatically the ability of selected Chlorella strains to grow on xylose mixotrophically. Growth on arabinose or xylitol was not changed in the xylose-selected strains.

Analysis of mixtures of fatty acids and fatty alcohols in fermentation broth.

PubMed

Liu, Yilan; Chen, Ting; Yang, Maohua; Wang, Caixia; Huo, Weiyan; Yan, Daojiang; Chen, Jinjin; Zhou, Jiemin; Xing, Jianmin

2014-01-03

Microbial production of fatty acids and fatty alcohols has attracted increasing concerns because of energy crisis and environmental impact of fossil fuels. Therefore, simple and efficient methods for the extraction and quantification of these compounds become necessary. In this study, a high-performance liquid chromatography-refractive index detection (HPLC-RID) method was developed for the simultaneous quantification of fatty acids and fatty alcohols in these samples. The optimum chromatographic conditions are C18 column eluted with methanol:water:acetic acid (90:9.9:0.1, v/v/v); column temperature, 26°C; flow rate, 1.0mL/min. Calibration curves of all selected analytes showed good linearity (r(2)≥0.9989). The intra-day and inter-day relative standard deviations (RSDs) of the 10 compounds were less than 4.46% and 5.38%, respectively, which indicated that the method had good repeatability and precision. Besides, a method for simultaneous extraction of fatty acids and fatty alcohols from fermentation broth was optimized by orthogonal design. The optimal extraction conditions were as follows: solvent, ethyl acetate; solvent to sample ratio, 0.5:1; rotation speed, 2min at 260rpm; extraction temperature, 10°C. This study provides simple and fast methods to simultaneously extract and quantify fatty acids and fatty alcohols for the first time. It will be useful for the study of microbial production of these products. Copyright © 2013 Elsevier B.V. All rights reserved.

Impact of alternative technique to ageing using oak chips in alcoholic or in malolactic fermentation on volatile and sensory composition of red wines.

PubMed

Gómez García-Carpintero, E; Gómez Gallego, M A; Sánchez-Palomo, E; González Viñas, M A

2012-09-15

This paper reports on a complete study of the effect of wood, in the form of oak chips, on the volatile composition and sensory characteristics of Moravia Agria wines added at different stages of the fermentation process. Aroma compounds were analyzed by gas chromatography-mass spectrometry (GC-MS). Sensory profile was evaluated by experienced wine-testers. Oak chips were added to wines in two dose rates at different stages of the winemaking process: during alcoholic fermentation (AF), during malolactic fermentation (MLF) and in young, red Moravia Agria wine. Wines fermented with oak chips during AF showed higher concentrations of the ethyl esters of straight-chain fatty acids, ethyl, hexyl, isoamyl acetates and superior alcohols than the control wines. The higher concentrations of benzene compound, oak lactones and furanic compounds were found in wines in contact with oak chips during MLF. The use of oak chips gives rise to a different sensorial profile of wines depending of the point of addition. Higher intensities of woody, coconut, vanilla and sweet spices descriptors were obtained when a large dose rate of chips was employed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Experiments with Fungi Part 2: Fermentation.

ERIC Educational Resources Information Center

Dale, Michele; Hetherington, Shane

1996-01-01

Gives details of three experiments with alcoholic fermentation by yeasts which yield carbon dioxide and ethanol. Lists procedures for making cider, vinegar, and fermentation gases. Provides some historical background and detailed equipment requirements. (DDR)

Evaluation of nonionic adsorbent resins for removal of inhibitory compounds from corncob hydrolysate for ethanol fermentation.

PubMed

Hatano, Ken-ichi; Aoyagi, Naokazu; Miyakawa, Takuya; Tanokura, Masaru; Kubota, Kenji

2013-12-01

The aim of this study was to investigate the effect of XAD4-column treatment on removal of several fermentation inhibitors from corncob hydrolysate (CH). From analysis using a model hydrolysate, more than 99% of 5-hydroxy-methyl furfural, furfural and vanillin were removed by this treatment, and more than 97% of the total xylose, glucose and arabinose remained in the detoxified CH (DCH). The resulting DCH was tested as a substrate for ethanol production by Saccharomyces cerevisiae and Pichia stipitis. The highest ethanol levels for S. cerevisiae were 1.40 and 4.92 g l(-1) in CH and DCH, respectively. For P. stipitis, the levels were 0 and 4.73 g l(-1) in the CH and DCH media, respectively. The levels of alcohol volumetric productivity in the DCH medium were 0.374 and 0.200 g l(-1)h(-1) for S. cerevisiae and P. stipitis, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

The impact of oxygen on the final alcohol content of wine fermented by a mixed starter culture.

PubMed

Morales, Pilar; Rojas, Virginia; Quirós, Manuel; Gonzalez, Ramon

2015-05-01

We have developed a wine fermentation procedure that takes advantage of the metabolic features of a previously characterized Metschnikowia pulcherrima strain in order to reduce ethanol production. It involves the use of M. pulcherrima/Saccharomyces cerevisiae mixed cultures, controlled oxygenation conditions during the first 48 h of fermentation, and anaerobic conditions thereafter. The influence of different oxygenation regimes and initial inoculum composition on yeast physiology and final ethanol content was studied. The impact of oxygenation on yeast physiology goes beyond the first aerated step and influences yields and survival rates during the anaerobic stage. The activity of M. pulcherrima in mixed oxygenated cultures resulted in a clear reduction in ethanol yield, as compared to S. cerevisiae. Despite relatively low initial cell numbers, S. cerevisiae always predominated in mixed cultures by the end of the fermentation process. Strain replacement was faster under low oxygenation levels. M. pulcherrima confers an additional advantage in terms of dissolved oxygen, which drops to zero after a few hours of culture, even under highly aerated conditions, and this holds true for mixed cultures. Alcohol reduction values about 3.7 % (v/v) were obtained for mixed cultures under high aeration, but they were associated to unacceptable volatile acidity levels. In contrast, under optimized conditions, only 0.35 g/L acetic acid was produced, for an alcohol reduction of 2.2 % (v/v), and almost null dissolved oxygen during the process.

Cell wall polysaccharides released during the alcoholic fermentation by Schizosaccharomyces pombe and S. japonicus: quantification and characterization

PubMed Central

Domizio, P.; Liu, Y.; Bisson, L.F.; Barile, D.

2016-01-01

The present work demonstrates that yeasts belonging to the Schizosaccharomyces genus release a high quantity of polysaccharides of cell wall origin starting from the onset of the alcoholic fermentation. By the end of the alcoholic fermentation, all of the Schizosaccharomyces yeast strains released a quantity of polysaccharides approximately 3-7 times higher than that released by a commercial Saccharomyces cerevisiae yeast strain under the same fermentative conditions of synthetic juice. A higher content of polysaccharide was found in media fermented by Schizosaccharomyces japonicus with respect to that of Schizosaccharomyces pombe. Some of the strains evaluated were also able to produce high levels of pyruvic acid, which has been shown to be an important compound for color stability of wine. The presence of strains with different malic acid consumption patterns along with high polysaccharide release would enable production of naturally modified wines with enhanced mouth feel and reduced acidity. The chemical analysis of the released polysaccharides demonstrated divergence between the two yeast species S. pombe and S. japonicus. A different mannose/galactose ratio and a different percentage of proteins was observed on the polysaccharides released by S. pombe as compared to S. japonicus. Analysis of the proteins released in the media revealed the presence of a glycoprotein with a molecular size around 32-33 kDa only for the species S. japonicus. Mass spectrometry analysis of carbohydrate moieties showed similar proportions among the N-glycan chains released in the media by both yeast species but differences between the two species were also observed. These observations suggest a possible role of rapid MALDI-TOF screening of N-glycans compositional fingerprint as a taxonomic tool for this genus. Polysaccharides release in the media, in particular galactomannoproteins in significant amounts, could make these yeasts particularly interesting also for the industrial

[Determination of sugars, organic acids and alcohols in microbial consortium fermentation broth from cellulose using high performance liquid chromatography].

PubMed

Jiang, Yan; Fan, Guifang; Du, Ran; Li, Peipei; Jiang, Li

2015-08-01

A high performance liquid chromatographic method was established for the determination of metabolites (sugars, organic acids and alcohols) in microbial consortium fermentation broth from cellulose. Sulfate was first added in the samples to precipitate calcium ions in microbial consortium culture medium and lower the pH of the solution to avoid the dissociation of organic acids, then the filtrates were effectively separated using high performance liquid chromatography. Cellobiose, glucose, ethanol, butanol, glycerol, acetic acid and butyric acid were quantitatively analyzed. The detection limits were in the range of 0.10-2.00 mg/L. The linear correlation coefficients were greater than 0.999 6 in the range of 0.020 to 1.000 g/L. The recoveries were in the range of 85.41%-115.60% with the relative standard deviations of 0.22% -4.62% (n = 6). This method is accurate for the quantitative analysis of the alcohols, organic acids and saccharides in microbial consortium fermentation broth from cellulose.

Volatile flavour profile of reduced alcohol wines fermented with the non-conventional yeast species Metschnikowia pulcherrima and Saccharomyces uvarum.

PubMed

Varela, C; Sengler, F; Solomon, M; Curtin, C

2016-10-15

Production of quality wines with decreased alcohol concentration continues to be one of the major challenges facing wine producers. Therefore, there is considerable interest in the isolation or generation of wine yeasts less efficient at transforming grape sugars into ethanol. We recently demonstrated that Metschnikowia pulcherrima AWRI1149 and Saccharomyces uvarum AWRI2846 were both able to produce reduced alcohol wine when used in sequential inoculation with Saccharomyces cerevisiae. This effect is additive when both strains are co-inoculated in grape must. Here we describe the volatile flavour profile of Chardonnay and Shiraz wines produced with these two strains. Wines fermented with M. pulcherrima showed concentrations of ethyl acetate likely to affect negatively wine aroma. Wines fermented with S. uvarum and with a combination of M. pulcherrima and S. uvarum were characterised by increased concentrations of 2-phenyl ethanol and 2-phenylethyl acetate, both associated with positive sensory attributes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

Treesearch

Yong-Su Jin; Thomas W. Jeffries

2003-01-01

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes(XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into...

Engineering genome-reduced Bacillus subtilis for acetoin production from xylose.

PubMed

Yan, Panpan; Wu, Yuanqing; Yang, Li; Wang, Zhiwen; Chen, Tao

2018-02-01

To investigate the capacity of a genome-reduced Bacillus subtilis strain as chassis cell for acetoin production from xylose. To endow the genome-reduced Bacillus subtilis strain BSK814 with the ability to utilize xylose, we inserted a native xyl operon into its genome and deleted the araR gene. The resulting strain BSK814A2 produced 2.94 g acetoin/l from 10 g xylose/l, which was 39% higher than control strain BSK19A2. The deletion of the bdhA and acoA genes further improved xylose utilization efficiency and increased acetoin production to 3.71 g/l in BSK814A4. Finally, BSK814A4 produced up to 23.3 g acetoin/l from 50 g xylose/l, with a yield of 0.46 g/g xylose. Both the titer and yield were 39% higher than those of control strain BSK19A4. As a chassis cell, genome-reduced B. subtilis showed significantly improved capacity for the production of the overflow product acetoin from xylose compared with wild-type strain.

Succinic acid production on xylose-enriched biorefinery streams by Actinobacillus succinogenes in batch fermentation

DOE PAGES

Salvachua, Davinia; Mohagheghi, Ali; Smith, Holly; ...

2016-02-02

Co-production of chemicals from lignocellulosic biomass alongside fuels holds promise for improving the economic outlook of integrated biorefineries. In current biochemical conversion processes that use thermochemical pretreatment and enzymatic hydrolysis, fractionation of hemicellulose-derived and cellulose-derived sugar streams is possible using hydrothermal or dilute acid pretreatment (DAP), which then offers a route to parallel trains for fuel and chemical production from xylose- and glucose-enriched streams. Succinic acid (SA) is a co-product of particular interest in biorefineries because it could potentially displace petroleum-derived chemicals and polymer precursors for myriad applications. Furthermore, SA production from biomass-derived hydrolysates has not yet been fully exploredmore » or developed.« less

Butanol production from wood pulping hydrolysate in an integrated fermentation-gas stripping process

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

Lu, CC; Dong, J; Yang, ST

2013-09-01

Wood pulping hydrolysate (WPH) containing mainly xylose and glucose as a potential substrate for acetone-butanol-ethanol (ABE) fermentation was studied. Due to the inhibitors present in the hydrolysate, several dilution levels and detoxification treatments, including overliming, activated charcoal adsorption, and resin adsorption, were evaluated for their effectiveness in relieving the inhibition on fermentation. Detoxification using resin and evaporation was found to be the most effective method in reducing the toxicity of WPH. ABE production in batch fermentation by Clostridium beijerinckii increased 68%, from 6.73 g/L in the non-treated and non-diluted WPH to 11.35 g/L in the resin treated WPH. With gasmore » stripping for in situ product removal, ABE production from WPH increased to 17.73 g/L, demonstrating that gas stripping was effective in alleviating butanol toxicity by selectively separating butanol from the fermentation broth, which greatly improved solvents production and sugar conversion in the fermentation. (C) 2013 Elsevier Ltd. All rights reserved.« less

Butanol production from wood pulping hydrolysate in an integrated fermentation-gas stripping process.

PubMed

Lu, Congcong; Dong, Jie; Yang, Shang-Tian

2013-09-01

Wood pulping hydrolysate (WPH) containing mainly xylose and glucose as a potential substrate for acetone-butanol-ethanol (ABE) fermentation was studied. Due to the inhibitors present in the hydrolysate, several dilution levels and detoxification treatments, including overliming, activated charcoal adsorption, and resin adsorption, were evaluated for their effectiveness in relieving the inhibition on fermentation. Detoxification using resin and evaporation was found to be the most effective method in reducing the toxicity of WPH. ABE production in batch fermentation by Clostridium beijerinckii increased 68%, from 6.73 g/L in the non-treated and non-diluted WPH to 11.35 g/L in the resin treated WPH. With gas stripping for in situ product removal, ABE production from WPH increased to 17.73 g/L, demonstrating that gas stripping was effective in alleviating butanol toxicity by selectively separating butanol from the fermentation broth, which greatly improved solvents production and sugar conversion in the fermentation. Copyright © 2013 Elsevier Ltd. All rights reserved.

RNAi assisted genome evolution unveils yeast mutants with improved xylose utilization.

PubMed

HamediRad, Mohammad; Lian, Jiazhang; Li, Hejun; Zhao, Huimin

2018-06-01

Xylose is a major component of lignocellulosic biomass, one of the most abundant feedstocks for biofuel production. Therefore, efficient and rapid conversion of xylose to ethanol is crucial in the viability of lignocellulosic biofuel plants. In this study, RNAi Assisted Genome Evolution (RAGE) was used to improve the xylose utilization rate in SR8, one of the most efficient publicly available xylose utilizing Saccharomyces cerevisiae strains. To identify gene targets for further improvement, we created a genome-scale library consisting of both genetic over-expression and down-regulation mutations in SR8. Followed by screening in media containing xylose as the sole carbon source, yeast mutants with 29% faster xylose utilization, and 45% higher ethanol productivity were obtained relative to the parent strain. Two known and two new effector genes were identified in these mutant strains. Notably, down-regulation of CDC11, an essential gene, resulted in faster xylose utilization, and this gene target cannot be identified in genetic knock-out screens. © 2018 Wiley Periodicals, Inc.

Population size drives industrial Saccharomyces cerevisiae alcoholic fermentation and is under genetic control.

PubMed

Albertin, Warren; Marullo, Philippe; Aigle, Michel; Dillmann, Christine; de Vienne, Dominique; Bely, Marina; Sicard, Delphine

2011-04-01

Alcoholic fermentation (AF) conducted by Saccharomyces cerevisiae has been exploited for millennia in three important human food processes: beer and wine production and bread leavening. Most of the efforts to understand and improve AF have been made separately for each process, with strains that are supposedly well adapted. In this work, we propose a first comparison of yeast AFs in three synthetic media mimicking the dough/wort/grape must found in baking, brewing, and wine making. The fermentative behaviors of nine food-processing strains were evaluated in these media, at the cellular, populational, and biotechnological levels. A large variation in the measured traits was observed, with medium effects usually being greater than the strain effects. The results suggest that human selection targeted the ability to complete fermentation for wine strains and trehalose content for beer strains. Apart from these features, the food origin of the strains did not significantly affect AF, suggesting that an improvement program for a specific food processing industry could exploit the variability of strains used in other industries. Glucose utilization was analyzed, revealing plastic but also genetic variation in fermentation products and indicating that artificial selection could be used to modify the production of glycerol, acetate, etc. The major result was that the overall maximum CO(2) production rate (V(max)) was not related to the maximum CO(2) production rate per cell. Instead, a highly significant correlation between V(max) and the maximum population size was observed in all three media, indicating that human selection targeted the efficiency of cellular reproduction rather than metabolic efficiency. This result opens the way to new strategies for yeast improvement.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

PubMed

Zepeda, S; Monasterio, O; Ureta, T

1990-03-15

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

Transformation of chemical constituents of lychee wine by simultaneous alcoholic and malolactic fermentations.

PubMed

Chen, Dai; Liu, Shao-Quan

2016-04-01

This work examined for the first time the impact of malolactic fermentation (MLF) on the chemical constituents of lychee wine. Oenococcus oeni Viniflora Oenos (MLF inducer) and Saccharomyces cerevisiae MERIT.ferm were co-inoculated into lychee juice to induce simultaneous alcoholic fermentation (AF) and MLF. MLF did not affect sugar utilisation and ethanol production statistically (8.54% v/v for MLF and 9.27% v/v for AF). However, MLF resulted in dramatic degradation of malic and citric acids with concomitant increases of lactic acid, ethyl lactate and pH. The final concentrations of acetic and succinic acids between AF and MLF wines had no significant difference. The MLF wine contained significantly higher amounts of amino acids than the AF wine. More importantly, MLF significantly elevated the levels of potent aroma-active compounds including isoamyl acetate, linalool, geraniol and cis-rose oxide (to levels above or near respective detection thresholds), suggesting that MLF is an effective way of retaining the original lychee flavour. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Effect of phenolic ketones on ethanol fermentation and cellular lipid composition of Pichia stipitis].

PubMed

Yang, Jinlong; Cheng, Yichao; Zhu, Yuanyuan; Zhu, Junjun; Chen, Tingting; Xu, Yong; Yong, Qiang; Yu, Shiyuan

2016-02-01

Lignin degradation products are toxic to microorganisms, which is one of the bottlenecks for fuel ethanol production. We studied the effects of phenolic ketones (4-hydroxyacetophenone, 4-hydroxy-3-methoxy-acetophenone and 4-hydroxy-3,5-dimethoxy-acetophenone) derived from lignin degradation on ethanol fermentation of xylose and cellular lipid composition of Pichia stipitis NLP31. Ethanol and the cellular fatty acid of yeast were analyzed by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Results indicate that phenolic ketones negatively affected ethanol fermentation of yeast and the lower molecular weight phenolic ketone compound was more toxic. When the concentration of 4-hydroxyacetophenone was 1.5 g/L, at fermentation of 24 h, the xylose utilization ratio, ethanol yield and ethanol concentration decreased by 42.47%, 5.30% and 9.76 g/L, respectively, compared to the control. When phenolic ketones were in the medium, the ratio of unsaturated fatty acids to saturated fatty acids (UFA/SFA) of yeast cells was improved. When 1.5 g/L of three aforementioned phenolic ketones was added to the fermentation medium, the UFA/SFA ratio of yeast cells increased to 3.03, 3.06 and 3.61, respectively, compared to 2.58 of the control, which increased cell membrane fluidity and instability. Therefore, phenolic ketones can reduce the yeast growth, increase the UFA/SFA ratio of yeast and lower ethanol productivity. Effectively reduce or remove the content of lignin degradation products is the key to improve lignocellulose biorefinery.

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production.

PubMed

Jo, Suah; Yoon, Jinkyung; Lee, Sun-Mi; Um, Youngsoon; Han, Sung Ok; Woo, Han Min

2017-09-20

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization. Copyright © 2017 Elsevier B.V. All rights reserved.

27 CFR 24.212 - High fermentation wine.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., DEPARTMENT OF THE TREASURY LIQUORS WINE Production of Other Than Standard Wine § 24.212 High fermentation wine. High fermentation wine is wine made with the addition of sugar within the limitations prescribed... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false High fermentation wine. 24...

27 CFR 24.212 - High fermentation wine.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., DEPARTMENT OF THE TREASURY LIQUORS WINE Production of Other Than Standard Wine § 24.212 High fermentation wine. High fermentation wine is wine made with the addition of sugar within the limitations prescribed... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false High fermentation wine. 24...

27 CFR 24.212 - High fermentation wine.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false High fermentation wine. 24..., DEPARTMENT OF THE TREASURY LIQUORS WINE Production of Other Than Standard Wine § 24.212 High fermentation wine. High fermentation wine is wine made with the addition of sugar within the limitations prescribed...

Simultaneous Alcoholic and Malolactic Fermentations by Saccharomyces cerevisiae and Oenococcus oeni Cells Co-immobilized in Alginate Beads

PubMed Central

Bleve, Gianluca; Tufariello, Maria; Vetrano, Cosimo; Mita, Giovanni; Grieco, Francesco

2016-01-01

Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF). However, the inoculation of lactic acid bacteria together with yeast starter cultures is a promising system to enhance the quality and safety of wine. In recent years, the use of immobilized cell systems has been investigated, with interesting results, for the production of different fermented foods and beverages. In this study we have carried out the simultaneous immobilization of Saccharomyces cerevisiae and Oenococcus oeni in alginate beads and used them in microvinifications tests to produce Negroamaro wine. The process was monitored by chemical and sensorial analyses and dominance of starters and cell leaking from beads were also checked. Co-immobilization of S. cerevisiae and O. oeni allowed to perform an efficient fermentation process, producing low volatile acidity levels and ethanol and glycerol concentrations comparable with those obtained by cell sequential inoculum and co-inoculum of yeast and bacteria cells in free form. More importantly, co-immobilization strategy produced a significant decrease of the time requested to complete AF and MLF. The immobilized cells could be efficiently reused for the wine fermentation at least three times without any apparent loss of cell metabolic activities. This integrated biocatalytic system is able to perform simultaneously AF and MLF, producing wines similar in organoleptic traits in comparison with wines fermented following traditional sequential AF and MLF with free cell starters. The immobilized-cell system, that we here describe for the first time in our knowledge, offers many advantages over conventional free cell fermentations, including: (i) elimination of non-productive cell growth phases; (ii) feasibility of continuous processing; (iii) re-use of the biocatalyst. PMID:27379072

Genetic engineering of Pichia stipitis for fermentation of xylose

Treesearch

Thomas W. Jeffries; N. Q. Shi; J. Y. Cho; P. Lu; K. Dahn; J. Hendrick; H. K. Sreenath

1998-01-01

A useful genetic system has been developed for the transformation of Pichia stipitis. This includes two selectable markers (URA3 and LEU2), integrating and autonomous replication vectors, a pop-out cassette that enables multiple targeted disruptions, and a genomic X-library for rapid cloning. Using this system we have cloned two genes for alcohol dehydrogenase (PsADH1...

Saccharomyces cerevisiae engineered for xylose metabolism exhibits a respiratory response

Treesearch

Yong-Su Jin; Jose M. Laplaza; Thomas W. Jeffries

2004-01-01

Native strains of Saccharomyces cerevisiae do not assimilate xylose. S. cerevisiae engineered for D-xylose utilization through the heterologous expression of genes for aldose reductase ( XYL1), xylitol dehydrogenase (XYL2), and D-xylulokinase ( XYL3 or XKS1) produce only limited amounts of ethanol in xylose medium. In recombinant S. cerevisiae expressing XYL1, XYL2,...

Fermentation Industry.

ERIC Educational Resources Information Center

Grady, C. P. L., Jr.; Grady, J. K.

1978-01-01

Presents a literature review of wastes from the fermentation industry, covering publications of 1976-77. This review focuses on: (1) alcoholic beverage production; (2) pharmaceuticals and biochemicals production; and (3) biomass production. A list of 62 references is also presented. (HM)

The Fermentative and Aromatic Ability of Kloeckera and Hanseniaspora Yeasts

NASA Astrophysics Data System (ADS)