Benzoate fermentation by the anaerobic bacterium Syntrophus aciditrophicus in the absence of hydrogen-using microorganisms

Abstract

The anaerobic bacterium Syntrophus aciditrophicus metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of S. aciditrophicus produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of S. aciditrophicus with the hydrogen-using methanogen Methanospirillum hungatei produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the S. aciditrophicus pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the S. aciditrophicus-M. hungatei coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by S. aciditrophicus only in a coculture with a hydrogen user and was not metabolized by S. aciditrophicus pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by S. aciditrophicus pure cultures until a free energy change (DeltaG') of -9.2 kJ/mol was reached (-4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an S. aciditrophicus pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by S. aciditrophicus pure cultures. These results suggest that benzoate fermentation by S. aciditrophicus in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO(2), and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than -20 kJ/mol, the postulated minimum free energy value for substrate metabolism.

FIG. 1

Metabolism of benzoate by pure cultures of S. aciditrophicus (A)and by S. aciditrophicus-M. hungatei cocultures (B). Symbols: ▪, benzoate; ●, cyclohexane carboxylate; ▴, acetate; ♦, methane; □, benzoate in autoclaved controls; ○, cyclohexane carboxylate in autoclaved controls; ▵, acetate in autoclaved controls; ⋄, methane in autoclaved controls. (Inset) Correlation between benzoate consumption and cyclohexane carboxylate production in S. aciditrophicus pure cultures. The data are averages ± standard deviations based on triplicate microcosms.

FIG. 2

(A) Effect of hydrogen on benzoate metabolism by S. aciditrophicus. Symbols: □ and ○, benzoate utilization and cyclohexane carboxylate production, respectively, in cultures receiving 10.1 kPa of hydrogen; ▪ and ●, benzoate production and cyclohexane carboxylate production, respectively, in cultures receiving 10.1 kPa of nitrogen. (B) Effect of acetate on benzoate metabolism by S. aciditrophicus. Symbols: □ and ○, benzoate utilization and cyclohexane carboxylate production, respectively, in cultures receiving 60 mM sodium acetate; ▪ and ●, benzoate production and cyclohexane carboxylate production, respectively, in cultures receiving 60 mM sodium chloride. The data are averages ± standard deviations based on triplicate microcosms.

FIG. 3

Metabolism of cyclohex-1-ene carboxylate by S. aciditrophicus pure cultures. Symbols: ▪, cyclohex-1-ene carboxylate; ●, cyclohexane carboxylate; ▴, acetate; □, cyclohex-1-ene carboxylate in autoclaved controls; ○, cyclohexane carboxylate in autoclaved controls; ▵, acetate in autoclaved controls. The data are averages ± standard deviations based on triplicate microcosms.