Gliding motility, ultrastructure and nutrition of two newly isolated filamentous sulfate-reducing bacteria, strains 5ac10 and 4be13, were investigated. The filaments were always attached to surfaces. Growth was supported by addition of insoluble aluminium phosphate or agar as substrata for gliding movement. Electron microscopy of ultrathin sections revealed cell walls characteristic of Gramnegative bacteria; the undulated structure of the outer membrane may pertain to the translocation mechanism. Intracytoplasmic membranes were present. Acetate, higher fatty acids, succinate or fumarate served as electron donors and carbon sources. Strain 5ac10 grew also with lactate, but not with benzoate that was used only by strain 4be13. Strain 5ac10 was able to grow slowly on H2 plus CO2 or formate in the presence of sulfate without additional organic carbon source. The capacity of complete oxidation was shown by stoichiometric measurements with acetate plus sulfate. Both strains contained b- and c-type cytochromes. Desulfoviridin was detected only in strain 5ac10. The two filamentous gliding sulfate reducers are described as new species of a new genus, Desulfonema limicola and Desulfonema magnum.

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids

Methanosarcina barkeri and Desulfobacter postgatei are ubiquitous anaerobic bacteria which grow on acetate or acetate plus sulfate, respectively, as sole energy sources. Their apparent K
s values for acetate were determined and found to be approximately 0.2 mM for the sulfate-reducing bacterium and 3 mM for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V
max) the rate of acetate consumption by D. postgatei approached 15-fold the rate of M. barkeri at low acetate concentrations. The apparent inhibition of methanogenesis was of the same order as expected from the different K
s value for acetate. Difference in substrate affinities can thus account for the inhibition of methanogenesis from acetate in sulfate-rich environments, where the acetate concentration is well below 1 mM.

Kinetic mechanism for the ability of sulfate reducers to out-compete methanogens for acetate

The effect of different substrates and different levels of sulfate and sulfide on methane production relative to sulfate reduction in high-rate anaerobic digestion was evaluated. Reactors could be acclimated so that sulfate up to a concentration of 5 g of sulfate S per liter did not significantly affect methanogenesis. Higher levels gave inhibition because of salt toxicity. Sulfate reduction was optimal at a relatively low level of sulfate, i.e., 0.5 g of sulfate S per liter, but was also not significantly affected by higher levels. Both acetoclastic and hydrogenotrophic methane-producing bacteria adapted to much higher levels of free H2S than the values reported in the literature (50% inhibition occurred only at free H2S levels of more than 1,000 mg/liter). High levels of free H2S affected the sulfate-reducing bacteria only slightly. Formate and acetate supported the sulfate-reducing bacteria very poorly. In the high-rate reactors studied, intensive H2S formation occurred only when H2 gas or an H2 precursor such as ethanol was supplied.

Sulfate reduction relative to methane production in high-rate anaerobic digestion: technical aspects.

The kinetics of sulfate and acetate uptake was studied in the sulfate-reducing bacterium Desulfobacter postgatei (DSM 2034). Kinetic parameters (Km and Vmax) were estimated from substrate consumption curves by resting cell suspensions with [35S]sulfate and [14C]acetate. Both sulfate and acetate consumption followed Michaelis-Menten saturation kinetics. The half-saturation constant (Km) for acetate uptake was 70 μM with cells from either long-term sulfate- or long-term acetate-limited chemostat cultures. The average Km value for sulfate uptake by D. postgatei was about 200 μM. Km values for sulfate uptake did not differ significantly when determined with cells derived either from batch cultures or sulfate- or acetate-limited chemostat cultures. Acetate consumption was observed at acetate concentrations of ≤1 μM, whereas sulfate uptake usually ceased at 5 to 20 μM. The results show that D. postgatei is not freely permeable to sulfate ions and further indicate that sulfate uptake is an energy-requiring process.

Kinetics of Sulfate and Acetate Uptake by Desulfobacter postgatei

Summary

The phylogenetic diversity of Bacteria and Archaea within a biodegraded, mesothermic petroleum reservoir in the Schrader Bluff Formation of Alaska was examined by two culture-independent methods based on fosmid and small-subunit rRNA gene PCR clone libraries. Despite the exclusion of certain groups by each method, there was overall no significant qualitative difference in the diversity of phylotypes recovered by the two methods. The resident Bacteria belonged to at least 14 phylum-level lineages, including the polyphyletic Firmicutes, which accounted for 36.2% of all small-subunit rRNA gene-containing (SSU+) fosmid clones identified. Members of uncultured divisions were also numerous and made up 35.2% of the SSU+ fosmid clones. Clones from domain Archaea accounted for about half of all SSU+ fosmids, suggesting that their cell numbers were comparable to those of the Bacteria in this microbial community. In contrast to the Bacteria, however, nearly all archaeal clones recovered by both methods were related to methanogens, especially acetoclastic methanogens, while the plurality of bacterial fosmid clones was affiliated with Synergistes-like acetogenic Firmicutes that possibly degrade longer-chain carboxylic acid components in the crude oil to acetate. These data suggest that acetate may be a key intermediary metabolite in this subsurface anaerobic food chain, which leads to methane production as the primary terminal electron sink.

Characterizing microbial diversity in production water from an Alaskan mesothermic petroleum reservoir with two independent molecular methods.

Acetate oxidation by sulphate was studied with Desulfobacter postgatei. Cell extracts of the organism were found to contain high activities of the following enzymes: citrate synthase, aconitase, isocitrate dehydrogenase, $\alpha$ -ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase and pyruvate synthase. It is concluded that acetate oxidation with sulphate in D. postgatei proceeds via the citric acid cycle with the synthesis of pyruvate from acetyl CoA and CO $\_2$ as an anaplerotic reaction. The apparent K $\_S$ for acetate oxidation by D. postgatei as determined in vivo was near 0.2 mM. The apparent K $\_S$ for acetate fermentation to methane and CO $\_2$ by Methanosarcina barkeri was 3 mM. The significantly lower K $_S$ for acetate of the sulphate reducer explains why methane formation from acetate in natural habitats is apparently inhibited by sulphate.

Thauer Rk

Papers

Dissimilatory sulphate reduction with acetate as electron donor.