Engineering a cellulose degrader Clostridium thermocellum for hydrogen production

Clostridium thermocellum is a thermophilic anaerobe with one of the highest rates in degrading cellulose.  It produces cellulosome which allows the bacteria to adhere to solid substrate while performing simultaneous saccharification and fermentation.  During cellulose fermentation, the bacterium produces hydrogen (H₂), ethanol, acetate, formate, lactate, and CO₂ as byproducts.  To produce cost-competitive H₂ from renewable biomass, our goal is to increase H₂  molar yield (mol H₂/mol hexose) in C. thermocellum and optimize cellulose fermentation in bioreactors.  Current approaches include redirection of the carbon flux in favor of H₂ production by knocking out competing pathways.  Following the selection and counter selection scheme published in Agyros et al in AEM 2011, we constructed replicating plasmids bearing essential features to achieve targeted gene deletion via homologous recombination.  Our data showed up to 50 % increase in ethanol production in mutants lacking pyruvate formate lyase (Dpfl) and demonstrated the redirection of carbon flux.  Characterization of hydrogenase enzymes and hydrogen production in different pathway mutants are underway to improve the H₂ molar yield.  To optimize parameters to achieve high rates of cellulose conversion to H₂ in bioreactors, 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 rate and minimal lag phase, an advantage over the conventional continuous fermentation (chemostat). Work is ongoing to further engineer the bacterium and optimize cellulose fermentation to improve H₂ production in C. thermocellum.