(635e) Hydrogenase Inhibition AS the Mechanism of Product Selectivity by Clostridium Thermocellum IN High-Pressure Cultures

 Hsin-Fen Li ¹, Barbara L. Knutson¹, Sue E. Nokes ², Bert C. Lynn ⁴, Michael D. Flythe ^{4, 5}

 ¹University of Kentucky, Department of Chemical and Materials Engineering, Lexington, Kentucky 40506

²University of Kentucky, Department of Biosystems and Agricultural Engineering, Lexington, Kentucky 40506

³University of Kentucky, Department of Chemistry, Lexington, Kentucky 40506

⁴USDA, Agricultural Research Service, Forage-Animal Production Research Unit; Lexington, Kentucky 40546

⁵University of Kentucky, Department of Animal and Food Sciences, Lexington, Kentucky 40546

Clostridium thermocellum is a cellulose-degrading thermophile that produces ethanol, but its utility in fuel production from biomass is limited by co-production of acetate and lactate.  Our previous investigations of continuous fermentations of C. thermocellum at elevated pressure (7.0 MPa, and 13.0 MPa) reveal that when the solubility of product gas (H₂) increases under elevated hydrostatic pressure, the acetate:ethanol ratios decrease as a result of a simultaneous increase in ethanol production and a decrease in acetate production.  While the decreased acetate production is broadly attributed to the inhibition of bacterial hydrogenases by the end-product H₂, the dramatic increase in ethanol production is unexplained.  With a goal of probing the underlying mechanism of the effect of H₂ partial pressure on ethanol production and manipulating the fermentation towards high ethanol selectivity, we examine the growth and end product formation of C. thermocellum in cultures perturbed with H₂ and several known hydrogenase inhibitors (carbon monoxide and methyl viologen).   The effect of these inhibitors in continuous cultures, batch cultures and high-density washed cell cultures (in the absence of growth phase-dependent substrate and energy use) is compared to fermentations in the presence of N₂ headspace.  The addition of exogenous H₂ with a hydrogenase inhibitor (methyl viologen) caused approximately 80% decrease in acetate production.  Likewise, batch C. thermocellum grew in the presence of the hydrogenase inhibitor, carbon monoxide (CO), but less than 1% acetate was produced.  When the sparge gas used in continuous cultures was switched from N₂ to H₂, acetate production decreased approximately 35%, and ethanol production increased more than 350%.  The conversion of NADH to NAD⁺ is coupled to the production of H₂, lactic acid, or ethanol.  Thus, the result provides evidence for compensatory production of lactate and/or ethanol occurred when hydrogenases were inhibited.  The contribution of substrate uptake rate to enhanced ethanol production was examined.