Carbon monoxide cycling by Desulfovibrio vulgaris Hildenborough

Abstract

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, use the reduction of sulfate as a sink for electrons liberated in oxidation reactions of organic substrates. The rate of the latter exceeds that of sulfate reduction at the onset of growth, causing a temporary accumulation of hydrogen and other fermentation products (the hydrogen or fermentation burst). In addition to hydrogen, D. vulgaris was found to produce significant amounts of carbon monoxide during the fermentation burst. With excess sulfate, the hyd mutant (lacking periplasmic Fe-only hydrogenase) and hmc mutant (lacking the membrane-bound, electron-transporting Hmc complex) strains produced increased amounts of hydrogen from lactate and formate compared to wild-type D. vulgaris during the fermentation burst. Both hydrogen and CO were produced from pyruvate, with the hyd mutant producing the largest transient amounts of CO. When grown with lactate and excess sulfate, the hyd mutant also exhibited a temporary pause in sulfate reduction at the start of stationary phase, resulting in production of 600 ppm of headspace hydrogen and 6,000 ppm of CO, which disappeared when sulfate reduction resumed. Cultures with an excess of the organic electron donor showed production of large amounts of hydrogen, but no CO, from lactate. Pyruvate fermentation was diverse, with the hmc mutant producing 75,000 ppm of hydrogen, the hyd mutant producing 4,000 ppm of CO, and the wild-type strain producing no significant amount of either as a fermentation end product. The wild type was most active in transient production of an organic acid intermediate, tentatively identified as fumarate, indicating increased formation of organic fermentation end products in the wild-type strain. These results suggest that alternative routes for pyruvate fermentation resulting in production of hydrogen or CO exist in D. vulgaris. The CO produced can be reoxidized through a CO dehydrogenase, the presence of which is indicated in the genome sequence.

FIG. 1.

Model for ATP synthesis in Desulfovibrio spp. growing on medium containing lactate and sulfate, pyruvate and sulfate, hydrogen and sulfate, or formate and sulfate. The reactions indicated are catalyzed by the following: 1, periplasmic (e.g., Fe-only) hydrogenase; 2, periplasmic formate dehydrogenase; 3, the cytochrome c₃ network; 4, transmembrane electron transport (e.g., Hmc) complex; 5, enzymes that reduce sulfate to sulfide (ATP sulfurylase, adenosine phosphosulfate reductase, dissimilatory sulfite reductase, adenylate kinase, and pyrophosphatase); 6, ATP synthase; 7, lactate dehydrogenase; 8-i, pyruvate-ferredoxin oxidoreductase, 8-ii, CO formation from pyruvate (enzyme unknown), 8-iii, pyruvate-formate lyase; 9, phosphotransacetylase; 10, acetate kinase, 11, cytoplasmic, membrane-bound hydrogenase (e.g., Ech [16]); 12, CODH; 13, CO-dependent hydrogenase. CoA, coenzyme A.

FIG. 2.

Growth physiology of the wild-type (WT) and hyd and hmc mutant strains in WP medium containing lactate (38 mM), sulfate (28 mM), and a headspace of 10% (vol/vol) CO₂ and 90% N₂. Plotted as a function of time are cell density (A), lactate concentration (B), H₂ concentration in the headspace (C), sulfate concentration (D), acetate concentration (E), and CO concentration in the headspace (F). The symbol definitions in panel D apply to the entire figure.

FIG. 3.

Growth physiology of the wild-type (WT) and hyd and hmc mutant strains in WP medium containing lactate (38 mM), sulfate (7 mM), and a headspace of 10% (vol/vol) CO₂ and 90% N₂. Plotted as a function of time are cell density (A), lactate (open symbols) and acetate (filled symbols) concentrations (B), H₂ concentration in the headspace (C), and sulfate concentration (D). The symbol definitions in panel D apply to the entire figure.

FIG. 4.

Growth physiology of the wild-type (WT) and hyd and hmc mutant strains in WP medium containing pyruvate (30 mM), sulfate (28 mM), and a headspace of 10% (vol/vol) CO₂ and 90% N₂. Plotted as a function of time are cell density (A), pyruvate concentration determined enzymatically (open symbols) and acetate concentration (filled symbols) (B), H₂ concentration in the headspace (C), and CO concentration in the headspace (D). The symbol definitions in panel A apply to the entire figure.

FIG. 5.

Effect of fumarate and/or hydrogen on fermentative growth of wild-type D. vulgaris on pyruvate. Growth was in WP medium containing pyruvate (30 mM) and no sulfate. Fumarate (34 mM) was added where indicated. Addition of hydrogen was done by changing the headspace gas from 90% (vol/vol) N₂-10% CO₂ to 80% (vol/vol) H₂-20% CO₂. Cell density is plotted as a function of time.

FIG. 6.

Growth physiology of the wild-type (WT) and hyd and hmc mutant strains in WP medium containing pyruvate (30 mM), sulfate (3 mM), and a headspace of 10% (vol/vol) CO₂ and 90% N₂. Plotted as a function of time are concentrations of pyruvate (open symbols) and acetate (filled symbols) (A), H₂ concentration in the headspace (B), concentration of a putative fumarate intermediate (C), and CO concentration in the headspace (D). The symbol definitions in panel D apply to the entire figure.

FIG. 7.

Growth physiology of the wild-type (WT) and hyd and hmc mutant strains in WP medium containing formate (35 mM), sulfate (28 mM), and a headspace of 10% (vol/vol) CO₂ and 90% N₂. Plotted as a function of time are cell density (A), H₂ concentration in the headspace (B), sulfate concentration (C), and formate concentration (D). The symbol definitions in panel C apply to the entire figure.