|Li, Hsin-Fen -|
|Knutson, Barbara -|
|Nokes, Sue -|
|Lynn, Bert -|
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 1, 2011
Publication Date: January 5, 2012
Citation: Li, H., Knutson, B.L., Nokes, S.E., Lynn, B.C., Flythe, M.D. 2012. Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate. Applied Microbiology and Biotechnology. 93(4):1777-1784. Interpretive Summary: Direct fermentation of lignocellulosic biomass to ethanol with cellulolytic bacteria is called consolidated bioprocessing (CBP). One organism used for CBP is Clostridium thermocellum, an anaerobic, thermophilic bacterium that catabolizes cellulose into soluble sugars, and produces ethanol. Unfortunately, the ethanol yield is considerably lower than that of yeast. Glycolysis by C. thermocellum proceeds via the Embden-Meyerhof-Parnas pathway. In this metabolic pathway, NAD+-producing and NAD+-consuming reactions must be balanced; therefore, factors that affect this cycle can also affect product formation. Exogenous hydrogen gas, elevated hydrostatic pressure, methyl viologen, and carbon monoxide (CO) were used to manipulate the metabolism of C. thermocellum in batch growth, resting cells, and in a chemostat. Acetate production decreased when these hydrogen synthesis-inhibitors were used. However, ethanol production increased only when methyl viologen was added, or when substrate delivery was limited in the chemostat. It was discovered that methyl viologen decreased the rate of glucose transport across the cell membrane. The conclusion is that limiting, or partially inhibiting, metabolic substrate transport can favor ethanol production. This research highlights the complex effects of high concentrations of dissolved gases and soluble inhibitors in fermentation, which are increasingly envisioned in both CBP and microbial applications of H2 production for the conversion of synthetic gases to chemicals.
Technical Abstract: 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 later 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.