Sulphate-reducing bacteria (SRB) are anaerobic microorganisms that use sulphate as a terminal electron acceptor in, for example, the degradation of organic compounds. They are ubiquitous in anoxic habitats, where they have an important role in both the sulphur and carbon cycles. SRB can cause a serious problem for industries, such as the offshore oil industry, because of the production of sulphide, which is highly reactive, corrosive and toxic. However, these organisms can also be beneficial by removing sulphate and heavy metals from waste streams. Although SRB have been studied for more than a century, it is only with the recent emergence of new molecular biological and genomic techniques that we have begun to obtain detailed information on their way of life.

The ecology and biotechnology of sulphate-reducing bacteria

The gram-negative metal-reducing microorganism, previously known as strain GS-15, was further characterized. This strict anaerobe oxidizes several short-chain fatty acids, alcohols, and monoaromatic compounds with Fe(III) as the sole electron acceptor. Furthermore, acetate is also oxidized with the reduction of Mn(IV), U(VI), and nitrate. In whole cell suspensions, the c-type cytochrome(s) of this organism was oxidized by physiological electron acceptors and also by gold, silver, mercury, and chromate. Menaquinone was recovered in concentrations comparable to those previously found in gram-negative sulfate reducers. Profiles of the phospholipid ester-linked fatty acids indicated that both the anaerobic desaturase and the branched pathways for fatty acid biosynthesis were operative. The organism contained three lipopolysaccharide hydroxy fatty acids which have not been previously reported in microorganisms, but have been observed in anaerobic freshwater sediments. The 16S rRNA sequence indicated that this organism belongs in the delta proteobacteria. Its closest known relative is Desulfuromonas acetoxidans. The name Geobacter metallireducens is proposed.

Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals.

A dissimilatory metal- and sulfur-reducing microorganism was isolated from surface sediments of a hydrocarbon-contaminated ditch in Norman, Okla. The isolate, which was designated strain PCA, was an obligately anaerobic, nonfermentative nonmotile, gram-negative rod. PCA grew in a defined medium with acetate as an electron donor and ferric PPi, ferric oxyhydroxide, ferric citrate, elemental sulfur, Co(III)-EDTA, fumarate, or malate as the sole electron acceptor. PCA also coupled the oxidation of hydrogen to the reduction of Fe(III) but did not reduce Fe(III) with sulfur, glucose, lactate, fumarate, propionate, butyrate, isobutyrate, isovalerate, succinate, yeast extract, phenol, benzoate, ethanol, propanol, or butanol as an electron donor. PCA did not reduce oxygen, Mn(IV), U(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PCA exhibited dithionite-reduced minus air-oxidized difference spectra which were characteristic of c-type cytochromes. Phylogenetic analysis of the 16S rRNA sequence placed PCA in the delta subgroup of the proteobacteria. Its closest known relative is Geobacter metallireducens. The ability to utilize either hydrogen or acetate as the sole electron donor for Fe(III) reduction makes strain PCA a unique addition to the relatively small group of respiratory metal-reducing microorganisms available in pure culture. A new species name, Geobacter sulfurreducens, is proposed.

Geobacter sulfurreducens sp. nov. , a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism

Dissimilatory Sulfate- and Sulfur-Reducing Prokaryotes

n-Hexadecane added as electron donor and carbon source to an anaerobic enrichment culture from an oil production plant or to anoxic marine sediment samples allowed dissimilatory sulfate reduction to sulfide. The enrichment from the oil field was purified via serial dilutions in liquid medium under a hexadecane phase and in agar medium with caprylate. A pure culture of a sulfate-reducing bacterium, strain Hxd3, with relatively tiny cells (0.4–0.5 by 0.8–2 μm) was isolated that grew anaerobically on hexadecane without addition of further organic substrates. Most of the cells were found to adhere to the hydrocarbon phase. It was verified that neither organic impurities in hexadecane nor residual oxygen were responsible for growth. Strain Hxd3 was grown with n-hexadecane of high purity (≥99.5%) in anoxic glass ampoules sealed by fusion. Of 0.4 ml hexadecane added per l (1.4 mmol per l), 90% was degraded with concomitant reduction of sulfate. Controls with pasteurized cells or a common Desulfovibrio species neither consumed hexadecane nor reduced sulfate. Incubation of cell-free medium with low reducing capacity and a redox indicator showed that the ampoules were completely oxygen-tight. Measured degradation balances and enzyme activities suggested a complete oxidation of the alkane to CO2 via the carbon monoxide dehydrogenase pathway. However, the first step in anaerobic alkane oxidation is unknown. On hexadecane, strain Hxd3 produced as much as 15 to 20 mM H2S, but growth was rather slow; with 5% inoculum, cultures were fully grown after 5 to 7 weeks. The new sulfate reducer grew on alkanes from C12 to C20, 1-hexadecene, 1-hexadecanol, 2-hexadecanol, palmitate and stearate. Best growth occurred on stearate (doubling time around 26 h). Growth on soluble fatty acids such as caprylate was very poor. Alkanes with chains shorter than C12, lactate, ethanol or H2 were not used. Strain Hxd3 is the first anaerobe shown to grow definitely on saturated hydrocarbons.

Anaerobic oxidation of saturated hydrocarbons to CO2 by a new type of sulfate-reducing bacterium*

Il est montre que toutes les enzymes exigees pour l'oxydation de l'acetate par la voie du cycle tricarboxylique sont presentes chez cette bacterie. Les enzymes des reactions anaplerotiques sont egelement decrites

Anaerobic acetate oxidation to CO2 by Desulfobacter postgatei. I: Demonstration of all enzymes required for the operation of the citric acid cycle

All the enzymes required for the oxidation of acetate to CO2 via the citric acid cycle were found in Desulfobacter postgatei. To obtain in vivo evidence for the operation of this cycle, the sulfate reducing bacterium was grown on [14C]acetate in the presence of a large pool of 12CO2 and the incorporation of 14C into glutamate (≙ 2-oxoglutarate), aspartate (≙ oxaloacetate), and alanine (≙ pyruvate) was studied. The labelling data were found to be consistent with (i) the oxidation of acetate to CO2 via the reactions of the citric acid cycle, (ii) the synthesis of citrate via a citrate (si)-synthase, and (iii) the anaplerotic synthesis of oxaloacetate from acetate and 2 CO2 via pyruvate as intermediate.

Anaerobic acetate oxidation to CO2 by Desulfobacter postgatei

The strict anaerobe Desulfuromonas acetoxidans can oxidize acetate to CO2 with elemental sulfur as electron acceptor. 14C-labelling experiments and enzyme studies are described revealing that acetate oxidation proceeds via the citric acid cycle with the synthesis of oxaloacetate from acetate and 2 CO2 via pyruvate as anaplerotic reaction. An oxidation of acetate via one carbon unit intermediates as proposed for anaerobic bacteria fermenting acetate to 2 CO2 and 4 H2 was excluded.

Mechanism of acetate oxidation to CO2 with elemental sulfur in Desulfuromonas acetoxidans

Factor F430 is a nickel porphinoid present in all methanogenic bacteria. It is a component of methyl-CoM reductase to which it is tightly but not covalently bound. Evidence is presented that in Methanobacterium thermoautotrophicum grown on nickel sufficient medium only approximately 30% of total F430 is associated with methyl-CoM reductase and that 70% is present in a non-bound, free form. When such cells were transferred to a nickel deficient medium the bacteria continued to grow although synthesis of total F430 stopped. During growth in the absence of nickel the amount of total F430 per 1 culture remained constant and that per g cells decreased. The ratio of free F430 to bound F430, however, changed. Free F430 was converted into the protein-bound form until almost all of the free F430 had disappeared. The kinetics of labelling with 63Ni of free and bound F430 agreed rather well with that calculated for a precursor-product relationship between free and bound F430.

Norbert A. Gebhardt

Papers

Anaerobic acetate oxidation to CO2 by Desulfobacter postgatei. I: Demonstration of all enzymes required for the operation of the citric acid cycle

Anaerobic acetate oxidation to CO2 by Desulfobacter postgatei

Mechanism of acetate oxidation to CO2 with elemental sulfur in Desulfuromonas acetoxidans

Functional relationship between protein-bound and free factor F430 in Methanobacterium