Clostridium thermocellum is a thermophilic (55 – 60oC) and anaerobic bacterium displaying one of the highest growth rates on cellulose. By the use of cellulosome which is a structured network of extracellular cellulases/hemicelluases, C. thermocellum is capable of simultaneous biomass hydrolysis and fermentation in one integrated step, which eliminates the need of separate and costly processes to produce cellulases enzyme cocktail in the biomass-to-fuel conversion. During cellulose fermentation, the bacterium produces hydrogen (H2), ethanol, formate, lactate, acetate, and CO2. To produce cost-competitive H2 from renewable biomass, our goals include optimizing cellulose fermentation in bioreactors, and developing /expanding on the genetic tools to increase the H2 molar yield (mol H2/mol hexose) in C. thermocellum. Our current approaches include redirecting carbon flux in favor of H2 production via knocking out competing pathways. Following the selection and counter-selection strategy published in Agyros et al in AEM 2011, we constructed replicating plasmids bearing essential features to achieve targeted gene deletion via homologous recombination. Characterization of hydrogenase enzymes and hydrogen production in different mutants are underway to improve the H2 yield. To optimize parameters to achieve high rates of cellulose conversion to H2 in bioreactos, we operated fully automated fed-batch fermentation in a cyclic mode of settle, draw, and fill. Results demonstrated that this mode of operation allows the retention of acclimated bacteria to hydrolyze cellulose at high rates and minimal lag phase, an advantage over continuous fermentation (chemostat). Work is ongoing to further engineer the bacterium and optimize cellulose fermentation to improve H2 production in C. thermocellum.

Engineering Clostridium thermocellum for hydrogen production

249th National Meeting (2015)

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