Friedrich Widdel

Gram-Negative Mesophilic Sulfate-Reducing Bacteria

An overview of the sulfate-reduction process is given in Chapter 24. Most types of dissimilatory sulfate-reducing bacteria that have been isolated from nature and described so far are mesophilic, nonsporeforming anaerobes. They are members of the delta subdivision of the proteobacteria. The earliest known representative of this category is Desulfovibrio (Beijerinck, 1895). Further investigations have revealed a great morphological and nutritional diversity within this group. Various cell types have been described including cocci; oval or long straight rods; more or less curved rods or spirilla; cell packets; cells with gas vesicles; and gliding, multicellular filaments (Figs. 7–9). Electron donors used for sulfate reduction include H2, alcohols, fatty acids, other monocarboxylic acids, dicarboxylic acids, some amino acids, a few sugars, phenyl-substituted acids, and some other aromatic compounds (Table 2). Even long-chain alkanes can be anaerobically oxidized by a particular type of sulfate-reducing bacterium (Aeckersberg et al., 1991). The utilization of polysaccharides or polypeptides, such as has been observed with the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus (Stetter, 1988; Stetter et al., 1987), has not been reported for mesophilic sulfate reducers.

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids

Three strains (2ac9, 3ac10 and 4ac11) of oval to rodshaped, Gram negative, nonsporing sulfate-reducing bacteria were isolated from brackish water and marine mud samples with acetate as sole electron donor. All three strains grew in simple defined media supplemented with biotin and 4-aminobenzoic acid as growth factors. Acetate was the only electron donor utilized by strain 2ac9, while the other two strains used in addition ethanol and/or lactate. Sulfate served as electron acceptor and was reduced to H2S. Complete oxidation of acetate to CO2 was shown by stoichiometric measurements with strain 2ac9 in batch cultures using sulfate, sulfite or thiosulfate as electron acceptors. With sulfate an average growth yield of 4.8 g cell dry weight was obtained per mol of acetate oxidized; with sulfite or thiosulfate the growth yield on acetate was about twice as high. None of the strains contained desulfoviridin. In strain 2ac9 cytochromes of the b- and c-type were detected. Strain 2ac9 is described as type strain of the new species and genus, Desulfobacter postgatei.

Anaerobic biodegradation of saturated and aromatic hydrocarbons.

Saturated and aromatic hydrocarbons are wide-spread in our environment. These compounds exhibit low chemical reactivity and for many decades were thought to undergo biodegradation only in the presence of free oxygen. During the past decade, however, an increasing number of microorganisms have been detected that degrade hydrocarbons under strictly anoxic conditions.

Methane formation from long-chain alkanes by anaerobic microorganisms

Biological formation of methane is the terminal process of biomass degradation in aquatic habitats where oxygen, nitrate, ferric iron and sulphate have been depleted as electron acceptors. The pathway leading from dead biomass to methane through the metabolism of anaerobic bacteria and archaea is well understood for easily degradable biomolecules such as carbohydrates, proteins and lipids. However, little is known about the organic compounds that lead to methane in old anoxic sediments where easily degradable biomolecules are no longer available. One class of naturally formed long-lived compounds in such sediments is the saturated hydrocarbons (alkanes). Alkanes are usually considered to be inert in the absence of oxygen, nitrate or sulphate, and the analysis of alkane patterns is often used for biogeochemical characterization of sediments. However, alkanes might be consumed in anoxic sediments below the zone of sulphate reduction, but the underlying process has not been elucidated. Here we used enrichment cultures to show that the biological conversion of long-chain alkanes to the simplest hydrocarbon, methane, is possible under strictly anoxic conditions.

Phospholipid Ester-linked Fatty Acid Biomarkers of Acetate-oxidizing Sulphate-reducers and Other Sulphide-forming Bacteria

Summary: The phospholipid ester-linked fatty acids were examined in four Desulfobacter strains (2ac9, AcBa, 3ac10 and 4ac11), a Desulfobacter-like ‘fat vibrio’ (AcKo) and Desulfotomaculum acetoxidans (5575), which are all sulphate-reducing bacteria that oxidize acetate. A thermophilic sulphate reducer, Desulfovibrio thermophilus, and two sulphur-reducing bacteria, Desulfuromonas acetoxidans (11070) and a Campylobacter-like spirillum (5175), were also studied. The Desulfobacter spp. were characterized by significant quantities of 10-methylhexadecanoic acid. Other 10-methyl fatty acids were also detected in Desulfobacter spp. No 10-methyl fatty acids were detected in the other organisms examined, supporting the use of 10-methylhexadecanoic acid as a biomarker for Desulfobacter. High levels of cyclopropyl fatty acids, including two isomers of both methylenehexadecanoic (cy17:0) and methyleneheptadecanoic (cy18:0) acids, were also characteristic of Desulfobacter spp. The influence of the volatile fatty acids (VFA) propionate, isobutyrate, isovalerate and 2-methylbutyrate on the lipid fatty acid distribution was studied with two Desulfobacter strains (2ac9, AcBa) and Desulfotomaculum acetoxidans. Although these sulphate reducers cannot oxidize the VFA, their presence in the acetate growth medium caused a shift in the fatty acid distribution in favour of odd-numbered and branched chains by apparent direct incorporation into the fatty acids as chain initiators. The Desulfobacter strains were distinguished from other sulphide-forming bacteria by the percentage of unsaturated and the percentage of branched fatty acids.

Iron corrosion by novel anaerobic microorganisms.

Corrosion of iron presents a serious economic problem. Whereas aerobic corrosion is a chemical process, anaerobic corrosion is frequently linked to the activity of sulphate-reducing bacteria (SRB). SRB are supposed to act upon iron primarily by produced hydrogen sulphide as a corrosive agent and by consumption of ‘cathodic hydrogen’ formed on iron in contact with water. Among SRB, Desulfovibrio species—with their capacity to consume hydrogen effectively—are conventionally regarded as the main culprits of anaerobic corrosion; however, the underlying mechanisms are complex and insufficiently understood. Here we describe novel marine, corrosive types of SRB obtained via an isolation approach with metallic iron as the only electron donor. In particular, a Desulfobacterium-like isolate reduced sulphate with metallic iron much faster than conventional hydrogen-scavenging Desulfovibrio species, suggesting that the novel surface-attached cell type obtained electrons from metallic iron in a more direct manner than via free hydrogen. Similarly, a newly isolated Methanobacterium-like archaeon produced methane with iron faster than do known hydrogen-using methanogens, again suggesting a more direct access to electrons from iron than via hydrogen consumption.

Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria

Anaerobic degradation of alkylbenzenes with side chains longer than that of toluene was studied in freshwater mud samples in the presence of nitrate. Two new denitrifying strains, EbN1 and PbN1, were isolated on ethylbenzene and n-propylbenzene, respectively. For comparison, two further denitrifying strains, ToN1 and mXyN1, were isolated from the same mud with toluene and m-xylene, respectively. Sequencing of 16SrDNA revealed a close relationship of the new isolates to Thauera selenatis. The strains exhibited different specific capacities for degradation of alkylbenzenes. In addition to ethylbenzene, strain EbN1 utilized toluence, but not propylbenzene. In contrast, propylbenzene-degrading strain PbN1 did not grow on toluene, but was able to utilize ethylbenzene. Strain ToN1 used toluene as the only hydrocarbon substrate, whereas strain mXyN1 utilized both toluene and m-xylene. Measurement of the degradation balance demonstrated complete oxidation of ethylbenzene to CO2 by strain EbN1. Further characteristic substrates of strains EbN1 and PbN1 were 1-phenylethanol and acetophenone. In contrast to the other isolates, strain mXyN1 did not grow on benzyl alcohol. Benzyl alcohol (also m-methylbenzyl alcohol) was even a specific inhibitor of toluene and m-xylene utilization by strain mXyN1. None of the strains was able to grow on any of the alkylbenzenes with oxygen as electron acceptor. However, polar aromatic compounds such as benzoate were utilized under both oxic and anoxic conditions. All four isolates grew anaerobically on crude oil. Gas chromatographic analysis of crude oil after growth of strain ToN1 revealed specific depletion of toluene.

Zero-valent sulphur is a key intermediate in marine methane oxidation

Emissions of methane, a potent greenhouse gas, from marine sediments are controlled by anaerobic oxidation of methane coupled primarily to sulphate reduction (AOM). Sulphate-coupled AOM is believed to be mediated by a consortium of methanotrophic archaea (ANME) and sulphate-reducing Deltaproteobacteria but the underlying mechanism has not yet been resolved. Here we show that zero-valent sulphur compounds (S0) are formed during AOM through a new pathway for dissimilatory sulphate reduction performed by the methanotrophic archaea. Hence, AOM might not be an obligate syntrophic process but may be carried out by the ANME alone. Furthermore, we show that the produced S0—in the form of disulphide—is disproportionated by the Deltaproteobacteria associated with the ANME. Our observations expand the diversity of known microbially mediated sulphur transformations and have significant implications for our understanding of the biogeochemical carbon and sulphur cycles.

A conspicuous nickel protein in microbial mats that oxidize methane anaerobically

Anaerobic oxidation of methane (AOM) in marine sediments is an important microbial process in the global carbon cycle and in control of greenhouse gas emission. The responsible organisms supposedly reverse the reactions of methanogenesis, but cultures providing biochemical proof of this have not been isolated. Here we searched for AOM-associated cell components in microbial mats from anoxic methane seeps in the Black Sea. These mats catalyse AOM rather than carry out methanogenesis. We extracted a prominent nickel compound displaying the same absorption spectrum as the nickel cofactor F430 of methyl-coenzyme M reductase, the terminal enzyme of methanogenesis; however, the nickel compound exhibited a higher molecular mass than F430. The apparent variant of F430 was part of an abundant protein that was purified from the mat and that consists of three different subunits. Determined amino-terminal amino acid sequences matched a gene locus cloned from the mat. Sequence analyses revealed similarities to methyl-coenzyme M reductase from methanogenic archaea. The abundance of the nickel protein (7% of extracted proteins) in the mat suggests an important role in AOM.

Metabolism of alkylbenzenes, alkanes, and other hydrocarbons in anaerobic bacteria.

Aromatic and aliphatic hydrocarbons are the main constituents of petroleum and its refined products. Whereas degradation of hydrocarbons by oxygen-respiring microorganisms has been known for about a century, utilization of hydrocarbons under anoxic conditions has been investigated only during the past decade. Diverse strains of anaerobic bacteria have been isolated that degrade toluene anaerobically, using nitrate, iron(III), or sulfate as electron acceptors. Also, other alkylbenzenes such as m-xylene or ethylbenzene are utilized by a number of strains. The capacity for anaerobic utilization of alkylbenzenes has been observed in members of the α-, β-, γ- and δ-subclasses of the Proteobacteria. Furthermore, denitrifying bacteria and sulfate-reducing bacteria with the capacity for anaerobic alkane degradation have been isolated, which are members of the β- and δ-subclass, respectively. The mechanism of the activation of hydrocarbons as apolar molecules in the absence of oxygen is of particular interest.The biochemistry of anaerobic toluene degradation has been studied in detail. Toluene is activated by addition to fumarate to yield benzylsuccinate, which is then further metabolized via benzoyl-CoA. The toluene-activating enzyme presents a novel type of glycine radical protein. Another principle of anaerobic alkylbenzene activation has been observed in the anaerobic degradation of ethylbenzene. Ethylbenzene in denitrifying bacteria is dehydrogenated to 1-phenylethanol and further to acetophenone; the latter is also metabolized to benzoyl-CoA. Naphthalene is presumably activated under anoxic conditions by a carboxylation reaction. Investigations into the pathway of anaerobic alkane degradation are only at the beginning. The saturated hydrocarbons are mostlikely activated by addition of a carbon compound rather than by desaturation and hydration, as speculated about in some early studies. An anaerobic oxidation of methane with sulfate as electron acceptor has been documented in aquatic sediments. The process is assumed to involve a reversal of methanogenesis catalyzed by Archaea, and scavenge of an electron-carrying metabolite by sulfate-reducing bacteria. Among unsaturated non-aromatic hydrocarbons, anaerobic bacterial degradation has been demonstrated and investigated with n-alkenes, alkenoic terpenes and the alkyne, acetylene.