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The anaerobic, cellulolytic bacterium Clostridium thermocellum formed biofilms on cellulose consisting of a single layer of cells which did not secrete an extracellular polymeric matrix. Sporulation occurred under normal growth and was believed to assist with biofilm translocation to new substrates. Although the cell-substrate distance was less than 210 nm, the biofilm layer lost up to 29% of hydrolyzed oligomeric products when reactors were loaded with extreme concentrations of cellulose (up to 200 g/litre). This effect was much less severe at lower cellulose concentrations. Of the total cellulose carbon, 4% (gC/gC) was utilized for cell mass production and up to 75% was converted into primary metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). Increasing the starting cellulose concentration shifted the ethanol-to-acetic acid ratio from 0.91 g/g to 0.41 g/g. Such high substrate loadings and metabolite shifts have not been previously reported and may be of interest for consolidated bioprocessing strategies. Cellulose conversion was initially limited by microbial growth, with a biofilm development rate estimated at 0.46h-1 to 0.33h-1 and where up to 20% of the substrate was consumed. Subsequently, substrate-limited conditions determined the rate kinetics. Surface accessibility for microbial colonization was the dominant rate limiting factor, while mass imposed constraints very late towards the end-point fermentation. CO2 was found to be an excellent reporter molecule for cellulose consumption and biofilm growth. Online CO2 tracking may also be used to assess the digestibility of substrates with unknown surface properties. A mathematical model that described biofilm growth, substrate consumption and product formation was found to have an excellent fit with experimental data of CO2 production which reinforced the previous findings on the cellulolytic biofilm form and function. Together, these results demonstrate that biofilms are undeniably the key to understanding the effective microbial conversion of cellulosic substrates.

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Consolidated bioprocessing, a method that involves cellulase production, substrate hydrolysis, and fermentation all in one step, requires lower energy input and aims at achieving reduced biofuel production costs than traditional processes. It is an economically appealing strategy for the efficient production of biofuels such as ethanol or H2. At present, the yields of fermentative hydrogen and ethanol production are less than the theoretical maximum and vary between anaerobic Clostridia due to the presence of highly branched metabolic pathways. With the recent advancements in ‘Omic technologies, the selected cellulolytic species, in this case, C. termitidis, was extensively studied to identify the key enzymes that are involved in hydrogen and ethanol synthesis pathways in both the genome and proteome under different culture conditions. Metabolic characterization involving growth and end-product synthesis patterns were performed on 2 g L-1 cellobiose and α-cellulose under batch conditions to determine its metabolic potential for hydrogen and/or ethanol production. Initial characterization has shown the ability of C. termitidis to produce hydrogen, ethanol, and various other end-products on the two susbtrates. Continous N2 sparging in the pH-controlled bioreactors with cellobiose and α-cellulose showed a consistent increase in the H2 synthesis and lowered ethanol production compared to batch studies, with the H2 yields of 1.03 and 1.34 mol product per mol hexose equivalent added, respectively. Shotgun 2-D proteome analyses were performed to compare cellulose versus cellobiose grown cultures across exponential and stationary phases of growth. Most of the glycolytic proteins were detected in the proteome with some exceptions and no significant change was observed across both growth conditions. Hydrogen synthesis was regulatd via PFOR and ferredoxin-dependent hydrogenase, where as ethanol synthesis was regulated primarily via bifunctional AdhE activity. Proteomic analyses of C. termitidis cultured on hexose sugars in the absence of xylose suggested possible sequential utilization of xylose and cellobiose for the first time. Putative proteins consistent with xylose fermentation were observed at high levels. The hypothesis that C. termitidis can sequentially utilize xylose and cellobiose was further validated using batch fermentations tests on pure (xylose, cellobiose, xylan) and mixed substrates (xylose + cellobiose).

Consolidated bioprocessing, a method that involves cellulase production, substrate hydrolysis, and fermentation all in one step, requires lower energy input and aims at achieving reduced biofuel production costs than traditional processes. It is an economically appealing strategy for the efficient production of biofuels such as ethanol or H2. At present, the yields of fermentative hydrogen and ethanol production are less than the theoretical maximum and vary between anaerobic Clostridia due to the presence of highly branched metabolic pathways. With the recent advancements in ‘Omic technologies, the selected cellulolytic species, in this case, C. termitidis, was extensively studied to identify the key enzymes that are involved in hydrogen and ethanol synthesis pathways in both the genome and proteome under different culture conditions. Metabolic characterization involving growth and end-product synthesis patterns were performed on 2 g L-1 cellobiose and α-cellulose under batch conditions to determine its metabolic potential for hydrogen and/or ethanol production. Initial characterization has shown the ability of C. termitidis to produce hydrogen, ethanol, and various other end-products on the two susbtrates. Continous N2 sparging in the pH-controlled bioreactors with cellobiose and α-cellulose showed a consistent increase in the H2 synthesis and lowered ethanol production compared to batch studies, with the H2 yields of 1.03 and 1.34 mol product per mol hexose equivalent added, respectively. Shotgun 2-D proteome analyses were performed to compare cellulose versus cellobiose grown cultures across exponential and stationary phases of growth. Most of the glycolytic proteins were detected in the proteome with some exceptions and no significant change was observed across both growth conditions. Hydrogen synthesis was regulatd via PFOR and ferredoxin-dependent hydrogenase, where as ethanol synthesis was regulated primarily via bifunctional AdhE activity. Proteomic analyses of C. termitidis cultured on hexose sugars in the absence of xylose suggested possible sequential utilization of xylose and cellobiose for the first time. Putative proteins consistent with xylose fermentation were observed at high levels. The hypothesis that C. termitidis can sequentially utilize xylose and cellobiose was further validated using batch fermentations tests on pure (xylose, cellobiose, xylan) and mixed substrates (xylose + cellobiose).

No Brasil são abatidos por ano cerca de 34 milhões de cabeças de rebanho bovino, sendo que cada animal abatido pode produzir aproximadamente 25 kg de conteúdo ruminal. Este resíduo se não disposto adequadamente pode ser considerado como um material de risco ambiental, necessitando assim de especial atenção no seu gerenciamento. Uma forma sustentável de tratamento do resíduo ruminal é a transformação deste em uma fonte orgânica de nutrientes e de microrganismos potenciais para produção de enzimas que podem ser utilizadas de diversas formas em processos biotecnológicos. O objetivo do trabalho foi a obtenção de cultura mista a partir de bactérias celulolíticas oriundas do conteúdo ruminal bovino no processo de compostagem e fungos filamentosos. Duzentas e cinquenta bactérias isoladas aos 7, 21, 39 e 62 dias ao longo do processo de compostagem foram analisadas quanto ao potencial de produzir enzimas celulolíticas. Na primeira triagem foram selecionadas 16 bactérias. Após os testes enzimáticos em meio liquido contendo Xilana e CMC para determinar Xilanase e CMCase respectivamente, os melhores resultados para CMCase foram dados pelas bactérias: 1T54(0.72 U/mL), 2T47(0.9 U/mL), 2T43.1(1.48 U/mL), 3T56.1(0.9 U/mL) e 3T32(1.12 U/mL) e para xilanase: 1T54(9.67 U/mL), 2T42.1(5.21 U/mL), 2T43.1(5.58 U/mL), 2T03(5.33 U/mL), 3T56.1(29.77 U/mL), 2T37.1(29.06 U/mL) e 3T32(5.82 U/mL). As melhores combinações entre bactérias foram 1T54+2T43.1, 2T37.1+2T47 e 3T56.1+3T32. E entre fungos e bactérias respectivamente (Bactéria+Fungo), as linhagens 2T43.1 +1T1502 e 1T54+1T1502. As linhagens de bactérias e fungos filamentosos cultivados em culturas mistas promoveram 12 combinações entre as bactérias e 14 entrelaçamentos mútuos entre 13 fungos testados. As melhores combinações entre bactérias foram: 1T54+2T43.1, 2T37.1+2T47 e 3T56.1+3T32. E entre fungos e bactérias respectivamente (Bactéria+Fungo) as 2T43.1 +1T1502 e 1T54+1T1502. As bactérias isoladas de resíduo ruminal expressaram capacidade de produção enzimáticas de CMCase e xilanase assim como apresentaram entrelaçamentos mútuos entre si e com fungos, mostrando que os isolados podem ser combinados para a formação de cultura mista e usadas para testes em leiras de compostagem como aceleradores de decomposição. / In Brazil, about 34 million head of cattle are slaughtered each year, and each slaughtered animal can produce approximately 25 kg of ruminal contents. This residue, if not disposed of properly, can be considered as an environmental risk material, thus requiring special attention in its management. A sustainable form of treatment of the ruminal residue is the transformation of this into an organic source of nutrients and potential microorganisms for the production of enzymes that can be used in various forms in biotechnological processes. The objective of the work was to obtain a mixed culture from cellulolytic bacteria derived from bovine ruminal content in the composting process and filamentous fungi. Two hundred and fifty bacteria isolated at 7, 21, 39 and 62 days throughout the composting process were analyzed for the potential to produce cellulolytic enzymes. At the first screening, 16 bacteria were selected. After the enzymatic tests in liquid medium containing Xylan and CMC to determine Xylanase and CMCase respectively, the best results for CMCase were given by the bacteria: 1T54 (0.72 U/mL), 2T47 (0.9 U/mL), 2T43.1 (1.48 U/mL), 3T56.1 (0.9 U/mL) and 3T32 (1.12 U/mL) and for xylanase: 1T54 (9.67 U/mL), 2T42.1 (5.21 U/mL), 2T43.1 (5.58 U/mL), 2T03 (5.33 U/mL), 3T56.1 (29.77 U/mL), 2T37.1 (29.06 U/mL) and 3T32 (5.82 U/mL). The best combinations among bacteria were 1T54 + 2T43.1, 2T37.1 + 2T47 and 3T56.1 + 3T32. Among fungi and bacteria respectively (Bacteria + Fungus), the strains 2T43.1 + 1T1502 and 1T54 + 1T1502. Bacteria and filamentous fungi strains cultured in mixed cultures promoted 12 combinations between bacteria and 14 mutual interlacements among 13 fungi tested. The best combinations among bacteria were: 1T54 + 2T43.1, 2T37.1 + 2T47 and 3T56.1 + 3T32. Among fungi and bacteria, respectively, (Bacteria + Fungus) the 2T43.1 + 1T1502 and 1T54 + 1T1502. Bacteria isolated from ruminal residue expressed an enzymatic production capacity of CMCase and xylanase as well as showing mutual entanglement with each other and with fungi, showing that the isolates can be combined for mixed culture formation and used for composting as well as decomposition.