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Sulfate-reducing bacteria (SRB) often exist as cell aggregates and in biofilms surrounded by a matrix of extracellular polymeric substances (EPSs). The chemical composition of EPSs may facilitate hydrophobic substrate biodegradation and promote microbial influenced corrosion (MIC). Although EPSs from non-hydrocarbon-degrading SRB have been studied, the chemical composition of EPSs from hydrocarbon-degrading SRBs has not been reported. The isolated EPSs from the sulfate-reducing alkane-degrading bacterium Desulfoglaeba alkanexedens ALDC was characterized with scanning and fluorescent microscopy, nuclear magnetic resonance spectroscopy (NMR), and by colorimetric chemical assays. Specific fluorescent staining and 1H NMR spectroscopy revealed that the fundamental chemical structure of the EPS produced by D. alkanexedens is composed of pyranose polysaccharide and cyclopentanone in a 2:1 ratio. NMR analyses indicated that the pyranose ring structure is bonded by 1,4 connections with the cyclopentanone directly bonded to one pyranose ring. The presence of cyclopentanone presumably increases the hydrophobicity of the EPS that may facilitate the accessibility of hydrocarbon substrates to aggregating cells or cells in a biofilm. Weight loss and iron dissolution experiments demonstrated that the EPS did not contribute to the corrosivity of D. alkanexedens cells.

Anaerobic alkane metabolism is critical in multiple environmental and industrial sectors, including environmental remediation, energy production, refined fuel stability, and biocorrosion. Here, we report the complete gap-closed genome sequence for a model n-alkane-degrading anaerobe, Desulfoglaeba alkanexedens ALDC.

Offshore oil-producing platforms are designed for efficient and cost-effective separation of oil from water. However, design features and operating practices may create conditions that promote the proliferation and spread of biocorrosive microorganisms. The microbial communities and their potential for metal corrosion were characterized for three oil production platforms that varied in their oil-water separation processes, fluid recycling practices, and history of microbially influenced corrosion (MIC). Microbial diversity was evaluated by 16S rRNA gene sequencing, and numbers of total bacteria, archaea, and sulfate-reducing bacteria (SRB) were estimated by qPCR. The rates of 35S sulfate reduction assay (SRA) were measured as a proxy for metal biocorrosion potential. A variety of microorganisms common to oil production facilities were found, but distinct communities were associated with the design of the platform and varied with different locations in the processing stream. Stagnant, lower temperature (

Community compositional changes and the corrosion of carbon steel in the presence of different electron donor and acceptor combinations were examined with a methanogenic consortium enriched for its ability to mineralize paraffins. Despite cultivation in the absence of sulfate, metagenomic analysis revealed the persistence of several sulfate-reducing bacterial taxa. Upon sulfate amendment, the consortium was able to couple C28H58 biodegradation with sulfate reduction. Comparative analysis suggested that Desulforhabdus and/or Desulfovibrio likely supplanted methanogens as syntrophic partners needed for C28H58 mineralization. Further enrichment in the absence of a paraffin revealed that the consortium could also utilize carbon steel as a source of electrons. The severity of both general and localized corrosion increased in the presence of sulfate, regardless of the electron donor utilized. With carbon steel as an electron donor, Desulfobulbus dominated in the consortium and electrons from iron accounted for ∼92% of that required for sulfate reduction. An isolated Desulfovibrio spp. was able to extract electrons from iron and accelerate corrosion. Thus, hydrogenotrophic partner microorganisms required for syntrophic paraffin metabolism can be readily substituted depending on the availability of an external electron acceptor and a single paraffin-degrading consortium harbored microbes capable of both chemical and electrical microbially influenced iron corrosion.

Summary Anaerobic microbial biodegradation of recalcitrant, water-insoluble substrates, such as paraffins, presents unique metabolic challenges. To elucidate this process, a methanogenic consortium capable of mineralizing long-chain n-paraffins (C28-C50) was enriched from San Diego Bay sediment. Analysis of 16S rRNA genes indicated the dominance of Syntrophobacterales (43%) and Methanomicrobiales (26%). Metagenomic sequencing allowed draft genome assembly of dominant uncultivated community members belonging to the bacterial genus Smithella and the archaeal genera Methanoculleus and Methanosaeta. Five contigs encoding homologs of the catalytic subunit of alkylsuccinate synthase (assA) were detected. Additionally, mRNA transcripts for these genes, including a homolog binned within the ‘Smithella’ sp. SDB genome scaffold, were detected via RT-PCR, implying that paraffins are activated via ‘fumarate addition’. Metabolic reconstruction and comparison with genome scaffolds of uncultivated n-alkane degrading ‘Smithella’ spp. are consistent with the hypothesis that syntrophically growing ‘Smithella’ spp. may achieve reverse electron transfer by coupling the reoxidation of ETFred to a membrane-bound FeS oxidoreductase functioning as an ETF:menaquinone oxidoreductase. Subsequent electron transfer could proceed via a periplasmic formate dehydrogenase and/or hydrogenase, allowing energetic coupling to hydrogenotrophic methanogens such as Methanoculleus. Ultimately, these data provide fundamental insight into the energy conservation mechanisms that dictate interspecies interactions salient to methanogenic alkane mineralization.

Anaerobic hydrocarbon biodegradation not only diminishes fuel quality, but also exacerbates the biocorrosion of the metallic infrastructure. While successional events in marine microbial ecosystems impacted by petroleum are well documented, far less is known about the response of communities chronically exposed to hydrocarbons. Shipboard oily wastewater was used to assess the biotransformation of different diesel fuels and their propensity to impact carbon steel corrosion. When amended with sulfate and an F76 military diesel fuel, the sulfate removal rate in the assay mixtures was elevated (26.8 μM/d) relative to incubations receiving a hydroprocessed biofuel (16.1 μM/d) or a fuel-unamended control (17.8 μM/d). Microbial community analysis revealed the predominance of Anaerolineae and Deltaproteobacteria in F76-amended incubations, in contrast to the Beta- and Gammaproteobacteria in the original wastewater. The dominant Smithella-like sequences suggested the potential for syntrophic hydrocarbon metabolism. The general corrosion rate was relatively low (0.83 − 1.29 ± 0.12 mpy) and independent of the particular fuel, but pitting corrosion was more pronounced in F76-amended incubations. Desulfovibrionaceae constituted 50–77% of the sessile organisms on carbon steel coupons. Thus, chronically exposed microflora in oily wastewater were differentially acclimated to the syntrophic metabolism of traditional hydrocarbons but tended to resist isoalkane-laden biofuels.

Microbial activity associated with produced water from hydraulic fracturing operations can lead to gas souring and corrosion of carbon-steel equipment. We examined the microbial ecology of produced water and the prospective role of the prevalent microorganisms in corrosion in a gas production field in the Barnett Shale. The microbial community was mainly composed of halophilic, sulfidogenic bacteria within the order Halanaerobiales, which reflected the geochemical conditions of highly saline water containing sulfur species (S2O3 (2-), SO4 (2-), and HS(-)). A predominant, halophilic bacterium (strain DL-01) was subsequently isolated and identified as belonging to the genus Halanaerobium. The isolate could degrade guar gum, a polysaccharide polymer used in fracture fluids, to produce acetate and sulfide in a 10% NaCl medium at 37°C when thiosulfate was available. To mitigate potential deleterious effects of sulfide and acetate, a quaternary ammonium compound was found to be an efficient biocide in inhibiting the growth and metabolic activity of strain DL-01 relative to glutaraldehyde and tetrakis (hydroxymethyl) phosphonium sulfate. Collectively, our findings suggest that predominant halophiles associated with unconventional shale gas extraction could proliferate and produce sulfide and acetate from the metabolism of polysaccharides used in hydraulic fracturing fluids. These metabolic products might be returned to the surface and transported in pipelines to cause pitting corrosion in downstream infrastructure.

Experiments were designed to evaluate the corrosion-related consequences of storing/transporting fatty acid methyl ester (FAME) alternative diesel fuel in contact with natural seawater. Coastal Key West, FL (KW), and Persian Gulf (PG) seawaters, representing an oligotrophic and a more organic- and inorganic mineral-rich environment, respectively, were used in 60 day incubations with unprotected carbon steel. The original microflora of the two seawaters were similar with respect to major taxonomic groups but with markedly different species. After exposure to FAME diesel, the microflora of the waters changed substantially, with Clostridiales (Firmicutes) becoming dominant in both. Despite low numbers of sulphate-reducing bacteria in the original waters and after FAME diesel exposure, sulphide levels and corrosion increased markedly due to microbial sulphide production. Corrosion morphology was in the form of isolated pits surrounded by an intact, passive surface with the deepest pits associated with the fuel/seawater interface in the KW exposure. In the presence of FAME diesel, the highest corrosion rates measured by linear polarization occurred in the KW exposure correlating with significantly higher concentrations of sulphur and chlorine (presumed sulphide and chloride, respectively) in the corrosion products.

Summary Petrochemical and geological evidence suggest that petroleum in most reservoirs is anaerobically biodegraded to some extent. However, the conditions for this metabolism and the cultivation of the requisite microorganisms are rarely established. Here, we report on microbial hydrocarbon metabolism in two distinct oilfields on the North Slope of Alaska (designated Fields A and B). Signature anaerobic hydrocarbon metabolites were detected in produced water from the two oilfields offering evidence of in situ biodegradation activity. Rate measurements revealed that sulfate reduction was an important electron accepting process in Field A (6–807 µmol S l−1 day−1), but of lesser consequence in Field B (0.1–10 µmol S l−1 day−1). Correspondingly, enrichments established at 55°C with a variety of hydrocarbon mixtures showed relatively high sulfate consumption but low methane production in Field A incubations, whereas the opposite was true of the Field B enrichments. Repeated transfer of a Field B enrichment showed ongoing methane production in the presence of crude oil that correlated with ≥ 50% depletion of several component hydrocarbons. Molecular-based microbial community analysis of the methanogenic oil-utilizing consortium revealed five bacterial taxa affiliating with the orders Thermotogales, Synergistales, Deferribacterales (two taxa) and Thermoanaerobacterales that have known fermentative or syntrophic capability and one methanogen that is most closely affiliated with uncultured clones in the H2-using family Methanobacteriaceae. The findings demonstrate that oilfield-associated microbial assemblages can metabolize crude oil under the thermophilic and anaerobic conditions prevalent in many petroleum reservoirs.

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n -alkanes (C 6 to C 12 ) and 1- 13 C-labeled n -hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d 14 -hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n -hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1- 13 C-labeled n -hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that 13 C nuclei from 1- 13 C-labeled n -hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that 13 C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177: 235-243, 2002) for metabolism of deuterated n -hexane by a denitrifying bacterium.

The anaerobic biodegradation of n-alkanes has recently been demonstrated under nitrate-reducing (6, 11, 19), sulfate-reducing (1, 2, 7, 9, 20–22), and methanogenic (3, 25) conditions. Pure bacterial cultures that couple the reduction of the first two electron acceptors to the oxidation of alkanes have been isolated (1, 2, 11, 20–22), but little is known about the initial mechanism(s) of anaerobic activation and subsequent metabolism of these hydrocarbons. Previous studies of the total cellular fatty acid composition of pure sulfate-reducing cultures grown on alkanes have suggested that activation does not occur via dehydrogenation to the corresponding 1-alkene (2) but does occur by addition of an unknown organic carbon fragment to the C-2 position of the original molecule (22). Recent research has shown that the anaerobic bacterial metabolism of toluene and xylenes is initiated by addition of the methyl group to the double bond of fumarate, forming optically pure (R)-(+)-benzylsuccinic acid and methylbenzylsuccinic acids, respectively (4, 5, 12–14). These addition reactions occur with retention of the proton abstracted from the methyl group in the succinyl moiety of the metabolite (4, 5). The detection of 3-phenyl-1,2-butanedicarboxylic acid from ethylbenzene (L. M. Gieg and J. M. Suflita, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. Q-109, p. 554, 1999) may result from an analogous fumarate addition reaction of the methylene group. Alternatively, and as was suggested for toluene, this metabolite may be formed by successive additions of two carbon units from acetyl coenzyme A (acetyl-CoA), a mechanism that would result in the loss of two H atoms (8). Partially purified benzylsuccinate synthase from the denitrifying Azoarcus sp. strain T was shown to catalyze addition to fumarate by the methyl groups of toluene and all three isomers of xylene, as well as of 1-methyl-1-cyclohexene but not of 4-methyl-1-cyclohexene or methylcyclohexane (5). The nonreactivity of the latter two substrates led the researchers to conclude that the resonance stabilization offered to the radical transition state in the form of a conjugated π system by the adjacent aromatic ring or conjugated double bond was necessary for this bioconversion (5). The dodecylsuccinic acid metabolites observed in the present study are formed with retention of the deuterium from D26-dodecane on the succinyl moiety, suggesting that the bond between the dodecane and succinate was formed by addition across the double bond of fumarate. This is a novel bioconversion for alkanes; the nature of the enzymes that catalyze this conversion, as well as how it occurs without resonance stabilization offered by a conjugated π system, remains to be clarified.

The bioconversion of coal to methane in the San Juan Basin, New Mexico, was investigated. Production waters were analyzed via enrichment studies, metabolite-profiling, and culture-independent methods. Analysis of 16S rRNA gene sequences indicated the presence of methanogens potentially capable of acetoclastic, hydrogenotrophic, and methylotrophic metabolisms, predominantly belonging to the Methanosarcinales and Methanomicrobiales. Incubations of produced water and coal readily produced methane, but there was no correlation between the thermal maturity and methanogenesis. Coal methanogenesis was greater when samples with a greater richness of Firmicutes were utilized. A greater archaeal diversity was observed in the presence of several aromatic and short-chain fatty acid metabolites. Incubations amended with lactate, hydrogen, formate, and short-chain alcohols produced methane above un-amended controls. Methanogenesis from acetate was not observed. Metabolite profiling showed the widespread occurrence of putative aromatic ring intermediates including benzoate, toluic acids, phthalic acids, and cresols. The detection of saturated and unsaturated alkylsuccinic acids indicated n-alkane and cyclic alkane/alkene metabolism. Microarray analysis complemented observations based on hybridization to functional genes related to the anaerobic metabolism of aromatic and aliphatic substrates. These data suggest that coal methanogenesis is unlikely to be limited by methanogen biomass, but rather the activation and degradation of coal constituents.

Information on the susceptibility of higher molecular weight polynuclear aromatic hydrocarbons to anaerobic biodegradation is relatively rare. We obtained a sulfate-reducing bacterial enrichment capable of phenanthrene metabolism from a hydrocarbon-contaminated marine sediment. Phenanthrene degradation was in stoichiometric agreement with the theoretically expected amount of sulfate reduction and inhibited by molybdate. Mineralization of (14)C-phenanthrene by the enrichment was confirmed by the recovery of the expected amount of (14)CO(2). Stable isotope studies with protonated or deuterated phenanthrene resulted in the detection of the correspondingly labeled phenanthrene carboxylic acid by gas chromatography-mass spectrometry. Comparison of the metabolite profile with a synthesized standard confirmed that the parent molecule was carboxylated at the C-2 position. Incorporation of (13)C-bicarbonate into the carboxyl group implicated a direct carboxylation of phenanthrene as a likely key initial reaction. Denaturing gradient gel electrophoresis analysis of the enrichment showed only two major bands and 16S rRNA sequences obtained by cloning clustered with known hydrocarbon-degrading sulfate-reducing delta-proteobacteria, indicating their possible involvement in the anaerobic oxidation of phenanthrene via carboxylation as the initial activation reaction.

Two novel sulfate-reducing bacteria, strains ALDCTand Lake, which were able to oxidize n-alkanes, were isolated from a naval oily wastewater-storage facility (VA, USA) and from oilfield production water (OK, USA), respectively. The type strain (ALDCT) had a narrow substrate specificity and could grow only with n-alkanes (from C6to C12), pyruvate, butyrate, hexanoic acid and 4-methyloctanoic acid. Cells of strain ALDCTstained Gram-negative and were slightly curved, short rods with oval ends (2.5–3.0×1.0–1.4 μm), often occurring in pairs. Cells tended to form aggregates or large clusters and were non-motile and did not form endospores. Optimum growth occurred between 31 and 37 °C and at pH 6.5–7.2. NaCl was not required for growth, but salt concentrations up to 55 g l−1could be tolerated. The DNA G+C content was 53.6 mol%. Phylogenetic analysis of the 16S rRNA genes revealed that strains ALDCTand Lake were closely related, but not identical (99.9 % similarity). The two strains were not closely related to other known alkane-degrading, sulfate-reducing bacteria or to other genera of theDeltaproteobacteria. Therefore, it is proposed that strain ALDCT(=JCM 13588T=ATCC BAA-1302T) represents the type strain of a novel species and genus, with the nameDesulfoglaeba alkanexedensgen. nov., sp. nov.

Publisher Summary There is a biotechnological link between oil and natural gas that is the product of the relatively recent recognition that many hydrocarbons are susceptible to anaerobic biodegradation and can be converted into methane and carbon dioxide. Unlike the well-documented patterns of aerobic oil biodegradation, anaerobic hydrocarbon metabolism was dismissed as ecologically insignificant. This chapter reviews evidence for anaerobic hydrocarbon biodegradation and provide an overview of some of the more generalizing metabolic features. It explores whether these reactions can be predicted, identifies some of the implications for the ability of anaerobes to convert hydrocarbons into methane, and thereby, generates useful energy. There is ample evidence that anaerobic microbial processes occur under reservoir conditions. There is even evidence, albeit indirect, that such processes occur in situ . The most widely used indicator for biological methanogenesis comes from the carbon isotopic abundance signature of the methane in natural gas deposits. The chapter ends with a discussion on microbial enhanced energy recovery and carbon dioxide.