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Sample records for zymomonas mobilis zm4

  1. Systems Biology Analysis of Zymomonas mobilis ZM4 Ethanol Stress Responses

    PubMed Central

    Yang, Shihui; Pan, Chongle; Tschaplinski, Timothy J.; Hurst, Gregory B.; Engle, Nancy L.; Zhou, Wen; Dam, PhuongAn; Xu, Ying; Rodriguez, Miguel; Dice, Lezlee; Johnson, Courtney M.; Davison, Brian H.; Brown, Steven D.

    2013-01-01

    Background Zymomonas mobilis ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully. Methodology/Principal Findings In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update Z. mobilis ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. Conclusions Our study has provided insights into the responses of Z. mobilis to ethanol stress through an integrated “omics” approach for the first time. This systems biology study elucidated key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress. PMID:23874800

  2. Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4.

    PubMed

    Cao, Qing-Hua; Shao, Huan-Huan; Qiu, Hui; Li, Tao; Zhang, Yi-Zheng; Tan, Xue-Mei

    2017-03-01

    The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.

  3. Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032.

    PubMed

    Yang, Shihui; Vera, Jessica M; Grass, Jeff; Savvakis, Giannis; Moskvin, Oleg V; Yang, Yongfu; McIlwain, Sean J; Lyu, Yucai; Zinonos, Irene; Hebert, Alexander S; Coon, Joshua J; Bates, Donna M; Sato, Trey K; Brown, Steven D; Himmel, Michael E; Zhang, Min; Landick, Robert; Pappas, Katherine M; Zhang, Yaoping

    2018-01-01

    Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2-8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. In this study, we determined the complete chromosome and plasmid sequences of ZM4 and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1-2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion. The complete genome of ZM4 and two xylose

  4. Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

    SciT

    Yang, Shihui; Vera, Jessica M.; Grass, Jeff

    Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2-8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. In this study, we determined the complete chromosome and plasmid sequences of ZM4more » and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1-2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion. The complete genome of ZM4 and two

  5. Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

    DOE PAGES

    Yang, Shihui; Vera, Jessica M.; Grass, Jeff; ...

    2018-05-02

    Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2-8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. In this study, we determined the complete chromosome and plasmid sequences of ZM4more » and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1-2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion. The complete genome of ZM4 and two

  6. Isolation of noninhibitory strains of Zymomonas mobilis

    SciT

    Haffie, T.L.; Louie, P.W.; Khachatourians, G.G.

    1985-04-01

    Wild-type Zymomonas mobilis strains inhibit the growth of Escherichia coli. The authors report the first isolation of noninhibitory strains, called Zymomonas inhibition negative (Zin/sup -/), after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. A standardized soft-agar overlay procedure for detecting E. coli growth inhibition was also developed.

  7. 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.

  8. Improving furfural tolerance of Zymomonas mobilis by rewiring a sigma factor RpoD protein.

    PubMed

    Tan, Fu-Rong; Dai, Li-Chun; Wu, Bo; Qin, Han; Shui, Zong-Xia; Wang, Jing-Li; Zhu, Qi-Li; Hu, Qi-Chun; Ruan, Zhi-Yong; He, Ming-Xiong

    2015-06-01

    Furfural from lignocellulosic hydrolysates is the key inhibitor for bio-ethanol fermentation. In this study, we report a strategy of improving the furfural tolerance in Zymomonas mobilis on the transcriptional level by engineering its global transcription sigma factor (σ(70), RpoD) protein. Three furfural tolerance RpoD mutants (ZM4-MF1, ZM4-MF2, and ZM4-MF3) were identified from error-prone PCR libraries. The best furfural-tolerance strain ZM4-MF2 reached to the maximal cell density (OD600) about 2.0 after approximately 30 h, while control strain ZM4-rpoD reached its highest cell density of about 1.3 under the same conditions. ZM4-MF2 also consumed glucose faster and yield higher ethanol; expression levels and key Entner-Doudoroff (ED) pathway enzymatic activities were also compared to control strain under furfural stress condition. Our results suggest that global transcription machinery engineering could potentially be used to improve stress tolerance and ethanol production in Z. mobilis.

  9. Continuous production of ethanol by use of flocculent zymomonas mobilis

    DOEpatents

    Arcuri, Edward J.; Donaldson, Terrence L.

    1983-01-01

    Ethanol is produced by means of a floc-forming strain of Zymomonas mobilis bacteria. Gas is vented along the length of a column containing the flocculent bacteria to preclude disruption of liquid flow.

  10. The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors

    SciT

    Yang, Shihui; Pelletier, Dale A; Lu, Tse-Yuan

    Zymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. To date, few studies have examined its responses to various stresses at the gene level. Hfq is a conserved bacterial member of the Sm-like family of RNA-binding proteins, coordinating a broad array of responses including multiple stress responses. In a previous study, we observed Z. mobilis ZM4 gene ZMO0347 showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions. We have shown the utility of the pKNOCK suicide plasmid for mutant construction in Z. mobilis, and constructed a Gateway compatible expressionmore » plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.« less

  11. Inhibition of growth of Zymomonas mobilis by model compounds found in lignocellulosic hydrolysates

    PubMed Central

    2013-01-01

    Background During the pretreatment of biomass feedstocks and subsequent conditioning prior to saccharification, many toxic compounds are produced or introduced which inhibit microbial growth and in many cases, production of ethanol. An understanding of the toxic effects of compounds found in hydrolysate is critical to improving sugar utilization and ethanol yields in the fermentation process. In this study, we established a useful tool for surveying hydrolysate toxicity by measuring growth rates in the presence of toxic compounds, and examined the effects of selected model inhibitors of aldehydes, organic and inorganic acids (along with various cations), and alcohols on growth of Zymomonas mobilis 8b (a ZM4 derivative) using glucose or xylose as the carbon source. Results Toxicity strongly correlated to hydrophobicity in Z. mobilis, which has been observed in Escherichia coli and Saccharomyces cerevisiae for aldehydes and with some exceptions, organic acids. We observed Z. mobilis 8b to be more tolerant to organic acids than previously reported, although the carbon source and growth conditions play a role in tolerance. Growth in xylose was profoundly inhibited by monocarboxylic organic acids compared to growth in glucose, whereas dicarboxylic acids demonstrated little or no effects on growth rate in either substrate. Furthermore, cations can be ranked in order of their toxicity, Ca++ > > Na+ > NH4+ > K+. HMF (5-hydroxymethylfurfural), furfural and acetate, which were observed to contribute to inhibition of Z. mobilis growth in dilute acid pretreated corn stover hydrolysate, do not interact in a synergistic manner in combination. We provide further evidence that Z. mobilis 8b is capable of converting the aldehydes furfural, vanillin, 4-hydroxybenzaldehyde and to some extent syringaldehyde to their alcohol forms (furfuryl, vanillyl, 4-hydroxybenzyl and syringyl alcohol) during fermentation. Conclusions Several key findings in this report provide a

  12. Single Zymomonas mobilis strain for xylose and arabinose fermentation

    DOEpatents

    Zhang, M.; Chou, Y.C.; Picataggio, S.K.; Finkelstein, M.

    1998-12-01

    This invention relates to single microorganisms which normally do not ferment pentose sugars which are genetically altered to ferment the pentose sugars, xylose and arabinose, to produce ethanol, and a fermentation process utilizing the same. Examples include Zymomonas mobilis which has been transformed with a combination of E. coli genes for xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase and transketolase. Expression of added genes are under the control of Z. mobilis promoters. These newly created microorganisms are useful for fermenting glucose, xylose and arabinose, produced by hydrolysis of hemicellulose and cellulose or starch, to produce ethanol. 6 figs.

  13. Single zymomonas mobilis strain for xylose and arabinose fermentation

    DOEpatents

    Zhang, Min; Chou, Yat-Chen; Picataggio, Stephen K.; Finkelstein, Mark

    1998-01-01

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

  14. Can Zymomonas mobilis Substitute Saccharomyces cerevisiae in Cereal Dough Leavening?

    PubMed Central

    Musatti, Alida; Mapelli, Chiara

    2018-01-01

    Baker’s yeast intolerance is rising among Western populations, where Saccharomyces cerevisiae is spread in fermented food and food components. Zymomonas mobilis is a bacterium commonly used in tropical areas to produce alcoholic beverages, and it has only rarely been considered for dough leavening probably because it only ferments glucose, fructose and sucrose, which are scarcely present in flour. However, through alcoholic fermentation, similarly to S. cerevisiae, it provides an equimolar mixture of ethanol and CO2 that can rise a dough. Here, we propose Z. mobilis as a new leavening agent, as an alternative to S. cerevisiae, overcoming its technological limit with different strategies: (1) adding glucose to the dough formulation; and (2) exploiting the maltose hydrolytic activity of Lactobacillus sanfranciscensis associated with Z. mobilis. CO2 production, dough volume increase, pH value, microbial counts, sugars consumption and ethanol production were monitored. Results suggest that glucose addition to the dough lets Z. mobilis efficiently leaven a dough, while glucose released by L. sanfranciscensis is not so well fermented by Z. mobilis, probably due to the strong acidification. Nevertheless, the use of Z. mobilis as a leavening agent could contribute to increasing the variety of baked goods alternative to those leavened by S. cerevisiae. PMID:29659515

  15. Transcriptome profiling of Zymomonas mobilis under furfural stress.

    PubMed

    He, Ming-xiong; Wu, Bo; Shui, Zong-xia; Hu, Qi-chun; Wang, Wen-guo; Tan, Fu-rong; Tang, Xiao-yu; Zhu, Qi-li; Pan, Ke; Li, Qing; Su, Xiao-hong

    2012-07-01

    Furfural from lignocellulosic hydrolysates is the prevalent inhibitor to microorganisms during cellulosic ethanol production, but the molecular mechanisms of tolerance to this inhibitor in Zymomonas mobilis are still unclear. In this study, genome-wide transcriptional responses to furfural were investigated in Z. mobilis using microarray analysis. We found that 433 genes were differentially expressed in response to furfural. Furfural up- or down-regulated genes related to cell wall/membrane biogenesis, metabolism, and transcription. However, furfural has a subtle negative effect on Entner-Doudoroff pathway mRNAs. Our results revealed that furfural had effects on multiple aspects of cellular metabolism at the transcriptional level and that membrane might play important roles in response to furfural. This research has provided insights into the molecular response to furfural in Z. mobilis, and it will be helpful to construct more furfural-resistant strains for cellulosic ethanol production.

  16. Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

    PubMed

    Shui, Zong-Xia; Qin, Han; Wu, Bo; Ruan, Zhi-yong; Wang, Lu-shang; Tan, Fu-Rong; Wang, Jing-Li; Tang, Xiao-Yu; Dai, Li-Chun; Hu, Guo-Quan; He, Ming-Xiong

    2015-07-01

    Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

  17. Flocculating Zymomonas mobilis is a promising host to be engineered for fuel ethanol production from lignocellulosic biomass.

    PubMed

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

    2014-03-01

    Whereas Saccharomyces cerevisiae uses the Embden-Meyerhof-Parnas pathway to metabolize glucose, Zymomonas mobilis uses the Entner-Doudoroff (ED) pathway. Employing the ED pathway, 50% less ATP is produced, which could lead to less biomass being accumulated during fermentation and an improved yield of ethanol. Moreover, Z. mobilis cells, which have a high specific surface area, consume glucose faster than S. cerevisiae, which could improve ethanol productivity. We performed ethanol fermentations using these two species under comparable conditions to validate these speculations. Increases of 3.5 and 3.3% in ethanol yield, and 58.1 and 77.8% in ethanol productivity, were observed in ethanol fermentations using Z. mobilis ZM4 in media containing ∼100 and 200 g/L glucose, respectively. Furthermore, ethanol fermentation bythe flocculating Z. mobilis ZM401 was explored. Although no significant difference was observed in ethanol yield and productivity, the flocculation of the bacterial species enabled biomass recovery by cost-effective sedimentation, instead of centrifugation with intensive capital investment and energy consumption. In addition, tolerance to inhibitory byproducts released during biomass pretreatment, particularly acetic acid and vanillin, was improved. These experimental results indicate that Z. mobilis, particularly its flocculating strain, is superior to S. cerevisiae as a host to be engineered for fuel ethanol production from lignocellulosic biomass. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Zymomonas mobilis: a novel platform for future biorefineries

    PubMed Central

    2014-01-01

    Biosynthesis of liquid fuels and biomass-based building block chemicals from microorganisms have been regarded as a competitive alternative route to traditional. Zymomonas mobilis possesses a number of desirable characteristics for its special Entner-Doudoroff pathway, which makes it an ideal platform for both metabolic engineering and commercial-scale production of desirable bio-products as the same as Escherichia coli and Saccharomyces cerevisiae based on consideration of future biomass biorefinery. Z. mobilis has been studied extensively on both fundamental and applied level, which will provide a basis for industrial biotechnology in the future. Furthermore, metabolic engineering of Z. mobilis for enhancing bio-ethanol production from biomass resources has been significantly promoted by different methods (i.e. mutagenesis, adaptive laboratory evolution, specific gene knock-out, and metabolic engineering). In addition, the feasibility of representative metabolites, i.e. sorbitol, bionic acid, levan, succinic acid, isobutanol, and isobutanol produced by Z. mobilis and the strategies for strain improvements are also discussed or highlighted in this paper. Moreover, this review will present some guidelines for future developments in the bio-based chemical production using Z. mobilis as a novel industrial platform for future biofineries. PMID:25024744

  19. Zymomonas mobilis: a novel platform for future biorefineries.

    PubMed

    He, Ming Xiong; Wu, Bo; Qin, Han; Ruan, Zhi Yong; Tan, Fu Rong; Wang, Jing Li; Shui, Zong Xia; Dai, Li Chun; Zhu, Qi Li; Pan, Ke; Tang, Xiao Yu; Wang, Wen Guo; Hu, Qi Chun

    2014-01-01

    Biosynthesis of liquid fuels and biomass-based building block chemicals from microorganisms have been regarded as a competitive alternative route to traditional. Zymomonas mobilis possesses a number of desirable characteristics for its special Entner-Doudoroff pathway, which makes it an ideal platform for both metabolic engineering and commercial-scale production of desirable bio-products as the same as Escherichia coli and Saccharomyces cerevisiae based on consideration of future biomass biorefinery. Z. mobilis has been studied extensively on both fundamental and applied level, which will provide a basis for industrial biotechnology in the future. Furthermore, metabolic engineering of Z. mobilis for enhancing bio-ethanol production from biomass resources has been significantly promoted by different methods (i.e. mutagenesis, adaptive laboratory evolution, specific gene knock-out, and metabolic engineering). In addition, the feasibility of representative metabolites, i.e. sorbitol, bionic acid, levan, succinic acid, isobutanol, and isobutanol produced by Z. mobilis and the strategies for strain improvements are also discussed or highlighted in this paper. Moreover, this review will present some guidelines for future developments in the bio-based chemical production using Z. mobilis as a novel industrial platform for future biofineries.

  20. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell

    1992-01-01

    The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

  1. Stable zymomonas mobilis xylose and arabinose fermenting strains

    DOEpatents

    Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Taipei, TW

    2008-04-08

    The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided.The transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.

  2. Zymomonas mobilis as a model system for production of biofuels and biochemicals

    SciT

    Yang, Shihui; Fei, Qiang; Zhang, Yaoping

    Zymomonas mobilis is a natural ethanologen with many desirable industrial biocatalyst characteristics. In this review, we will discuss work to develop Z. mobilis as a model system for biofuel production from the perspectives of substrate utilization, development for industrial robustness, potential product spectrum, strain evaluation and fermentation strategies. Lastly, this review also encompasses perspectives related to classical genetic tools and emerging technologies in this context.

  3. Zymomonas mobilis as a model system for production of biofuels and biochemicals

    DOE PAGES

    Yang, Shihui; Fei, Qiang; Zhang, Yaoping; ...

    2016-09-15

    Zymomonas mobilis is a natural ethanologen with many desirable industrial biocatalyst characteristics. In this review, we will discuss work to develop Z. mobilis as a model system for biofuel production from the perspectives of substrate utilization, development for industrial robustness, potential product spectrum, strain evaluation and fermentation strategies. Lastly, this review also encompasses perspectives related to classical genetic tools and emerging technologies in this context.

  4. Improvement of ethanol productivity and energy efficiency by degradation of inhibitors using recombinant Zymomonas mobilis (pHW20a-fdh).

    PubMed

    Dong, Hong-Wei; Fan, Li-Qiang; Luo, Zichen; Zhong, Jian-Jiang; Ryu, Dewey D Y; Bao, Jie

    2013-09-01

    Toxic compounds, such as formic acid, furfural, and hydroxymethylfurfural (HMF) generated during pretreatment of corn stover (CS) at high temperature and low pH, inhibit growth of Zymomonas mobilis and lower the conversion efficiency of CS to biofuel and other products. The inhibition of toxic compounds is considered as one of the major technical barriers in the lignocellulose bioconversion. In order to detoxify and/or degrade these toxic compounds by the model ethanologenic strain Z. mobilis itself in situ the fermentation medium, we constructed a recombinant Z. mobilis ZM4 (pHW20a-fdh) strain that is capable of degrading toxic inhibitor, formate. This is accomplished by cloning heterologous formate dehydrogenase gene (fdh) from Saccharomyces cerevisiae and by coupling this reaction of NADH regeneration reaction system with furfural and HMF degradation in the recombinant Z. mobilis strain. The NADH regeneration reaction also improved both the energy efficiency and cell physiological activity of the recombinant organism, which were definitely confirmed by the improved cell growth, ethanol yield, and ethanol productivity during fermentation with CS hydrolysate. Copyright © 2013 Wiley Periodicals, Inc.

  5. Xylose utilizing zymomonas mobilis with improved ethanol production in biomass hydrolysate medium

    DOEpatents

    Caimi, Perry G; Hitz, William D; Stieglitz, Barry; Viitanen, Paul V

    2013-07-02

    Xylose-utilizing, ethanol producing strains of Zymomonas mobilis with improved performance in medium comprising biomass hydrolysate were isolated using an adaptation process. Independently isolated strains were found to have independent mutations in the same coding region. Mutation in this coding may be engineered to confer the improved phenotype.

  6. Xylose utilizing Zymomonas mobilis with improved ethanol production in biomass hydrolysate medium

    DOEpatents

    Caimi, Perry G; Hitz, William D; Viitanen, Paul V; Stieglitz, Barry

    2013-10-29

    Xylose-utilizing, ethanol producing strains of Zymomonas mobilis with improved performance in medium comprising biomass hydrolysate were isolated using an adaptation process. Independently isolated strains were found to have independent mutations in the same coding region. Mutation in this coding may be engineered to confer the improved phenotype.

  7. N2 gas is an effective fertilizer for bioethanol production by Zymomonas mobilis

    PubMed Central

    Kremer, Timothy A.; LaSarre, Breah; Posto, Amanda L.; McKinlay, James B.

    2015-01-01

    A nascent cellulosic ethanol industry is struggling to become cost-competitive against corn ethanol and gasoline. Millions of dollars are spent on nitrogen supplements to make up for the low nitrogen content of the cellulosic feedstock. Here we show for the first time to our knowledge that the ethanol-producing bacterium, Zymomonas mobilis, can use N2 gas in lieu of traditional nitrogen supplements. Despite being an electron-intensive process, N2 fixation by Z. mobilis did not divert electrons away from ethanol production, as the ethanol yield was greater than 97% of the theoretical maximum. In a defined medium, Z. mobilis produced ethanol 50% faster per cell and generated half the unwanted biomass when supplied N2 instead of ammonium. In a cellulosic feedstock-derived medium, Z. mobilis achieved a similar cell density and a slightly higher ethanol yield when supplied N2 instead of the industrial nitrogen supplement, corn steep liquor. We estimate that N2-utilizing Z. mobilis could save a cellulosic ethanol production facility more than $1 million/y. PMID:25646422

  8. N 2 gas is an effective fertilizer for bioethanol production by Zymomonas mobilis

    DOE PAGES

    Kremer, Timothy A.; LaSarre, Breah; Posto, Amanda L.; ...

    2015-02-02

    A nascent cellulosic ethanol industry is struggling to become cost-competitive against corn ethanol and gasoline. Millions of dollars are spent on nitrogen supplements to make up for the low nitrogen content of the cellulosic feedstock. In this paper, we show for the first time to our knowledge that the ethanol-producing bacterium, Zymomonas mobilis, can use N 2 gas in lieu of traditional nitrogen supplements. Despite being an electron-intensive process, N 2 fixation by Z. mobilis did not divert electrons away from ethanol production, as the ethanol yield was greater than 97% of the theoretical maximum. In a defined medium, Z.more » mobilis produced ethanol 50% faster per cell and generated half the unwanted biomass when supplied N 2 instead of ammonium. In a cellulosic feedstock-derived medium, Z. mobilis achieved a similar cell density and a slightly higher ethanol yield when supplied N 2 instead of the industrial nitrogen supplement, corn steep liquor. Finally, we estimate that N 2-utilizing Z. mobilis could save a cellulosic ethanol production facility more than $1 million/y.« less

  9. Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli.

    PubMed Central

    Yomano, L P; Scopes, R K; Ingram, L O

    1993-01-01

    Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined. Images PMID:8320209

  10. Continuous production of ethanol with Zymomonas mobilis growing on Jerusalem artichoke juice

    SciT

    Allais, J.J.; Torres, E.F.; Baratti, J.

    1987-04-01

    Recent work from the authors laboratory has shown that, compared to yeasts, much higher ethanol productivity and yield can be obtained in batch or continuous cultures using the bacterium Zymomonas mobilis grown on fructose media. In batch culture, hydrolyzed Jerusalem artichoke juice with sugar concentrations ranging from 100 to 250 g/L can be converted efficiently to ethanol. The present work describes the conversion of the hydrolyzed juice to ethanol in continuous culture. The extraction and enzymatic hydrolysis of inulin from the tubers of Jerusalem artichoke is also reported.

  11. Very high gravity ethanol and fatty acid production of Zymomonas mobilis without amino acid and vitamin.

    PubMed

    Wang, Haoyong; Cao, Shangzhi; Wang, William Tianshuo; Wang, Kaven Tianyv; Jia, Xianhui

    2016-06-01

    Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.

  12. Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment

    Background: Phenolic aldehydes generated from lignocellulose pretreatment exhibited severe toxic inhibitions on microbial growth and fermentation. Numerous tolerance studies against furfural, 5-hydroxymethyl-2-furaldehyde (HMF), acetate, and ethanol were reported, but studies on inhibition of phenol...

  13. Pre-treatment step with Leuconostoc mesenteroides or L. pseudomesenteroides strains removes furfural from Zymomonas mobilis ethanolic fermentation broth

    Furfural (furan-2-carboxaldehyde), formed during dilute acid hydrolysis of biomass, is an inhibitor of growth and ethanol production by Zymomonas mobilis. The present study used a biological pre-treatment to reduce that amount of furfural in a model biofuel fermentation broth. The pre-treatment in...

  14. Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars

    DOE PAGES

    Yang, Shihui; Mohagheghi, Ali; Franden, Mary Ann; ...

    2016-09-02

    To develop pathways for advanced biofuel production, and to understand the impact of host metabolism and environmental conditions on heterologous pathway engineering for economic advanced biofuels production from biomass, we seek to redirect the carbon flow of the model ethanologen Zymomonas mobilis to produce desirable hydrocarbon intermediate 2,3-butanediol (2,3-BDO). 2,3-BDO is a bulk chemical building block, and can be upgraded in high yields to gasoline, diesel, and jet fuel. 2,3-BDO biosynthesis pathways from various bacterial species were examined, which include three genes encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase. Bioinformatics analysis was carried out to pinpoint potential bottlenecks formore » high 2,3-BDO production. Different combinations of 2,3-BDO biosynthesis metabolic pathways using genes from different bacterial species have been constructed. Our results demonstrated that carbon flux can be deviated from ethanol production into 2,3-BDO biosynthesis, and all three heterologous genes are essential to efficiently redirect pyruvate from ethanol production for high 2,3-BDO production in Z. mobilis. The down-selection of best gene combinations up to now enabled Z. mobilis to reach the 2,3-BDO production of more than 10 g/L from glucose and xylose, as well as mixed C6/C5 sugar streams derived from the deacetylation and mechanical refining process. In conclusion, this study confirms the value of integrating bioinformatics analysis and systems biology data during metabolic engineering endeavors, provides guidance for value-added chemical production in Z. mobilis, and reveals the interactions between host metabolism, oxygen levels, and a heterologous 2,3-BDO biosynthesis pathway. Taken together, this work provides guidance for future metabolic engineering efforts aimed at boosting 2,3-BDO titer anaerobically.« less

  15. Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars

    SciT

    Yang, Shihui; Mohagheghi, Ali; Franden, Mary Ann

    To develop pathways for advanced biofuel production, and to understand the impact of host metabolism and environmental conditions on heterologous pathway engineering for economic advanced biofuels production from biomass, we seek to redirect the carbon flow of the model ethanologen Zymomonas mobilis to produce desirable hydrocarbon intermediate 2,3-butanediol (2,3-BDO). 2,3-BDO is a bulk chemical building block, and can be upgraded in high yields to gasoline, diesel, and jet fuel. 2,3-BDO biosynthesis pathways from various bacterial species were examined, which include three genes encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase. Bioinformatics analysis was carried out to pinpoint potential bottlenecks formore » high 2,3-BDO production. Different combinations of 2,3-BDO biosynthesis metabolic pathways using genes from different bacterial species have been constructed. Our results demonstrated that carbon flux can be deviated from ethanol production into 2,3-BDO biosynthesis, and all three heterologous genes are essential to efficiently redirect pyruvate from ethanol production for high 2,3-BDO production in Z. mobilis. The down-selection of best gene combinations up to now enabled Z. mobilis to reach the 2,3-BDO production of more than 10 g/L from glucose and xylose, as well as mixed C6/C5 sugar streams derived from the deacetylation and mechanical refining process. In conclusion, this study confirms the value of integrating bioinformatics analysis and systems biology data during metabolic engineering endeavors, provides guidance for value-added chemical production in Z. mobilis, and reveals the interactions between host metabolism, oxygen levels, and a heterologous 2,3-BDO biosynthesis pathway. Taken together, this work provides guidance for future metabolic engineering efforts aimed at boosting 2,3-BDO titer anaerobically.« less

  16. Impact of expression of EMP enzymes on glucose metabolism in Zymomonas mobilis.

    PubMed

    Chen, Rachel Ruizhen; Agrawal, Manoj; Mao, Zichao

    2013-06-01

    Zymomonas mobilis is the only known microorganism that utilizes the Entner-Doudoroff (ED) pathway anaerobically. In this work, we investigated whether the overexpression of a phosphofructokinase (PFK), the only missing Embden-Meyerhof-Parnas (EMP) pathway enzyme, could establish the pathway in this organism. Introduction of a pyrophosphate-dependent PFK, along with co-expression of homologous fructose-1,6-bisphosphate aldolase and triosephosphate isomerase, did not result in an EMP flux to any appreciable level. However, the metabolism of glucose was impacted significantly. Eight percent of glucose was metabolized to form a new metabolite, dihydroxyacetone. Reducing flux through the ED pathway by as much as 40 % through antisense of a key enzyme, ED aldolase, did not result in a fully functional EMP pathway, suggesting that the ED pathway, especially the lower arm, downstream from glyceraldehyde-3-phosphate, is very rigid, possibly due to redox balance.

  17. Crystal structure of cbbF from Zymomonas mobilis and its functional implication

    SciT

    Hwang, Hyo-Jeong; Park, Suk-Youl; Kim, Jeong-Sun, E-mail: jsunkim@chonnam.ac.kr

    2014-02-28

    Highlights: • The crystal structure of one cbbF from Zymomonas mobilis was revealed. • Scores of residues form two secondary structures with a non-polar protruded residue. • It exists as a dimeric form in solution. - Abstract: A phosphate group at the C1-atom of inositol-monophosphate (IMP) and fructose-1,6-bisphosphate (FBP) is hydrolyzed by a phosphatase IMPase and FBPase in a metal-dependent way, respectively. The two enzymes are almost indiscernible from each other because of their highly similar sequences and structures. Metal ions are bound to residues on the β1- and β2-strands and one mobile loop. However, FBP has another phosphate andmore » FBPases exist as a higher oligomeric state, which may discriminate FBPases from IMPases. There are three genes annotated as FBPases in Zymomonas mobilis, termed also cbbF (ZmcbbF). The revealed crystal structure of one ZmcbbF shows a globular structure formed by five stacked layers. Twenty-five residues in the middle of the sequence form an α-helix and a β-strand, which occupy one side of the catalytic site. A non-polar Leu residue among them is protruded to the active site, pointing out unfavorable access of a bulky charged group to this side. In vitro assays have shown its dimeric form in solution. Interestingly, two β-strands of β1 and β2 are disordered in the ZmcbbF structure. These data indicate that ZmcbbF might structurally belong to IMPase, and imply that its active site would be reorganized in a yet unreported way.« less

  18. Fermentation of Soybean Meal Hydrolyzates with Saccharomyces cerevisiae and Zymomonas mobilis for Ethanol Production.

    PubMed

    Luján-Rhenals, Deivis E; Morawicki, Rubén O; Gbur, Edward E; Ricke, Steven C

    2015-07-01

    Most of the ethanol currently produced by fermentation is derived from sugar cane, corn, or beets. However, it makes good ecological and economic sense to use the carbohydrates contained in by-products and coproducts of the food processing industry for ethanol production. Soybean meal, a co-product of the production of soybean oil, has a relatively high carbohydrate content that could be a reasonable substrate for ethanol production after fermentable sugars are released via hydrolysis. In this research, the capability of Saccharomyces cerevisiae NRRL Y-2233 and Zymomonas mobilis subsp. mobilis NRRL B-4286 to produce ethanol was evaluated using soybean meal hydrolyzates as substrates for the fermentation. These substrates were produced from the dilute-acid hydrolysis of soybean meal at 135 °C for 45 min with 0, 0.5%, 1.25%, and 2% H2 SO4 and at 120 °C for 30 min with 1.25% H2 SO4 . Kinetic parameters of the fermentation were estimated using the logistic model. Ethanol production using S. cerevisiae was highest with the substrates obtained at 135 °C, 45 min, and 0.5% H2 SO4 and fermented for 8 h, 8 g/L (4 g ethanol/100 g fresh SBM), while Z. mobilis reached its maximum ethanol production, 9.2 g/L (4.6 g ethanol/100 g fresh SBM) in the first 20 h of fermentation with the same hydrolyzate. © 2015 Institute of Food Technologists®

  19. Method of inactivation of an end product of energy metabolism in Zymomonas mobilis

    DOEpatents

    Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Lakewood, CO

    2008-05-20

    The present invention briefly provides a method of site-specific insertion in Zymomonas, comprising, providing a Zymomonas gene fragment, interrupting a DNA sequence the fragment, and transforming the Zymomonas through homologous recombination with the interrupted fragment.

  20. Effect of acetic acid on ethanol production by Zymomonas mobilis mutant strains through continuous adaptation.

    PubMed

    Liu, Yu-Fan; Hsieh, Chia-Wen; Chang, Yao-Sheng; Wung, Being-Sun

    2017-08-01

    Acetic acid is a predominant by-product of lignocellulosic biofuel process, which inhibits microbial biocatalysts. Development of bacterial strains that are tolerant to acetic acid is challenging due to poor understanding of the underlying molecular mechanisms. In this study, we generated and characterized two acetic acid-tolerant strains of Zymomonas mobilis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-acetate adaptive breeding. Two mutants, ZMA-142 and ZMA-167, were obtained, showing a significant growth rate at a concentration of 244 mM sodium acetate, while the growth of Z. mobilis ATCC 31823 were completely inhibited in presence of 195 mM sodium acetate. Our data showed that acetate-tolerance of ZMA-167 was attributed to a co-transcription of nhaA from ZMO0117, whereas the co-transcription was absent in ATCC 31823 and ZMA-142. Moreover, ZMA-142 and ZMA-167 exhibited a converstion rate (practical ethanol yield to theorical ethanol yield) of 90.16% and 86% at 195 mM acetate-pH 5 stress condition, respectively. We showed that acid adaptation of ZMA-142 and ZMA-167 to 146 mM acetate increased ZMA-142 and ZMA-167 resulted in an increase in ethanol yield by 32.21% and 21.16% under 195 mM acetate-pH 5 stress condition, respectively. The results indicate the acetate-adaptive seed culture of acetate-tolerant strains, ZMA-142 and ZMA-167, could enhance the ethanol production during fermentation.

  1. Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis

    PubMed Central

    Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M.; Bartlam, Mark

    2016-01-01

    Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate. PMID:26919520

  2. Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis.

    PubMed

    Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M; Bartlam, Mark

    2016-03-01

    Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.

  3. Fermentable metabolite of Zymomonas mobilis controls collagen reduction in photoaging skin by improving TGF-beta/Smad signaling suppression.

    PubMed

    Tanaka, Hiroshi; Yamaba, Hiroyuki; Kosugi, Nobuhiko; Mizutani, Hiroshi; Nakata, Satoru

    2008-04-01

    Solar ultraviolet (UV) irradiation causes damages on human skin and premature skin aging (photoaging). UV-induced reduction of type I collagen in dermis is widely considered primarily induction of wrinkled appearance of photoaging skin. Type I procollagen synthesis is reduced under UV irradiation by blocking transforming growth factor-beta (TGF-beta)/Smad signaling; more specifically, it is down-regulation of TGF-beta type II receptor (T beta RII). Therefore, preventing UV-induced loss of T beta RII results decreased type I collagen reduction in photoaging skin. Zymomonas mobilis is an alcohol fermentable, gram-negative facultative anaerobic bacterium whose effect on skin tissue is scarcely studied. We investigated the protective effects of fermentable metabolite of Z. mobilis (FM of Z. mobilis) against reduction of type I procollagen synthesis of UV-induced down-regulation of T beta RII in human dermal fibroblasts FM of Z. mobilis was obtained from lyophilization of bacterium culture supernatant. The levels of T beta RII and type I procollagen mRNA in human dermal fibroblasts were measured by quantitative real-time RT-PCR, and T beta RII protein levels were assayed by western blotting. T beta RII, type I procollagen, and type I collagen proteins in human dermal fibroblasts or hairless mouse skin were detected by immunostaining. FM of Z. mobilis inhibited down regulation of T beta RII mRNA, and protein levels in UVB irradiated human dermal fibroblasts consequently recover reduced type I procollagen synthesis. These results indicate UVB irradiation inhibits type I procollagen synthesis by suppression of TGF-beta/Smad signaling pathway, and FM of Z. mobilis has inhibitory effect on UVB-induced reduction of type I procollagen synthesis. While short period UVB irradiation decreased both T beta RII and type I procollagen protein levels in hairless mouse skin, topical application of FM of Z. mobilis prevented this decrease. Wrinkle formation in hairless mouse skin

  4. Evaluation and optimization of ethanol production from carob pod extract by Zymomonas mobilis using response surface methodology.

    PubMed

    Vaheed, Hossein; Shojaosadati, Seyed Abbas; Galip, Hasan

    2011-01-01

    In this research, ethanol production from carob pod extract (extract) using Zymomonas mobilis with medium optimized by Plackett-Burman (P-B) and response surface methodologies (RSM) was studied. Z. mobilis was recognized as useful for ethanol production from carob pod extract. The effects of initial concentrations of sugar, peptone, and yeast extract as well as agitation rate (rpm), pH, and culture time in nonhydrolyzed carob pod extract were investigated. Significantly affecting variables (P = 0.05) in the model obtained from RSM studies were: weights of bacterial inoculum, initial sugar, peptone, and yeast extract. Acid hydrolysis was useful to complete conversion of sugars to glucose and fructose. Nonhydrolyzed extract showed higher ethanol yield and residual sugar compared with hydrolyzed extract. Ethanol produced (g g(-1) initial sugar, as the response) was not significantly different (P = 0.05) when Z. mobilis performance was compared in hydrolyzed and nonhydrolyzed extract. The maximum ethanol of 0.34 ± 0.02 g g(-1) initial sugar was obtained at 30°C, initial pH 5.2, and 80 rpm, using concentrations (g per 50 mL culture media) of: inoculum bacterial dry weight, 0.017; initial sugar, 5.78; peptone, 0.43; yeast extract, 0.43; and culture time of 36 h.

  5. 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

  6. Furfural-tolerant Zymomonas mobilis derived from error-prone PCR-based whole genome shuffling and their tolerant mechanism.

    PubMed

    Huang, Suzhen; Xue, Tingli; Wang, Zhiquan; Ma, Yuanyuan; He, Xueting; Hong, Jiefang; Zou, Shaolan; Song, Hao; Zhang, Minhua

    2018-04-01

    Furfural-tolerant strain is essential for the fermentative production of biofuels or chemicals from lignocellulosic biomass. In this study, Zymomonas mobilis CP4 was for the first time subjected to error-prone PCR-based whole genome shuffling, and the resulting mutants F211 and F27 that could tolerate 3 g/L furfural were obtained. The mutant F211 under various furfural stress conditions could rapidly grow when the furfural concentration reduced to 1 g/L. Meanwhile, the two mutants also showed higher tolerance to high concentration of glucose than the control strain CP4. Genome resequencing revealed that the F211 and F27 had 12 and 13 single-nucleotide polymorphisms. The activity assay demonstrated that the activity of NADH-dependent furfural reductase in mutant F211 and CP4 was all increased under furfural stress, and the activity peaked earlier in mutant than in control. Also, furfural level in the culture of F211 was also more rapidly decreased. These indicate that the increase in furfural tolerance of the mutants may be resulted from the enhanced NADH-dependent furfural reductase activity during early log phase, which could lead to an accelerated furfural detoxification process in mutants. In all, we obtained Z. mobilis mutants with enhanced furfural and high concentration of glucose tolerance, and provided valuable clues for the mechanism of furfural tolerance and strain development.

  7. Pre-treatment step with Leuconostoc mesenteroides or L. pseudomesenteroides strains removes furfural from Zymomonas mobilis ethanolic fermentation broth.

    PubMed

    Hunter, William J; Manter, Daniel K

    2014-10-01

    Furfural is an inhibitor of growth and ethanol production by Zymomonas mobilis. This study used a naturally occurring (not GMO) biological pre-treatment to reduce that amount of furfural in a model fermentation broth. Pre-treatment involved inoculating and incubating the fermentation broth with strains of Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides. The Leuconostoc strains converted furfural to furfuryl alcohol without consuming large amounts of dextrose in the process. Coupling this pre-treatment to ethanolic fermentation reduced furfural in the broth and improved growth, dextrose uptake and ethanol formation. Pre-treatment permitted ethanol formation in the presence of 5.2 g L(-1) furfural, which was otherwise inhibitive. The pre-treatment and presence of the Leuconostoc strains in the fermentation broth did not interfere with Z. mobilis ethanolic fermentation or the amounts of ethanol produced. The method suggests a possible technique for reducing the effect that furfural has on the production of ethanol for use as a biofuel. Published by Elsevier Ltd.

  8. Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism

    SciT

    Barnell, W.O.; Kyung Cheol Yi; Conway, T.

    1990-12-01

    The Zymomonas mobilis genes that encode glucose-6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), and glucokinase (glk) were cloned independently by genetic complementation of specific defects in Escherichia coli metabolism. The identify of these cloned genes was confirmed by various biochemical means. Nucleotide sequence analysis established that these three genes are clustered on the genome and revealed an additional open reading frame in this region that has significant amino acid identity to the E.coli xylose-proton symporter and the human glucose transporter. On the basis of this evidence and structural analysis of the deduced primary amino acid sequence, this gene is believed tomore » encode the Z. mobilis glucose-facilitated diffusion protein, glf. The four genes in the 6-kb cluster are organized in the order glf, zwf, edd, glk. The glf and zwf genes are separated by 146 bp. The zwf and edd genes overlap by 8 bp, and their expression may be translationally coupled. The edd and glk genes are separated by 203 bp. The glk gene is followed by tandem transcriptional terminators. The four genes appear to be organized in an operon. Such an arrangement of the genes that govern glucose uptake and the first three steps of the Entner-Doudoroff glycolytic pathway provides the organism with a mechanism for carefully regulating the levels of the enzymes that control carbon flux into the pathway.« less

  9. Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase.

    PubMed

    Balodite, Elina; Strazdina, Inese; Galinina, Nina; McLean, Samantha; Rutkis, Reinis; Poole, Robert K; Kalnenieks, Uldis

    2014-09-01

    The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc1 complex via cytochrome c552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase. © 2014 The Authors.

  10. Detrimental effect of increasing sugar concentrations on ethanol production from maize or decorticated sorghum mashes fermented with Saccharomyces cerevisiae or Zymomonas mobilis: biofuels and environmental biotechnology.

    PubMed

    Pérez-Carrillo, Esther; Luisa Cortés-Callejas, M; Sabillón-Galeas, Luis E; Montalvo-Villarreal, Jorge L; Canizo, Jesica R; Georgina Moreno-Zepeda, M; Serna-Saldivar, Sergio O

    2011-02-01

    The efficiency of ethanol fermentation, as affected by grain source (maize and decorticated red sorghum), total sugar concentration (13 or 20° Plato) and type of microorganism (Saccharomyces cerevisiae or Zymomonas mobilis) was studied. Maize mashes yielded 0.32 l ethanol kg(-1) ground grain whereas mashes prepared with decorticated red sorghum produced 0.28 l ethanol kg(-1). Both microorganisms yielded similar amounts of ethanol. However, high-gravity mashes (20° Plato) yielded lower amounts of ethanol compared to counterparts adjusted to 13° Plato (0.28 vs. 0.22 l ethanol kg(-1) ground grains). In decorticated sorghum mashes adjusted to 20° P, Z. mobilis produced 40 ml kg(-1) more ethanol compared to S. cerevisiae. In addition, Z. mobilis had a lower dependency on nitrogenous compounds.

  11. Improving a recombinant Zymomonas mobilis strain 8b through continuous adaptation on dilute acid pretreated corn stover hydrolysate

    DOE PAGES

    Mohagheghi, Ali; Linger, Jeffrey G.; Yang, Shihui; ...

    2015-03-31

    Complete conversion of the major sugars of biomass including both the C 5 and C 6 sugars is critical for biofuel production processes. Several inhibitory compounds like acetate, hydroxymethylfurfural (HMF), and furfural are produced from the biomass pretreatment process leading to ‘hydrolysate toxicity,’ a major problem for microorganisms to achieve complete sugar utilization. Therefore, development of more robust microorganisms to utilize the sugars released from biomass under toxic environment is critical. In this study, we use continuous culture methodologies to evolve and adapt the ethanologenic bacterium Zymomonas mobilis to improve its ethanol productivity using corn stover hydrolysate. The results aremore » the following: A turbidostat was used to adapt the Z. mobilis strain 8b in the pretreated corn stover liquor. The adaptation was initiated using pure sugar (glucose and xylose) followed by feeding neutralized liquor at different dilution rates. Once the turbidostat reached 60% liquor content, the cells began washing out and the adaptation was stopped. Several ‘sub-strains’ were isolated, and one of them, SS3 (sub-strain 3), had 59% higher xylose utilization than the parent strain 8b when evaluated on 55% neutralized PCS (pretreated corn stover) liquor. Using saccharified PCS slurry generated by enzymatic hydrolysis from 25% solids loading, SS3 generated an ethanol yield of 75.5% compared to 64% for parent strain 8b. Furthermore, the total xylose utilization was 57.7% for SS3 versus 27.4% for strain 8b. To determine the underlying genotypes in these new sub-strains, we conducted genomic resequencing and identified numerous single-nucleotide mutations (SNPs) that had arisen in SS3. We further performed quantitative reverse transcription PCR (qRT-PCR) on genes potentially affected by these SNPs and identified significant down-regulation of two genes, ZMO0153 and ZMO0776, in SS3 suggesting potential genetic mechanisms behind SS3’s improved

  12. Improving a recombinant Zymomonas mobilis strain 8b through continuous adaptation on dilute acid pretreated corn stover hydrolysate

    SciT

    Mohagheghi, Ali; Linger, Jeffrey G.; Yang, Shihui

    Complete conversion of the major sugars of biomass including both the C 5 and C 6 sugars is critical for biofuel production processes. Several inhibitory compounds like acetate, hydroxymethylfurfural (HMF), and furfural are produced from the biomass pretreatment process leading to ‘hydrolysate toxicity,’ a major problem for microorganisms to achieve complete sugar utilization. Therefore, development of more robust microorganisms to utilize the sugars released from biomass under toxic environment is critical. In this study, we use continuous culture methodologies to evolve and adapt the ethanologenic bacterium Zymomonas mobilis to improve its ethanol productivity using corn stover hydrolysate. The results aremore » the following: A turbidostat was used to adapt the Z. mobilis strain 8b in the pretreated corn stover liquor. The adaptation was initiated using pure sugar (glucose and xylose) followed by feeding neutralized liquor at different dilution rates. Once the turbidostat reached 60% liquor content, the cells began washing out and the adaptation was stopped. Several ‘sub-strains’ were isolated, and one of them, SS3 (sub-strain 3), had 59% higher xylose utilization than the parent strain 8b when evaluated on 55% neutralized PCS (pretreated corn stover) liquor. Using saccharified PCS slurry generated by enzymatic hydrolysis from 25% solids loading, SS3 generated an ethanol yield of 75.5% compared to 64% for parent strain 8b. Furthermore, the total xylose utilization was 57.7% for SS3 versus 27.4% for strain 8b. To determine the underlying genotypes in these new sub-strains, we conducted genomic resequencing and identified numerous single-nucleotide mutations (SNPs) that had arisen in SS3. We further performed quantitative reverse transcription PCR (qRT-PCR) on genes potentially affected by these SNPs and identified significant down-regulation of two genes, ZMO0153 and ZMO0776, in SS3 suggesting potential genetic mechanisms behind SS3’s improved

  13. Open fermentative production of fuel ethanol from food waste by an acid-tolerant mutant strain of Zymomonas mobilis.

    PubMed

    Ma, Kedong; Ruan, Zhiyong; Shui, Zongxia; Wang, Yanwei; Hu, Guoquan; He, Mingxiong

    2016-03-01

    The aim of present study was to develop a process for open ethanol fermentation from food waste using an acid-tolerant mutant of Zymomonas mobilis (ZMA7-2). The mutant showed strong tolerance to acid condition of food waste hydrolysate and high ethanol production performance. By optimizing fermentation parameters, ethanol fermentation with initial glucose concentration of 200 g/L, pH value around 4.0, inoculum size of 10% and without nutrient addition was considered as best conditions. Moreover, the potential of bench scales fermentation and cell reusability was also examined. The fermentation in bench scales (44 h) was faster than flask scale (48 h), and the maximum ethanol concentration and ethanol yield (99.78 g/L, 0.50 g/g) higher than that of flask scale (98.31 g/L, 0.49 g/g). In addition, the stable cell growth and ethanol production profile in five cycles successive fermentation was observed, indicating the mutant was suitable for industrial ethanol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Genetic engineering and improvement of a Zymomonas mobilis for arabinose utilization and its performance on pretreated corn stover hydrolyzate

    SciT

    Chou, Yat -Chen; Linger, Jeffrey; Yang, Shihui

    In this paper, a glucose, xylose and arabinose utilizing Zymomonas mobilis strain was constructed by incorporating arabinose catabolic pathway genes, araBAD encoding L-ribulokinase, L-arabinose isomerase and L-ribulose-5-phosphate- 4-epimerase in a glucose, xylose co-fermenting host, 8b, using a transposition integration approach. Further improvement on this arabinose-capable integrant, 33C was achieved by applying a second transposition to create a genomic knockout (KO) mutant library. Using arabinose as a sole carbon source and a selection pressure, the KO library was subjected to a growth-enrichment process involving continuous sub-culturing for over 120 generations. Strain 13-1-17, isolated from such process demonstrated significant improvement in metabolizingmore » arabinose in a dilute acid pretreated, saccharified corn stover slurry. Through Next Generation Sequencing (NGS) analysis, integration sites of the transposons were identified. Furthermore, multiple additional point mutations (SNPs: Single Nucleotide Polymorphisms) were discovered in 13-1-17, affecting genes araB and RpiB in the genome. Finally, we speculate that these mutations may have impacted the expression of the enzymes coded by these genes, ribulokinase and Ribose 5-P-isomerase, thus attributing to the improvement of the arabinose utilization.« less

  15. Genetic engineering and improvement of a Zymomonas mobilis for arabinose utilization and its performance on pretreated corn stover hydrolyzate

    DOE PAGES

    Chou, Yat -Chen; Linger, Jeffrey; Yang, Shihui; ...

    2015-04-28

    In this paper, a glucose, xylose and arabinose utilizing Zymomonas mobilis strain was constructed by incorporating arabinose catabolic pathway genes, araBAD encoding L-ribulokinase, L-arabinose isomerase and L-ribulose-5-phosphate- 4-epimerase in a glucose, xylose co-fermenting host, 8b, using a transposition integration approach. Further improvement on this arabinose-capable integrant, 33C was achieved by applying a second transposition to create a genomic knockout (KO) mutant library. Using arabinose as a sole carbon source and a selection pressure, the KO library was subjected to a growth-enrichment process involving continuous sub-culturing for over 120 generations. Strain 13-1-17, isolated from such process demonstrated significant improvement in metabolizingmore » arabinose in a dilute acid pretreated, saccharified corn stover slurry. Through Next Generation Sequencing (NGS) analysis, integration sites of the transposons were identified. Furthermore, multiple additional point mutations (SNPs: Single Nucleotide Polymorphisms) were discovered in 13-1-17, affecting genes araB and RpiB in the genome. Finally, we speculate that these mutations may have impacted the expression of the enzymes coded by these genes, ribulokinase and Ribose 5-P-isomerase, thus attributing to the improvement of the arabinose utilization.« less

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

    SciT

    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

  17. Use of a Tn5-based transposon system to create a cost-effective Zymomonas mobilis for ethanol production from lignocelluloses

    PubMed Central

    2013-01-01

    Background Current methods of ethanol production from lignocelluloses generate a mixture of sugars, primarily glucose and xylose; the fermentation cells are always exposed to stresses like high temperature and low nutritional conditions that affect their growth and productivity. Stress-tolerant strains capable of using both glucose and xylose to produce ethanol with high yield are highly desirable. Results A recombinant Zymomonas mobilis (Z. mobilis) designated as HYMX was constructed by integrating seven genes (Pfu-sHSP, yfdZ, metB, xylA, xylB, tktA and talB) into the genome of Z. mobilis CP4 (CP4) via Tn5 transposon in the present study. The small heat shock protein gene (Pfu-sHSP) from Pyrococcus furious (P. furious) was used to increase the heat-tolerance, the yfdZ and metB genes from E. coli were used to decrease the nutritional requirement. To overcome the bottleneck of CP4 being unable to use pentose, xylose catabolic genes (xylA, xylB, tktA and talB) from E. coli were integrated into CP4 also for construction of the xylose utilizing metabolic pathway. Conclusions The genomic integration confers on Z. mobilis the ability to grow in medium containing xylose as the only carbon source, and to grow in simple chemical defined medium without addition of amino acid. The HYMX demonstrated not only the high tolerance to unfavorable stresses like high temperature and low nutrient, but also the capability of converting both glucose and xylose to ethanol with high yield at high temperature. What’s more, these genetic characteristics were stable up to 100 generations on nonselective medium. Although significant improvements were achieved, yeast extract is needed for ethanol production. PMID:23635356

  18. Development of corn silk as a biocarrier for Zymomonas mobilis biofilms in ethanol production from rice straw.

    PubMed

    Todhanakasem, Tatsaporn; Tiwari, Rashmi; Thanonkeo, Pornthap

    2016-01-01

    Z. mobilis cell immobilization has been proposed as an effective means of improving ethanol production. In this work, polystyrene and corn silk were used as biofilm developmental matrices for Z. mobilis ethanol production with rice straw hydrolysate as a substrate. Rice straw was hydrolyzed by dilute sulfuric acid (H2SO4) and enzymatic hydrolysis. The final hydrolysate contained furfural (271.95 ± 76.30 ppm), 5-hydroxymethyl furfural (0.07 ± 0.00 ppm), vanillin (1.81 ± 0.00 ppm), syringaldehyde (5.07 ± 0.83 ppm), 4-hydroxybenzaldehyde (4-HB) (2.39 ± 1.20 ppm) and acetic acid (0.26 ± 0.08%). Bacterial attachment or biofilm formation of Z. mobilis strain TISTR 551 on polystyrene and delignified corn silk carrier provided significant ethanol yields. Results showed up to 0.40 ± 0.15 g ethanol produced/g glucose consumed when Z. mobilis was immobilized on a polystyrene carrier and 0.51 ± 0.13 g ethanol produced/g glucose consumed when immobilized on delignified corn silk carrier under batch fermentation by Z. mobilis TISTR 551 biofilm. The higher ethanol yield from immobilized, rather than free living, Z. mobilis could possibly be explained by a higher cell density, better control of anaerobic conditions and higher toxic tolerance of Z. mobilis biofilms over free cells.

  19. 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.

  20. 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.

  1. Development of a high-throughput method to evaluate the impact of inhibitory compounds from lignocellulosic hydrolysates on the growth of Zymomonas mobilis.

    PubMed

    Franden, Mary Ann; Pienkos, Philip T; Zhang, Min

    2009-12-01

    Overcoming the effects of hydrolysate toxicity towards ethanologens is a key technical barrier in the biochemical conversion process for biomass feedstocks to ethanol. Despite its importance, the complexity of the hydrolysate toxicity phenomena and the lack of systematic studies, analysis and tools surrounding this issue have blocked a full understanding of relationships involving toxic compounds in hydrolysates and their effects on ethanologen growth and fermentation. In this study, we developed a quantitative, high-throughput biological growth assay using an automated turbidometer to obtain detailed inhibitory kinetics for individual compounds present in lignocellulosic biomass hydrolysate. Information about prolonged lag time and final cell densities can also be obtained. The effects of furfural, hydroxymethylfurfural (HMF), acetate and ethanol on growth rate and final cell densities of Zymomonas mobilis 8b on glucose are presented. This method was also shown to be of value in toxicity studies of hydrolysate itself, despite the highly colored nature of this material. Using this approach, we can generate comprehensive inhibitory profiles with many individual compounds and develop models that predict and examine toxic effects in the complex mixture of hydrolysates, leading to the development of improved pretreatment and conditioning processes as well as fermentation organisms.

  2. Biotransformation of pineapple juice sugars into dietetic derivatives by using a cell free oxidoreductase from Zymomonas mobilis together with commercial invertase.

    PubMed

    Aziz, M G; Michlmayr, H; Kulbe, K D; Del Hierro, A M

    2011-01-05

    An easy procedure for cell free biotransformation of pineapple juice sugars into dietetic derivatives was accomplished using a commercial invertase and an oxidoreductase from Zymomonas mobilis. First, pineapple juice sucrose was quantitatively converted into glucose and fructose by invertase, thus increasing the concentration of each monosaccharide in the original juice to almost twice. In a second step, glucose-fructose oxidoreductase (GFOR) transformed glucose into gluconolactone, and fructose into the low calorie sweetener sorbitol. The advantage of using GFOR is simultaneous reduction of fructose and oxidation of glucose, allowing the continuous regeneration of the essential coenzyme NADP(H), that is tightly bound to the enzyme. The yield of GFOR catalyzed sugar conversion depends on initial pH and control of pH during the reaction. At optimal conditions (pH control at 6.2) a maximum of 80% (w/v) sugar conversion was obtained. Without pH control, GFOR is inactivated rapidly due to gluconic acid formation. Therefore, conversion yields are relatively low at the natural pH of pineapple juice. The application of this process might be more advantageous on juices of other tropical fruits (papaya, jackfruit, mango) due to their naturally given higher pH. Copyright © 2010 Elsevier Inc. All rights reserved.

  3. 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

  4. High expression Zymomonas promoters

    DOEpatents

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

    2011-08-02

    Identified are mutants of the promoter of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene, which direct improved expression levels of operably linked heterologous nucleic acids. These are high expression promoters useful for expression of chimeric genes in Zymomonas, Zymobacter, and other related bacteria.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. From lin-benzoguanines to lin-benzohypoxanthines as ligands for Zymomonas mobilis tRNA-guanine transglycosylase: replacement of protein-ligand hydrogen bonding by importing water clusters.

    PubMed

    Barandun, Luzi Jakob; Immekus, Florian; Kohler, Philipp C; Tonazzi, Sandro; Wagner, Björn; Wendelspiess, Severin; Ritschel, Tina; Heine, Andreas; Kansy, Manfred; Klebe, Gerhard; Diederich, François

    2012-07-23

    The foodborne illness shigellosis is caused by Shigella bacteria that secrete the highly cytotoxic Shiga toxin, which is also formed by the closely related enterohemorrhagic Escherichia coli (EHEC). It has been shown that tRNA-guanine transglycosylase (TGT) is essential for the pathogenicity of Shigella flexneri. Herein, the molecular recognition properties of a guanine binding pocket in Zymomonas mobilis TGT are investigated with a series of lin-benzohypoxanthine- and lin-benzoguanine-based inhibitors that bear substituents to occupy either the ribose-33 or the ribose-34 pocket. The three inhibitor scaffolds differ by the substituent at C(6) being H, NH(2), or NH-alkyl. These differences lead to major changes in the inhibition constants, pK(a) values, and binding modes. Compared to the lin-benzoguanines, with an exocyclic NH(2) at C(6), the lin-benzohypoxanthines without an exocyclic NH(2) group have a weaker affinity as several ionic protein-ligand hydrogen bonds are lost. X-ray cocrystal structure analysis reveals that a new water cluster is imported into the space vacated by the lacking NH(2) group and by a conformational shift of the side chain of catalytic Asp102. In the presence of an N-alkyl group at C(6) in lin-benzoguanine ligands, this water cluster is largely maintained but replacement of one of the water molecules in the cluster leads to a substantial loss in binding affinity. This study provides new insight into the role of water clusters at enzyme active sites and their challenging substitution by ligand parts, a topic of general interest in contemporary structure-based drug design. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes.

    PubMed

    Turner, Peter C; Yomano, Lorraine P; Jarboe, Laura R; York, Sean W; Baggett, Christy L; Moritz, Brélan E; Zentz, Emily B; Shanmugam, K T; Ingram, Lonnie O

    2012-04-01

    Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.

  13. Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae

    SciT

    Conway, T.; Ingram, L.O.

    1989-07-01

    The gene that encodes 1,2-propanediol oxidoreductase (fucO) from Escherichia coli was sequenced. The reading frame specified a protein of 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,642. The induction of fucO transcription, which occurred in the presence of fucose, was confirmed by Northern blot analysis. In E. coli, the primary fucO transcript was approximately 2.1 kilobases in length. The 5{prime} end of the transcript began more than 0.7 kilobase upstream of the fucO start codon within or beyond the fucA gene. Propanediol oxidoreductase exhibited 41.7% identity with the iron-containing alcohol dehydrogenase II from Zymomonasmore » mobilis and 39.5% identity with ADH4 from Saccharomyces cerevisiae. These three proteins did not share homology with either short-chain or long-chain zinc-containing alcohol dehydrogenase enzymes. We propose that these three unusual alcohol dehydrogenases define a new family of enzymes.« less

  14. 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.

  15. Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell; Alterthum, Flavio

    1991-01-01

    A novel operon and plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase activities of Zymomonas mobilis are described. Also disclosed are methods for increasing the growth of microorganisms or eukaryotic cells and methods for reducing the accumulation of undesirable metabolic products in the growth medium of microorganisms or cells.

  16. Pantothenic acid biosynthesis in zymomonas

    DOEpatents

    Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V.

    2014-07-01

    Zymomonas is unable to synthesize pantothenic acid and requires this essential vitamin in growth medium. Zymomonas strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pantothenic acid supplementation in seed culture and fermentation media.

  17. 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.

  18. 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.

  19. Insights into Acetate Toxicity in Zymomonas mobilis 8b using Different Substrates

    DOE PAGES

    Yang, Shihui; Franden, M. A.; Brown, S. D.; ...

    2014-09-30

    The lignocellulosic biomass is a promising renewable feedstock for biofuel production. Acetate is one of the major inhibitors liberated from hemicelluloses during hydrolysis. Likewise, an understanding of the toxic effects of acetate on the fermentation microorganism and the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production.

  20. 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.

  1. Zymomonas with improved ethanol production in medium containing concentrated sugars and acetate

    DOEpatents

    Caimi, Perry G.; Chou, Yat-Chen; Franden, Mary Ann; Knoke, Kyle; Tao, Luan; Viitanen, Paul V.; Zhang, Min; Zhang, Yuying

    2010-09-28

    Through screening of a Zymomonas mutant library the himA gene was found to be involved in the inhibitory effect of acetate on Zymomonas performance. Xylose-utilizing Zymomonas further engineered to reduce activity of the himA gene were found to have increased ethanol production in comparison to a parental strain, when cultured in medium comprising xylose and acetate.

  2. Investigation of potential of agro-industrial residues for ethanol production by using Candida tropicalis and Zymomonas mobilis

    NASA Astrophysics Data System (ADS)

    Patle, Sonali

    India is becoming more susceptible regarding energy security with increasing world prices of crude oil and increasing dependence on imports. Based on experiments by the Indian Institute of Petroleum, a 10% ethanol blend with gasoline is being considered for use in vehicles in at least one state and it will be mandatory for all oil companies to blend petrol with 10% ethanol from October 2008. In view of the above, the Government has already started supply of 5% ethanol blended petrol from 2003 in nine states and four contiguous Union Territories. Currently, fuel ethanol is produced mainly from molasses, corn, wheat and sugar beets. The production cost of ethanol from these agro-feedstocks is more than twice the price of gasoline. The high feedstock cost poses a major obstacle to large scale implementation of ethanol as a transportation fuel. Molasses could be in short supply due to the implementation of 10% blending norm. A reduction in import duty for industrial alcohol from7.5% to 5% has been suggested. The use of lignocellulosic energy crops, and particularly low cost biomass residues, offers excellent perspectives for application of ethanol in transportation fuels (Ridder, 2000). These materials will increase the ethanol production capacity and reduce the production cost to a competitive level. There is a huge demand (500 million litres) of ethanol to meet the 5% blending in India. With the present infrastructure, only 90 million litres of ethanol was produced till November 2006 and could reach up to 140 million litres (around) till October 2007. Bioethanol from these materials provides a highly cost effective option for CO2 emission reduction in the transportation sector. The aim of the present investigation was to evaluate the potential of biomass as feedstock for ethanol production. The dedicated energy crops would require thorough support as well as planning efforts such as assessing resources, availability and utilization. Furthermore, applied research is needed to develop environmentally and socially acceptable low-cost, high quality crops and cropping systems for producing sufficient quantities of value added biomass feedstock on substantially larger areas. This would require taking a look at environmental implications and economic assessments as over 70% of Indian population directly or indirectly depends on agricultural income sources. In other words, a long term strategy of intensive research would be required to get the desired level of acceptance both by the researchers and the farmers. This would mean long term field trials with the newly developed energy crops, awareness creation, and demonstration of visual benefits to farmers leading to change in mind-set towards greater flexibility for cropping patterns. This holds enormous promising research and development opportunities, but substantially longer period might be required to achieve these goals. The petroleum industry is now committed to the use of ethanol as fuel, as it is expected to benefit sugarcane farmers as well as the oil industry in the long run. Production of ethanol from agricultural and biodegradable wastes provides a viable solution to multiple environmental problems simultaneously creating a sink for waste and renewable energy production as well. Using ethanol-blended fuels for automobiles can significantly reduce petroleum use. Ethanol is one of the best tools to fight vehicular pollution, contains more oxygen that helps complete combustion of fuel and thus reduces harmful tailpipe emissions. It also reduces particulate emissions that pose a health hazard. Currently, fuel grade ethanol is produced from sugarcane, corn, wheat and sugar beets but the ethanol production cost from these substrates is very high as compared to gasoline. This high feedstock cost is the biggest hindrance in large scale implementation of ethanol as a transportation fuel. To counter the high feedstock costs, use of lignocellulosic materials, such as crop residues, grasses, sawdust, wood chips etc., can be promoted, which presents an inexpensive and abundant renewable source for ethanol production. Also there is an enormous production of fruits and vegetables in India and a very huge amount goes waste due to post-harvest losses and a large quantity of unused portion is also generated from processing industries. These substrates can be used as a potential source for ethanol production. These substrates are complex and are required to be broken down into simple sugars by acid, alkaline or enzymatic treatment. Two common methods for converting complex substrates to fermentable sugars are dilute acid hydrolysis and concentrated acid hydrolysis, both of which use either HCl or H2 So4. Since, acid hydrolysis has few disadvantages enzymatic hydrolysis was explored and found to be a better and more economic option. After substrate selection and its hydrolysis, it is very important to optimize the fermentation parameters and scale up the process. Different agro-industrial substrates were explored for this process. (Abstract shortened by UMI.)

  3. Zymomonas with improved ethanol production in medium containing concentrated sugars and acetate

    DOEpatents

    Caimi, Perry G [Kennett Square, PA; Chou, Yat-Chen [Lakewood, CO; Franden, Mary Ann [Centennial, CO; Knoke, Kyle [Newark, DE; Tao, Luan [Havertown, PA; Viitanen, Paul V [West Chester, PA; Zhang, Min [Lakewood, CO; Zhang, Yuying [New Hope, PA

    2011-03-01

    Through screening of a Zymomonas mutant library the himA gene was found to be involved in the inhibitory effect of acetate on Zymomonas performance. Xylose-utilizing Zymomonas strains further engineered to reduce activity of the himA gene were found to have increased ethanol production in comparison to a parental strain, when cultured in mixed-sugars medium comprising xylose, and, in particular, in the presence of acetate.

  4. Ultrastructure of the enteromonad flagellate Caviomonas mobilis.

    PubMed

    Brugerolle, G; Regnault, J P

    2001-08-01

    Caviomonas mobilis was collected from the caecum of mice harbouring a controlled fauna. Phase contrast and immunofluorescence microscopy using an anti-tubulin antibody and electron microscopy demonstrated the presence of one basal body bearing a flagellum and a second barren basal body, both inserted in the face of two cup-like depressions in the nuclear surface, as in other enteromonad/diplomonad genera. Three microtubular fibres arise close to the main basal body: the first, composed of three microtubules cross-linked with a dense structure, lies within a groove above the nuclear surface; the second is oriented antero-dorsally and corresponds to the peristyle as observed by light microscopy; and the third is situated ventrally, below the proximal part of the recurrent flagellum, and corresponds to the funis. There is no mitochondrion, no Golgi body, the endoplasmic reticulum is reduced, there is no cytostome, the cell feeds by pinocytosis and phagocytosis and the division spindle is intranuclear. The cytological characters of Caviomonas are homologous to those of genera which comprise the enteromonad/diplomonad evolutionary lineage, as previously presumed.

  5. Pnp gene modification for improved xylose utilization in Zymomonas

    DOEpatents

    Caimi, Perry G G; Qi, Min; Tao, Luan; Viitanen, Paul V; Yang, Jianjun

    2014-12-16

    The endogenous pnp gene encoding polynucleotide phosphorylase in the Zymomonas genome was identified as a target for modification to provide improved xylose utilizing cells for ethanol production. The cells are in addition genetically modified to have increased expression of ribose-5-phosphate isomerase (RPI) activity, as compared to cells without this genetic modification, and are not limited in xylose isomerase activity in the absence of the pnp modification.

  6. Fuel alcohol biosynthesis by Zymomonas anaerobia: optimization studies

    SciT

    Kosaric, N.; Ong, S.L.; Davnjak, Z.

    1982-03-01

    The optimum operating conditions for growth and ethanol production of Zymomonas anaerobia ATCC 29501 were established. The optimum pH range and temperature were found to be 5.0-6.0 and 35/sup 0/C, respectively. Based on the results obtained from the temperature optimization study, an Arrhenius-type temperature relationship for the specific growth rate was developed. The growth and ethanol production of this microbe also have been optimized in terms of concentrations of glucose, essential nutrients, and minerals. With optimum medium and operating conditions, an ethanol concentration of 96 g/L was obtained in 23h. Both growth and ethanol yield coefficients in dependence on initialmore » glucose concentrations were determined.« less

  7. Fuel alcohol biosynthesis by Zymomonas anaerobia: optimization studies

    SciT

    Kosaric, N.; Ong, S.L.; Duvnjak, Z.

    1982-03-01

    The optimum operating conditions for growth and ethanol production of Zymomonas anaerobia ATCC 29501 were established. The optimum pH range and temperature were found to be 5.0-6.0 and 35 degrees C, respectively. Based on the results obtained from the temperature optimization study, an Arrhenius-type temperature relationship for the specific growth rate was developed. The growth and ethanol production of this microbe also have been optimized in terms of concentrations of glucose, essential nutrients, and minerals. With optimum medium and operating conditions, an ethanol concentration of 96 g/L was obtained in 23 hours. Both growth and ethanol yield coefficients in dependencemore » on initial glucose concentrations were determined. (Refs. 16).« less

  8. A comparison of simple rheological parameters and simulation data for Zymomonas mobilis fermentation broths with high substrate loading in a 3-L bioreactor.

    PubMed

    Um, Byung-Hwan; Hanley, Thomas R

    2008-03-01

    Traditionally, as much as 80% or more of an ethanol fermentation broth is water that must be removed. This mixture is not only costly to separate but also produces a large aqueous stream that must then be disposed of or recycled. Integrative approaches to water reduction include increasing the biomass concentration during fermentation. In this paper, experimental results are presented for the rheological behavior of high-solids enzymatic cellulose hydrolysis and ethanol fermentation for biomass conversion using Solka Floc as the model feedstock. The experimental determination of the viscosity, shear stress, and shear rate relationships of the 10 to 20% slurry concentrations with constant enzyme concentrations are performed with a variable speed rotational viscometer (2.0 to 200 rpm) at 40 degrees C. The viscosities of enzymatic suspension observed were in range of 0.0418 to 0.0144, 0.233 to 0.0348, and 0.292 to 0.0447 Pa s for shear rates up to 100 reciprocal seconds at 10, 15, and 20% initial solids (w/v), respectively. Computational fluid dynamics analysis of bioreactor mixing demonstrates the change in bioreactor mixing with increasing biomass concentration. The portion-loading method is shown to be effective for processing high-solids slurries.

  9. A Comparison of Simple Rheological Parameters and Simulation Data for Zymomonas mobilis Fermentation Broths with High Substrate Loading in a 3-L Bioreactor

    NASA Astrophysics Data System (ADS)

    Um, Byung-Hwan; Hanley, Thomas R.

    Traditionally, as much as 80% or more of an ethanol fermentation broth is water that must be removed. This mixture is not only costly to separate but also produces a large aqueous stream that must then be disposed of or recycled. Integrative approaches to water reduction include increasing the biomass concentration during fermentation. In this paper, experimental results are presented for the rheological behavior of high-solids enzymatic cellulose hydrolysis and ethanol fermentation for biomass conversion using Solka Floc as the model feedstock. The experimental determination of the viscosity, shear stress, and shear rate relationships of the 10 to 20% slurry concentrations with constant enzyme concentrations are performed with a variable speed rotational viscometer (2.0 to 200 rpm) at 40 °C. The viscosities of enzymatic suspension observed were in range of 0.0418 to 0.0144, 0.233 to 0.0348, and 0.292 to 0.0447 Pa s for shear rates up to 100 reciprocal seconds at 10, 15, and 20% initial solids (w/v), respectively. Computational fluid dynamics analysis of bioreactor mixing demonstrates the change in bioreactor mixing with increasing biomass concentration. The portion-loading method is shown to be effective for processing highsolids slurries.

  10. Comparative fermentation behaviour and chemical characteristics of Saccharomyces and Zymomonas fermented culled apple juice.

    PubMed

    Sandhu, D K; Joshi, V K

    1994-12-01

    Ethanol production from culled apple juice showed that fermentability of the juice could be enhanced by addition of DAHP or ammonium sulphate in Saccharomyces and DAHP in Zymomonas fermentation. Addition of trace elements inhibited both the fermentations and ethanol, consequently. With respect to by-products of fermentation, no clear advantage of Zymomnas fermentation of culled apple juice could be observed. Differences in physico-chemical characteristics of the fermented apple juice were also noted. Saccharomyces cerevisiae proved to be better than Zymomonas in most of the parameters and is preferrable from handling and spoilage point of view.

  11. 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.

  12. 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.

  13. Global occurrence and heterogeneity of the Roseobacter-clade species Ruegeria mobilis

    PubMed Central

    Sonnenschein, Eva C; Nielsen, Kristian F; D'Alvise, Paul; Porsby, Cisse H; Melchiorsen, Jette; Heilmann, Jens; Kalatzis, Panos G; López-Pérez, Mario; Bunk, Boyke; Spröer, Cathrin; Middelboe, Mathias; Gram, Lone

    2017-01-01

    Tropodithietic acid (TDA)-producing Ruegeria mobilis strains of the Roseobacter clade have primarily been isolated from marine aquaculture and have probiotic potential due to inhibition of fish pathogens. We hypothesized that TDA producers with additional novel features are present in the oceanic environment. We isolated 42 TDA-producing R. mobilis strains during a global marine research cruise. While highly similar on the 16S ribosomal RNA gene level (99–100% identity), the strains separated into four sub-clusters in a multilocus sequence analysis. They were further differentiated to the strain level by average nucleotide identity using pairwise genome comparison. The four sub-clusters could not be associated with a specific environmental niche, however, correlated with the pattern of sub-typing using co-isolated phages, the number of prophages in the genomes and the distribution in ocean provinces. Major genomic differences within the sub-clusters include prophages and toxin-antitoxin systems. In general, the genome of R. mobilis revealed adaptation to a particle-associated life style and querying TARA ocean data confirmed that R. mobilis is more abundant in the particle-associated fraction than in the free-living fraction occurring in 40% and 6% of the samples, respectively. Our data and the TARA data, although lacking sufficient data from the polar regions, demonstrate that R. mobilis is a globally distributed marine bacterial species found primarily in the upper open oceans. It has preserved key phenotypic behaviors such as the production of TDA, but contains diverse sub-clusters, which could provide new capabilities for utilization in aquaculture. PMID:27552638

  14. Permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, a thermoacidophilic sulfur-reducing crenarchaeon isolated from acidic hot springs of Hveravellir, Iceland.

    PubMed

    Susanti, Dwi; Johnson, Eric F; Lapidus, Alla; Han, James; Reddy, T B K; Pilay, Manoj; Ivanova, Natalia N; Markowitz, Victor M; Woyke, Tanja; Kyrpides, Nikos C; Mukhopadhyay, Biswarup

    2016-01-01

    This report presents the permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, an obligate anaerobic hyperthermophilic crenarchaeon that was isolated from acidic hot springs in Hveravellir, Iceland. D. mobilis utilizes peptides as carbon and energy sources and reduces elemental sulfur to H2S. A metabolic construction derived from the draft genome identified putative pathways for peptide degradation and sulfur respiration in this archaeon. Existence of several hydrogenase genes in the genome supported previous findings that H2 is produced during the growth of D. mobilis in the absence of sulfur. Interestingly, genes encoding glucose transport and utilization systems also exist in the D. mobilis genome though this archaeon does not utilize carbohydrate for growth. The draft genome of D. mobilis provides an additional mean for comparative genomic analysis of desulfurococci. In addition, our analysis on the Average Nucleotide Identity between D. mobilis and Desulfurococcus mucosus suggested that these two desulfurococci are two different strains of the same species.

  15. Fine structure and ecdysis of mandibular sensilla associated with the lacinia mobilis in Neomysis integer (Leach, 1814) (Crustacea, Malacostraca, Peracarida).

    PubMed

    Geiselbrecht, Hannes; Melzer, Roland R

    2014-05-01

    The external and internal structures of adult Neomysis integer mandibles were studied using light and electron microscopy with special reference to the lacinia mobilis, a highly specialized appendage on the gnathal edge of many crustaceans. The right and left lacinia mobilis are equipped with ciliary primary sensory cells revealing that both laciniae are also mechanosensory organs in addition to their mechanical function during mastication. A detailed character analyses indicated that the right lacinia is probably a highly derived sensory seta, whereas two alternative interpretations are considered for the left lacinia; it could be a sensillar appendage equipped with two mechanosensory units, or it could be a movable appendage of the incisor process containing two sensilla deprived of external appendages. The ecdysis of the lacinia mobilis corresponds very well to type I sensillar ecdysis, suggesting classification as a sensillar appendage. These features support a possible homology of the right lacinia mobilis in Peracarida and Decapoda, tracing them to an origin as a member of the setal row. Whether the left lacinia mobilis is a sensillum or an appendage with sensilla cannot be resolved presently. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Towards an informative mutant phenotype for every bacterial gene

    DOE PAGES

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; ...

    2014-08-11

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, inmore » Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.« less

  17. SciT

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, inmore » Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.« less

  18. Isolation, characterization, and primary structure of rubredoxin from the photosynthetic bacterium, Heliobacillus mobilis

    NASA Technical Reports Server (NTRS)

    Lee, W. Y.; Brune, D. C.; LoBrutto, R.; Blankenship, R. E.

    1995-01-01

    Rubredoxin is a small nonheme iron protein that serves as an electron carrier in bacterial systems. Rubredoxin has now been isolated and characterized from the strictly anaerobic phototroph, Heliobacillus mobilis. THe molecular mass (5671.3 Da from the amino acid sequence) was confirmed and partial formylation of the N-terminal methionyl residue was established by matrix-assisted laser desorption mass spectroscopy. The complete 52-amino-acid sequence was determined by a combination of N-terminal sequencing by Edman degradation and C-terminal sequencing by a novel method using carboxypeptidase treatment in conjunction with amino acid analysis and laser desorption time of flight mass spectrometry. The molar absorption coefficient of Hc. mobilis rubredoxin at 490 nm is 6.9 mM-1 cm-1 and the midpoint redox potential at pH 8.0 is -46 mV. The EPR spectrum of the oxidized form shows resonances at g = 9.66 and 4.30 due to a high-spin ferric iron. The amino acid sequence is homologous to those of rubredoxins from other species, in particular, the gram-positive bacteria, and the phototrophic green sulfur bacteria, and the evolutionary implications of this are discussed.

  19. Permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, a thermoacidophilic sulfur-reducing crenarchaeon isolated from acidic hot springs of Hveravellir, Iceland

    DOE PAGES

    Susanti, Dwi; Johnson, Eric F.; Lapidus, Alla; ...

    2016-01-13

    Our report presents the permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, an obligate anaerobic hyperthermophilic crenarchaeon that was isolated from acidic hot springs in Hveravellir, Iceland. D. mobilis utilizes peptides as carbon and energy sources and reduces elemental sulfur to H 2S. A metabolic construction derived from the draft genome identified putative pathways for peptide degradation and sulfur respiration in this archaeon. Existence of several hydrogenase genes in the genome supported previous findings that H 2 is produced during the growth of D. mobilis in the absence of sulfur. Interestingly, genes encoding glucose transport and utilizationmore » systems also exist in the D. mobilis genome though this archaeon does not utilize carbohydrate for growth. The draft genome of D. mobilis provides an additional mean for comparative genomic analysis of desulfurococci. In addition, our analysis on the Average Nucleotide Identity between D. mobilis and Desulfurococcus mucosus suggested that these two desulfurococci are two different strains of the same species.« less

  20. Permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, a thermoacidophilic sulfur-reducing crenarchaeon isolated from acidic hot springs of Hveravellir, Iceland

    SciT

    Susanti, Dwi; Johnson, Eric F.; Lapidus, Alla

    Our report presents the permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, an obligate anaerobic hyperthermophilic crenarchaeon that was isolated from acidic hot springs in Hveravellir, Iceland. D. mobilis utilizes peptides as carbon and energy sources and reduces elemental sulfur to H 2S. A metabolic construction derived from the draft genome identified putative pathways for peptide degradation and sulfur respiration in this archaeon. Existence of several hydrogenase genes in the genome supported previous findings that H 2 is produced during the growth of D. mobilis in the absence of sulfur. Interestingly, genes encoding glucose transport and utilizationmore » systems also exist in the D. mobilis genome though this archaeon does not utilize carbohydrate for growth. The draft genome of D. mobilis provides an additional mean for comparative genomic analysis of desulfurococci. In addition, our analysis on the Average Nucleotide Identity between D. mobilis and Desulfurococcus mucosus suggested that these two desulfurococci are two different strains of the same species.« less

  1. Host and symbiont intraspecific variability: The case of Paramecium calkinsi and "Candidatus Trichorickettsia mobilis".

    PubMed

    Sabaneyeva, E; Castelli, M; Szokoli, F; Benken, K; Lebedeva, N; Salvetti, A; Schweikert, M; Fokin, S; Petroni, G

    2018-02-01

    Newly isolated strains of the ciliate Paramecium calkinsi and their cytoplasmic bacterial endosymbionts were characterized by a multidisciplinary approach, including live observation, ultrastructural investigation, and molecular analysis. Despite morphological resemblance, the characterized P. calkinsi strains showed a significant molecular divergence compared to conspecifics, possibly hinting for a cryptic speciation. The endosymbionts were clearly found to be affiliated to the species "Candidatus Trichorickettsia mobilis" (Rickettsiales, Rickettsiaceae), currently encompassing only bacteria retrieved in an obligate intracellular association with other ciliates. However, a relatively high degree of intraspecific divergence was observed as well, thus it was possible to split "Candidatus Trichorickettsia" into three subspecies, one of which represented so far only by the newly characterized endosymbionts of P. calkinsi. Other features distinguished the members of each different subspecies. In particular, the endosymbionts of P. calkinsi resided in the cytoplasm and possessed numerous peritrichous flagella, although no motility was evidenced, whereas their conspecifics in other hosts were either cytoplasmic and devoid of flagella, or macronuclear, displaying flagellar-driven motility. Moreover, contrarily to previously analyzed "Candidatus Trichorickettsia" hosts, infected P. calkinsi cells frequently became amicronucleate and demonstrated abnormal cell division, eventually leading to decline of the laboratory culture. Copyright © 2017 Elsevier GmbH. All rights reserved.

  2. Alcanivorax mobilis sp. nov., a new hydrocarbon-degrading bacterium isolated from deep-sea sediment.

    PubMed

    Yang, Shuo; Li, Meiqing; Lai, Qiliang; Li, Guizhen; Shao, Zongze

    2018-05-01

    A taxonomic study was carried out on strain MT13131 T , which was isolated from deep-sea sediment of the Indian Ocean during the screening of oil-degrading bacteria. The chain length range of n-alkanes (C8 to C32) oxidized by strain MT13131 T was determined in this study. The bacterium was Gram-negative, oxidase- and catalase-positive, single rod shaped, and motile by peritrichous flagella. Growth was observed at salinities of 1-12 % and at temperatures of 10-42 °C. The isolate was capable of Tween 20, 40 and 80 hydrolysis, but incapable of gelatin, cellulose or starch hydrolysis. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MT13131 T belonged to the genus Alcanivorax, with highest sequence similarity to Alcanivorax marinus R8-12 T (96.92 %), other species of genus Alcanivorax shared 92.96-96.69 % sequence similarity. The principal fatty acids were summed feature 3 (C16 : 1ω6c/ω7c), summed feature 8 (C18 : 1ω7c/ω6c), C16 : 0 and C12 : 0 3OH. The G+C content of the chromosomal DNA was 64.2 mol%. Phosphatidylglycerol, phosphatidylethanolamine, three aminolipids and three phospholipids were present. The combined genotypic and phenotypic data showed that strain MT13131 T represents a novel species within the genus Alcanivorax, for which the name Alcanivorax mobilis sp. nov. is proposed, with the type strain MT13131 T (=MCCC 1A11581 T =KCTC 52985 T ).

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

    SciT

    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

  4. Enterobacter aerogenes Hormaeche and Edwards 1960 (Approved Lists 1980) and Klebsiella mobilis Bascomb et al. 1971 (Approved Lists 1980) share the same nomenclatural type (ATCC 13048) on the Approved Lists and are homotypic synonyms, with consequences for the name Klebsiella mobilis Bascomb et al. 1971 (Approved Lists 1980).

    PubMed

    Tindall, B J; Sutton, G; Garrity, G M

    2017-02-01

    Enterobacter aerogenes Hormaeche and Edwards 1960 (Approved Lists 1980) and Klebsiella mobilis Bascomb et al. 1971 (Approved Lists 1980) were placed on the Approved Lists of Bacterial Names and were based on the same nomenclatural type, ATCC 13048. Consequently they are to be treated as homotypic synonyms. However, the names of homotypic synonyms at the rank of species normally are based on the same epithet. Examination of the Rules of the International Code of Nomenclature of Bacteria in force at the time indicates that the epithet mobilis in Klebsiella mobilis Bascomb et al. 1971 (Approved Lists 1980) was illegitimate at the time the Approved Lists were published and according to the Rules of the current International Code of Nomenclature of Prokaryotes continues to be illegitimate.

  5. 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.

  6. Ethanol production in Gram-positive microbes

    DOEpatents

    Ingram, L.O.; Barbosa-Alleyne, M.D.F.

    1999-06-29

    The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase. 2 figs.

  7. Ethanol production in recombinant hosts

    DOEpatents

    Ingram, Lonnie O'Neal; Barbosa-Alleyne, Maria D.

    2005-02-01

    The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

  8. Ethanol production in gram-positive microbes

    DOEpatents

    Ingram, Lonnie O'Neal; Barbosa-Alleyne, Maria D. F.

    1999-01-01

    The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

  9. Ethanol production in Gram-positive microbes

    DOEpatents

    Ingram, Lonnie O'Neal; Barbosa-Alleyne, Maria D. F.

    1996-01-01

    The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

  10. Ethanol production in Gram-positive microbes

    DOEpatents

    Ingram, L.O.; Barbosa-Alleyne, M.D.F.

    1996-01-09

    The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase. 2 figs.

  11. Biological conversion system

    DOEpatents

    Scott, C.D.

    A system for bioconversion of organic material comprises a primary bioreactor column wherein a biological active agent (zymomonas mobilis) converts the organic material (sugar) to a product (alcohol), a rejuvenator column wherein the biological activity of said biological active agent is enhanced, and means for circulating said biological active agent between said primary bioreactor column and said rejuvenator column.

  12. Identification of yeast and bacteria involved in the mezcal fermentation of Agave salmiana.

    PubMed

    Escalante-Minakata, P; Blaschek, H P; Barba de la Rosa, A P; Santos, L; De León-Rodríguez, A

    2008-06-01

    To identify the yeast and bacteria present in the mezcal fermentation from Agave salmiana. The restriction and sequence analysis of the amplified region, between 18S and 28S rDNA and 16S rDNA genes, were used for the identification of yeast and bacteria, respectively. Eleven different micro-organisms were identified in the mezcal fermentation. Three of them were the following yeast: Clavispora lusitaniae, Pichia fermentans and Kluyveromyces marxianus. The bacteria found were Zymomonas mobilis subsp. mobilis and Zymomonas mobilis subsp. pomaceae, Weissella cibaria, Weissella paramesenteroides, Lactobacillus pontis, Lactobacillus kefiri, Lactobacillus plantarum and Lactobacillus farraginis. The phylogenetic analysis of 16S rDNA and ITS sequences showed that microbial diversity present in mezcal is dominated by bacteria, mainly lactic acid bacteria species and Zymomonas mobilis. Pichia fermentans and K. marxianus could be micro-organisms with high potential for the production of some volatile compounds in mezcal. We identified the community of bacteria and yeast present in mezcal fermentation from Agave salmiana.

  13. Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

    PubMed Central

    Hensgens, C M; Vonck, J; Van Beeumen, J; van Bruggen, E F; Hansen, T A

    1993-01-01

    A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH. Images PMID:8491707

  14. Importance of stability study of continuous systems for ethanol production.

    PubMed

    Paz Astudillo, Isabel Cristina; Cardona Alzate, Carlos Ariel

    2011-01-10

    Fuel ethanol industry presents different problems during bioreactors operation. One of them is the unexpected variation in the output ethanol concentration from the bioreactor or a drastic fall in the productivity. In this paper, a compilation of concepts and relevant results of several experimental and theoretical studies about dynamic behavior of fermentation systems for bioethanol production with Saccharomyces cerevisiae and Zymomonas mobilis is done with the purpose of understanding the stability phenomena that could affect the productivity of industries producing fuel ethanol. It is shown that the design of high scale biochemical processes for fuel ethanol production must be done based on stability studies. © 2010 Elsevier B.V. All rights reserved.

  15. Bio-based extraction and stabilization of anthocyanins.

    PubMed

    Roy, Anirban; Mukherjee, Rudra Palash; Howard, Luke; Beitle, Robert

    2016-05-01

    This work reports a novel method of recovering anthocyanin compounds from highly-pigmented grapes via a fermentation based approach. It was hypothesized that batch growth of Zymomonas mobilis on simple medium would produce both ethanol and enzymes/biomass-acting materials, the combination of which will provide a superior extraction when compared to simple alcohol extraction. To examine this hypothesis, Z. mobilis was fermented in a batch consisting of mashed Vitis vinifera and glucose, and the recovered anthocyanin pool was compared to that recovered via extraction with ethanol. Data indicated higher amounts of anthocyanins were recovered when compared to simple solvent addition. Additionally, the percent polymeric form of the anthocyanins could be manipulated by the level of aeration maintained in the fermentation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:601-605, 2016. © 2016 American Institute of Chemical Engineers.

  16. Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism

    SciT

    Kosina, Suzanne M.; Danielewicz, Megan A.; Mohammed, Mujahid

    Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. In this paper, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the testmore » species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.« less

  17. Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism

    DOE PAGES

    Kosina, Suzanne M.; Danielewicz, Megan A.; Mohammed, Mujahid; ...

    2016-02-17

    Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. In this paper, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the testmore » species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.« less

  18. Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism.

    PubMed

    Kosina, Suzanne M; Danielewicz, Megan A; Mohammed, Mujahid; Ray, Jayashree; Suh, Yumi; Yilmaz, Suzan; Singh, Anup K; Arkin, Adam P; Deutschbauer, Adam M; Northen, Trent R

    2016-07-15

    Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. Here, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the test species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.

  19. Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli

    SciT

    Martinez, A.; York, S.W.; Yomano, L.P.

    1999-10-01

    Previous studies have shown an unexpectedly high nutrient requirement for efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. The basis for this requirement has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopymore » plasmids which express pdc and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: {sigma}{sup 70}, {sigma}{sup 38}, and {sigma}{sup 28}. The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pdc and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation.« less

  20. Bioethanol production from leafy biomass of mango (Mangifera indica) involving naturally isolated and recombinant enzymes.

    PubMed

    Das, Saprativ P; Ravindran, Rajeev; Deka, Deepmoni; Jawed, Mohammad; Das, Debasish; Goyal, Arun

    2013-01-01

    The present study describes the usage of dried leafy biomass of mango (Mangifera indica) containing 26.3% (w/w) cellulose, 54.4% (w/w) hemicellulose, and 16.9% (w/w) lignin, as a substrate for bioethanol production from Zymomonas mobilis and Candida shehatae. The substrate was subjected to two different pretreatment strategies, namely, wet oxidation and an organosolv process. An ethanol concentration (1.21 g/L) was obtained with Z. mobilis in a shake-flask simultaneous saccharification and fermentation (SSF) trial using 1% (w/v) wet oxidation pretreated mango leaves along with mixed enzymatic consortium of Bacillus subtilis cellulase and recombinant hemicellulase (GH43), whereas C. shehatae gave a slightly higher (8%) ethanol titer of 1.31 g/L. Employing 1% (w/v) organosolv pretreated mango leaves and using Z. mobilis and C. shehatae separately in the SSF, the ethanol titers of 1.33 g/L and 1.52 g/L, respectively, were obtained. The SSF experiments performed with 5% (w/v) organosolv-pretreated substrate along with C. shehatae as fermentative organism gave a significantly enhanced ethanol titer value of 8.11 g/L using the shake flask and 12.33 g/L at the bioreactor level. From the bioreactor, 94.4% (v/v) ethanol was recovered by rotary evaporator with 21% purification efficiency.

  1. Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates

    PubMed Central

    Skerker, Jeffrey M; Leon, Dacia; Price, Morgan N; Mar, Jordan S; Tarjan, Daniel R; Wetmore, Kelly M; Deutschbauer, Adam M; Baumohl, Jason K; Bauer, Stefan; Ibáñez, Ana B; Mitchell, Valerie D; Wu, Cindy H; Hu, Ping; Hazen, Terry; Arkin, Adam P

    2013-01-01

    The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4-fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate-derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance. PMID:23774757

  2. Novel reaction for new fuels

    SciT

    Clyde, R.A.

    Independent investigators have found that sugar can be fermented to alcohol for gasohol in 15 minutes by immobilizing organisms such as Zymomonas mobilis on rotating fibers. Half a pound of Celite has the surface area of a football field and it can be entrapped in the fibers to hold many cells. Many others take several hours. In another design, methanol can be dissociated to CO and H{sub 2} and 30% more energy obtained because waste heat is utilized. When one mol goes to three there is an expansion, and this is provided for. Both these designs result in clean fuelsmore » and are covered by U.S. patents.« less

  3. Accounting for all sugars produced during integrated production of ethanol from lignocellulosic biomass

    SciT

    Schell, Daniel J.; Dowe, Nancy; Chapeaux, Alexandre

    This study explored integrated conversion of corn stover to ethanol and highlights techniques for accurate yield calculations. Acid pretreated corn stover (PCS) produced in a pilot-scale reactor was enzymatically hydrolyzed and the resulting sugars were fermented to ethanol by the glucose–xylose fermenting bacteria, Zymomonas mobilis 8b. The calculations account for high solids operation and oligomeric sugars produced during pretreatment, enzymatic hydrolysis, and fermentation, which, if not accounted for, leads to overestimating ethanol yields. The calculations are illustrated for enzymatic hydrolysis and fermentation of PCS at 17.5% and 20.0% total solids achieving 80.1% and 77.9% conversion of cellulose and xylan tomore » ethanol and ethanol titers of 63 g/L and 69 g/L, respectively. In the future, these techniques, including the TEA results, will be applied to fully integrated pilot-scale runs.« less

  4. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

    1998-10-13

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

  5. Starch saccharification and fermentation of uncooked sweet potato roots for fuel ethanol production.

    PubMed

    Zhang, Peng; Chen, Caifa; Shen, Yanhu; Ding, Tielin; Ma, Daifu; Hua, Zichun; Sun, Dongxu

    2013-01-01

    An energy-saving ethanol fermentation technology was developed using uncooked fresh sweet potato as raw material. A mutant strain of Aspergillus niger isolated from mildewed sweet potato was used to produce abundant raw starch saccharification enzymes for treating uncooked sweet potato storage roots. The viscosity of the fermentation paste of uncooked sweet potato roots was lower than that of the cooked roots. The ethanol fermentation was carried out by Zymomonas mobilis, and 14.4 g of ethanol (87.2% of the theoretical yield) was produced from 100g of fresh sweet potato storage roots. Based on this method, an energy-saving, high efficient and environment-friendly technology can be developed for large-scale production of fuel ethanol from sweet potato roots. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Mathematical modeling of ethanol production in solid-state fermentation based on solid medium' dry weight variation.

    PubMed

    Mazaheri, Davood; Shojaosadati, Seyed Abbas; Zamir, Seyed Morteza; Mousavi, Seyyed Mohammad

    2018-04-21

    In this work, mathematical modeling of ethanol production in solid-state fermentation (SSF) has been done based on the variation in the dry weight of solid medium. This method was previously used for mathematical modeling of enzyme production; however, the model should be modified to predict the production of a volatile compound like ethanol. The experimental results of bioethanol production from the mixture of carob pods and wheat bran by Zymomonas mobilis in SSF were used for the model validation. Exponential and logistic kinetic models were used for modeling the growth of microorganism. In both cases, the model predictions matched well with the experimental results during the exponential growth phase, indicating the good ability of solid medium weight variation method for modeling a volatile product formation in solid-state fermentation. In addition, using logistic model, better predictions were obtained.

  7. Simultaneous saccharification of inulin and starch using commercial glucoamylase and the subsequent bioconversion to high titer sorbitol and gluconic acid.

    PubMed

    An, Kehong; Hu, Fengxian; Bao, Jie

    2013-12-01

    A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.

  8. Conceptual design of cost-effective and environmentally-friendly configurations for fuel ethanol production from sugarcane by knowledge-based process synthesis.

    PubMed

    Sánchez, Óscar J; Cardona, Carlos A

    2012-01-01

    In this work, the hierarchical decomposition methodology was used to conceptually design the production of fuel ethanol from sugarcane. The decomposition of the process into six levels of analysis was carried out. Several options of technological configurations were assessed in each level considering economic and environmental criteria. The most promising alternatives were chosen rejecting the ones with a least favorable performance. Aspen Plus was employed for simulation of each one of the technological configurations studied. Aspen Icarus was used for economic evaluation of each configuration, and WAR algorithm was utilized for calculation of the environmental criterion. The results obtained showed that the most suitable synthesized flowsheet involves the continuous cultivation of Zymomonas mobilis with cane juice as substrate and including cell recycling and the ethanol dehydration by molecular sieves. The proposed strategy demonstrated to be a powerful tool for conceptual design of biotechnological processes considering both techno-economic and environmental indicators. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Accounting for all sugars produced during integrated production of ethanol from lignocellulosic biomass

    DOE PAGES

    Schell, Daniel J.; Dowe, Nancy; Chapeaux, Alexandre; ...

    2016-01-19

    This study explored integrated conversion of corn stover to ethanol and highlights techniques for accurate yield calculations. Acid pretreated corn stover (PCS) produced in a pilot-scale reactor was enzymatically hydrolyzed and the resulting sugars were fermented to ethanol by the glucose–xylose fermenting bacteria, Zymomonas mobilis 8b. The calculations account for high solids operation and oligomeric sugars produced during pretreatment, enzymatic hydrolysis, and fermentation, which, if not accounted for, leads to overestimating ethanol yields. The calculations are illustrated for enzymatic hydrolysis and fermentation of PCS at 17.5% and 20.0% total solids achieving 80.1% and 77.9% conversion of cellulose and xylan tomore » ethanol and ethanol titers of 63 g/L and 69 g/L, respectively. In the future, these techniques, including the TEA results, will be applied to fully integrated pilot-scale runs.« less

  10. Single-species microbial biofilm screening for industrial applications.

    PubMed

    Li, Xuan Zhong; Hauer, Bernhard; Rosche, Bettina

    2007-10-01

    While natural microbial biofilms often consist of multiple species, single-species biofilms are of great interest to biotechnology. The current study evaluates biofilm formation for common industrial and laboratory microorganisms. A total of 68 species of biosafety level one bacteria and yeasts from over 40 different genera and five phyla were screened by growing them in microtiter plates and estimating attached biomass by crystal violet staining. Most organisms showed biofilm formation on surfaces of polystyrene within 24 h. By changing a few simple conditions such as substratum characteristics, inoculum and nutrient availability, 66 strains (97%) demonstrated biofilm formation under at least one of the experimental conditions and over half of these strains were classified as strong biofilm formers, potentially suitable as catalysts in biofilm applications. Many non-motile bacteria were also strong biofilm formers. Biofilm morphologies were visualized for selected strains. A model organism, Zymomonas mobilis, easily established itself as a biofilm on various reactor packing materials, including stainless steel.

  11. Inhibition of microbial biofuel production in drought-stressed switchgrass hydrolysate

    DOE PAGES

    Ong, Rebecca Garlock; Higbee, Alan; Bottoms, Scott; ...

    2016-11-08

    Here, interannual variability in precipitation, particularly drought, can affect lignocellulosic crop biomass yields and composition, and is expected to increase biofuel yield variability. However, the effect of precipitation on downstream fermentation processes has never been directly characterized. In order to investigate the impact of interannual climate variability on biofuel production, corn stover and switchgrass were collected during 3 years with significantly different precipitation profiles, representing a major drought year (2012) and 2 years with average precipitation for the entire season (2010 and 2013). All feedstocks were AFEX (ammonia fiber expansion)-pretreated, enzymatically hydrolyzed, and the hydrolysates separately fermented using xylose-utilizing strainsmore » of Saccharomyces cerevisiae and Zymomonas mobilis. As a result, a chemical genomics approach was also used to evaluate the growth of yeast mutants in the hydrolysates.« less

  12. 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.

  13. [Optimization of fuel ethanol production from kitchen waste by Plackett-Burman design].

    PubMed

    Ma, Hong-Zhi; Gong, Li-Juan; Wang, Qun-Hui; Zhang, Wen-Yu; Xu, Wen-Long

    2008-05-01

    Kitchen garbage was chosen to produce ethanol through simultaneous saccharification and fermentation (SSF) by Zymomonas mobilis. Plackett-Burman design was employed to screen affecting parameters during SSF process. The parameters were divided into two parts, enzymes and nutritions. None of the nutritions added showed significant effect during the experiment, which demonstrated that the kitchen garbage could meet the requirement of the microorganism without extra supplementation. Protease and glucoamylase were determined to be affecting factors for ethanol production. Single factor experiment showed that the optimum usage of these two enzymes were both 100 U/g and the corresponding maximum ethanol was determined to be 53 g/L. The ethanol yield could be as high as 44%. The utilization of kitchen garbage to produce ethanol could reduce threaten of waste as well as improve the protein content of the spent. This method could save the ethanol production cost and benefit for the recycle of kitchen garbage.

  14. Semicontinuous production of ethanol from agricultural wastes by immobilised coculture in a two stage bioreactor.

    PubMed

    Dey, Sabita

    2002-10-01

    The seed testing laboratories of Maharashtra discard 10 tonnes of grains and oil seeds treated with pesticides per annum. These agricultural wastes could be converted to reducing sugar and ethanol in a two stage semicontinuous fluidised bed bioreactor containing immobilised, Bacillus sp. and Zymomonas mobilis in the 1st stage and Saccharomyces diastaticus and S. cerevisae in the 2nd stage. The optimum temperature and pH for fermentation in both the stages were 30 degrees C and 7.2 respectively. In this process 600 g (approximately 400 g starch) waste seeds could yield 402 g of reducing sugar in the 1st stage after 20 h and 205 g ethanol in the 2nd stage after 40 h incubation with a yield factor 0.51. Using these system 10 tonnes of agricultural wastes could be converted to 3.4 x 107 g of ethanol.

  15. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    1998-01-01

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  16. Recombinant cells that highly express chromosomally-integrated heterologous gene

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  17. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2000-08-22

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  18. 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

  19. A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass

    PubMed Central

    Vija, Heiki; Aasamets, Anneli; Viigand, Katrin

    2017-01-01

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed. PMID:28103254

  20. Bioethanol Production From Banana Stem By Using Simultaneous Saccharification and Fermentation (SSF)

    NASA Astrophysics Data System (ADS)

    Kusmiyati; Mustofa, A.; Jumarmi

    2018-05-01

    The rapid growth and development of industries in the world result in a greater energy needs. Some studies show that ethanol can be used as an alternative energy. However, bioethanol production from food raw materials such as sugar and starch has drawback that cause the food crisis. This aim of this study was to convert banana stem into bioethanol. Banana stem contained of 44.6% cellulose, 36.0% hemicellulose and 19.4% lignin. After banana stems were pretreated with acid (H2SO4) and alkaline (NaOH) at a concentration of 2% w/v at 121 °C for 30 minutes, then subsequently the simultaneous saccharification and fermentation (SSF) were carried out by using mixed cultures of Aspergillus niger, Trichoderma reesei and Zymomonas mobilis at various enzymes ratios of (1:1:1), (1:2:1), (1:2:2), (1:1:2) and various pH (4, 5 and 6) with SSF time for 144 hours and temperature of 30°C. The results show that acid pretreatment showed better results than the alkali pretreatment. After acid pretreatment and alkali pretreatment, lignin content of pretreted banana stem reduced to 15.92% and 16.34%, respectively, cellulose increased to 52.11% and 50.6% respectively, hemicellulose reduced to 28.45% and 28.83%, respectively The SSF showed that pH 5 gave the highest bioethanol. The highest concentration of bioethanol (8.51 g/L) was achieved at the SSF process at pH 5 with a ratio Aspergillus niger, Trichoderma reesei and Zymomonas mobilis enzymes of (1:1:2).

  1. A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass.

    PubMed

    Mardo, Karin; Visnapuu, Triinu; Vija, Heiki; Aasamets, Anneli; Viigand, Katrin; Alamäe, Tiina

    2017-01-01

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5-6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

  2. Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels

    PubMed Central

    Jung, Sang-Kyu; Parisutham, Vinuselvi; Jeong, Seong Hun; Lee, Sung Kuk

    2012-01-01

    A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies. PMID:22911272

  3. OptSSeq: High-throughput sequencing readout of growth enrichment defines optimal gene expression elements for homoethanologenesis

    SciT

    Ghosh, Indro Neil; Landick, Robert

    The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosomebinding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate tomore » ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe 2+-dependent AdhB and Zn 2+- dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. Furthermore, OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.« less

  4. Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production.

    PubMed

    Kannuchamy, Saranyah; Mukund, Nisha; Saleena, Lilly M

    2016-05-11

    The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis. The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain. Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

  5. OptSSeq: High-throughput sequencing readout of growth enrichment defines optimal gene expression elements for homoethanologenesis

    DOE PAGES

    Ghosh, Indro Neil; Landick, Robert

    2016-07-16

    The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosomebinding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate tomore » ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe 2+-dependent AdhB and Zn 2+- dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. Furthermore, OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.« less

  6. Technique of ethanol food grade production with batch distillation and dehydration using starch-based adsorbent

    NASA Astrophysics Data System (ADS)

    Widjaja, Tri; Altway, Ali; Ni'mah, Hikmatun; Tedji, Namira; Rofiqah, Umi

    2015-12-01

    Development and innovation of ethanol food grade production are becoming the reasearch priority to increase economy growth. Moreover, the government of Indonesia has established regulation for increasing the renewable energy as primary energy. Sorghum is cerealia plant that contains 11-16% sugar that is optimum for fermentation process, it is potential to be cultivated, especially at barren area in Indonesia. The purpose of this experiment is to learn about the effect of microorganisms in fermentation process. Fermentation process was carried out batchwise in bioreactor and used 150g/L initial sugar concentration. Microorganisms used in this experiment are Zymomonas mobilis mutation (A3), Saccharomyces cerevisiae and mixed of Pichia stipitis. The yield of ethanol can be obtained from this experiment. For ethanol purification result, distillation process from fermentation process has been done to search the best operation condition for efficiency energy consumption. The experiment for purification was divided into two parts, which are distillation with structured packing steel wool and adsorption (dehydration) sequencely. In distillation part, parameters evaluation (HETP and pressure drop) of distillation column that can be used for scale up are needed. The experiment was operated at pressure of 1 atm. The distillation stage was carried out at 85 °C and reflux ratio of 0.92 with variety porosities of 20%, 40%, and 60%. Then the adsorption process was done at 120°C and two types of adsorbent, which are starch - based adsorbent with ingredient of cassava and molecular sieve 3A, were used. The adsorption process was then continued to purify the ethanol from impurities by using activated carbon. This research shows that the batch fermentation process with Zymomonas mobilis A3 obtain higher % yield of ethanol of 40,92%. In addition to that, for purification process, the best operation condition is by using 40% of porosity of stuctured packing steel wool in distillation

  7. Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B

    SciT

    Zhou, S.; Yomano, L.P.; Saleh, A.Z.

    1999-06-01

    Escherichia coli B has been engineered as a biocatalyst for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of E. coli B can produce high levels of Erwinia chrysanthemi endoglucanase (encoded by celZ) as a periplasmic product and that this enzyme can function with commercial fungal cellulase to increase ethanol production. In this study, the authors have demonstrated two methods that improve celZ expression in E. coli B. Initially, with a low-copy-number vector, two E. coli glycolytic gene promoters (gap and eno) were tested and found to be less effective than the original celZ promoter. Bymore » screening 18,000 random fragments of Zymomonas mobilis DNA, a surrogate promoter was identified which increased celZ expression up to sixfold. With this promoter, large polar inclusion bodies were clearly evident in the periplasmic space. Sequencing revealed that the most active surrogate promoter is derived from five Sau3A1 fragments, one of which was previously sequenced in Z. mobilis. Visual inspection indicated that this DNA fragment contains at least five putative promoter regions, two of which were confirmed by primer extension analysis. Addition of the out genes from E. chrysanthemi EC16 caused a further increase in the production of active enzyme and facilitated secretion or release of over half of the activity into the extracellular environment. With the most active construct, of a total of 13,000 IU of active enzyme per liter of culture, 7,800 IU was in the supernatant. The total active endoglucanase was estimated to represent 4 to 6% of cellular protein.« less

  8. Increase in Furfural Tolerance in Ethanologenic Escherichia coli LY180 by Plasmid-Based Expression of thyA

    PubMed Central

    Zheng, Huabao; Wang, Xuan; Yomano, Lorraine P.; Shanmugam, Keelnatham T.

    2012-01-01

    Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886, Escherichia coli NC3, and Zymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing the thyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in the de novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression of thyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA in E. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA in E. coli would be expected to increase the cellular requirement for dTMP. Increased expression of thyA (E. coli, B. subtilis, or Z. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair. PMID:22504824

  9. Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA.

    PubMed

    Zheng, Huabao; Wang, Xuan; Yomano, Lorraine P; Shanmugam, Keelnatham T; Ingram, Lonnie O

    2012-06-01

    Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886, Escherichia coli NC3, and Zymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing the thyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in the de novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression of thyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA in E. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA in E. coli would be expected to increase the cellular requirement for dTMP. Increased expression of thyA (E. coli, B. subtilis, or Z. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

  10. Replacing process water and nitrogen sources with biogas slurry during cellulosic ethanol production.

    PubMed

    You, Yang; Wu, Bo; Yang, Yi-Wei; Wang, Yan-Wei; Liu, Song; Zhu, Qi-Li; Qin, Han; Tan, Fu-Rong; Ruan, Zhi-Yong; Ma, Ke-Dong; Dai, Li-Chun; Zhang, Min; Hu, Guo-Quan; He, Ming-Xiong

    2017-01-01

    Environmental issues, such as the fossil energy crisis, have resulted in increased public attention to use bioethanol as an alternative renewable energy. For ethanol production, water and nutrient consumption has become increasingly important factors being considered by the bioethanol industry as reducing the consumption of these resources would decrease the overall cost of ethanol production. Biogas slurry contains not only large amounts of wastewater, but also the nutrients required for microbial growth, e.g., nitrogen, ammonia, phosphate, and potassium. Therefore, biogas slurry is an attractive potential resource for bioethanol production that could serve as an alternative to process water and nitrogen sources. In this study, we propose a method that replaces the process water and nitrogen sources needed for cellulosic ethanol production by Zymomonas mobilis with biogas slurry. To test the efficacy of these methods, corn straw degradation following pretreatment with diluted NaOH and enzymatic hydrolysis in the absence of fresh water was evaluated. Then, ethanol fermentation using the ethanologenic bacterial strain Z. mobilis ZMT2 was conducted without supplementing with additional nitrogen sources. After pretreatment with 1.34% NaOH (w/v) diluted in 100% biogas slurry and continuous enzymatic hydrolysis for 144 h, 29.19 g/L glucose and 12.76 g/L xylose were generated from 30 g dry corn straw. The maximum ethanol concentration acquired was 13.75 g/L, which was a yield of 72.63% ethanol from the hydrolysate medium. Nearly 94.87% of the ammonia nitrogen was depleted and no nitrate nitrogen remained after ethanol fermentation. The use of biogas slurry as an alternative to process water and nitrogen sources may decrease the cost of cellulosic ethanol production by 10.0-20.0%. By combining pretreatment with NaOH diluted in biogas slurry, enzymatic hydrolysis, and ethanol fermentation, 56.3 kg of ethanol was produced by Z. mobilis ZMT-2 through fermentation of

  11. Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments.

    PubMed

    Wiechert, W; de Graaf, A A

    1997-07-05

    The extension of metabolite balancing with carbon labeling experiments, as described by Marx et al. (Biotechnol. Bioeng. 49: 11-29), results in a much more detailed stationary metabolic flux analysis. As opposed to basic metabolite flux balancing alone, this method enables both flux directions of bidirectional reaction steps to be quantitated. However, the mathematical treatment of carbon labeling systems is much more complicated, because it requires the solution of numerous balance equations that are bilinear with respect to fluxes and fractional labeling. In this study, a universal modeling framework is presented for describing the metabolite and carbon atom flux in a metabolic network. Bidirectional reaction steps are extensively treated and their impact on the system's labeling state is investigated. Various kinds of modeling assumptions, as usually made for metabolic fluxes, are expressed by linear constraint equations. A numerical algorithm for the solution of the resulting linear constrained set of nonlinear equations is developed. The numerical stability problems caused by large bidirectional fluxes are solved by a specially developed transformation method. Finally, the simulation of carbon labeling experiments is facilitated by a flexible software tool for network synthesis. An illustrative simulation study on flux identifiability from available flux and labeling measurements in the cyclic pentose phosphate pathway of a recombinant strain of Zymomonas mobilis concludes this contribution.

  12. Fermentation of xylose to ethanol by genetically modified enteric bacteria

    SciT

    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

  13. Fermentation of D-xylose and L-arabinose to ethanol by Erwinia chrysanthemi

    SciT

    Tolan, J.S.; Finn, R.K.

    1987-09-01

    Erwinia spp. are gram-negative facultative anaerobes within the family Enterobacteriacae which possess several desirable traits for the conversion of pentose sugars to ethanol, such as the ability to ferment a broad range of carbohydrates and the ease with which they can be genetically modified. Twenty-eight strains of Erwinia carotovora and E. chrysanthemi were screened for the ability to ferment D-xylose to ethanol. E. chrysanthemi B374 was chosen for further study on the basis of its superior (4%) ethanol tolerance. They have characterized the fermentation of D-xylose and L-arabinose by the wild type and mutants which bear plasmids containing the pyruvatemore » decarboxylase gene from Zymomonas mobilis. Expression of the gene markedly increased the yields of ethanol (from 0.7 up to 1.45 mol/mol of xylose) and decreased the yields of formate, acetate, and lactate. However, the cells with pyruvate decarboxylase grew only one-fourth as fast as the wild type and tolerated only 2% ethanol. Alcohol tolerance was stimulated by the addition of yeast extract to the growth medium. Xylose catabolism was characterized by a high saturation constant K/sub s/ (4.5 mM).« less

  14. 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.

  15. Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria

    PubMed Central

    Flynn, Jeffrey M.; Ross, Daniel E.; Hunt, Kristopher A.; Bond, Daniel R.; Gralnick, Jeffrey A.

    2010-01-01

    Cellular metabolism is a series of tightly linked oxidations and reductions that must be balanced. Recycling of intracellular electron carriers during fermentation often requires substrate conversion to undesired products, while respiration demands constant addition of electron acceptors. The use of electrode-based electron acceptors to balance biotransformations may overcome these constraints. To test this hypothesis, the metal-reducing bacterium Shewanella oneidensis was engineered to stoichiometrically convert glycerol into ethanol, a biotransformation that will not occur unless two electrons are removed via an external reaction, such as electrode reduction. Multiple modules were combined into a single plasmid to alter S. oneidensis metabolism: a glycerol module, consisting of glpF, glpK, glpD, and tpiA from Escherichia coli, and an ethanol module containing pdc and adh from Zymomonas mobilis. A further increase in product yields was accomplished through knockout of pta, encoding phosphate acetyltransferase, shifting flux toward ethanol and away from acetate production. In this first-generation demonstration, conversion of glycerol to ethanol required the presence of an electrode to balance the reaction, and electrode-linked rates were on par with volumetric conversion rates observed in engineered E. coli. Linking microbial biocatalysis to current production can eliminate redox constraints by shifting other unbalanced reactions to yield pure products and serve as a new platform for next-generation bioproduction strategies. PMID:21060736

  16. Simultaneous saccharification and cofermentation of peracetic acid-pretreated biomass.

    PubMed

    Teixeira, L C; Linden, J C; Schroeder, H A

    2000-01-01

    Previous work in our laboratories has demonstrated the effectiveness of peracetic acid for improving enzymatic digestibility of lignocellulosic materials. The use of dilute alkali solutions as a pre-pretreatment prior to peracetic acid lignin oxidation increased carbohydrate hydrolysis yields in a synergistic as opposed to additive manner. Deacetylation of xylan is easily achieved using dilute alkali solutions under mild conditions. In this article, we evaluate the effectiveness of peracetic acid combined with an alkaline pre-pretreatment through simultaneous saccharification and cofermentation (SSCF) of pretreated hybrid poplar wood and sugar cane bagasse. Respective ethanol yields of 92.8 and 91.9% of theoretical are achieved using 6% NaOH/15% peracetic acid-pretreated substrates and recombinant Zymomonas mobilis CP4/pZB5. Reduction of acetyl groups of the lignocellulosic materials is demonstrated following alkaline pre-pretreatments. Such processing may be helpful in reducing peracetic acid requirements. The influence of deacetylation is more significant in combined pretreatments using lower peracetic acid loadings.

  17. De novo Biosynthesis of Biodiesel by Escherichia coli in Optimized Fed-Batch Cultivation

    PubMed Central

    Cai, Ke; Tan, Xiaoming; Lu, Xuefeng

    2011-01-01

    Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L−1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate. PMID:21629774

  18. Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol.

    PubMed

    Mohagheghi, Ali; Schell, Daniel J

    2010-04-01

    Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute-acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose-xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute-acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical-based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently. (c) 2009 Wiley Periodicals, Inc.

  19. Bioethanol production from fermentable sugar juice.

    PubMed

    Zabed, Hossain; Faruq, Golam; Sahu, Jaya Narayan; Azirun, Mohd Sofian; Hashim, Rosli; Boyce, Amru Nasrulhaq

    2014-01-01

    Bioethanol production from renewable sources to be used in transportation is now an increasing demand worldwide due to continuous depletion of fossil fuels, economic and political crises, and growing concern on environmental safety. Mainly, three types of raw materials, that is, sugar juice, starchy crops, and lignocellulosic materials, are being used for this purpose. This paper will investigate ethanol production from free sugar containing juices obtained from some energy crops such as sugarcane, sugar beet, and sweet sorghum that are the most attractive choice because of their cost-effectiveness and feasibility to use. Three types of fermentation process (batch, fed-batch, and continuous) are employed in ethanol production from these sugar juices. The most common microorganism used in fermentation from its history is the yeast, especially, Saccharomyces cerevisiae, though the bacterial species Zymomonas mobilis is also potentially used nowadays for this purpose. A number of factors related to the fermentation greatly influences the process and their optimization is the key point for efficient ethanol production from these feedstocks.

  20. Bacterial supersystem for alginate import/metabolism and its environmental and bioenergy applications.

    PubMed

    Hashimoto, Wataru; Kawai, Shigeyuki; Murata, Kousaku

    2010-01-01

    Distinct from most alginate-assimilating bacteria that secrete polysaccharide lyases extracellularly, a gram-negative bacterium, Sphingomonas sp. A1 (strain A1), can directly incorporate alginate into its cytoplasm, without degradation, through a "superchannel" consisting of a mouth-like pit on the cell surface, periplasmic binding proteins, and a cytoplasmic membrane-bound ATP-binding cassette transporter. Flagellin homologues function as cell surface alginate receptors essential for expressing the superchannel. Cytoplasmic alginate lyases with different substrate specificities and action modes degrade the polysaccharide to its constituent monosaccharides. The resultant monosaccharides, α-keto acids, are converted to a reduced form by NADPH-dependent reductase, and are finally metabolized in the TCA cycle. Transplantation of the strain A1 superchannel to xenobiotic-degrading sphingomonads enhances bioremediation through the propagation of bacteria with an elevated transport activity. Furthermore, strain A1 cells transformed with Zymomonas mobilis genes for pyruvate decarboxylase and alcohol dehydrogenase II produce considerable amounts of biofuel ethanol from alginate when grown statically. © 2010 Landes Bioscience

  1. An in vivo, label-free quick assay for xylose transport in Escherichia coli.

    PubMed

    Chen, Tingjian; Zhang, Jingqing; Liang, Ling; Yang, Rong; Lin, Zhanglin

    2009-07-01

    Efficient use of xylose is necessary for economic production of biochemicals and biofuels from lignocellulosic materials. Current studies on xylose uptake for various microorganisms have been hampered by the lack of a facile assay for xylose transport. In this work, a rapid in vivo, label-free method for measuring xylose transport in Escherichia coli was developed by taking advantage of the Bacillus pumilus xylosidase (XynB), which cleaved a commercially available xylose analog, p-nitrophenyl-beta-d-xylopyranoside (pNPX), to release a chromogenic group, p-nitrophenol (pNP). XynB was expressed alone or in conjunction with a Zymomonas mobilis glucose facilitator protein (Glf) capable of transporting xylose. This XynB-mediated transport assay was demonstrated in test tubes and 96-well plates with submicromolar concentrations of pNPX. Kinetic inhibition experiments validated that pNPX and xylose were competitive substrates for the transport process, and the addition of glucose (20 g/L) in the culture medium clearly diminished the transmembrane transport of pNPX and, thus, mimicked its inhibitory action on xylose uptake. This method should be useful for engineering of the xylose transport process in E. coli, and similar assay schemes can be extended to other microorganisms.

  2. Pulque production from fermented agave sap as a dietary supplement in Prehispanic Mesoamerica.

    PubMed

    Correa-Ascencio, Marisol; Robertson, Ian G; Cabrera-Cortés, Oralia; Cabrera-Castro, Rubén; Evershed, Richard P

    2014-09-30

    Although in modern societies fermented beverages are associated with socializing, celebration, and ritual, in ancient times they were also importa`nt sources of essential nutrients and potable water. In Mesoamerica, pulque, an alcoholic beverage produced from the fermented sap of several species of maguey plants (Agavaceae; Fig. 1) is hypothesized to have been used as a dietary supplement and risk-buffering food in ancient Teotihuacan (150 B.C. to A.D. 650). Although direct archaeological evidence of pulque production is lacking, organic residue analysis of pottery vessels offers a new avenue of investigation. However, the chemical components of alcoholic beverages are water-soluble, greatly limiting their survival over archaeological timescales compared with hydrophobic lipids widely preserved in food residues. Hence, we apply a novel lipid biomarker approach that considers detection of bacteriohopanoids derived from the ethanol-producing bacterium Zymomonas mobilis for identifying pulque production/consumption in pottery vessels. Gas chromatography-mass spectrometry selected ion monitoring (m/z 191) of lipid extracts of >300 potsherds revealed characteristic bacteriohopanoid distributions in a subset of 14 potsherds. This hopanoid biomarker approach offers a new means of identifying commonly occurring bacterially fermented alcoholic beverages worldwide, including palm wine, beer, cider, perry, and other plant sap- or fruit-derived beverages [Swings J, De Ley J (1977) Bacteriol Rev 41(1):1-46].

  3. Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin.

    PubMed

    Chionis, Kostas; Krikorian, Dimitrios; Koukkou, Anna-Irini; Sakarellos-Daitsiotis, Maria; Panou-Pomonis, Eugenia

    2016-11-01

    Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G 1 LLKR 5 IKT 8 LL-NH 2 , it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly 1 , Arg 5 , and Thr 8 and lipophilic groups with different lengths in the N-terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF 5 IKK 8 LL-NH 2 exhibited high activity against Gram-negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N-terminus increased the antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α-helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  4. Bioethanol production by heterologous expression of Pdc and AdhII in Streptomyces lividans.

    PubMed

    Lee, Jae Sun; Chi, Won-Jae; Hong, Soon-Kwang; Yang, Ji-Won; Chang, Yong Keun

    2013-07-01

    Two genes from Zymomonas mobilis that are responsible for ethanol production, pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhII), were heterologously expressed in the Gram-positive bacterium Streptomyces lividans TK24. An examination of carbon distribution revealed that a significant portion of carbon metabolism was switched from biomass and organic acid biosynthesis to ethanol production upon the expression of pdc and adhII. The recombinant S. lividans TK24 produced ethanol from glucose with a yield of 23.7% based on the carbohydrate consumed. The recombinant was able to produce ethanol from xylose, L-arabinose, mannose, L-rhamnose, galactose, ribose, and cellobiose with yields of 16.0, 25.6, 21.5, 33.6, 30.6, 14.6, and 33.3%, respectively. Polymeric substances such as starch and xylan were directly converted to ethanol by the recombinant with ethanol yields of 18.9 and 8.8%, respectively. The recombinant S. lividans TK24/Tpet developed in this study is potentially a useful microbial resource for ethanol production from various sources of biomasses, especially microalgae.

  5. Three immobilized-cell columnar bioreactors for enhanced production of commodity chemicals

    SciT

    Davison, B.H.; Scott, C.D.; Kaufman, E.N.

    1993-12-31

    Immobilized-cell fluidized-bed bioreactors (FBRs) can be used with a variety of fermentations to increase production of fuels, solvents, organic acids, and other fermentation products. Part of the increased rates and yields are due to the immobilization of the biocatalyst at high concentrations. This FBR system with immobilized Zymomonas mobilis increased ethanol productivity more than tenfold with 99% conversion and near stoichiometric yields. FBRs also offer several additional modes of operation for simultaneous fermentation and separation to further increase production by removing the inhibitory products directly from the continuous fermentation. The production of lactic acid by immobilized Lactobacillus was augmented withmore » the addition and removal of solid adsorbent particles to the FBR. An immiscible organic extractant also was used to extract butanol from the acetone-butanol fermentation by Clostridium acetobutylicum. Demonstrations with these FBR systems have already shown definite advantages by improved overall product yields (decreasing feed costs) and by increased rates (decreasing capital and operating costs). Further demonstration and scale-up continue.« less

  6. Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

    PubMed

    Yang, Mingfeng; Li, Xuefeng; Bu, Chunya; Wang, Hui; Shi, Guanglu; Yang, Xiushan; Hu, Yong; Wang, Xiaoqin

    2014-11-01

    Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

  7. Bioethanol Production from Fermentable Sugar Juice

    PubMed Central

    Zabed, Hossain; Faruq, Golam; Sahu, Jaya Narayan; Azirun, Mohd Sofian; Hashim, Rosli; Nasrulhaq Boyce, Amru

    2014-01-01

    Bioethanol production from renewable sources to be used in transportation is now an increasing demand worldwide due to continuous depletion of fossil fuels, economic and political crises, and growing concern on environmental safety. Mainly, three types of raw materials, that is, sugar juice, starchy crops, and lignocellulosic materials, are being used for this purpose. This paper will investigate ethanol production from free sugar containing juices obtained from some energy crops such as sugarcane, sugar beet, and sweet sorghum that are the most attractive choice because of their cost-effectiveness and feasibility to use. Three types of fermentation process (batch, fed-batch, and continuous) are employed in ethanol production from these sugar juices. The most common microorganism used in fermentation from its history is the yeast, especially, Saccharomyces cerevisiae, though the bacterial species Zymomonas mobilis is also potentially used nowadays for this purpose. A number of factors related to the fermentation greatly influences the process and their optimization is the key point for efficient ethanol production from these feedstocks. PMID:24715820

  8. Metabolically engineered glucose-utilizing Shewanella strains under anaerobic conditions.

    PubMed

    Choi, Donggeon; Lee, Sae Bom; Kim, Sohyun; Min, Byoungnam; Choi, In-Geol; Chang, In Seop

    2014-02-01

    Comparative genome analysis of Shewanella strains predicted that the strains metabolize preferably two- and three-carbon carbohydrates as carbon/electron source because many Shewanella genomes are deficient of the key enzymes in glycolysis (e.g., glucokinase). In addition, all Shewanella genomes are known to have only one set of genes associated with the phosphotransferase system required to uptake sugars. To engineer Shewanella strains that can utilize five- and six-carbon carbohydrates, we constructed glucose-utilizing Shewanella oneidensis MR-1 by introducing the glucose facilitator (glf; ZMO0366) and glucokinase (glk; ZMO0369) genes of Zymomonas mobilis. The engineered MR-1 strain was able to grow on glucose as a sole carbon/electron source under anaerobic conditions. The glucose affinity (Ks) and glucokinase activity in the engineered MR-1 strain were 299.46 mM and 0.259 ± 0.034 U/g proteins. The engineered strain was successfully applied to a microbial fuel cell system and exhibited current generation using glucose as the electron source. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Pulque production from fermented agave sap as a dietary supplement in Prehispanic Mesoamerica

    PubMed Central

    Correa-Ascencio, Marisol; Robertson, Ian G.; Cabrera-Cortés, Oralia; Cabrera-Castro, Rubén; Evershed, Richard P.

    2014-01-01

    Although in modern societies fermented beverages are associated with socializing, celebration, and ritual, in ancient times they were also importa`nt sources of essential nutrients and potable water. In Mesoamerica, pulque, an alcoholic beverage produced from the fermented sap of several species of maguey plants (Agavaceae; Fig. 1) is hypothesized to have been used as a dietary supplement and risk-buffering food in ancient Teotihuacan (150 B.C. to A.D. 650). Although direct archaeological evidence of pulque production is lacking, organic residue analysis of pottery vessels offers a new avenue of investigation. However, the chemical components of alcoholic beverages are water-soluble, greatly limiting their survival over archaeological timescales compared with hydrophobic lipids widely preserved in food residues. Hence, we apply a novel lipid biomarker approach that considers detection of bacteriohopanoids derived from the ethanol-producing bacterium Zymomonas mobilis for identifying pulque production/consumption in pottery vessels. Gas chromatography–mass spectrometry selected ion monitoring (m/z 191) of lipid extracts of >300 potsherds revealed characteristic bacteriohopanoid distributions in a subset of 14 potsherds. This hopanoid biomarker approach offers a new means of identifying commonly occurring bacterially fermented alcoholic beverages worldwide, including palm wine, beer, cider, perry, and other plant sap- or fruit-derived beverages [Swings J, De Ley J (1977) Bacteriol Rev 41(1):1–46]. PMID:25225408

  10. Classification of Rhizomonas suberifaciens, an unnamed Rhizomonas species, and Sphingomonas spp. in rRNA superfamily IV.

    PubMed

    van Bruggen, A H; Jochimsen, K N; Steinberger, E M; Segers, P; Gillis, M

    1993-01-01

    Thermal melting profiles of hybrids between 3H-labeled rRNA of Rhizomonas suberifaciens, the causal agent of corky root of lettuce, and chromosomal DNAs from 27 species of gram-negative bacteria indicated that the genus Rhizomonas belongs to superfamily IV of De Ley. On the basis of the melting temperatures of DNA hybrids with rRNAs from the type strains of R. suberifaciens, Sphingomonas paucimobilis, and Sphingomonas capsulata, Rhizomonas strains constitute a separate branch in superfamily IV, which is closely related to but separate from branches containing Zymomonas mobilis, Sphingomonas spp., and S. capsulata. Sphingomonas yanoikuyae and Rhizomonas sp. strain WI4 are located toward the base of the Rhizomonas rRNA branch. DNA-DNA hybridization indicated that S. yanoikuyae is equidistant from Rhizomonas sp. strain WI4 and S. paucimobilis. Sequences of 270 bp of 16S ribosomal DNAs from eight strains of Rhizomonas spp., eight strains of Sphingomonas spp., and Agrobacterium tumefaciens indicated that S. yanoikuyae and Rhizomonas sp. strains WI4 and CA16 are genetically more closely related to R. suberifaciens than to Sphingomonas spp. Thus, S. yanoikuyae may need to be transferred to the genus Rhizomonas on the basis of the results of further study.

  11. Conditioning of dilute-acid pretreated corn stover hydrolysate liquors by treatment with lime or ammonium hydroxide to improve conversion of sugars to ethanol.

    PubMed

    Jennings, Edward W; Schell, Daniel J

    2011-01-01

    Dilute-acid pretreatment of lignocellulosic biomass enhances the ability of enzymes to hydrolyze cellulose to glucose, but produces many toxic compounds that inhibit fermentation of sugars to ethanol. The objective of this study was to compare the effectiveness of treating hydrolysate liquor with Ca(OH)2 and NH4OH for improving ethanol yields. Corn stover was pretreated in a pilot-scale reactor and then the liquor fraction (hydrolysate) was extracted and treated with various amounts of Ca(OH)2 or NH4OH at several temperatures. Glucose and xylose in the treated liquor were fermented to ethanol using a glucose-xylose fermenting bacteria, Zymomonas mobilis 8b. Sugar losses up to 10% occurred during treatment with Ca(OH)2, but these losses were two to fourfold lower with NH4OH treatment. Ethanol yields for NH4OH-treated hydrolysate were 33% greater than those achieved in Ca(OH)2-treated hydrolysate and pH adjustment to either 6.0 or 8.5 with NH4OH prior to fermentation produced equivalent ethanol yields. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Accounting for all sugars produced during integrated production of ethanol from lignocellulosic biomass.

    PubMed

    Schell, Daniel J; Dowe, Nancy; Chapeaux, Alexandre; Nelson, Robert S; Jennings, Edward W

    2016-04-01

    Accurate mass balance and conversion data from integrated operation is needed to fully elucidate the economics of biofuel production processes. This study explored integrated conversion of corn stover to ethanol and highlights techniques for accurate yield calculations. Acid pretreated corn stover (PCS) produced in a pilot-scale reactor was enzymatically hydrolyzed and the resulting sugars were fermented to ethanol by the glucose-xylose fermenting bacteria, Zymomonas mobilis 8b. The calculations presented here account for high solids operation and oligomeric sugars produced during pretreatment, enzymatic hydrolysis, and fermentation, which, if not accounted for, leads to overestimating ethanol yields. The calculations are illustrated for enzymatic hydrolysis and fermentation of PCS at 17.5% and 20.0% total solids achieving 80.1% and 77.9% conversion of cellulose and xylan to ethanol and ethanol titers of 63g/L and 69g/L, respectively. These procedures will be employed in the future and the resulting information used for techno-economic analysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Thermophilic Bacillus coagulans requires less cellulases for simultaneous saccharification and fermentation of cellulose to products than mesophilic microbial biocatalysts.

    PubMed

    Ou, Mark S; Mohammed, Nazimuddin; Ingram, L O; Shanmugam, K T

    2009-05-01

    Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g(-1) cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g(-1) cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.

  14. Monitoring microbial growth and activity using spectral induced polarization and low-field nuclear magnetic resonance

    NASA Astrophysics Data System (ADS)

    Zhang, Chi; Keating, Kristina; Revil, Andre

    2015-04-01

    Microbes and microbial activities in the Earth's subsurface play a significant role in shaping subsurface environments and are involved in environmental applications such as remediation of contaminants in groundwater and oil fields biodegradation. Stimulated microbial growth in such applications could cause wide variety of changes of physical/chemical properties in the subsurface. It is critical to monitor and determine the fate and transportation of microorganisms in the subsurface during such applications. Recent geophysical studies demonstrate the potential of two innovative techniques, spectral induced polarization (SIP) and low-field nuclear magnetic resonance (NMR), for monitoring microbial growth and activities in porous media. The SIP measures complex dielectric properties of porous media at low frequencies of exciting electric field, and NMR studies the porous structure of geologic media and characterizes fluids subsurface. In this laboratory study, we examined both SIP and NMR responses from bacterial growth suspension as well as suspension mixed with silica sands. We focus on the direct contribution of microbes to the SIP and NMR signals in the absence of biofilm formation or biomineralization. We used Zymomonas mobilis and Shewanella oneidensis (MR-1) for SIP and NMR measurements, respectively. The SIP measurements were collected over the frequency range of 0.1 - 1 kHz on Z. mobilis growth suspension and suspension saturated sands at different cell densities. SIP data show two distinct peaks in imaginary conductivity spectra, and both imaginary and real conductivities increased as microbial density increased. NMR data were collected using both CPMG pulse sequence and D-T2 mapping to determine the T2-distribution and diffusion properties on S. oneidensis suspension, pellets (live and dead), and suspension mixed with silica sands. NMR data show a decrease in the T2-distribution in S. oneidensis suspension saturated sands as microbial density increase. A

  15. 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

  16. Controlling microbial contamination during hydrolysis of AFEX-pretreated corn stover and switchgrass: Effects on hydrolysate composition, microbial response and fermentation

    DOE PAGES

    Serate, Jose; Xie, Dan; Pohlmann, Edward; ...

    2015-11-14

    Microbial conversion of lignocellulosic feedstocks into biofuels remains an attractive means to produce sustainable energy. It is essential to produce lignocellulosic hydrolysates in a consistent manner in order to study microbial performance in different feedstock hydrolysates. Because of the potential to introduce microbial contamination from the untreated biomass or at various points during the process, it can be difficult to control sterility during hydrolysate production. In this study, we compared hydrolysates produced from AFEX-pretreated corn stover and switchgrass using two different methods to control contamination: either by autoclaving the pretreated feedstocks prior to enzymatic hydrolysis, or by introducing antibiotics duringmore » the hydrolysis of non-autoclaved feedstocks. We then performed extensive chemical analysis, chemical genomics, and comparative fermentations to evaluate any differences between these two different methods used for producing corn stover and switchgrass hydrolysates. Autoclaving the pretreated feedstocks could eliminate the contamination for a variety of feedstocks, whereas the antibiotic gentamicin was unable to control contamination consistently during hydrolysis. Compared to the addition of gentamicin, autoclaving of biomass before hydrolysis had a minimal effect on mineral concentrations, and showed no significant effect on the two major sugars (glucose and xylose) found in these hydrolysates. However, autoclaving elevated the concentration of some furanic and phenolic compounds. Chemical genomics analyses using Saccharomyces cerevisiae strains indicated a high correlation between the AFEX-pretreated hydrolysates produced using these two methods within the same feedstock, indicating minimal differences between the autoclaving and antibiotic methods. Comparative fermentations with S. cerevisiae and Zymomonas mobilis also showed that autoclaving the AFEX-pretreated feedstocks had no significant effects on microbial

  17. Sucrose dependent mineral phosphate solubilization in Enterobacter asburiae PSI3 by heterologous overexpression of periplasmic invertases.

    PubMed

    Kumar, Chanchal; Wagh, Jitendra; Archana, G; Naresh Kumar, G

    2016-12-01

    Enterobacter asburiae PSI3 solubilizes mineral phosphates in the presence of glucose by the secretion of gluconic acid generated by the action of a periplasmic pyrroloquinoline quinone dependent glucose dehydrogenase. In order to achieve mineral phosphate solubilization phenotype in the presence of sucrose, plasmids pCNK4 and pCNK5 containing genes encoding the invertase enzyme of Zymomonas mobilis (invB) and of Saccharomyces cerevisiae (suc2) under constitutive promoters were constructed with malE signal sequence (in case of invB alone as the suc2 is secreted natively). When introduced into E. asburiae PSI3, E. a. (pCNK4) and E. a. (pCNK5) transformants secreted 21.65 ± 0.94 and 22 ± 1.3 mM gluconic acid, respectively, in the presence of 75 mM sucrose and they also solubilized 180 ± 4.3 and 438 ± 7.3 µM P from the rock phosphate. In the presence of a mixture of 50 mM sucrose and 25 mM glucose, E. a. (pCNK5) secreted 34 ± 2.3 mM gluconic acid and released 479 ± 8.1 µM P. Moreover, in the presence of a mixture of eight sugars (10 mM each) in the medium, E. a. (pCNK5) released 414 ± 5.3 µM P in the buffered medium. Thus, this study demonstrates incorporation of periplasmic invertase imparted P solubilization ability to E. asburiae PSI3 in the presence of sucrose and mixture of sugars.

  18. 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

  19. 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.

  20. Controlling microbial contamination during hydrolysis of AFEX-pretreated corn stover and switchgrass: Effects on hydrolysate composition, microbial response and fermentation

    SciT

    Serate, Jose; Xie, Dan; Pohlmann, Edward

    Microbial conversion of lignocellulosic feedstocks into biofuels remains an attractive means to produce sustainable energy. It is essential to produce lignocellulosic hydrolysates in a consistent manner in order to study microbial performance in different feedstock hydrolysates. Because of the potential to introduce microbial contamination from the untreated biomass or at various points during the process, it can be difficult to control sterility during hydrolysate production. In this study, we compared hydrolysates produced from AFEX-pretreated corn stover and switchgrass using two different methods to control contamination: either by autoclaving the pretreated feedstocks prior to enzymatic hydrolysis, or by introducing antibiotics duringmore » the hydrolysis of non-autoclaved feedstocks. We then performed extensive chemical analysis, chemical genomics, and comparative fermentations to evaluate any differences between these two different methods used for producing corn stover and switchgrass hydrolysates. Autoclaving the pretreated feedstocks could eliminate the contamination for a variety of feedstocks, whereas the antibiotic gentamicin was unable to control contamination consistently during hydrolysis. Compared to the addition of gentamicin, autoclaving of biomass before hydrolysis had a minimal effect on mineral concentrations, and showed no significant effect on the two major sugars (glucose and xylose) found in these hydrolysates. However, autoclaving elevated the concentration of some furanic and phenolic compounds. Chemical genomics analyses using Saccharomyces cerevisiae strains indicated a high correlation between the AFEX-pretreated hydrolysates produced using these two methods within the same feedstock, indicating minimal differences between the autoclaving and antibiotic methods. Comparative fermentations with S. cerevisiae and Zymomonas mobilis also showed that autoclaving the AFEX-pretreated feedstocks had no significant effects on microbial

  1. Production of C4 and C5 branched-chain alcohols by engineered Escherichia. coli.

    PubMed

    Chen, Xiaoyan; Xu, Jingliang; Yang, Liu; Yuan, Zhenhong; Xiao, Shiyuan; Zhang, Yu; Liang, Cuiyi; He, Minchao; Guo, Ying

    2015-11-01

    Higher alcohols, longer chain alcohols, contain more than 3 carbon atoms, showed close energy advantages as gasoline, and were considered as the next generation substitution for chemical fuels. Higher alcohol biosynthesis by native microorganisms mainly needs gene expression of heterologous keto acid decarboxylase and alcohol dehydrogenases. In the present study, branched-chain α-keto acid decarboxylase gene from Lactococcus lactis subsp. lactis CICC 6246 (Kivd) and alcohol dehydrogenases gene from Zymomonas mobilis CICC 41465 (AdhB) were transformed into Escherichia coli for higher alcohol production. SDS-PAGE results showed these two proteins were expressed in the recombinant strains. The resulting strain was incubated in LB medium at 37 °C in Erlenmeyer flasks and much more 3-methyl-1-butanol (104 mg/L) than isobutanol (24 mg/L) was produced. However, in 5 g/L glucose-containing medium, the production of two alcohols was similar, 156 and 161 mg/L for C4 (isobutanol) and C5 (3-methyl-1-butanol) alcohol, respectively. Effects of fermentation factors including temperature, glucose content, and α-keto acid on alcohol production were also investigated. The increase of glucose content and the adding of α-keto acids facilitated the production of C4 and C5 alcohols. The enzyme activities of pure Kivd on α-ketoisovalerate and α-ketoisocaproate were 26.77 and 21.24 μmol min(-1) mg(-1), respectively. Due to its ability on decarboxylation of α-ketoisovalerate and α-ketoisocaproate, the recombinant E. coli strain showed potential application on isoamyl alcohol and isobutanol production.

  2. A novel cost-effective technology to convert sucrose and homocelluloses in sweet sorghum stalks into ethanol.

    PubMed

    Li, Jihong; Li, Shizhong; Han, Bing; Yu, Menghui; Li, Guangming; Jiang, Yan

    2013-11-29

    Sweet sorghum is regarded as a very promising energy crop for ethanol production because it not only supplies grain and sugar, but also offers lignocellulosic resource. Cost-competitive ethanol production requires bioconversion of all carbohydrates in stalks including of both sucrose and lignocellulose hydrolyzed into fermentable sugars. However, it is still a main challenge to reduce ethanol production cost and improve feasibility of industrial application. An integration of the different operations within the whole process is a potential solution. An integrated process combined advanced solid-state fermentation technology (ASSF) and alkaline pretreatment was presented in this work. Soluble sugars in sweet sorghum stalks were firstly converted into ethanol by ASSF using crushed stalks directly. Then, the operation combining ethanol distillation and alkaline pretreatment was performed in one distillation-reactor simultaneously. The corresponding investigation indicated that the addition of alkali did not affect the ethanol recovery. The effect of three alkalis, NaOH, KOH and Ca(OH)2 on pretreatment were investigated. The results indicated the delignification of lignocellulose by NaOH and KOH was more significant than that by Ca(OH)2, and the highest removal of xylan was caused by NaOH. Moreover, an optimized alkali loading of 10% (w/w DM) NaOH was determined. Under this favorable pretreatment condition, enzymatic hydrolysis of sweet sorghum bagasse following pretreatment was investigated. 92.0% of glucan and 53.3% of xylan conversion were obtained at enzyme loading of 10 FPU/g glucan. The fermentation of hydrolyzed slurry was performed using an engineered stain, Zymomonas mobilis TSH-01. A mass balance of the overall process was calculated, and 91.9 kg was achieved from one tonne of fresh sweet sorghum stalk. A low energy-consumption integrated technology for ethanol production from sweet sorghum stalks was presented in this work. Energy consumption for raw

  3. Alkaline/peracetic acid as a pretreatment of lignocellulosic biomass for ethanol fuel production

    NASA Astrophysics Data System (ADS)

    Teixeira, Lincoln Cambraia

    Peracetic acid is a lignin oxidation pretreatment with low energy input by which biomass can be treated in a silo type system for improving enzymatic digestibility of lignocellulosic materials for ethanol production. Experimentally, ground hybrid poplar wood and sugar cane bagasse are placed in plastic bags and a peracetic acid solution is added to the biomass in different concentrations based on oven-dry biomass. The ratio of solution to biomass is 6:1; after initial mixing of the resulting paste, a seven-day storage period at about 20°C is used in this study. As a complementary method, a series of pre-pretreatments using stoichiometric amounts of sodium hydroxide and ammonium hydroxide based on 4-methyl-glucuronic acid and acetyl content in the biomass is been performed before addition of peracetic acid. The alkaline solutions are added to the biomass in a ratio of 14:1 solution to biomass; the slurry is mixed for 24 hours at ambient temperature. The above procedures give high xylan content substrates. Consequently, xylanase/beta-glucosidase combinations are more effective than cellulase preparations in hydrolyzing these materials. The pretreatment effectiveness is evaluated using standard enzymatic hydrolysis and simultaneous saccharification and cofermentation (SSCF) procedures. Hybrid poplar wood pretreated with 15 and 21% peracetic acid based on oven-dry weight of wood gives glucan conversion yields of 76.5 and 98.3%, respectively. Sugar cane bagasse pretreated with the same loadings gives corresponding yields of 85.9 and 93.1%. Raw wood and raw bagasse give corresponding yields of 6.8 and 28.8%, respectively. The combined 6% NaOH/15% peracetic acid pretreatments increase the glucan conversion yields from 76.5 to 100.0% for hybrid poplar wood and from 85.9 to 97.6% for sugar cane bagasse. Respective ethanol yields of 92.8 and 91.9% are obtained from 6% NaOH/15% peracetic acid pretreated materials using recombinant Zymomonas mobilis CP4/pZB5. Peracetic acid

  4. 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

  5. A novel cost-effective technology to convert sucrose and homocelluloses in sweet sorghum stalks into ethanol

    PubMed Central

    2013-01-01

    Background Sweet sorghum is regarded as a very promising energy crop for ethanol production because it not only supplies grain and sugar, but also offers lignocellulosic resource. Cost-competitive ethanol production requires bioconversion of all carbohydrates in stalks including of both sucrose and lignocellulose hydrolyzed into fermentable sugars. However, it is still a main challenge to reduce ethanol production cost and improve feasibility of industrial application. An integration of the different operations within the whole process is a potential solution. Results An integrated process combined advanced solid-state fermentation technology (ASSF) and alkaline pretreatment was presented in this work. Soluble sugars in sweet sorghum stalks were firstly converted into ethanol by ASSF using crushed stalks directly. Then, the operation combining ethanol distillation and alkaline pretreatment was performed in one distillation-reactor simultaneously. The corresponding investigation indicated that the addition of alkali did not affect the ethanol recovery. The effect of three alkalis, NaOH, KOH and Ca(OH)2 on pretreatment were investigated. The results indicated the delignification of lignocellulose by NaOH and KOH was more significant than that by Ca(OH)2, and the highest removal of xylan was caused by NaOH. Moreover, an optimized alkali loading of 10% (w/w DM) NaOH was determined. Under this favorable pretreatment condition, enzymatic hydrolysis of sweet sorghum bagasse following pretreatment was investigated. 92.0% of glucan and 53.3% of xylan conversion were obtained at enzyme loading of 10 FPU/g glucan. The fermentation of hydrolyzed slurry was performed using an engineered stain, Zymomonas mobilis TSH-01. A mass balance of the overall process was calculated, and 91.9 kg was achieved from one tonne of fresh sweet sorghum stalk. Conclusions A low energy-consumption integrated technology for ethanol production from sweet sorghum stalks was presented in this work

  6. A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

    PubMed

    Wiebe, Marilyn G; Nygård, Yvonne; Oja, Merja; Andberg, Martina; Ruohonen, Laura; Koivula, Anu; Penttilä, Merja; Toivari, Mervi

    2015-11-01

    An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l(-1) and 11.5 g D-xylitol l(-1) from 26 g D-xylose l(-1)), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l(-1)). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.

  7. Enhanced production of nargenicin A1 and creation of a novel derivative using a synthetic biology platform.

    PubMed

    Dhakal, Dipesh; Chaudhary, Amit Kumar; Yi, Jeong Sang; Pokhrel, Anaya Raj; Shrestha, Biplav; Parajuli, Prakash; Shrestha, Anil; Yamaguchi, Tokutaro; Jung, Hye Jin; Kim, Seung-Young; Kim, Byung-Gee; Sohng, Jae Kyung

    2016-12-01

    Nargenicin A1, an antibacterial produced by Nocardia sp. CS682 (KCTC 11297BP), demonstrates effective activity against various Gram-positive bacteria. Hence, we attempted to enhance nargenicin A1 production by utilizing the cumulative effect of synthetic biology, metabolic engineering and statistical media optimization strategies. To facilitate the modular assembly of multiple genes for genetic engineering in Nocardia sp. CS682, we constructed a set of multi-monocistronic vectors, pNV18L1 and pNV18L2 containing hybrid promoter (derived from ermE* and promoter region of neo r ), ribosome binding sites (RBS), and restriction sites for cloning, so that each cloned gene was under its own promoter and RBS. The multi-monocistronic vector, pNV18L2 containing transcriptional terminator showed better efficiency in reporter gene assay. Thus, multiple genes involved in the biogenesis of pyrrole moiety (ngnN2, ngnN3, ngnN4, and ngnN5 from Nocardia sp. CS682), glucose utilization (glf and glk from Zymomonas mobilis), and malonyl-CoA synthesis (accA2 and accBE from Streptomyces coelicolor A3 (2)), were cloned in pNV18L2. Further statistical optimization of specific precursors (proline and glucose) and their feeding time led to ~84.9 mg/L nargenicin from Nocardia sp. GAP, which is ~24-fold higher than Nocardia sp. CS682 (without feeding). Furthermore, pikC from Streptomyces venezuelae was expressed to generate Nocardia sp. PikC. Nargenicin A1 acid was characterized as novel derivative of nargenicin A1 produced from Nocardia sp. PikC by mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. We also performed comparative analysis of the anticancer and antibacterial activities of nargenicin A1 and nargenicin A1 acid, which showed a reduction in antibacterial potential for nargenicin A1 acid. Thus, the development of an efficient synthetic biological platform provided new avenues for enhancing or structurally diversifying nargenicin A1 by means of pathway designing

  8. Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics.

    PubMed

    Gold, Nicholas D; Gowen, Christopher M; Lussier, Francois-Xavier; Cautha, Sarat C; Mahadevan, Radhakrishnan; Martin, Vincent J J

    2015-05-28

    L-tyrosine is a common precursor for a wide range of valuable secondary metabolites, including benzylisoquinoline alkaloids (BIAs) and many polyketides. An industrially tractable yeast strain optimized for production of L-tyrosine could serve as a platform for the development of BIA and polyketide cell factories. This study applied a targeted metabolomics approach to evaluate metabolic engineering strategies to increase the availability of intracellular L-tyrosine in the yeast Saccharomyces cerevisiae CEN.PK. Our engineering strategies combined localized pathway engineering with global engineering of central metabolism, facilitated by genome-scale steady-state modelling. Addition of a tyrosine feedback resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase Aro4 from S. cerevisiae was combined with overexpression of either a tyrosine feedback resistant yeast chorismate mutase Aro7, the native pentafunctional arom protein Aro1, native prephenate dehydrogenase Tyr1 or cyclohexadienyl dehydrogenase TyrC from Zymomonas mobilis. Loss of aromatic carbon was limited by eliminating phenylpyruvate decarboxylase Aro10. The TAL gene from Rhodobacter sphaeroides was used to produce coumarate as a simple test case of a heterologous by-product of tyrosine. Additionally, multiple strategies for engineering global metabolism to promote tyrosine production were evaluated using metabolic modelling. The T21E mutant of pyruvate kinase Cdc19 was hypothesized to slow the conversion of phosphoenolpyruvate to pyruvate and accumulate the former as precursor to the shikimate pathway. The ZWF1 gene coding for glucose-6-phosphate dehydrogenase was deleted to create an NADPH deficiency designed to force the cell to couple its growth to tyrosine production via overexpressed NADP(+)-dependent prephenate dehydrogenase Tyr1. Our engineered Zwf1(-) strain expressing TYRC ARO4(FBR) and grown in the presence of methionine achieved an intracellular L-tyrosine accumulation up to 520

  9. Process development and modeling of fluidized-bed reactor with coimmobilized biocatalyst for fuel ethanol production

    NASA Astrophysics Data System (ADS)

    Sun, May Yongmei

    This research focuses on two steps of commercial fuel ethanol production processes: the hydrolysis starch process and the fermentation process. The goal of this research is to evaluate the performance of co-immobilized biocatalysts in a fluidized bed reactor with emphasis on economic and engineering aspects and to develop a predictive mathematical model for this system. The productivity of an FBR is higher than productivity of a traditional batch reactor or CSTR. Fluidized beds offer great advantages over packed beds for immobilized cells when small particles are used or when the reactant feed contains suspended solids. Plugging problems, excessive pressure drops (and thus attrition), or crushing risks may be avoided. No mechanical stirring is required as mixing occurs due to the natural turbulence in the fluidized process. Both enzyme and microorganism are immobilized in one catalyst bead which is called co-immobilization. Inside this biocatalyst matrix, starch is hydrolyzed by the enzyme glucoamylase to form glucose and then converted to ethanol and carbon dioxide by microorganisms. Two biocatalysts were evaluated: (1) co-immobilized yeast strain Saccharomyces cerevisiae and glucoamylase. (2) co-immobilized Zymomonas mobilis and glucoamylase. A co-immobilized biocatalyst accomplishes the simultaneous saccharification and fermentation (SSF process). When compared to a two-step process involving separate saccharification and fermentation stages, the SSF process has productivity values twice that given by the pre-saccharified process when the time required for pre-saccharification (15--25 h) was taken into account. The SSF process should also save capital cost. The information about productivity, fermentation yield, concentration profiles along the bed, ethanol inhibition, et al., was obtained from the experimental data. For the yeast system, experimental results showed that: no apparent decrease of productivity occurred after two and half months, the productivity

  10. SciT

    Teymouri, Farzaneh

    were demonstrated; >94% ammonia recovery, >75% sugar yields at high solid loading, and complete utilization of the sugars for ethanol production at the 2500 liter scale. Fermentation tests were performed using Zymomonas mobilis 8b and densified AFEX-treated corn stover at >20% solid loading. The obtained titer (~60g/l), productivity (2.5 g/L-h), and yield (330 L/tonne of biomass) exceeded the performance targets set out by NREL. The key findings from these efforts are: no contamination was observed, no cleanup of the sugar stream was required, and no major nutrient addition was required. Our economic model shows that using a packed bed design for the AFEX process and pelleted AFEX-treated biomass reduces the ethanol production cost by 24% when compared to using the traditional AFEX design.« less

  11. Indiana state highway cost allocation and revenue attribution study and estimation of travel by out\\0x2010of\\0x2010state vehicles on Indiana highways.

    DOT National Transportation Integrated Search

    2015-06-01

    This study was commissioned by INDOT to investigate the cost responsibility and the revenue contribution of highway users with regard to the : upkeep of Indianas state and local highway infrastructure (pavements, bridges, safety assets, and mobili...

  12. Yat-Chen Chou | NREL

    Patents "Xylitol synthesis mutant of xylose-utilizing Zymomonas for ethanol production," U.S . Patent No. 7,741,119 (2010) "Ethanol production using xylitol synthesis mutant of xylose-utilizing formulation using cellobiohydrolase-1 from Penicillium funiculosum," U.S. Patent No. 7,449,550 (2008

  13. 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.

  14. Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis▿

    PubMed Central

    Fernandes, Chantal; Mendes, Vitor; Costa, Joana; Empadinhas, Nuno; Jorge, Carla; Lamosa, Pedro; Santos, Helena; da Costa, Milton S.

    2010-01-01

    The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis. PMID:20061481

  15. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  16. Vehicle Mobility Assessment for Project Wheels Study Group

    DTIC Science & Technology

    1972-07-01

    cash savings p’Asible through the elimination of special military automotive features, such as front-wheel 1 Idrive , or the use of commercial vehicles...E12. (12) off-,,,d mobiliY profile 4x4 0ruck, c 2Z4< 20 ,ernn It Iot "- III IIIII III I I I IIII A%. West Germany 0A * s ps ArizonazD • Ř’.. • D

  17. The effects of salinity on nitrification using halophilic nitrifiers in a Sequencing Batch Reactor treating hypersaline wastewater.

    PubMed

    Cui, You-Wei; Zhang, Hong-Yu; Ding, Jie-Ran; Peng, Yong-Zhen

    2016-04-25

    With annual increases in the generation and use of saline wastewater, the need to avoid environmental problems such as eutrophication is critical. A previous study identified ways to start up a halophilic sludge domesticated from estuarine sediments to remove nitrogen from wastewater with a salinity of 30 g/L. This investigation expands that work to explore the impact of salinity on nitrogen removal. This study demonstrated that the mixed halophilic consortia removed nitrogen from wastewater with a salinity of 30-85 g/L. A kinetic analysis showed that halophilic nitrifiers selected based on hypersalinity were characterized by low Ks, μmax and specific ammonium oxidization rates. This explains the decrease in ammonium removal efficiency in the high salinity operational phases. Salinity inhibited ammonia oxidizing bacteria (AOB) activity, as well as the number of dominant AOB, but did not significantly affect the AOB dominant species. Three most dominant AOB lineages in the halophilic sludge were Nitrosomonas marina, Nitrosomonas europaea, and Nitrosococcus mobilis. Nitrosomonas europaea and Nitrosococcus mobilis were mainly affected by salinity, while nitrite accumulation and ammonia loading played the key role in determining the abundance of Nitrosococcus mobilis and Nitrosococcus europaea. The study contributes insights about shifts in halophilic nitrifying bacterial populations.

  18. The effects of salinity on nitrification using halophilic nitrifiers in a Sequencing Batch Reactor treating hypersaline wastewater

    PubMed Central

    Cui, You-Wei; Zhang, Hong-Yu; Ding, Jie-Ran; Peng, Yong-Zhen

    2016-01-01

    With annual increases in the generation and use of saline wastewater, the need to avoid environmental problems such as eutrophication is critical. A previous study identified ways to start up a halophilic sludge domesticated from estuarine sediments to remove nitrogen from wastewater with a salinity of 30 g/L. This investigation expands that work to explore the impact of salinity on nitrogen removal. This study demonstrated that the mixed halophilic consortia removed nitrogen from wastewater with a salinity of 30–85 g/L. A kinetic analysis showed that halophilic nitrifiers selected based on hypersalinity were characterized by low Ks, μmax and specific ammonium oxidization rates. This explains the decrease in ammonium removal efficiency in the high salinity operational phases. Salinity inhibited ammonia oxidizing bacteria (AOB) activity, as well as the number of dominant AOB, but did not significantly affect the AOB dominant species. Three most dominant AOB lineages in the halophilic sludge were Nitrosomonas marina, Nitrosomonas europaea, and Nitrosococcus mobilis. Nitrosomonas europaea and Nitrosococcus mobilis were mainly affected by salinity, while nitrite accumulation and ammonia loading played the key role in determining the abundance of Nitrosococcus mobilis and Nitrosococcus europaea. The study contributes insights about shifts in halophilic nitrifying bacterial populations. PMID:27109617

  19. [Predominant strains of polycyclic aromatic hydrocarbon-degrading consortia from deep sea of the Middle Atlantic Ridge].

    PubMed

    Cui, Zhisong; Shao, Zongze

    2009-07-01

    In order to identify the predominant strains of polycyclic aromatic hydrocarbon (PAH)-degrading consortia harboring in sea water and surface sediment collected from deep sea of the Middle Atlantic Ridge. We employed enrichment method and spread-plate method to isolate cultivable bacteria and PAHs degraders from deep sea samples. Phylogenetic analysis was conducted by 16S rRNA gene sequencing of the bacteria. Then we analyzed the dominant bacteria in the PAHs-degrading consortia by denaturing gradient gel electrophoresis (DGGE) combined with DNA sequencing. Altogether 16 cultivable bacteria were obtained, including one PAHs degrader Novosphingobium sp. 4D. Phylogenetic analysis showed that strains closely related to Alcanivorax dieselolei NO1A (5/16) and Tistrella mobilis TISTR 1108T (5/16) constituted two biggest groups among the cultivable bacteria. DGGE analysis showed that strain 4L (also 4M and 4N, Alcanivorax dieselolei NO1A, 99.21%), 4D (Novosphingobium pentaromativorans US6-1(T), 97.07%) and 4B (also 4E, 4H and 4K, Tistrella mobilis TISTR 1108T, > 99%) dominated the consortium MC2D. While in consortium MC3CO, the predominant strains were strain 5C (also 5H, Alcanivorax dieselolei NO1A, > 99%), uncultivable strain represented by band 5-8 (Novosphingobium aromaticivorans DSM 12444T, 99.41%), 5J (Tistrella mobilis TISTR 1108T, 99.52%) and 5F (also 5G, Thalassospira lucentensis DSM 14000T, < 97%). We found that strains of genus Alcanivorax, Novosphingobium, Tistrella and Thalassospira were predominant bacteria of PAHs-degrading consortia in sea water and surface sediment of Middle Atlantic Ridge deep sea, with Novosphingobium spp. as their main PAHs degraders.

  20. Evaluation of an Automated Swallow-Detection Algorithm Using Visual Biofeedback in Healthy Adults and Head and Neck Cancer Survivors.

    PubMed

    Constantinescu, Gabriela; Kuffel, Kristina; Aalto, Daniel; Hodgetts, William; Rieger, Jana

    2018-06-01

    Mobile health (mHealth) technologies may offer an opportunity to address longstanding clinical challenges, such as access and adherence to swallowing therapy. Mobili-T ® is an mHealth device that uses surface electromyography (sEMG) to provide biofeedback on submental muscles activity during exercise. An automated swallow-detection algorithm was developed for Mobili-T ® . This study evaluated the performance of the swallow-detection algorithm. Ten healthy participants and 10 head and neck cancer (HNC) patients were fitted with the device. Signal was acquired during regular, effortful, and Mendelsohn maneuver saliva swallows, as well as lip presses, tongue, and head movements. Signals of interest were tagged during data acquisition and used to evaluate algorithm performance. Sensitivity and positive predictive values (PPV) were calculated for each participant. Saliva swallows were compared between HNC and controls in the four sEMG-based parameters used in the algorithm: duration, peak amplitude ratio, median frequency, and 15th percentile of the power spectrum density. In healthy participants, sensitivity and PPV were 92.3 and 83.9%, respectively. In HNC patients, sensitivity was 92.7% and PPV was 72.2%. In saliva swallows, HNC patients had longer event durations (U = 1925.5, p < 0.001), lower median frequency (U = 2674.0, p < 0.001), and lower 15th percentile of the power spectrum density [t(176.9) = 2.07, p < 0.001] than healthy participants. The automated swallow-detection algorithm performed well with healthy participants and retained a high sensitivity, but had lowered PPV with HNC patients. With respect to Mobili-T ® , the algorithm will next be evaluated using the mHealth system.

  1. A new species of seagrass-boring Limnoria (Limnoriidae, Isopoda, Crustacea) from Japan.

    PubMed

    Yoshino, Hiroki; Watabe, Hikari; Ohsawa, Takeshi A

    2017-02-15

    The marine seagrass-boring isopod, Limnoria rhombipunctata sp. nov. (Limnoriidae) is described from the rhizome of Phyllospadix iwatensis seagrass, in shallow coastal waters off Chiba Prefecture, Japan. L. rhombipunctata sp. nov. is distinguished from other Limnoria species by the unique carinae of pleonite 5 and pleotelson, two branched lacinia mobilis of the right mandible, 3 flagellar articles of antenna 1, and triangular epipod of the maxilliped. Specimens of L. magadanensis, a species similar to L. rhombipunctata sp. nov., are re-examined and compared with L. rhombipunctata sp. nov.

  2. Facilities Maintenance in the U.S. Navy

    DTIC Science & Technology

    1986-01-01

    Row’a9IVI s..Nl,I I~de 14. 11111116104 It. safeI MeAI. 5,124 AN (:lgnd) JOHN SMITH I765 IIf Figure 111-1 Work Request is EMIROENCY/SERVICE WORK...Eliminate fire, health and active life of less than 3 years safety hazards o Infrequently or only partially o Patch and reinforce instead used of...safety or health hazards and to permit reactivation within the period prescribed under mobili- zation plans o Surplus facilities a Fliminate fire, safety

  3. The state of autotrophic ethanol production in Cyanobacteria.

    PubMed

    Dexter, J; Armshaw, P; Sheahan, C; Pembroke, J T

    2015-07-01

    Ethanol production directly from CO2 , utilizing genetically engineered photosynthetic cyanobacteria as a biocatalyst, offers significant potential as a renewable and sustainable source of biofuel. Despite the current absence of a commercially successful production system, significant resources have been deployed to realize this goal. Utilizing the pyruvate decarboxylase from Zymomonas species, metabolically derived pyruvate can be converted to ethanol. This review of both peer-reviewed and patent literature focuses on the genetic modifications utilized for metabolic engineering and the resultant effect on ethanol yield. Gene dosage, induced expression and cassette optimizat-ion have been analyzed to optimize production, with production rates of 0·1-0·5 g L(-1) day(-1) being achieved. The current 'toolbox' of molecular manipulations and future directions focusing on applicability, addressing the primary challenges facing commercialization of cyanobacterial technologies are discussed. © 2015 The Society for Applied Microbiology.

  4. Relation entre les caractéristiques des table-bancs et les mesures anthropométriques des écoliers au Benin

    PubMed Central

    Falola, Stève Marjelin; Gouthon, Polycarpe; Falola, Jean-Marie; Fiogbe, Michel Armand; Nigan, Issiako Bio

    2014-01-01

    Introduction Le mobilier scolaire et la posture assise en classe sont souvent impliqués dans l'apparition des douleurs rachidiennes, influant de fait sur la qualité des tâches réalisées par les apprenants. Aucune étude n'a encore vérifié le degré d'adéquation entre les caractéristiques du mobilier et celles des écoliers au Bénin. L'objectif de cette étude transversale est donc de déterminer la relation entre les dimensions des table-bancs utilisées en classe et les mesures anthropométriques des écoliers au Bénin. Methods Elle a été réalisée avec un échantillon probabiliste de 678 écoliers, âgés de 4 à 17 ans. Les mesures anthropométriques des écoliers et les mensurations relatives aux longueurs, largeurs et hauteurs des table-bancs ont été mesurées, puis intégrées aux équations proposées dans la littérature. Les pourcentages des valeurs situées hors des limitesacceptables, dérivées de l'application des équations ont été calculés. Results La largeur et la hauteur des table-bancs utilisées par les écoliers étaient plus élevées (p < 0,05) que les valeurs de référence recommandées par les structures officielles de contrôle et de production des mobiliers scolaires au Bénin. Quel que soit le sexe, il y avait une inadéquation entre la largeur du banc et la longueur fesse-poplité, puis entre la hauteur de la table et la distance coude-bancdes écoliers. Conclusion Les résultats suggèrent de prendre en compte l’évolution des mesures anthropométriques des écoliers dans la confection des table-bancs, afin de promouvoir de bonnes postures assises en classe et de réduire le risque de troubles du rachis. PMID:25317232

  5. Description of Pseudingolfiella possessionis n. sp. (Crustacea, Amphipoda) from sub-Antarctic Île de La Possession, Crozet archipelago: the second freshwater amphipod known from the Antarctic biome, a human introduction of Gondwanan ancestry?

    PubMed

    Smet, Willem H De

    2015-03-31

    A new species of freshwater amphipod, Pseudingolfiella possessionis n. sp. (Senticaudata, Pseudingolfiellidae), is described from the submerged moss vegetation of small brooklets at sub-Antarctic Île de La Possession, Crozet archipelago. It constitutes the second freshwater amphipod species known for the sub-Antarctic and Antarctic region, and the fourth member of the genus. The main characters distinguishing it from all congeners are: the spine on the posterior margin of the dactylus, incisor and lamina mobilis of mandible each with 5 teeth, the setation of the maxilliped, the vestigial second article of pleopod 3 in the female, the undulate and laterally notched posterolateral margin of the external ramus of uropods 1 and 2 in the male, the spinulate dorsomedian projection of the telson.

  6. Structure and dynamics of mesophilic variants from the homing endonuclease I-DmoI

    NASA Astrophysics Data System (ADS)

    Alba, Josephine; Marcaida, Maria Jose; Prieto, Jesus; Montoya, Guillermo; Molina, Rafael; D'Abramo, Marco

    2017-12-01

    I-DmoI, from the hyperthermophilic archaeon Desulfurococcus mobilis, belongs to the LAGLIDADG homing endonuclease protein family. Its members are highly specific enzymes capable of recognizing long DNA target sequences, thus providing potential tools for genome manipulation. Working towards this particular application, many efforts have been made to generate mesophilic variants of I-DmoI that function at lower temperatures than the wild-type. Here, we report a structural and computational analysis of two I-DmoI mesophilic mutants. Despite very limited structural variations between the crystal structures of these variants and the wild-type, a different dynamical behaviour near the cleavage sites is observed. In particular, both the dynamics of the water molecules and the protein perturbation effect on the cleavage site correlate well with the changes observed in the experimental enzymatic activity.

  7. The Type 1 Homodimeric Reaction Center in Heliobacterium modesticaldum

    SciT

    Golbeck, John

    In this funding period, we (i) found that strong illumination of Heliobacterium modesticaldum cells results in saturation of the electron acceptor pool, leading to reduction of the acceptor side and the creation of a back-reacting state that gives rise to delayed fluorescence; (ii) noted that when the FX cluster is reduced in purified reaction centers, no electron transfer occurs beyond A0, even though a quinone is present; (iii) observed by photochemically induced dynamic nuclear polarization (photo-CIDNP) studies of whole cells of Heliobacterium mobilis that primary charge separation is retained even after conversion of the majority of BChl g to Chlmore » aF. ; and (iv) purified a homogeneous preparation of reaction center cores, which led to promising crystallization trials to obtain a three-dimensional structure.« less

  8. Enlisted MOS Suitable for the Physically Handicapped

    DTIC Science & Technology

    1958-12-01

    highly motivated in a mobili- zation situation. The pr.mary reason for using this approach was the need for a standardized classification system which...the classification process. The solution was to consider all disabili- ties as "severe" and motivation to perform "good." Thus any individual disabled...MC+e0)H 4E-4)OOHe~~~ A A A P44 O0O)HCeHl H’d e (d *Hc.3d,4-4 HO) HQ .r4 to’- L 4 Xe 𔃺- H r4 Ps4 p) HO -i -i--I<-) rdr r d 90 P 4-- >< d0I - d a)0SoSS

  9. Fast coliform detection in portable microbe enrichment unit (PMEU) with Colilert(®) medium and bubbling.

    PubMed

    Hakalehto, Elias; Heitto, Anneli; Heitto, Lauri

    2013-09-01

    Laboratory strains of coliforms Escherichia coli and Klebsiella mobilis were used to artificially contaminate water samples in two different cultivation and detection systems, without and with bubble flow. Samples were collected with an automated system (ASCS). The positive coliform signal caused the color change into yellow (at 550-570nm). This signal could also be transmitted on-line to cell phones. E. coli containing samples emitted UV fluorescence at 480-560nm when activated by UV light. If cultivation was started with inocula varying from 10,000 to 1cfu/ml, the positive detection was obtained between 2 and 18h, respectively, in Colilert medium using Coline PMEU device without gas bubbling. Accordingly, a single K. mobilis cell produced detectable growth in 18h. Various clinical E. coli strains were compared to each other with equal inoculum sizes, and they showed slight variations in the initiation and speed of growth. The gas bubble flow in PMEU Spectrion promoted the mixing and interaction of bacteria and indicator media and speeded the onset of growth. Carbon dioxide also accelerated bacterial growth. In the presence of vancomycin, the onset of E. coli culture growth was speeded up by the volatile outlet flow from previous cultures. In the last cultivation syringe in a series of five, the lag phase disappeared and the growth of the inoculum continued without major interruption. the stimulation of the cultures by the gas flow turned out to be a useful means for improving the detection of indicator bacteria. It could also be used in combination with antibiotic selection in the broth medium. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Protein sequences and redox titrations indicate that the electron acceptors in reaction centers from heliobacteria are similar to Photosystem I

    NASA Technical Reports Server (NTRS)

    Trost, J. T.; Brune, D. C.; Blankenship, R. E.

    1992-01-01

    Photosynthetic reaction centers isolated from Heliobacillus mobilis exhibit a single major protein on SDS-PAGE of 47 000 Mr. Attempts to sequence the reaction center polypeptide indicated that the N-terminus is blocked. After enzymatic and chemical cleavage, four peptide fragments were sequenced from the Heliobacillus mobilis apoprotein. Only one of these sequences showed significant specific similarity to any of the protein and deduced protein sequences in the GenBank data base. This fragment is identical with 56% of the residues, including both cysteines, found in highly conserved region that is proposed to bind iron-sulfur center Fx in the Photosystem I reaction center peptide that is the psaB gene product. The similarity to the psaA gene product in this region is 48%. Redox titrations of laser-flash-induced photobleaching with millisecond decay kinetics on isolated reaction centers from Heliobacterium gestii indicate a midpoint potential of -414 mV with n = 2 titration behavior. In membranes, the behavior is intermediate between n = 1 and n = 2, and the apparent midpoint potential is -444 mV. This is compared to the behavior in Photosystem I, where the intermediate electron acceptor A1, thought to be a phylloquinone molecule, has been proposed to undergo a double reduction at low redox potentials in the presence of viologen redox mediators. These results strongly suggest that the acceptor side electron transfer system in reaction centers from heliobacteria is indeed analogous to that found in Photosystem I. The sequence similarities indicate that the divergence of the heliobacteria from the Photosystem I line occurred before the gene duplication and subsequent divergence that lead to the heterodimeric protein core of the Photosystem I reaction center.

  11. Bacterial diversity in the active stage of a bioremediation system for mineral oil hydrocarbon-contaminated soils.

    PubMed

    Popp, Nicole; Schlömann, Michael; Mau, Margit

    2006-11-01

    Soils contaminated with mineral oil hydrocarbons are often cleaned in off-site bioremediation systems. In order to find out which bacteria are active during the degradation phase in such systems, the diversity of the active microflora in a degrading soil remediation system was investigated by small-subunit (SSU) rRNA analysis. Two sequential RNA extracts from one soil sample were generated by a procedure incorporating bead beating. Both extracts were analysed separately by generating individual SSU rDNA clone libraries from cDNA of the two extracts. The sequencing results showed moderate diversity. The two clone libraries were dominated by Gammaproteobacteria, especially Pseudomonas spp. Alphaproteobacteria and Betaproteobacteria were two other large groups in the clone libraries. Actinobacteria, Firmicutes, Bacteroidetes and Epsilonproteobacteria were detected in lower numbers. The obtained sequences were predominantly related to genera for which cultivated representatives have been described, but were often clustered together in the phylogenetic tree, and the sequences that were most similar were originally obtained from soils and not from pure cultures. Most of the dominant genera in the clone libraries, e.g. Pseudomonas, Acinetobacter, Sphingomonas, Acidovorax and Thiobacillus, had already been detected in (mineral oil hydrocarbon) contaminated environmental samples. The occurrence of the genera Zymomonas and Rhodoferax was novel in mineral oil hydrocarbon-contaminated soil.

  12. Three immobilized-cell columnar bioreactors for enhanced production of commodity chemicals

    SciT

    Davison, B.H.; Scott, C.D.; Kaufman, E.N.

    1993-07-01

    Immobilized-cell fluidized-bed bioreactors (FBRS) can be used with a variety of fermentations to increase production of fuels, solvents, organic acids, and other fermentation products. Part of the increased rates and yields are due to the immobilization of the biocatalyst at high concentrations. This FBR system with immobilized Zymomonas mobiles increased ethanol productivity more than tenfold with 99% conversion and near stoichiometric yields. FBRs also offer several additional modes of operation for simultaneous fermentation and separation to further increase production by removing the inhibitory products directly from the continuous fermentation. The production of lactic acid by immobilized Lactobacillus was augmented withmore » the addition and removal of solid adsorbent particles to the FBR. An immiscible organic extractant also was used to extract butanol from the acetone-butanol fermentation by Clostridium acetobutylicum. Demonstrations with these FBR systems have already shown definite advantages by improved overall product yields (decreasing feed costs) and by increased rates (decreasing capital and operating costs). Further demonstration and scale-up continue.« less

  13. Sequence-based analysis of the microbial composition of water kefir from multiple sources.

    PubMed

    Marsh, Alan J; O'Sullivan, Orla; Hill, Colin; Ross, R Paul; Cotter, Paul D

    2013-11-01

    Water kefir is a water-sucrose-based beverage, fermented by a symbiosis of bacteria and yeast to produce a final product that is lightly carbonated, acidic and that has a low alcohol percentage. The microorganisms present in water kefir are introduced via water kefir grains, which consist of a polysaccharide matrix in which the microorganisms are embedded. We aimed to provide a comprehensive sequencing-based analysis of the bacterial population of water kefir beverages and grains, while providing an initial insight into the corresponding fungal population. To facilitate this objective, four water kefirs were sourced from the UK, Canada and the United States. Culture-independent, high-throughput, sequencing-based analyses revealed that the bacterial fraction of each water kefir and grain was dominated by Zymomonas, an ethanol-producing bacterium, which has not previously been detected at such a scale. The other genera detected were representatives of the lactic acid bacteria and acetic acid bacteria. Our analysis of the fungal component established that it was comprised of the genera Dekkera, Hanseniaspora, Saccharomyces, Zygosaccharomyces, Torulaspora and Lachancea. This information will assist in the ultimate identification of the microorganisms responsible for the potentially health-promoting attributes of these beverages. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  14. Atmospheric anthropic impacts tracked by the French atmospheric mobile observatory

    NASA Astrophysics Data System (ADS)

    Cuesta, J.; Chazette, P.; Flamant, P. H.

    2009-04-01

    A new ATmospheric Mobile ObServatory, so called "ATMOS", has been developed by the LiMAG "Lidar, Meteorology and Geophysics" team of the Institut Pierre Simon Laplace (IPSL) in France, in order to contribute to international field campaigns for studying atmospheric physico-chemistry, air quality and climate (i.e. aerosols, clouds, trace gazes, atmospheric dynamics and energy budget) and the ground-based validation of satellite observations. ATMOS has been deployed in the framework of i) LISAIR, for monitoring air quality in Paris in 2005, ii) AMMA "African Monsoon Multidisciplinary Analysis", in Tamanrasset and in Niamey for observing the aerosols and the atmospheric boundary layer in the Sahara and in the Sahel in 2006, iii) COPS "Convectively and Orographycally driven Precipitation Study" in the Rhin Valley in 2007 and iv) the validation of the spatial mission CALIPSO, launched in April 2006. In the coming years, ATMOS will be deployed i) in the Paris Megacity, in the framework of MEGAPOLI (2009-2010), ii) in southern France (near Marseille) for the Chemistry-Aerosol Mediterranean Experiment CHARMEX (2011-2012) and iii) the validation of ADM-Aeolus in 2010-2011 and Earth-Care in 2012. ATMOS payload is modular, accounting for the different platforms, instruments and measuring techniques. The deployment of ATMOS is an essential contribution to field campaigns, complementing the fixed sites, and a potential alternative of airborne platforms, heavier and more expensive. ATMOS mobile payload comprises both the remote sensing platform MOBILIS ("Moyens mOBIles de téLédetection de l'IPSL") and the in-situ physico-chemical station SAMMO ("Station Aérosols et chiMie MObile"). MOBILIS is an autonomous and high-performance system constituted by a full set of active and passive remote sensing instrumentation (i.e. Lidars and radiometers), whose payload may be adapted for either i) long term fixed monitoring in a maritime container or a shelter, ii) ground-based transect

  15. Coadministration of the Campylobacter jejuni N-Glycan-Based Vaccine with Probiotics Improves Vaccine Performance in Broiler Chickens

    PubMed Central

    Perez-Muñoz, M. E.; Gouveia, G. J.; Wanford, J. J.; Lango-Scholey, L.; Panagos, C. G.; Srithayakumar, V.; Plastow, G. S.; Coros, C.; Bayliss, C. D.; Edison, A. S.; Walter, J.

    2017-01-01

    ABSTRACT Source attribution studies report that the consumption of contaminated poultry is the primary source for acquiring human campylobacteriosis. Oral administration of an engineered Escherichia coli strain expressing the Campylobacter jejuni N-glycan reduces bacterial colonization in specific-pathogen-free leghorn chickens, but only a fraction of birds respond to vaccination. Optimization of the vaccine for commercial broiler chickens has great potential to prevent the entry of the pathogen into the food chain. Here, we tested the same vaccination approach in broiler chickens and observed similar efficacies in pathogen load reduction, stimulation of the host IgY response, the lack of C. jejuni resistance development, uniformity in microbial gut composition, and the bimodal response to treatment. Gut microbiota analysis of leghorn and broiler vaccine responders identified one member of Clostridiales cluster XIVa, Anaerosporobacter mobilis, that was significantly more abundant in responder birds. In broiler chickens, coadministration of the live vaccine with A. mobilis or Lactobacillus reuteri, a commonly used probiotic, resulted in increased vaccine efficacy, antibody responses, and weight gain. To investigate whether the responder-nonresponder effect was due to the selection of a C. jejuni “supercolonizer mutant” with altered phase-variable genes, we analyzed all poly(G)-containing loci of the input strain compared to nonresponder colony isolates and found no evidence of phase state selection. However, untargeted nuclear magnetic resonance (NMR)-based metabolomics identified a potential biomarker negatively correlated with C. jejuni colonization levels that is possibly linked to increased microbial diversity in this subgroup. The comprehensive methods used to examine the bimodality of the vaccine response provide several opportunities to improve the C. jejuni vaccine and the efficacy of any vaccination strategy. IMPORTANCE Campylobacter jejuni is a common

  16. Response to UVB radiation and oxidative stress of marine bacteria isolated from South Pacific Ocean and Mediterranean Sea.

    PubMed

    Matallana-Surget, S; Villette, C; Intertaglia, L; Joux, F; Bourrain, M; Lebaron, P

    2012-12-05

    Marine bacterial strains isolated from South Pacific and Mediterranean Sea were studied for their resistance to UVB radiation, their repair capacity under photoreactivating light, as well as their oxidative stress response using concentrated hydrogen peroxide (H(2)O(2)), as an oxidizer. A total of 30 marine bacteria were isolated from the hyper-oligotrophic waters of the South Pacific Gyre to the eutrophic waters of the Chilean coast during the BIOSOPE cruise (2004), and 10 strains from surface Mediterranean coastal waters. One third of bacteria presented a high resistance to UVB and almost all isolates presented an efficient post-irradiation recovery. Only few strains showed cell survival to high concentration of H(2)O(2). No correlation between the sampling sites and the bacterial UVB resistance was observed. Two marine bacteria, Erythrobacter flavus and Ruegeria mobilis, were of particular interest, presenting a good response to the three parameters (UVB and H(2)O(2) resistance/efficient repair). Unexpectedly, two resistant strains were again identified as Ruegeria species underlining that this geographically widespread genus, resist to UVB regardless the environment from which the isolates originate. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Description of two ubiquitous species of Desmosomatidae (Isopoda: Asellota) from the Northwest Pacific Basin east of the Kuril-Kamchatka Trench.

    PubMed

    Golovan, Olga A

    2015-11-05

    Two new species of Desmosomatidae, Eugerdella kurabyssalis sp. nov. and Parvochelus serricaudis sp. nov., are described from the Northwest Pacific open abyssal plain to the east of the Kuril-Kamchatka Trench. These species constituted about one fourth of all collected desmosomatid specimens of the KuramBio expedition (2012), indicating it may be the most abundant species of the family in this area. E. kurabyssalis sp. nov. is rather similar to E. minutula Mezhov, 1986 and E. theodori Brix, 2007, resembling them in the shape of its body segments and appendages, including the absence of the mandibular palp and morphology of the pereopod I. The new species can be distinguished from the mentioned species by the number of cusps of the mandibular incisor process and lacinia mobilis, the relatively stouter pereopods and different setation of the pereopod I ischium. P. serricaudis sp. nov. is the second species of the genus Parvochelus Brix & Kihara, 2015 (see Brix et al. 2015) and the first record of the genus from the Pacific Ocean and from the Northern Hemisphere. P. serricaudis sp. nov. can be distinguished from P. russus Brix & Kihara, 2015 by its relatively stouter pereopod I, presence of the mandibular palp and the shape of the maxilliped palp articles and female operculum. A discussion of the taxonomic characters and the modified diagnosis of the genus Parvochelus are presented.

  18. Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo.

    PubMed

    Limpiyakorn, Tawan; Shinohara, Yuko; Kurisu, Futoshi; Yagi, Osami

    2005-10-01

    We investigated ammonia-oxidizing bacteria in activated sludge collected from 12 sewage treatment systems, whose ammonia removal and treatment processes differed, during three different seasons. We used real-time PCR quantification to reveal total bacterial numbers and total ammonia oxidizer numbers, and used specific PCR followed by denaturing gel gradient electrophoresis, cloning, and sequencing of 16S rRNA genes to analyze ammonia-oxidizing bacterial communities. Total bacterial numbers and total ammonia oxidizer numbers were in the range of 1.6 x 10(12) - 2.4 x 10(13) and 1.0 x 10(9) - 9.2 x 10(10)cellsl(-1), respectively. Seasonal variation was observed in the total ammonia oxidizer numbers, but not in the ammonia-oxidizing bacterial communities. Members of the Nitrosomonas oligotropha cluster were found in all samples, and most sequences within this cluster grouped within two of the four sequence types identified. Members of the clusters of Nitrosomonas europaea-Nitrosococcus mobilis, Nitrosomonas cryotolerans, and unknown Nitrosomonas, occurred solely in one anaerobic/anoxic/aerobic (A2O) system. Members of the Nitrosomonas communis cluster occurred almost exclusively in association with A2O and anaerobic/aerobic systems. Solid residence time mainly influenced the total numbers of ammonia-oxidizing bacteria, whereas dissolved oxygen concentration primarily affected the ammonia-oxidizing activity per ammonia oxidizer cell.

  19. Shifts in Nitrification Kinetics and Microbial Community during Bioaugmentation of Activated Sludge with Nitrifiers Enriched on Sludge Reject Water

    PubMed Central

    Yu, Lifang; Peng, Dangcong; Pan, Ruiling

    2012-01-01

    This study used two laboratory-scale sequencing batch reactors (SBRs) to evaluate the shifts in nitrification kinetics and microbial communities of an activated sludge sewage treatment system (main stream) during bioaugmentation with nitrifiers cultivated on real sludge reject water (side stream). Although bioaugmentation exerted a strong influence on the microbial community and the nitrification kinetics in the main stream, there was 58% of maximum ammonia uptake rate (AUR) and 80% of maximum nitrite uptake rate (NUR) loss of the seed source after bioaugmentation. In addition, nitrite accumulation occurred during bioaugmentation due to the unequal and asynchronous increase of the AUR (from 2.88 to 13.36 mg N/L·h) and NUR (from 0.76 to 4.34 mg N/L·h). FISH results showed that ammonia oxidizing bacteria (AOB) was inclined to be washed out with effluent in contrast to nitrite oxidizing bacteria (NOB), and Nitrosococcus mobilis lineage was the dominant AOB, while the dominant NOB in the main stream gradually transferred from Nitrospira to Nitrobacter. Nitrospina and Nitrococcus which existed in the seed source could not be detected in the main stream. It can be inferred that nitrite accumulation occurred due to the mismatch of NOB structure but washed out with effluent. PMID:23091354

  20. Identification of Bacteria Responsible for Ammonia Oxidation in Freshwater Aquaria

    PubMed Central

    Burrell, Paul C.; Phalen, Carol M.; Hovanec, Timothy A.

    2001-01-01

    Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to the Nitrosomonas marina cluster, the Nitrosospira cluster, or the Nitrosomonas europaea-Nitrosococcus mobilis cluster of the β subdivision of the class Proteobacteria. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Enrichments containing the Nitrosomonas marina-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Furthermore, if the Nitrosomonas marina-like AOB strain was present in the original enrichment, even one with other AOB, only the Nitrosomonas marina-like AOB strain was present in aquaria after nitrification was established. Nitrosomonas marina-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established. PMID:11722936

  1. Toxicity of Bioactive and Probiotic Marine Bacteria and Their Secondary Metabolites in Artemia sp. and Caenorhabditis elegans as Eukaryotic Model Organisms

    PubMed Central

    Neu, Anna Katrin; Månsson, Maria; Prol-García, María J.

    2014-01-01

    We have previously reported that some strains belonging to the marine Actinobacteria class, the Pseudoalteromonas genus, the Roseobacter clade, and the Photobacteriaceae and Vibrionaceae families produce both antibacterial and antivirulence compounds, and these organisms are interesting from an applied point of view as fish probiotics or as a source of pharmaceutical compounds. The application of either organisms or compounds requires that they do not cause any side effects, such as toxicity in eukaryotic organisms. The purpose of this study was to determine whether these bacteria or their compounds have any toxic side effects in the eukaryotic organisms Artemia sp. and Caenorhabditis elegans. Arthrobacter davidanieli WX-11, Pseudoalteromonas luteoviolacea S4060, P. piscicida S2049, P. rubra S2471, Photobacterium halotolerans S2753, and Vibrio coralliilyticus S2052 were lethal to either or both model eukaryotes. The toxicity of P. luteoviolacea S4060 could be related to the production of the antibacterial compound pentabromopseudilin, while the adverse effect observed in the presence of P. halotolerans S2753 and V. coralliilyticus S2052 could not be explained by the production of holomycin nor andrimid, the respective antibiotic compounds in these organisms. In contrast, the tropodithietic acid (TDA)-producing bacteria Phaeobacter inhibens DSM17395 and Ruegeria mobilis F1926 and TDA itself had no adverse effect on the target organisms. These results reaffirm TDA-producing Roseobacter bacteria as a promising group to be used as probiotics in aquaculture, whereas Actinobacteria, Pseudoalteromonas, Photobacteriaceae, and Vibrionaceae should be used with caution. PMID:24141121

  2. An Integrated Proteomics/Transcriptomics Approach Points to Oxygen as the Main Electron Sink for Methanol Metabolism in Methylotenera mobilis▿†

    PubMed Central

    Beck, David A. C.; Hendrickson, Erik L.; Vorobev, Alexey; Wang, Tiansong; Lim, Sujung; Kalyuzhnaya, Marina G.; Lidstrom, Mary E.; Hackett, Murray; Chistoserdova, Ludmila

    2011-01-01

    Methylotenera species, unlike their close relatives in the genera Methylophilus, Methylobacillus, and Methylovorus, neither exhibit the activity of methanol dehydrogenase nor possess mxaFI genes encoding this enzyme, yet they are able to grow on methanol. In this work, we integrated a genome-wide proteomics approach, shotgun proteomics, and a genome-wide transcriptomics approach, shotgun transcriptome sequencing (RNA-seq), of Methylotenera mobilis JLW8 to identify genes and enzymes potentially involved in methanol oxidation, with special attention to alternative nitrogen sources, to address the question of whether nitrate could play a role as an electron acceptor in place of oxygen. Both proteomics and transcriptomics identified a limited number of genes and enzymes specifically responding to methanol. This set includes genes involved in oxidative stress response systems, a number of oxidoreductases, including XoxF-type alcohol dehydrogenases, a type II secretion system, and proteins without a predicted function. Nitrate stimulated expression of some genes in assimilatory nitrate reduction and denitrification pathways, while ammonium downregulated some of the nitrogen metabolism genes. However, none of these genes appeared to respond to methanol, which suggests that oxygen may be the main electron sink during growth on methanol. This study identifies initial targets for future focused physiological studies, including mutant analysis, which will provide further details into this novel process. PMID:21764938

  3. VizieR Online Data Catalog: Scheiner drawing sunspot areas and tilt angles (Arlt+, 2016)

    NASA Astrophysics Data System (ADS)

    Arlt, R.; Senthamizh Pavai, V.; Schmiel, C.; Spada, F.

    2016-09-01

    Christoph Scheiner and his collaborators observed the sunspots from 1611-1631 at five different locations of Rome in Italy, Ingolstadt in Germany, Douai (Duacum in Latin) in France, Freiburg im Breisgau, Germany and Vienna, Austria. However, most of his published drawings were made in Rome. These sunspot drawings are important because they can tell us how the solar activity declined to a very low-activity phase which lasted for nearly five decades. The three sources used for the sunspot data extraction are Scheiner (1630rour.book.....S, Rosa Ursina sive solis), Scheiner (1651ppsm.book.....S, Prodromus pro sole mobili et terra stabili contra Academicum Florentinum Galilaeum a Galilaeis), and Reeves & Van Helden (2010, On sunspots. Galileo Galilei and Christoph Scheiner (University of Chicago Press)). The suspot drawings show the sunspot groups traversing the solar disk in a single full-disk drawing. The positions and areas of the sunspots were measured using 13 circular cursor shapes with different diameters. Umbral areas for 8167 sunspots and tilt angles for 697 manually selected, supposedly bipolar groups were obtained from Scheiner's sunspot drawings. The database does not contain spotless days. There is, of course, no polarity information in the sunspot drawings, so the tilt angles are actually pseudo-tilt angles. Both an updated sunspot database and a tilt angle database may be available at http://www.aip.de/Members/rarlt/sunspots for further study. (2 data files).

  4. Discovery and Description of Giant Submarine Smectite Cones on the Seafloor in Eyjafjordur, Northern Iceland, and a Novel Thermal Microbial Habitat

    PubMed Central

    Marteinsson, Viggó Thór; Kristjánsson, Jakob K.; Kristmannsdóttir, Hrefna; Dahlkvist, Maria; Sæmundsson, Kristján; Hannington, Mark; Pétursdóttir, Sólveig K.; Geptner, Alfred; Stoffers, Peter

    2001-01-01

    With the submersible JAGO and by scuba diving we discovered three remarkable geothermal cones, rising 33, 25, and 45 m from the seafloor at a depth of 65 m in Eyjafjordur, northern Iceland. The greatest geothermal activity was on the highest cone, which discharged up to 50 liters of freshwater per s at 72°C and pH 10.0. The cones were built up from precipitated smectite, formed by mixing of the hot SiO2-rich geothermal fluid with the cold Mg-rich seawater. By connecting a rubber hose to one outflow, about 240 liters of pure geothermal fluids was concentrated through a 0.2-μm-pore-size filter. Among 50 thermophilic isolates, we found members of Bacillus and Thermonema and a new unidentified low-G+C gram-positive member of the Bacteria as well as one member of the Archaea, Desulfurococcus mobilis. Analysis of small-subunit rRNA genes PCR amplified and cloned directly from environmental DNA showed that 41 out of 45 Bacteria sequences belonged to members of the Aquificales, whereas all of the 10 Archaea sequences belonged to the Korarchaeota. The physiological characteristics of isolates from different parts of the cones indicate a completely freshwater habitat, supporting the possibility of subterranean transmittance of terrestrial organisms. PMID:11157250

  5. Toxicity of bioactive and probiotic marine bacteria and their secondary metabolites in Artemia sp. and Caenorhabditis elegans as eukaryotic model organisms.

    PubMed

    Neu, Anna Katrin; Månsson, Maria; Gram, Lone; Prol-García, María J

    2014-01-01

    We have previously reported that some strains belonging to the marine Actinobacteria class, the Pseudoalteromonas genus, the Roseobacter clade, and the Photobacteriaceae and Vibrionaceae families produce both antibacterial and antivirulence compounds, and these organisms are interesting from an applied point of view as fish probiotics or as a source of pharmaceutical compounds. The application of either organisms or compounds requires that they do not cause any side effects, such as toxicity in eukaryotic organisms. The purpose of this study was to determine whether these bacteria or their compounds have any toxic side effects in the eukaryotic organisms Artemia sp. and Caenorhabditis elegans. Arthrobacter davidanieli WX-11, Pseudoalteromonas luteoviolacea S4060, P. piscicida S2049, P. rubra S2471, Photobacterium halotolerans S2753, and Vibrio coralliilyticus S2052 were lethal to either or both model eukaryotes. The toxicity of P. luteoviolacea S4060 could be related to the production of the antibacterial compound pentabromopseudilin, while the adverse effect observed in the presence of P. halotolerans S2753 and V. coralliilyticus S2052 could not be explained by the production of holomycin nor andrimid, the respective antibiotic compounds in these organisms. In contrast, the tropodithietic acid (TDA)-producing bacteria Phaeobacter inhibens DSM17395 and Ruegeria mobilis F1926 and TDA itself had no adverse effect on the target organisms. These results reaffirm TDA-producing Roseobacter bacteria as a promising group to be used as probiotics in aquaculture, whereas Actinobacteria, Pseudoalteromonas, Photobacteriaceae, and Vibrionaceae should be used with caution.

  6. Bacterial community changes in copper and PEX drinking water pipeline biofilms under extra disinfection and magnetic water treatment.

    PubMed

    Inkinen, J; Jayaprakash, B; Ahonen, M; Pitkänen, T; Mäkinen, R; Pursiainen, A; Santo Domingo, J W; Salonen, H; Elk, M; Keinänen-Toivola, M M

    2018-02-01

    To study the stability of biofilms and water quality in pilot scale drinking water copper and PEX pipes in changing conditions (extra disinfection, magnetic water treatment, MWT). Next-generation sequencing (NGS) of 16S ribosomal RNA genes (rDNA) to describe total bacterial community and ribosomal RNA (rRNA) to describe active bacterial members in addition to traditional microbiological methods were applied. Biofilms from control copper and PEX pipes shared same most abundant bacteria (Methylobacterium spp., Sphingomonas spp., Zymomonas spp.) and average species diversities (Shannon 3·8-4·2) in rDNA and rRNA libraries, whereas few of the taxa differed by their abundance such as lower total Mycobacterium spp. occurrence in copper (<0·02%) to PEX (<0·2%) pipes. Extra disinfection (total chlorine increase from c. 0·5 to 1 mg l -1 ) affected total and active population in biofilms seen as decrease in many bacterial species and diversity (Shannon 2·7, P < 0·01, rRNA) and increase in Sphingomonas spp. as compared to control samples. Furthermore, extra-disinfected copper and PEX samples formed separate clusters in unweighted non-metric multidimensional scaling plot (rRNA) similarly to MWT-treated biofilms of copper (but not PEX) pipes that instead showed higher species diversity (Shannon 4·8, P < 0·05 interaction). Minor chlorine dose addition increased selection pressure and many species were sensitive to chlorination. Pipe material seemed to affect mycobacteria occurrence, and bacterial communities with MWT in copper but not in PEX pipes. This study using rRNA showed that chlorination affects especially active fraction of bacterial communities. Copper and PEX differed by the occurrence of some bacterial members despite similar community profiles. © 2017 The Society for Applied Microbiology.

  7. Coassimilation of Organic Substrates via the Autotrophic 3-Hydroxypropionate Bi-Cycle in Chloroflexus aurantiacus▿†

    PubMed Central

    Zarzycki, Jan; Fuchs, Georg

    2011-01-01

    Chloroflexus aurantiacus is a facultative autotrophic green nonsulfur bacterium that grows phototrophically in thermal springs and forms microbial mats with cyanobacteria. Cyanobacteria produce glycolate during the day (photorespiration) and excrete fermentation products at night. C. aurantiacus uses the 3-hydroxypropionate bi-cycle for autotrophic carbon fixation. This pathway was thought to be also suited for the coassimilation of various organic substrates such as glycolate, acetate, propionate, 3-hydroxypropionate, lactate, butyrate, or succinate. To test this possibility, we added these compounds at a 5 mM concentration to autotrophically pregrown cells. Although the provided amounts of H2 and CO2 allowed continuing photoautotrophic growth, cells immediately consumed most substrates at rates equaling the rate of autotrophic carbon fixation. Using [14C]acetate, half of the labeled organic carbon was incorporated into cell mass. Our data suggest that C. aurantiacus uses the 3-hydroxypropionate bi-cycle, together with the glyoxylate cycle, to channel organic substrates into the central carbon metabolism. Enzyme activities of the 3-hydroxypropionate bi-cycle were marginally affected when cells were grown heterotrophically with such organic substrates. The 3-hydroxypropionate bi-cycle in Chloroflexi is unique and was likely fostered in an environment in which traces of organic compounds can be coassimilated. Other bacteria living under oligotrophic conditions acquired genes of a rudimentary 3-hydroxypropionate bi-cycle, possibly for the same purpose. Examples are Chloroherpeton thalassium, Erythrobacter sp. strain NAP-1, Nitrococcus mobilis, and marine gammaproteobacteria of the OM60/NOR5 clade such as Congregibacter litoralis. PMID:21764971

  8. Influence of Iron on Production of the Antibacterial Compound Tropodithietic Acid and Its Noninhibitory Analog in Phaeobacter inhibens

    PubMed Central

    D'Alvise, Paul W.; Phippen, Christopher B. W.; Nielsen, Kristian F.

    2015-01-01

    Tropodithietic acid (TDA) is an antibacterial compound produced by some Phaeobacter and Ruegeria spp. of the Roseobacter clade. TDA production is studied in marine broth or agar since antibacterial activity in other media is not observed. The purpose of this study was to determine how TDA production is influenced by substrate components. High concentrations of ferric citrate, as present in marine broth, or other iron sources were required for production of antibacterially active TDA. However, when supernatants of noninhibitory, low-iron cultures of Phaeobacter inhibens were acidified, antibacterial activity was detected in a bioassay. The absence of TDA in nonacidified cultures and the presence of TDA in acidified cultures were verified by liquid chromatography–high-resolution mass spectrometry. A noninhibitory TDA analog (pre-TDA) was produced by P. inhibens, Ruegeria mobilis F1926, and Phaeobacter sp. strain 27-4 under low-iron concentrations and was instantaneously converted to TDA when pH was lowered. Production of TDA in the presence of Fe3+ coincides with formation of a dark brown substance, which could be precipitated by acid addition. From this brown pigment TDA could be liberated slowly with aqueous ammonia, and both direct-infusion mass spectrometry and elemental analysis indicated a [FeIII(TDA)2]x complex. The pigment could also be produced by precipitation of pure TDA with FeCl3. Our results raise questions about how biologically active TDA is produced in natural marine settings where iron is typically limited and whether the affinity of TDA to iron points to a physiological or ecological function of TDA other than as an antibacterial compound. PMID:26519388

  9. Comparative morphology of the mouthparts of the megadiverse South African monkey beetles (Scarabaeidae: Hopliini): feeding adaptations and guild structure

    PubMed Central

    Hansal, Teresa; Krenn, Harald W.; Colville, Jonathan F.

    2016-01-01

    Although anthophilous Coleoptera are regarded to be unspecialised flower-visiting insects, monkey beetles (Scarabaeidae: Hopliini) represent one of the most important groups of pollinating insects in South Africa’s floristic hotspot of the Greater Cape Region. South African monkey beetles are known to feed on floral tissue; however, some species seem to specialise on pollen and/or nectar. The present study examined the mouthpart morphology and gut content of various hopliine species to draw conclusions on their feeding preferences. According to the specialisations of their mouthparts, the investigated species were classified into different feeding groups. Adaptations to pollen-feeding included a well-developed, toothed molar and a lobe-like, setose lacinia mobilis on the mandible as well as curled hairs or sclerotized teeth on the galea of the maxillae. Furthermore, elongated mouthparts were interpreted as adaptations for nectar feeding. Floral- and folial-tissue feeding species showed sclerotized teeth on the maxilla, but the lacinia was mostly found to be reduced to a sclerotized ledge. While species could clearly be identified as floral or folial tissue feeding, several species showed intermediate traits suggesting both pollen and nectar feeding adaptations. Mismatches found between mouthpart morphology and previously reported flower visiting behaviours across different genera and species requires alternative explanations, not necessarily associated with feeding preferences. Although detailed examinations of the mouthparts allowed conclusions about the feeding preference and flower-visiting behaviour, additional morphological and behavioural investigations, combined with greater taxon sampling and phylogenetic data, are still necessary to fully understand hopliine host plant relationships, related to monkey beetle diversity. PMID:26819850

  10. Sunspot positions, areas, and group tilt angles for 1611-1631 from observations by Christoph Scheiner

    NASA Astrophysics Data System (ADS)

    Arlt, R.; Senthamizh Pavai, V.; Schmiel, C.; Spada, F.

    2016-11-01

    Aims: Digital images of observations printed in the books Rosa Ursina sive solis and Prodromus pro sole mobili by Christoph Scheiner, as well as the drawings from Scheiner's letters to Marcus Welser, are analysed to obtain information on the positions and sizes of sunspots that appeared before the Maunder minimum. Methods: In most cases, the given orientation of the ecliptic is used to set up the heliographic coordinate system for the drawings. Positions and sizes are measured manually on screen. Very early drawings have no indication of their orientation. A rotational matching using common spots of adjacent days is used in some cases, while in other cases, the assumption that images were aligned with a zenith-horizon coordinate system appeared to be the most probable. Results: In total, 8167 sunspots were measured. A distribution of sunspot latitudes versus time (butterfly diagram) is obtained for Scheiner's observations. The observations of 1611 are very inaccurate, the drawings of 1612 have at least an indication of their orientation, while the remaining part of the spot positions from 1618-1631 have good to very good accuracy. We also computed 697 tilt angles of apparently bipolar sunspot groups observed in the period 1618-1631. We find that the average tilt angle of nearly 4 degrees is not significantly different from 20th-century values. Data on the sunspot position and area are only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (http://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/595/A104

  11. Phylogenetic Diversity Analysis of Subterranean Hot Springs in Iceland

    PubMed Central

    Marteinsson, Viggó Thór; Hauksdóttir, Sigurbjörg; Hobel, Cédric F. V.; Kristmannsdóttir, Hrefna; Hreggvidsson, Gudmundur Oli; Kristjánsson, Jakob K.

    2001-01-01

    Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130°C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-μm-pore-size filter. Cells were observed in wells RG-39 (91.4°C) and MG-18 (71.8°C) and in hot tap water (76°C), but no cells were detected in wells SN-4, SN-5 (95 to 117°C), and RV-5 (130°C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85°C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles. PMID:11526029

  12. Phylogenetic diversity analysis of subterranean hot springs in Iceland.

    PubMed

    Marteinsson, V T; Hauksdóttir, S; Hobel, C F; Kristmannsdóttir, H; Hreggvidsson, G O; Kristjánsson, J K

    2001-09-01

    Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130 degrees C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-microm-pore-size filter. Cells were observed in wells RG-39 (91.4 degrees C) and MG-18 (71.8 degrees C) and in hot tap water (76 degrees C), but no cells were detected in wells SN-4, SN-5 (95 to 117 degrees C), and RV-5 (130 degrees C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85 degrees C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.

  13. Mobilisporobacter senegalensis gen. nov., sp. nov., an anaerobic bacterium isolated from tropical shea cake.

    PubMed

    Mbengue, Malick; Thioye, Abdoulaye; Labat, Marc; Casalot, Laurence; Joseph, Manon; Samb, Abdoulaye; Ben Ali Gam, Zouhaier

    2016-03-01

    A Gram-stain positive, endospore-forming, strictly anaerobic bacterium, designated strain Gal1 T , was isolated from shea cake, a waste material from the production of shea butter, originating from Saraya, Senegal. The cells were rod-shaped, slightly curved, and motile with peritrichous flagella. The strain was oxidase-negative and catalase-negative. Growth was observed at temperatures ranging from 15 to 45 °C (optimum 30 °C) and at pH 6.5-9.3 (optimum pH 7.8). The salinity range for growth was 0-3.5 % NaCl (optimum 1 %). Yeast extract was required for growth. Strain Gal1 T fermented various carbohydrates such as mannose, mannitol, arabinose, cellobiose, fructose, glucose, maltose, sucrose, trehalose and lactose and the major end-products were ethanol and acetate. The only major cellular fatty acid was C16 : 0 (19.6 %). The DNA base G+C content of strain Gal1 T was 33.8 mol%. Analysis of the 16S rRNA gene sequence of the isolate indicated that this strain was related to Mobilitalea sibirica DSM 26468 T with 94.27 % similarity, Clostridium populeti ATTC 35295 T with 93.94 % similarity, and Clostridium aminovalericum DSM 1283 T and Anaerosporobacter mobilis DSM 15930 T with 93.63 % similarity. On the basis of phenotypic characteristics, phylogenetic analysis and the results of biochemical and physiological tests, strain Gal1 T was clearly distinguished from closely related genera, and strain Gal1 T can be assigned to a novel species of a new genus for which the name Mobilisporobacter senegalensis gen. nov., sp. nov. is proposed. The type strain is Gal1 T ( = DSM 26537 T  = JCM 18753 T ).

  14. Fasting serum insulin and the homeostasis model of insulin resistance (HOMA-IR) in the monitoring of lifestyle interventions in obese persons.

    PubMed

    Vogeser, Michael; König, Daniel; Frey, Ingrid; Predel, Hans-Georg; Parhofer, Klaus Georg; Berg, Aloys

    2007-09-01

    Lifestyle changes with increased physical activity and balanced energy intake are recognized as the principal interventions in obesity and insulin resistance. Only few prospective studies, however, have so far addressed the potential role of routine biochemical markers of insulin sensitivity in the monitoring of respective interventions. Fasting insulin and glucose was measured in 33 obese individuals undergoing a lifestyle modification program (MOBILIS) at baseline and after 1 year. The HOMA-IR index (homeostasis model of insulin resistance) was calculated as [fasting serum glucose*fasting serum insulin/22.5], with lower values indicating a higher degree of insulin sensitivity. While the median body mass index (BMI) and waist circumference decreased by 10% and 11%, respectively, the HOMA-IR index decreased in an over-proportional manner by 45% within 1 year (BMI baseline, median 35.7, interquartile range (IQR) 33.7-37.7; after 1 year, median 32.2, IQR 29.6-35.1. HOMA-IR baseline, median 2.9, IQR 1.5-4.6; after 1 year 1.6, IQR 0.9-2.7). In contrast to HOMA-IR and fasting serum insulin, no significant changes in fasting serum glucose were observed. Baseline and post-intervention HOMA-IR showed a high degree of inter-individual variation with eight individuals maintaining high HOMA-IR values despite weight loss after 1 year of intervention. Individual changes in the carbohydrate metabolism achieved by a lifestyle intervention program were displayed by fasting serum insulin concentrations and the HOMA-IR but not by fasting glucose measurement alone. Therefore, assessment of the HOMA-IR may help to individualize lifestyle interventions in obesity and to objectify improvements in insulin sensitivity after therapeutic lifestyle changes.

  15. The Response of a 16S Ribosomal RNA Gene Fragment Amplified Community to Lead, Zinc, and Copper Pollution in a Shanghai Field Trial.

    PubMed

    Kou, Shumeng; Vincent, Gilles; Gonzalez, Emmanuel; Pitre, Frederic E; Labrecque, Michel; Brereton, Nicholas J B

    2018-01-01

    methylotrophus , Nitrosospira , and Lysobacter mobilis . The capacity to track alterations of an amplified microbial community at high taxonomic resolution using modern bioinformatic approaches, as well as identifying where that resolution is lost for technical or biological reasons, provides an insight into the complexity of the microbial world resisting anthropogenic pollution. While functional assessment of uncharacterized organisms within environmental samples is technically challenging, an important step is observing those organisms able to tolerate extreme stress and to recognize the extent to which important amplifiable community members still require characterization.

  16. Co-production of acetone and ethanol with molar ratio control enables production of improved gasoline or jet fuel blends.

    PubMed

    Baer, Zachary C; Bormann, Sebastian; Sreekumar, Sanil; Grippo, Adam; Toste, F Dean; Blanch, Harvey W; Clark, Douglas S

    2016-10-01

    The fermentation of simple sugars to ethanol has been the most successful biofuel process to displace fossil fuel consumption worldwide thus far. However, the physical properties of ethanol and automotive components limit its application in most cases to 10-15 vol% blends with conventional gasoline. Fermentative co-production of ethanol and acetone coupled with a catalytic alkylation reaction could enable the production of gasoline blendstocks enriched in higher-chain oxygenates. Here we demonstrate a synthetic pathway for the production of acetone through the mevalonate precursor hydroxymethylglutaryl-CoA. Expression of this pathway in various strains of Escherichia coli resulted in the co-production of acetone and ethanol. Metabolic engineering and control of the environmental conditions for microbial growth resulted in controllable acetone and ethanol production with ethanol:acetone molar ratios ranging from 0.7:1 to 10.0:1. Specifically, use of gluconic acid as a substrate increased production of acetone and balanced the redox state of the system, predictively reducing the molar ethanol:acetone ratio. Increases in ethanol production and the molar ethanol:acetone ratio were achieved by co-expression of the aldehyde/alcohol dehydrogenase (AdhE) from E. coli MG1655 and by co-expression of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) from Z. mobilis. Controlling the fermentation aeration rate and pH in a bioreactor raised the acetone titer to 5.1 g L(-1) , similar to that obtained with wild-type Clostridium acetobutylicum. Optimizing the metabolic pathway, the selection of host strain, and the physiological conditions employed for host growth together improved acetone titers over 35-fold (0.14-5.1 g/L). Finally, chemical catalysis was used to upgrade the co-produced ethanol and acetone at both low and high molar ratios to higher-chain oxygenates for gasoline and jet fuel applications. Biotechnol. Bioeng. 2016;113: 2079-2087. © 2016 Wiley

  17. Biotechnological experiments in space flights on board of space stations

    NASA Astrophysics Data System (ADS)

    Nechitailo, Galina S.

    2012-07-01

    Space flight conditions are stressful for any plant and cause structural-functional transition due to mobiliation of adaptivity. In space flight experiments with pea tissue, wheat and arabidopsis we found anatomical-morphological transformations and biochemistry of plants. In following experiments, tissue of stevia (Stevia rebaudiana), potato (Solanum tuberosum), callus culture and culture and bulbs of suffron (Crocus sativus), callus culture of ginseng (Panax ginseng) were investigated. Experiments with stevia carried out in special chambers. The duration of experiment was 8-14 days. Board lamp was used for illumination of the plants. After experiment the plants grew in the same chamber and after 50 days the plants were moved into artificial ionexchange soil. The biochemical analysis of plants was done. The total concentration of glycozides and ratio of stevioside and rebauside were found different in space and ground plants. In following generations of stevia after flight the total concentration of stevioside and rebauside remains higher than in ground plants. Experiments with callus culture of suffron carried out in tubes. Duration of space flight experiment was 8-167 days. Board lamp was used for illumination of the plants. We found picrocitina pigment in the space plants but not in ground plants. Tissue culture of ginseng was grown in special container in thermostate under stable temperature of 22 ± 0,5 C. Duration of space experiment was from 8 to 167 days. Biological activity of space flight culutre was in 5 times higher than the ground culture. This difference was observed after recultivation of space flight samples on Earth during year after flight. Callus tissue of potato was grown in tubes in thermostate under stable temperature of 22 ± 0,5 C. Duration of space experiment was from 8 to 14 days. Concentration of regenerates in flight samples was in 5 times higher than in ground samples. The space flight experiments show, that microgravity and other

  18. Experimental determination of the speciation, partitioning, and release of perrhenate as a chemical surrogate for pertechnetate from a sodalite-bearing multiphase ceramic waste form

    SciT

    Pierce, Eric M.; Lukens, Wayne W.; Fitts, Jeff. P.

    2013-12-01

    -bearing sodalites contained in the multiphase ceramic matrix are present as mixed-anion sodalite phases. These results suggest the multiphase FBSR NAS material may be a viable host matrix for long-lived, highly mobilie radionuclides which is a critical aspect in the management of nuclear waste.« less

  19. The Response of a 16S Ribosomal RNA Gene Fragment Amplified Community to Lead, Zinc, and Copper Pollution in a Shanghai Field Trial

    PubMed Central

    Kou, Shumeng; Vincent, Gilles; Gonzalez, Emmanuel; Pitre, Frederic E.; Labrecque, Michel; Brereton, Nicholas J. B.

    2018-01-01

    methylotrophus, Nitrosospira, and Lysobacter mobilis. The capacity to track alterations of an amplified microbial community at high taxonomic resolution using modern bioinformatic approaches, as well as identifying where that resolution is lost for technical or biological reasons, provides an insight into the complexity of the microbial world resisting anthropogenic pollution. While functional assessment of uncharacterized organisms within environmental samples is technically challenging, an important step is observing those organisms able to tolerate extreme stress and to recognize the extent to which important amplifiable community members still require characterization. PMID:29545788

  20. Relevé postnatal Rourke 2014

    PubMed Central

    Riverin, Bruno; Li, Patricia; Rourke, Leslie; Leduc, Denis; Rourke, James

    2015-01-01

    Résumé Objectif Mettre à jour la version de 2011 du Relevé postnatal Rourke (RPR) à la suite d’une révision des meilleures données probantes récentes sur le suivi de la santé des nourrissons et des enfants de la naissance jusqu’à l’âge de 5 ans. Qualité des données La qualité des données a été cotée en fonction de l’ancien système de classification du Groupe d’étude canadien sur les soins de santé préventifs (jusqu’à 2006) et l’approche de détermination, d’élaboration et d’évaluation des recommandations (GRADE). Message principal De nouveaux faits scientifiques ont été pris en compte dans les recommandations du RPR 2014 en ce qui a trait au suivi de la croissance, à la nutrition, à l’éducation et aux conseils, au développement, à l’examen physique et à l’immunisation. La croissance est surveillée à l’aide des courbes de l’Organisation mondiale de la Santé qui ont été révisées en 2014. On devrait introduire les aliments solides en fonction de l’état de préparation du nourrisson et ces produits devraient contenir du fer. Il n’est actuellement plus recommandé de retarder l’introduction des allergènes alimentaires courants pour prévenir les allergies. Il faut promouvoir l’utilisation d’une tasse sans couvercle au lieu d’une tasse à bec dès l’âge de 12 mois. La section sur l’éducation et les conseils porte sur les blessures causées par du mobilier instable, ainsi que l’utilisation d’un siège d’auto orienté vers l’arrière jusqu’à 2 ans. Elle comporte aussi de l’information sur les saines habitudes de sommeil, la prévention de la maltraitance des enfants, la vie saine et active et la sédentarité de la famille, de même que l’hygiène buccale. On a aussi ajouté à cette section une nouvelle catégorie consacrée à la santé environnementale pour tenir compte des effets des dangers environnementaux sur la santé des enfants. Le RPR a recours à une

  1. Préface

    NASA Astrophysics Data System (ADS)

    Aspect, Alain

    2004-11-01

    ouverture sur les recherches de base d'aujourd'hui qui seront peut-être les outils quotidiens de l'optique de demain. Quant aux 169 communications par affiches elles ont, suivant la règle, couvert sans restriction tous les sujets de l'optique actuelle. Ce volume spécial du Journal de Physique IV regroupe la majorité des conférences invitées et des communications par affiches. Profitant de la participation des mondes académique et industriel, un forum sur “innovation et nouveaux débouchés pour l'optique” a permis de dégager des raisons d'espoir pour l'emploi des chercheurs en optique. Enfin, renouvelant l'expérience d'éditions précédentes, la dernière demi journée a été consacrée à deux conférences qui étaient ouvertes à un public élargi, notamment les étudiants de l'université et des classes préparatoires ainsi que leurs enseignants. Si Antoine Labeyrie nous a laissés sans voix avec ses projets “hyperfuturistes” d'hypertélescopes à l'échelle du million de kilomètres, Pierre Lena nous a donné un moment de vrai bonheur en célébrant le mariage sans cesse renouvelé du toujours jeune couple que sont l'optique et l'astronomie : qu'il soit remercié aussi pour le magnifique teste publié dans ce volume. COLOQ se devait d'honorer la mémoire de deux personnalités hors du commun de l'optique, Jean Brossel et Gilbert Grynberg. Tous ceux qui avaient connu l'un ou l'autre ont pu apprécier la justesse des évocations, par d'anciens collaborateurs, de l'oeuvre scientifique mais aussi des personnalités de ces deux grands physiciens. On trouvera les textes de ces interventions émouvantes dans ce recueil. La réussite de COLOQ 8 est évidemment due; avant tout, à la qualité scientifique des présentations, mais elle repose aussi sur l'action conjuguée des comités d'organisation national et local. Au niveau local, Françoise Lozes et Jacques Vigué ont mobilisé les membres du LCAR-IRSAMC et du LAAS pour l'organisation conjointe - particuli