Carboxydotrophic growth of Geobacter sulfurreducens

Abstract

This study shows that Geobacter sulfurreducens grows on carbon monoxide (CO) as electron donor with fumarate as electron acceptor. Geobacter sulfurreducens was tolerant to high CO levels, with up to 150 kPa in the headspace tested. During growth, hydrogen was detected in very slight amounts (∼5 Pa). In assays with cell-free extract of cells grown with CO and fumarate, production of hydrogen from CO was not observed, and hydrogenase activity with benzyl viologen as electron acceptor was very low. Taken together, this suggested that CO is not utilized via hydrogen as intermediate. In the presence of CO, reduction of NADP(+) was observed at a rate comparable to CO oxidation coupled to fumarate reduction in vivo. The G. sulfurreducens genome contains a single putative carbon monoxide dehydrogenase-encoding gene. The gene is part of a predicted operon also comprising a putative Fe-S cluster-binding subunit (CooF) and a FAD-NAD(P) oxidoreductase and is preceded by a putative CO-sensing transcription factor. This cluster may be involved in a novel pathway for CO oxidation, but further studies are necessary to ascertain this. Similar gene clusters are present in several other species belonging to the Deltaproteobacteria and Firmicutes, for which CO utilization is currently not known.

Keywords: Carbon monoxide; Carboxydotrophic growth; Fumarate reduction; Geobacter sulfurreducens; Microbial metabolism.

Fig. 1

Growth of Geobacter sulfurreducens with CO and fumarate. a The oxidation of CO is coupled to the reduction of fumarate to succinate, resulting in an increase in cell protein in the culture liquid. Slight amounts of H₂ are also produced. Data points are averages (±SD) for two cultures. b Fumarate is partially hydrolyzed to malate, which accumulates transiently. Over the course of the experiment, a decrease in total dicarboxylic acid concentration in the culture liquid was observed. ΔSuc, succinate produced. c G. sulfurreducens tolerates high concentrations of CO. The observed rate of CO oxidation increased with larger CO pressure in the headspace. d Uptake of CO was observed in the absence of fumarate. Small amounts of H₂ and acetate were produced, but the protein concentration in the culture decreased strongly

Fig. 2

Geobacter sulfurreducens cells metabolize fumarate to succinate. In the absence of an added electron donor, fumarate conversion to succinate occurred in a ratio ∼1:0.8 and did not yield energy for growth. Transient production of malate was observed. Cells were pre-grown with acetate as electron donor which was depleted at t = 0. [Fumarate + malate] consumption and succinate production could be described with an exponentional function using k = 1 (g protein l⁻¹)⁻¹ day⁻¹ (inset)

Fig. 3

Specific enzyme activities of cell-free extracts of cultures grown with CO, acetate, or formate. a Carbon monoxide dehydrogenase, formate dehydrogenase, and hydrogenase activity measured as hydrogen consumption and as hydrogen production. b NADPH and NADH oxidase activity and CO-dependent reduction of NADP⁺ and NAD⁺. Bars with the same color indicate the activities (average ± SD) for multiple cell-free extract preparations of cultures grown with the same electron donor, see Table S1

Fig. 4

Carbon monoxide dehydrogenase gene cluster in Geobacter sulfurreducens and similar gene clusters present in other organisms. Putatively encoded gene functions: CO-sensing transcriptional regulator (rcoM); CO dehydrogenase catalytic subunit (cooS); accessory protein (cooC); Fe–S cluster - binding protein (cooF); FAD–NAD oxidoreductase (FNOR); BadM/Rrf2 type transcriptional regulator (b/r); rubrerythrin (rub). Clostridium sp. denotes Cl. carboxidivorans, Cl. autoethanogenum, and Cl. ljungdahlii