Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Consolidated bioprocessing is a one-step process that allows the direct microbial conversion of cellulosic substrates to ethanol and hydrogen. The fermentation was initially performed in batch cultures, in a pH and temperature controlled reactor using Clostridium thermocellum ATCC 27405. With an objective of increasing the production of ethanol and hydrogen, various types of fed-batch fermentations were investigated: variable volume (VV) fed-batch, fixed volume (FV) fed-batch, and semi-continuous fermentation. Semi-continuous processes were carried out at low (10-15 g/L) and high (20-25 g/L) cellulose concentrations. The maximum ethanol production obtained in batch, VV, FV, semi-continuous with low concentrations and high concentrations were 554 mmol, 336 mmol, 477 mmol, 695 mmol and 741 mmol respectively. In the same order, the total hydrogen production was 288 mmol, 364 mmol, 231 mmol, 434 mmol, and 387 mmol. Overall, the semi-continuous fermentation showed more promise in terms of large-scale deployment compared to batch, VV, and FV fed-batch. / October 2015

Consolidated bioprocessing, a process in which cellulase production, substrate hydrolysis, and fermentation occur simultaneously, offers the potential for lower biofuel production costs than traditional approaches and is an economically attractive near-term goal for fermentative production of ethanol and/or hydrogen (H2) as biofuels. Current yields fall short of theoretical maxima, vary considerably between species, and are influenced by the highly branched metabolic pathways utilized by fermentative organisms. For fermentative ethanol/ H2 production to become practical, yields must be increased either through intelligent species selection, a manipulation of culture conditions, or via the implementation of rational metabolic engineering strategies. A comparative genomics approach amoungst select members of the Firmicutes, Euryarchaeota, and Thermotogae was used to identify genes relevent to ethanol and H2 production. Growth, end-product synthesis, enzyme activities and the associated transcription of select genes were studied in the cellulolytic anaerobe, Clostridium thermocellum ATCC 27405, during batch fermentation of cellobiose to determine the effect of elevated N2 and H2 sparging on end-product distribution. The absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) correlates with elevated H2 yields and low ethanol production. The type(s) of encoded hydrogenases appear to have minimal impact on H2 production in organisms that do not encode ethanologenic pathways, however, they do influence reduced end-product yields in those that do. We also find that while gas sparging can be used to effectively shift carbon and electron flow, the observed shifts at the pyruvate branch-point are likely principally influenced by the availability of reduced electron carriers (NAD, NADP, ferredoxin) and thermodynamic considerations. Finally, both electrotransformation and conjugative plasmid protocols were developed and evaluated for thermophilic species C. thermocellum and Thermoanaerobacter pseudethanolicus 39E, and the mesophilic bacterium, Clostridium termitidis CT1112. The efficiency of transformation for C. thermocellum strain ATCC 27405 is consistently low whereas transformation frequencies were ~100-fold higher in C. termitidis. Observed frequencies of plasmid transfer, via conjugation, were similar in both C. thermocellum and C. termitidis suggesting the transfer of single stranded DNA may circumvent aggressive restriction methylation systems.

Consolidated bioprocessing, a process in which cellulase production, substrate hydrolysis, and fermentation occur simultaneously, offers the potential for lower biofuel production costs than traditional approaches and is an economically attractive near-term goal for fermentative production of ethanol and/or hydrogen (H2) as biofuels. Current yields fall short of theoretical maxima, vary considerably between species, and are influenced by the highly branched metabolic pathways utilized by fermentative organisms. For fermentative ethanol/ H2 production to become practical, yields must be increased either through intelligent species selection, a manipulation of culture conditions, or via the implementation of rational metabolic engineering strategies. A comparative genomics approach amoungst select members of the Firmicutes, Euryarchaeota, and Thermotogae was used to identify genes relevent to ethanol and H2 production. Growth, end-product synthesis, enzyme activities and the associated transcription of select genes were studied in the cellulolytic anaerobe, Clostridium thermocellum ATCC 27405, during batch fermentation of cellobiose to determine the effect of elevated N2 and H2 sparging on end-product distribution. The absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) correlates with elevated H2 yields and low ethanol production. The type(s) of encoded hydrogenases appear to have minimal impact on H2 production in organisms that do not encode ethanologenic pathways, however, they do influence reduced end-product yields in those that do. We also find that while gas sparging can be used to effectively shift carbon and electron flow, the observed shifts at the pyruvate branch-point are likely principally influenced by the availability of reduced electron carriers (NAD, NADP, ferredoxin) and thermodynamic considerations. Finally, both electrotransformation and conjugative plasmid protocols were developed and evaluated for thermophilic species C. thermocellum and Thermoanaerobacter pseudethanolicus 39E, and the mesophilic bacterium, Clostridium termitidis CT1112. The efficiency of transformation for C. thermocellum strain ATCC 27405 is consistently low whereas transformation frequencies were ~100-fold higher in C. termitidis. Observed frequencies of plasmid transfer, via conjugation, were similar in both C. thermocellum and C. termitidis suggesting the transfer of single stranded DNA may circumvent aggressive restriction methylation systems.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2015. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2015-12-21T13:54:05Z No. of bitstreams: 1 2015_KarenOfujiOsiro.pdf: 2867423 bytes, checksum: 9d23fe29816120dc28d973b7de26c02f (MD5) / Approved for entry into archive by Patrícia Nunes da Silva(patricia@bce.unb.br) on 2016-01-25T15:53:17Z (GMT) No. of bitstreams: 1 2015_KarenOfujiOsiro.pdf: 2867423 bytes, checksum: 9d23fe29816120dc28d973b7de26c02f (MD5) / Made available in DSpace on 2016-01-25T15:53:17Z (GMT). No. of bitstreams: 1 2015_KarenOfujiOsiro.pdf: 2867423 bytes, checksum: 9d23fe29816120dc28d973b7de26c02f (MD5) / O presente trabalho tem como propósito a purificação de celulossomas, sua caracterização e do secretoma de um novo isolado de Clostridium thermocellum B8, obtido do rúmen de caprino. Para obtenção das amostras proteicas C. thermocellum foi cultivada em meio líquido contendo celulose como fonte de carbono. O secretoma que constitui a amostra de Proteínas Ligadas á Celulose (PLC) foi eluído a partir da celulose residual da cultura. As frações cromatográficas do principal pico de proteína resultante do fracionamento da amostra PLC em uma cromatografia de exclusão molecular (Superdex S-200) compõe a amostra de Celulossomas Parcialmente Purificados (CPP). Ambas as amostras apresentaram atividades enzimáticas celulolíticas e xilanolíticas. As análises de SDS-PAGE, DLS e espectrometria de massa confirmaram que a amostra CPP se trata de um complexo celulossomal, com uma massa molecular estimada de 11,3 MDa ± 4,4 MDa. As proteínas constituintes de CPP foram identificadas por espectrometria de massa LC-MS/MS, levando a identificação de: proteína estrutural (CipA), glicosil hidrolases das famílias 5, 8, 9, 10 e 48 e transportadores do tipo ABC. Na amostra PLC foram identificadas principalmente proteínas envolvidas no catabolismo de polissacarídeos, além de proteínas relacionadas com o mecanismo de transporte de moléculas e processos metabólicos. Dentre as proteínas que têm a função no catabolismo de polissacarídeos, foram identificados glicosil hidrolases das famílias 5, 8, 9, 10, 11, 26, 30, 43 e 53, com atividades de endoglucanase, celobiohidrolases, endoxilanases, endoxilanases com esterase feruloil e acetil com função de xilana esterase, xiloglucanases, arabinoxilanase, glucoxilana xilanahidrolase, arabinofuranosidase, exogalactanase, endogalactanase (arabinogalactana), quitinase, mananase. CPP e PLC apresentaram atividade máxima no intervalo de temperatura entre 60°C-70°C, pH 5.0, e estabilidade térmica a 50, 60 e 70°C, principalmente, no que diz respeito a amostra de PLC. As atividades holocelulolíticas de PLC foram inibidas por compostos fenólicos, enquanto que CPP apresentou um aumento nas atividades de CMCase e xilanase na presença de vários fenóis. A partir dos resultados da sacarificação dos substratos de celulose, palha de cana e piolho de algodão por PLC e CPP, foi observado maiores quantidades de açúcares liberados principalmente a 50°C, em comparação com os dados de 60°C, após 10 dias de incubação. Entretanto, foi observado que após o segundo dia de sacarificação, que os produtos obtidos pela desconstrução de materiais lignocelulósicos, acabam por interferir na velocidade de hidrólise das enzimas de C. thermocellum B8. Em conclusão, este estudo demonstrou potencial para utilização de ambas as amostras (CPP e PLC) de C. thermocellum B8 para hidrolisar biomassas lignocelulósicas, destacando-se o aumento da atividade celulolítica e xilanolítica de CPP na presença de compostos fenólicos, além da termoestabilidade de PLC. ____________________________________________________________________________________ ABSTRACT / The present work aimed the purification and characterization of the secretome produced by Clostridium thermocellum B8, a novel isolate obtained from goat rumen, after growth on liquid medium containing cellulose as carbon source. The secretome were eluted from the residual substrate, constituting the Protein Linked on Cellulose (PLC) sample. The main protein peak of PLC fractionation onto a molecular exclusion (Superdex S-200) composes the Cellulosomes Partially Purified (CPP) sample. Both samples presented enzymatic activities of cellulases and xylanases. It was elucidated through SDS-PAGE, DLS and mass spectrometry that CPP sample is a cellulosome complex, with an estimated mass of 11,3 MDa ± 4,4 MDa. CPP’s constituting proteins were identified by mass spectrometry LC-MS/MS leading the identification of: scaffolding protein (CipA), glycoside hydrolase proteins classified on the families 5, 8, 9, 10 and 48 and ABC transporter substrate-binding protein. In the PLC sample were identified mostly proteins involved in the catabolism of polysaccharides, besides proteins related to the transport mechanism molecules and metabolic processes. Among the proteins which have catabolism of polysaccharides function, were identified glycosyl hydrolase families 5, 8, 9, 10, 11, 26, 30, 43 and 53 with endoglucanase activity, cellobiohydrolases, endoxylanases, endoxylanases with feruloil esterase and acetyl xylan esterase function, xyloglucanases, arabinoxylanase, glucoxylana xylanhydrolase, arabinofuranosidase, exogalactanase, endogalactanase (arabinogalactan), chitinase, mannanase. CPP and PLC presented maximal activity in the range of 60° to 70°C and pH 5.0, and also those samples have a high thermostability at 50, 60 and 70°C mainly for PLC sample. PLC holocelullolytic activities were inhibited by phenolic compounds, while CPP showed improvement or was less inhibited on its xylanase and CMCase activity in phenols presence. Saccharification results of cellulose, sugarcane straw and cotton gin waste by PLC and CPP, showed highest amounts of sugar released mostly at 50°C in comparison to 60°C and after 10 days of incubation. In summary, this research demonstrated the potential of using CPP and PLC samples of C. thermocellum B8 to hydrolyze lignocellulosic biomasses, with the ability of CPP increase its holocelullolytic activities in the presence of phenolic compounds, and the interesting thermostability of PLC sample, both being valuable for second generation production of biofuels.

Clostridium thermocellum is a fermentative, Gram-positive, thermophile capable of cellulosome-mediated breakdown of hemicellulose and simultaneous biofuels (ethanol and H2) production, and is thus an excellent candidate for consolidated bioprocessing. However, ethanol and/or H2 production yields are below theoretical maxima due to branched product pathways. Biofuel yields may be improved by manipulation of fermentation conditions or implementation of rational metabolic engineering strategies. However, the latter relies on a thorough understanding of gene content, gene product expression, enzyme activity, and intracellular metabolite levels, which can all influence carbon and electron flux. The thesis work represents the first large-scale attempt in combining bioinformatic, enzymatic, proteomic, and culture perturbation approaches to systematically understand these interactions. C. thermocellum was used to investigate how these parameters affect end-product yields. Enzyme activities involved in conversion of pyruvate to end-products were consistent with end-product profiles and draft genome annotation. NADH and NADPH-dependent alcohol dehydrogenase (ADH) activities were comparable, whereas NADPH-dependent hydrogenase activities were higher than NADH and ferredoxin-dependent hydrogenase activities. While product yields changed in response to exogenous end-product additions, most core fermentative enzyme activities did not, suggesting that these changes may be governed by thermodynamics. The lack of major changes (>2-fold) in expression in response to growth and gas sparging was further confirmed by proteomics and RT-qPCR, respectively, although the latter revealed that ADH expression changes in response to gas sparging. Improved genome curation allowed refinement of metabolic pathways. A genomic and end-product meta-analysis of ethanol and/or H2 producing fermentative bacteria revealed that presence/absence of genes encoding hydrogenases and aldehyde dehydrogenases/ADHs had the greatest impacts on biofuel yields. However, genome content alone did not necessarily explain end-product yields. Given that genomic analysis of C. thermocellum revealed the presence of redundant genes encoding enzymes with analogous functions, shotgun and multiple reaction monitoring proteomics was used to refine which proteins are expressed. Absence/low expression of aldehyde dehydrogenase, ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase suggest that these enzymes may not play a significant role in metabolism. An alternative electron flow pathway is proposed to explain end-product synthesis patterns in response to pyruvate addition or presence of protein inhibitors (CO, hypophosphite).

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 264-277).

Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2017. / Submitted by Gabriela Lima (gabrieladaduch@gmail.com) on 2017-11-28T14:00:16Z No. of bitstreams: 1 2017_BrendaRabellodeCamargo.pdf: 4900807 bytes, checksum: c27bb67d00e82428ce979800193440bd (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2018-01-22T20:36:24Z (GMT) No. of bitstreams: 1 2017_BrendaRabellodeCamargo.pdf: 4900807 bytes, checksum: c27bb67d00e82428ce979800193440bd (MD5) / Made available in DSpace on 2018-01-22T20:36:24Z (GMT). No. of bitstreams: 1 2017_BrendaRabellodeCamargo.pdf: 4900807 bytes, checksum: c27bb67d00e82428ce979800193440bd (MD5) Previous issue date: 2018-01-22 / O Brasil possui biomassa de origem lignocelulósica, de abundância, como os resíduos da indústria da cana de açúcar que, podem ser convertidos em produtos de alto valor agregado. Clostridium thermocellum é um microrganismo com grande potencial para degradação de celulose e biomassa lignocelulósica, devido à capacidade dessa bactéria em secretar um complexo multi-enzimático (celulossoma), formado por um arsenal de enzimas entre glicosil hidrolases, carboidrato esterases, e polissacarídeo liases, considerado carboidrato-ativas. Além disso, essa bactéria fermenta hexoses (C6) e não pentoses (C5), produzindo principalmente etanol, acetato e lactato. A regulação da expressão de genes, regulados pela fonte de carbono, é uma temática de contínuo estudo. Assim, foi realizado o proteoma descritivo de Clostridium thermocellum quando crescido por 96 horas em diferentes fontes de carbono: celulose, bagaço e palha de cana. Foram analisadas diferentes frações de conteúdo de proteína; sobrenadante (FS), ligado ao substrato (FES), e parcialmente purificado em coluna de exclusão molecular (CPP). Em todas as frações foram encontradas proteínas relacionadas à degradação da biomassa, em maior número em amostras de CPP de celulose, seguido pela amostra FES de palha. As exoglucanases CelS, CelK, CbhA, e proteínas estruturais CipA e OlpB foram encontradas em todos os substratos. Na amostra de bagaço foram identificadas as endoglucanases CelN, CelA, CelB, CelR, CelJ, CelQ, CelW, CelG e hemicelulases, Xgh74A, XynC, XynZ, Cthe_0798, sendo a última, presente também em palha. O transcritoma (RNAseq) e análise diferencial dos genes nas mesmas condições, porém em 37 horas, foi realizado. Das proteínas encontradas no proteoma, os genes cthe_0798 e xgh74A foram encontrados diferencialmente expressos em bagaço quando comparado com celulose, seguido pelos genes codificadores de CelP, CtMan5A, CelC, XynA, ManA, Cthe_1257, Cthe_3163 e Orf2p. Genes envolvidos na mobilidade celular, incluindo proteínas de formação de flagelos, motor e regulação, foram encontrados como a categoria (COG N) de maior regulação positiva em bagaço e palha. Na mesma categoria estão genes envolvidos na quimiotaxia e quorum sensing. Durante as análises do transcritoma foi verificada a presença de outra bactéria, Moorella thermoacetica, conhecida por não ser celulolítica, porém com a habilidade de fermentar açúcares C5 e C6. A análise de expressão diferencial de genes nos diferentes tratamentos, mostrou moth_0612 e moth_0699 regulados positivamente em bagaço, ambos codificando transportadores de ribose (C5). A análise de proteínas ortólogas entre as duas bactérias revelou que M.thermoacética não possui ortólogos com potenciais de atuar como enzimas carboidrato ativas de C.thermocellum. Relativo à C.thermocellum, o gene cthe_2196, ausente de caracterização, contendo os domínios GH43, CBM6 e Doquerina tipo I, foi encontrado expresso em bagaço e palha, ausente em celulose, além de ser predito como fazendo parte de um operon juntamente com cthe_2195. Cthe_2196, foi clonado em sistema pET, expresso em E.coli, e a proteína denominada AxB8 foi caracterizada bioquimicamente. AxB8 possui habilidade de degradar arabinoxilana e substratos sintéticos pNP-α-Larabinofuranosidase, e pNP-α-L-arabinofuranosidase. AxB8 apresenta domínio Glicosil hidrolase família 43, subfamília 29, e sua sequência mostrou maior similaridade com outras proteínas de termófilos não caracterizadas. Concluindo, os resultados desse trabalho demonstraram proteínas que são essenciais à degradação de bagaço e palha de cana, além de revelar importantes mecanismos regulados nessas condições que facilitam a proximidade da bactéria com o substrato, como os genes envolvidos na motilidade da bactéria. Essas informações podem ser utilizadas para desenvolvimento de novas linhagens e enzimas que aumentem o processo de degradação de biomassas lignocelulósicas, com o concomitante uso de produtos formados na geração de novas tecnologias. / Lignocellulosic biomass is in abundance in Brazil, as the residues of the sugar cane industry, which can be converted into products with high aggregated value. Clostridium thermocellum is a microorganism with great potential for degradation of cellulose and lignocellulosic biomass due to its ability to secrete a multi-enzymatic complex (cellulosome), formed by an arsenal of enzymes between glycosyl hydrolases, carbohydrate esterases, and polysaccharide lyases, considered Carbohydrate-active. In addition, this bacterium ferments hexoses (C6) and not pentoses (C5), mainly producing ethanol, acetate, and lactate. Metabolism gene expression, regulated by the carbon source, is a subject of continuous study. Thus, the descriptive proteome of Clostridium thermocellum was carried out when grown for 96 hours in different carbon sources: cellulose, bagasse and cane straw. Different fractions of protein content were analyzed; Supernatant (FS), bound to the substrate (FES) and partially purified by size exclusion chromatography (CPP). In all the fractions were found proteins related to the degradation of the biomass, in greater number in samples of cellulose CPP, followed by straw FES. The exoglucanases CelS, CelK, CbhA, and structural proteins CipA and OlpB were found in all substrates. In the bagasse sample the endoglucanases CelN, CelA, CelB, CelR, CelJ, CelQ, CelW, CelW, CelG and hemicellulases, Xgh74A, XynC, XynZ, Cthe_0798 were identified, the latter being also present in straw. Transcriptomic (RNAseq) and differential analysis of the genes under the same conditions, but growth at 37 hours, was performed. From the proteins found in the proteome, the encoding genes cthe_0798 and xgh74A were differentially expressed as bagasse when compared to cellulose, followed by the genes encoding CelP, CtMan5A, CelC, XynA, ManA, Cthe_1257, Cthe_3163 and Orf2p. Genes involved in cell motility(COG N), including flagella, motor and regulation proteins, were found in the highest category of up-regulation in bagasse and straw. In the same category were genes involved in chemotaxis and quorum sensing. During ranscriptomic analysis, was detected the presence of another bacterium, Moorella thermoacetica, knowing to be not ellulolytic, but having the ability to ferment C5 and C6 sugars. Differential gene expression in different treatments showed moth_0612 and moth_0699 up-regulated in bagasse, both encoding ribose (C5) transporters. Orthologous proteins analysis between the two bacteria revealed that M.thermoacética did not have orthologs with the potential to act as active carbohydrate enzymes from C.thermocellum. Regarding C.thermocellum, the cthe_2196 gene, absent from characterization, containing the domains GH43, CBM6 and Dockerin type I, was found expressed in bagasse and straw, but absent in cellulose, besides being predicted as part of an operon together with cthe_2195. Cthe_2196 was cloned into pET system, further expressed in E. coli, and the protein named AxB8 was characterized biochemically. AxB8 has the ability to degrade arabinoxylan and synthetic substrates pNP-α-L-arabinofuranosidase, and pNP-α-Larabinofuranosidase. AxB8 contains Glycosyl hydrolase family domain 43, subfamily 29, and its sequence showed greater similarity with other non-characterized thermophilic proteins. In conclusion, the results of this work demonstrated proteins that are essential for the degradation of sugarcane bagasse and straw, besides revealing important regulated mechanisms due to these conditions that facilitate the proximity of the bacterium with the substrate, as the genes involved in the motility of the bacteria. This information can be used to develop new strains and enzymes that increase the degradation process of lignocellulosic biomasses, with the concomitant use of products formed in the generation of new technologies.

No description available.

Clostridium thermocellum is a thermophilic bacterium that converts biomass to ethanol directly; however, high sensitivity of this bacterium toward ethanol limits its commercial utility. To elucidate the effect of ethanol on the growth of this bacterium a metabolite analysis of C. thermocellum was performed. The hypothesis of the project was that exogenous ethanol alters the metabolite profile of C. thermocellum. For metabolite analysis, capillary electrophoresis-electrospray ionization-mass spectrometry method (CE-ESI-MS) was developed due to highly polar and charged nature of metabolites. To increase the sensitivity of CE-ESI-MS, several parameters at the ESI interface were optimized. The application of 50% isopropanol as a sheath liquid increased sensitivity for metabolite analysis dramatically. Trimethylamine acetate (pH 10) was used as background electrolyte (BGE) due to its ability to separate the structural isomers of glucose phosphate. For metabolite sample preparation, novel methods for quenching and CE compatible metabolite extraction protocols were developed. Newly developed protocols were applied to metabolite analysis of wild type (WT) and ethanol adapted (EA) strains of C. thermocellum grown in batch cultures. Significant differences were found in key intracellular metabolites such as NAD+ and pyruvic acid. Intracellular concentrations of NAD+ were low in EA cells compared to WT cells and pyruvic acid was only detected in EA cells. To further understand the effect of ethanol on metabolite fluxes, WT and EA cells were grown in increasing concentrations of ethanol and the metabolite profile for each ethanol treatment was obtained. Significant changes were found in intracellular metabolite concentrations. Metabolic data showed that the glycolysis process in WT cells was obstructed due to exogenous ethanol which was evident from accumulation of G6P. On the other hand, no such accumulation of G6P was observed in the EA strain; however pyruvate began to accumulate in EA strain. These changes in intracellular metabolite concentrations due to perturbation of exogenous ethanol supported the hypothesis. Also, this investigation revealed a correlation between ethanol and metabolite profile changes and was able to explain a possible mechanism of growth inhibition of C. thermocellum which will certainly help genetic engineers to develop superior strains of C. thermocellum for commercial cellulosic ethanol production.