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Exogenous H2/CO2 and glucose were consumed simultaneously by Butyribacterium methylotrophicum when grown under glucose-limited conditions. CO2 reduction to acetate was coupled to H2 consumption. The addition of either H2 or CO2 to glucose batch fermentation resulted in an increase in cell density, hydrogenase (H2-consuming and -producing) activities and fatty acid production by B. methylotrophicum as compared to when N2 was the feed gas. Hydrogenase activities appeared to be tightly regulated and were produced at higher rates during the exponential phase when CO2 was the feed gas as compared to H2 or N2. The increase in H2-consuming activity and decrease in H2-producing activity was correlated with an increase in butyrate synthesis. H2-consuming and ferredoxin (Fd)–NAD reductase activities increased while H2-producing and NADH–Fd reductase activities decreased in cells grown at pH 5.5 compared to those at pH 7.0. The molar ratio of butyrate/acetate was shifted from 0.35 at pH 7.0 to 1.22 at pH 5.5. The addition of exogenous H2 did not decrease the butyrate/acetate ratio at pH 7.0 nor at pH 5.5. The results indicated that growth pH values regulated both hydrogenase and Fd–NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards butyrate synthesis than H2 production.

Butyribacterium methylotrophicum produced more butyrate when grown on lactate than when grown on glucose, and only acetate was detected during growth on pyruvate. Higher levels of NADH were found in butyrate-producing than in acetate-producing cells. The addition of neutral red, an electron-flow modulator, to cells growing on pyruvate altered the carbon and electron flow from acetate plus H2 synthesis to butyrate synthesis. Enzymatic analysis suggested that pyruvate was produced from glucose via an Embden-Meyerhof-Parnas pathway. Pyruvate was further metabolized to butyryl-CoA via, β-hydroxybutyryl-CoA and butyryl-CoA dehydrogenases. Lactate dehydrogenase, unlike butyryl-CoA dehydrogenase, was inducible and detected only in lactate-grown cells. Both of these dehydrogenases utilized 2,6-dichloroindophenol and other artificial electron acceptors but not NAD(P). Ferredoxin–NAD oxidoreductase levels were highest in lactate and lowest in pyruvate-grown cells. Cells contained both a ferredoxin–neutral-red reductase activity and a neutral-red–NAD reductase activity that coupled electron flow to butyrate synthesis. These results showed that butyrate synthesis by B. methylotrophicum was regulated by the carbon source and was dependent on the cellular NADH/NAD ratios, and the levels and direction of ferredoxin- and NAD-linked oxidoreductases.