Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate
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Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H2) are also produced. The effect of hydrogenase inhibitors (H2, carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N2 to H2, acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H2 or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD+ from NADH by H2, lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H2 production for the conversion of synthetic gases to chemicals.
KeywordsChemostat Consolidated bioprocessing Cellulosic biomass conversion Cellulose Metabolic engineering Product selectivity Continuous culture Paraquat
The authors gratefully acknowledge the financial support of the Southeastern Sun Grant Center, administered by the University of Tennessee, grant number DTOS59-07-G-0050. MF was supported by the Agricultural Research Service, USDA. The authors would like to thank Gloria Gellin and Jerry Vice for the technical assistance.
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