Heinz Wilkes

Anaerobic oxidation of short-chain hydrocarbons by marine sulphate-reducing bacteria

The short-chain hydrocarbons ethane, propane and butane are constituents of natural gas. They are usually assumed to be of thermochemical origin, but biological formation of ethane and propane has been also observed. Microbial utilization of short-chain hydrocarbons has been shown in some aerobic species but not in anaerobic species of bacteria. On the other hand, anaerobic utilization of short-chain hydrocarbons would in principle be expected because various anaerobic bacteria grow with higher homologues (≥C6). Indeed, chemical analyses of hydrocarbon-rich habitats with limited or no access of oxygen indicated in situ biodegradation of short-chain hydrocarbons. Here we report the enrichment of sulphate-reducing bacteria (SRB) with such capacity from marine hydrocarbon seep areas. Propane or n-butane as the sole growth substrate led to sediment-free sulphate-reducing enrichment cultures growing at 12, 28 or 60 °C. With ethane, a slower enrichment with residual sediment was obtained at 12 °C. Isolation experiments resulted in a mesophilic pure culture (strain BuS5) that used only propane and n-butane (methane, isobutane, alcohols or carboxylic acids did not support growth). Complete hydrocarbon oxidation to CO2 and the preferential oxidation of 12C-enriched alkanes were observed with strain BuS5 and other cultures. Metabolites of propane included iso- and n-propylsuccinate, indicating a subterminal as well as an unprecedented terminal alkane activation with involvement of fumarate. According to 16S ribosomal RNA analyses, strain BuS5 affiliates with Desulfosarcina/Desulfococcus, a cluster of widespread marine SRB. An enrichment culture with propane growing at 60 °C was dominated by Desulfotomaculum-like SRB. Our results suggest that diverse SRB are able to thrive in seep areas and gas reservoirs on propane and butane, thus altering the gas composition and contributing to sulphide production.

Anaerobic Initial Reaction of n-Alkanes in a Denitrifying Bacterium: Evidence for (1-Methylpentyl)succinate as Initial Product and for Involvement of an Organic Radical in n-Hexane Metabolism

A novel type of denitrifying bacterium (strain HxN1) with the capacity to oxidize n-alkanes anaerobically with nitrate as the electron acceptor to CO(2) formed (1-methylpentyl)succinate (MPS) during growth on n-hexane as the only organic substrate under strict exclusion of air. Identification of MPS by gas chromatography-mass spectrometry was based on comparison with a synthetic standard. MPS was not formed during anaerobic growth on n-hexanoate. Anaerobic growth with [1-(13)C]n-hexane or d(14)-n-hexane led to a 1-methylpentyl side chain in MPS with one (13)C atom or 13 deuterium atoms, respectively. This indicates that the 1-methylpentyl side chain originates directly from n-hexane. Electron paramagnetic resonance spectroscopy revealed the presence of an organic radical in n-hexane-grown cells but not in n-hexanoate-grown cells. Results point at a mechanistic similarity between the anaerobic initial reaction of n-hexane and that of toluene, even though n-hexane is much less reactive; the described initial reaction of toluene in anaerobic bacteria is an addition to fumarate via a radical mechanism yielding benzylsuccinate. We conclude that n-hexane is activated at its second carbon atom by a radical reaction and presumably added to fumarate as a cosubstrate, yielding MPS as the first stable product. When 2,3-d(2)-fumarate was added to cultures growing on unlabeled n-hexane, 3-d(1)-MPS rather than 2,3-d(2)-MPS was detected, indicating loss of one deuterium atom by an as yet unknown mechanism.

Anaerobic Degradation of Ethylbenzene by a New Type of Marine Sulfate-Reducing Bacterium

ABSTRACT Anaerobic degradation of the aromatic hydrocarbon ethylbenzene was studied with sulfate as the electron acceptor. Enrichment cultures prepared with marine sediment samples from different locations showed ethylbenzene-dependent reduction of sulfate to sulfide and always contained a characteristic cell type that formed gas vesicles towards the end of growth. A pure culture of this cell type, strain EbS7, was isolated from sediment from Guaymas Basin (Gulf of California). Complete mineralization of ethylbenzene coupled to sulfate reduction was demonstrated in growth experiments with strain EbS7. Sequence analysis of the 16S rRNA gene revealed a close relationship between strain EbS7 and the previously described marine sulfate-reducing strains NaphS2 and mXyS1 (similarity values, 97.6 and 96.2%, respectively), which grow anaerobically with naphthalene and m-xylene, respectively. However, strain EbS7 did not oxidize naphthalene, m-xylene, or toluene. Other compounds utilized by strain EbS7 were phenylacetate, 3-phenylpropionate, formate, n-hexanoate, lactate, and pyruvate. 1-Phenylethanol and acetophenone, the characteristic intermediates in anaerobic ethylbenzene degradation by denitrifying bacteria, neither served as growth substrates nor were detectable as metabolites by gas chromatography-mass spectrometry in ethylbenzene-grown cultures of strain EbS7. Rather, (1-phenylethyl)succinate and 4-phenylpentanoate were detected as specific metabolites in such cultures. Formation of these intermediates can be explained by a reaction sequence involving addition of the benzyl carbon atom of ethylbenzene to fumarate, carbon skeleton rearrangement of the succinate moiety (as a thioester), and loss of one carboxyl group. Such reactions are analogous to those suggested for anaerobic n-alkane degradation and thus differ from the initial reactions in anaerobic ethylbenzene degradation by denitrifying bacteria which employ dehydrogenations.

Anaerobic degradation of n-hexane in a denitrifying bacterium: further degradation of the initial intermediate (1-methylpentyl)succinate via C-skeleton rearrangement.

Abstract. The anaerobic degradation pathway of the saturated hydrocarbon n-hexane in a denitrifying strain (HxN1) was examined by gas chromatography-mass spectrometry of derivatized extracts from cultures grown with unlabeled and deuterated substrate; several authentic standard compounds were included for comparison. The study was focused on possible reaction steps that follow the initial formation of (1-methylpentyl)succinate from n-hexane and fumarate. 4-Methyloctanoic, 4-methyloct-2-enoic, 2-methylhexanoic, 2-methylhex-2-enoic and 3-hydroxy-2-methylhexanoic acids (in addition to a few other methyl-branched acids) were detected in n-hexane-grown but not in n-hexanoate-grown cultures. Labeling indicated preservation of the original carbon chain of n-hexane in these acids. Tracing of the deuterium label of 3-d1-(1-methylpentyl)succinate in tentative subsequent products indicated a deuterium/carboxyl carbon exchange in the succinate moiety. This suggests that the metabolism of (1-methylpentyl)succinate employs reactions analogous to those in the established conversion of succinyl-CoA via methylmalonyl-CoA to propionyl-CoA. Accordingly, a pathway is proposed in which (1-methylpentyl)succinate is converted to the CoA-thioester, rearranged to (2-methylhexyl)malonyl-CoA and decarboxylated (perhaps by a transcarboxylase) to 4-methyloctanoyl-CoA. The other identified fatty acids match with a further degradation of 4-methyloctanoyl-CoA via rounds of conventional β-oxidation. Such a pathway would also allow regeneration of fumarate (for n-hexane activation) from propionyl-CoA formed as intermediate and hence present a cyclic process.

Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria

The anaerobic biodegradation of naphthalene, an aromatic hydrocarbon in tar and petroleum, has been repeatedly observed in environments but scarcely in pure cultures. To further explore the relationships and physiology of anaerobic naphthalene-degrading microorganisms, sulfate-reducing bacteria (SRB) were enriched from a Mediterranean sediment with added naphthalene. Two strains (NaphS3, NaphS6) with oval cells were isolated which showed naphthalene-dependent sulfate reduction. According to 16S rRNA gene sequences, both strains were Deltaproteobacteria and closely related to each other and to a previously described naphthalene-degrading sulfate-reducing strain (NaphS2) from a North Sea habitat. Other close relatives were SRB able to degrade alkylbenzenes, and phylotypes enriched anaerobically with benzene. If in adaptation experiments the three naphthalene-grown strains were exposed to 2-methylnaphthalene, this compound was utilized after a pronounced lag phase, indicating that naphthalene did not induce the capacity for 2-methylnaphthalene degradation. Comparative denaturing gel electrophoresis of cells grown with naphthalene or 2-methylnaphthalene revealed a striking protein band which was only present upon growth with the latter substrate. Peptide sequences from this band perfectly matched those of a protein predicted from genomic libraries of the strains. Sequence similarity (50% identity) of the predicted protein to the large subunit of the toluene-activating enzyme (benzylsuccinate synthase) from other anaerobic bacteria indicated that the detected protein is part of an analogous 2-methylnaphthalene-activating enzyme. The absence of this protein in naphthalene-grown cells together with the adaptation experiments as well as isotopic metabolite differentiation upon growth with a mixture of d(8)-naphthalene and unlabelled 2-methylnaphthalene suggest that the marine strains do not metabolize naphthalene by initial methylation via 2-methylnaphthalene, a previously suggested mechanism. The inability to utilize 1-naphthol or 2-naphthol also excludes these compounds as free intermediates. Results leave open the possibility of naphthalene carboxylation, another previously suggested activation mechanism.

Anaerobic degradation and carbon isotopic fractionation of alkylbenzenes in crude oil by sulphate-reducing bacteria

Abstract A mesophilic enrichment culture of sulphate-reducing bacteria isolated from the water phase of a North Sea oil tank using oil from the same tank as sole source of carbon and energy specifically depletes certain C1–C5 alkylbenzenes in crude oil during growth. The enrichment culture grows on oils of different origin and composition resulting in similar patterns of alkylbenzene depletion. Two pure cultures of sulphate-reducing bacteria, strain oXyS1 and mXyS1 which were isolated on o-xylene and m-xylene, respectively, also grow on crude oil. Strain oXyS1 preferentially depletes o-xylene and o-ethyltoluene while strain mXyS1 preferentially depletes m-xylene and m-ethyltoluene. Both strains also utilize toluene. The degradative patterns of the pure cultures are complementary and their combination results in the degradative pattern of the enrichment culture. During growth of the enrichment culture and the pure strains on crude oil alkylated benzoic acids were the main metabolic products, which were isolated from the water phases of the incubation experiments. The patterns of alkylated benzoic acids produced by the pure cultures are again complementary with respect to the pattern observed for the enrichment culture. The spectrum of alkylated benzoic acids suggests that partial oxidation of alkylbenzenes, which do not support growth, takes place resulting in the formation of dead-end metabolites. Alkylphenylsuccinic and fumaric acids were produced in trace amounts only. The portion of alkylbenzenes remaining in crude oil becomes enriched in 13C during growth of bacteria. From the data obtained in this study it can be estimated that the carbon isotopic fractionation of the initial reaction of alkylbenzene degradation by the present bacteria is between −26 and −33‰. We suggest that the variability in alkylbenzene concentrations and their carbon isotopic signature together with the occurrence of alkylated benzoic acids may be used as a specific indicator of initial biodegradation of crude oils and fossil fuel products by sulphate-reducing bacteria in various environments.

Intact phospholipids—microbial “life markers” in marine deep subsurface sediments

AbstractDeepsubsurfacesedimentsfromtheNankaiTrough,JapanSea,ODPLeg190,sites1173,1174,1177,andnear-surfacesedimentsfromHydrateRidge,NE-Pacifichavebeenanalysedbyhighperformanceliquidchromatography(HPLC)–electrosprayionisation(ESI)-massspectrometry(MS).Themainobjectivewastoutilizethepresenceofintactphospholipidsasadirectindicatorofviablemicroorganisms.TheextractsofNankaiTroughsedimentswerefoundtocontainavarietyofphospholipid(PL)structures,well-knowntostemfrommicroorganisms,todepthsasgreatas745mbsfandinsitutemperaturesashighas85C.Inaddition,highrelativeamountsoflysophospholipids(e.g.lysopho-sphatidylcholines)exceedingthoseoftheregularphospholipidsweredetected.DiglyceridemassfragmentsofvariousPLshavebeenassignedtofattyacylside-chainsoftypicalchainlength(C 14 ,C 16 ,C 18 ,C 20 )anddegreeofunsaturation(zero,oneortwodoublebonds).Similarresultswereobtainedforthephospholipiddistributioninextractsoforganicmatter-richHydrateRidgesediments.Todate,theenhancedoccurrenceoflysophospholipidscannotbeexplainedcompletelybutaresponsetoincreasingthermalandecologicalstressseemsprobable.# 2003ElsevierScienceLtd.Allrightsreserved.1.IntroductionBiologicalmarkersprovideinformationonthepre-cursorbiotaandpost-depositionalhistoryoforganicmatterpreservedinsediments,therebyenablingbiolo-gicalevolution,palaeoclimateandpetroleum-formingprocessestobedocumentedindetail(Brassell,1993;PetersandMoldowan,1993).Thisinformation,whetherbasedonhydrocarbonsorfunctionalisedcompounds,haslargelybeengatheredusinggaschromatography-massspectrometry,andwasinitiallymadepossiblebyrapidtechnicaladvancesinthemid-1960s.Inthelatterpartofthelastcentury,anewandcompellingchallengebegantotakeshape,thisbeingtodefinethetempera-ture-anddepthlimitstowhichlifecanoccurwithintheEarth.Thiswasmotivatedbythediscoveryofmicro-bially-basedecosystemsfuelledbygeothermalenergyathydrothermalvents,andtheoccurrenceofdiversemicrobialpopulationstohundredsofmetresdepthinclasticsediments(Craggetal.,1992;Parkesetal.,1994,2000;FredricksonandOnstott,1996;Wellsburyetal.,1997;Boetiusetal.,2000).Carbonatemoundswhoseformationmaybeinitiatedbymethanotrophicbacteria,gashydrates,andbiodegradedpetroleumreservoirsalsobearwitnesstothefactthatthisso-calleddeepbio-spheremaybeubiquitousandinterceptfluxesfromthegeosphere.Bacterialcommunitiesinsubsurfaceenvironmentsaremostcommonlyquantifiedbycellcounting(Cragget

Alkane degradation under anoxic conditions by a nitrate-reducing bacterium with possible involvement of the electron acceptor in substrate activation

Microorganisms can degrade saturated hydrocarbons (alkanes) not only under oxic but also under anoxic conditions. Three denitrifying isolates (strains HxN1, OcN1, HdN1) able to grow under anoxic conditions by coupling alkane oxidation to CO2 with NO3− reduction to N2 were compared with respect to their alkane metabolism. Strains HxN1 and OcN1, which are both Betaproteobacteria, utilized n-alkanes from C6 to C8 and C8 to C12 respectively. Both activate alkanes anaerobically in a fumarate-dependent reaction yielding alkylsuccinates, as suggested by present and previous metabolite and gene analyses. However, strain HdN1 was unique in several respects. It belongs to the Gammaproteobacteria and was more versatile towards alkanes, utilizing the range from C6 to C30. Neither analysis of metabolites nor analysis of genes in the complete genome sequence of strain HdN1 hinted at fumarate-dependent alkane activation. Moreover, whereas strains HxN1 and OcN1 grew with alkanes and NO3−, NO2− or N2O added to the medium, strain HdN1 oxidized alkanes only with NO3− or NO2− but not with added N2O; but N2O was readily used for growth with long-chain alcohols or fatty acids. Results suggest that NO2− or a subsequently formed nitrogen compound other than N2O is needed for alkane activation in strain HdN1. From an energetic point of view, nitrogen–oxygen species are generally rather strong oxidants. They may enable enzymatic mechanisms that are not possible under conditions of sulfate reduction or methanogenesis and thus allow a special mode of alkane activation.

Formation of n-alkane- and cycloalkane-derived organic acids during anaerobic growth of a denitrifying bacterium with crude oil

Abstract The formation of metabolites during anaerobic biodegradation of saturated hydrocarbons directly from crude oil in the absence of oxygen was investigated using a denitrifying bacterium, the Azoarcus -like strain HxN1, which can utilise C 6 –C 8 n -alkanes anaerobically as growth substrates. Various alkylsuccinates (apparently diastereomers) with alkyl chains (probably linked at C-2) ranging from C 4 to C 8 were detected by gas chromatography–mass spectrometry. These metabolites apparently result from the activation reaction of C 4 –C 8 alkanes with cellular fumarate, analogous to the recently established reaction of pure n -hexane with fumarate in strain HxN1 to yield (1-methylpentyl)succinate. Other succinates carried substituents derived from cyclopentane and methylcyclopentane and hence indicated an activation of such cycloalkanes. Since n -butane, n -pentane or cycloalkanes as single compounds did not support growth of strain HxN1, their apparent products point to co-metabolic reactions during utilisation of the C 6 –C 8 n -alkanes. Furthermore, methyl-branched and cyclopentyl-substituted fatty acids were detected. This finding is explained by a further metabolism of the substituted succinates via carbon skeleton rearrangement and decarboxylation. All metabolites detected in the oil-grown cultures were also identified in cultures grown with defined mixtures of saturated hydrocarbons. Results are of potential value for an understanding of metabolite formation in hydrocarbon-rich anoxic environments from the viewpoint of bacterial physiology.

Carbazole distributions in carbonate and clastic source rocks

Abstract Carbazole, the benzocarbazoles, and their alkyl derivatives have been studied from rock extracts to assess the contribution of facies and maturity in controlling their distribution. Carbonates from the Keg River Formation (Elk Point Group, Middle Devonian, Western Canada) consisting of two different facies reflecting deposition under trangressive and regressive events were used to assess facies influences. To study maturation, a uniform organo-facies of organic rich Posidonia Shale (Lower Toarcian, Jurassic, Northern Germany), from 0.48 to 1.45% Rr was used. The organic rich Lower Keg Member, deposited under photic zone anoxia, contained predominantly C 4 C 5 carbazoles. By contrast, the Upper Keg River Member deposited under regressive higher salinity environment contains predominantly carbazole itself, 1-methylcarbazole, and a high abundance of benzo-[c] carbazole relative to benzo[a]carbazole. The distribution of the C 1 to C 3 carbazoles was seen to vary with facies. An examination of the carbazole derivatives within the Posidonia Shale reveals that maturation strongly influences the distribution of carbazole, methylcarbazoles, 1,8-dimethylcarbazole, 1-ethylcarbazole, benzo[a]carbazole, and benzo[c]carbazole. Quantitative determination of these compounds indicates a maximum yield at 0.88% Rr for all derivatives, with the exception of carbazole which displays as a maximum at 0.53% Rr. a systematic variation of compound ratios with maturity to 0.88% Rr suggests that selective isomerisation/destruction/generation of carbazole derivatives may occur. In contrast to earlier publications which stress the importance of carbazoles as migration markers in migrated oils, the results presented here indicate that facies and maturation do play a significant role in controlling the distribution of carbazole derivatives in source rocks and other sediments.