Methanogenesis, sulfate reduction and crude oil biodegradation in hot Alaskan oilfields

Methanogenesis, sulfate reduction and crude oilbiodegradation in hot Alaskan oilfieldsemi_2282 1..13

Lisa M. Gieg,1,2† Irene A. Davidova,1,2

Kathleen E. Duncan1,2 and Joseph M. Suflita1,2*1Institute for Energy & the Environment and2Department of Botany & Microbiology, University ofOklahoma, Norman, OK 73019, USA.

Summary

Petrochemical and geological evidence suggest thatpetroleum in most reservoirs is anaerobically biode-graded to some extent. However, the conditions forthis metabolism and the cultivation of the requisitemicroorganisms are rarely established. Here, wereport on microbial hydrocarbon metabolism in twodistinct oilfields on the North Slope of Alaska (desig-nated Fields A and B). Signature anaerobic hydrocar-bon metabolites were detected in produced waterfrom the two oilfields offering evidence of in situ bio-degradation activity. Rate measurements revealedthat sulfate reduction was an important electronaccepting process in Field A (6–807 mmol S l-1

day-1), but of lesser consequence in Field B(0.1–10 mmol S l-1 day-1). Correspondingly, enrich-ments established at 55°C with a variety of hydro-carbon mixtures showed relatively high sulfateconsumption but low methane production in Field Aincubations, whereas the opposite was true of theField B enrichments. Repeated transfer of a Field Benrichment showed ongoing methane production inthe presence of crude oil that correlated with � 50%depletion of several component hydrocarbons.Molecular-based microbial community analysis of themethanogenic oil-utilizing consortium revealed fivebacterial taxa affiliating with the orders Thermoto-gales, Synergistales, Deferribacterales (two taxa) andThermoanaerobacterales that have known fermenta-tive or syntrophic capability and one methanogen thatis most closely affiliated with uncultured clones in theH2-using family Methanobacteriaceae. The findings

demonstrate that oilfield-associated microbial assem-blages can metabolize crude oil under the thermo-philic and anaerobic conditions prevalent in manypetroleum reservoirs.

Introduction

Petroleum reservoirs are deep subsurface biospheresthat are of global economic importance as > 35% of theworld’s energy supply comes from oil. Most of the oilfound in petroleum reservoirs has been biodegraded tosome extent over geological time, a phenomenon thatleads to the formation of heavy oil that is of lower eco-nomic value and can pose problems for recovery (Headet al., 2003). Evidence for in situ crude oil biodegrada-tion in reservoirs comes from numerous petrochemical,geological and microbiological studies. Petrochemicaldata collected from diverse reservoirs have shown thatn-alkanes, isoprenoids, aromatic hydrocarbons, and N,O and S-heterocycles have been depleted in many oils(Huang and Larter, 2005). Such data led to the formu-lation of quantitative scales to describe the level of bio-degradation of oil, with increased degradation correlatingwith an increase in API gravity, viscosity and total acidnumber (TAN) (Peters and Moldowan, 1993; Wengeret al., 2002; Head et al., 2003). It is reasonable topresume that many of the acidic components character-izing an oil’s TAN value are formed via the microbialdegradation of hydrocarbon components (Barth et al.,2004). Indeed, anaerobic microorganisms are believedto be the key catalysts of oil biodegradation in deeppetroleum reservoirs where oxygen is absent, tempera-tures have remained below 85°C, and where oil andwater interfaces exist (Stetter et al., 1993; Head et al.,2003; Röling et al., 2003). Anaerobes have beencultivated from oilfield systems since the 1920s andinclude diverse fermentative, nitrate-reducing, iron- andmanganese-reducing, and sulfate-reducing bacteria aswell as sulfate-reducing and methanogenic archaea (e.g.Ollivier and Magot, 2005). Within the last decade,culture-independent methods have confirmed the broaddiversity of microorganisms found in mesophilic andthermophilic reservoirs and continually reveal novel bac-terial and archaeal species (Orphan et al., 2000; 2003;Grabowski et al., 2005; Nazina et al., 2006; Li et al.,

Received 22 February, 2010; accepted 11 May, 2010. *For correspon-dence. E-mail [email protected]; Tel. (+1) 405 325 5761; Fax (+1) 405325 7541. †Present address: Department of Biological Sciences, Uni-versity of Calgary, Calgary, AB, Canada T2N 1N4.

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© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd

2007a,b; Dahle et al., 2008; Duncan et al., 2009; Kasteret al., 2009; Pham et al., 2009). The gas caps overlyingthe oil legs in many reservoirs are frequently enriched inmethane that is biogenic in nature as determined bystable isotope studies (Rice and Claypool, 1981; Larterand di Primo, 2005; Jones et al., 2008). This finding pin-points the importance of methanogenesis in the reser-voir ecosystem, with methane being a major end-productof in situ crude oil biodegradation (Head et al., 2003;Gray et al., 2009). Recent metabolic evidence has alsosupported the notion of anaerobic biodegradation in oilreservoirs by extant microbial populations. Aitken andcolleagues (2004) identified known metabolites ofanaerobic naphthalene degradation in biodegraded oilsamples from across the globe, and Duncan andcolleagues (2009) detected low-molecular-weight alkyl-succinates in anaerobic oilfield fluids indicating theanaerobic metabolism of low-molecular-weight n-alkanes. Using intact phospholipids as indicators ofliving microbes in oil reservoirs, Hallmann and col-leagues (2008) showed that indigenous microbialcommunities differed depending on the composition ofavailable hydrocarbons altered through biodegradationactivities.

Despite the overwhelming biogeochemical evidencethat crude oil biodegradation occurs in deep petroleumreservoirs and the widespread detection of anaerobes insuch systems, little is known about how the associatedmicrobial populations utilize oil hydrocarbons under theprevailing conditions. Only a handful of reports existshowing the cultivation of anaerobic hydrocarbondegraders from oilfield fluids. For example, Connan andcolleagues (1995) found that low-molecular-weight aro-matic compounds in oil were preferentially used byanaerobic populations derived from an oil-producingwell, Chen and Taylor (1997) reported the enrichment ofa sulfate-reducing consortium from produced water fromAlaska’s Kuparuk field that biodegraded BTEX com-pounds under thermophilic conditions, and xylene- andn-alkane-degrading sulphate reducers were isolatedfrom oilfield separators (Harms et al., 1999; Davidovaet al., 2006). However, the cultivation of anaerobicpopulations from reservoirs able to metabolize wholecrude oil has remained elusive despite attempts (Headet al., 2003; Grabowski et al., 2005; Magot, 2005; Grayet al., 2009). In this study, we investigated two distinctoilfields on Alaska’s North Slope for the predominantanaerobic processes occurring in the presence of hydro-carbon substrates and for evidence of anaerobic hydro-carbon biodegradation by the associated microbialcommunities. We document the successful cultivationof a crude oil-degrading methanogenic consortiumunder thermophilic conditions from petroleum reservoirfluids.

Results

Field evidence for in situ anaerobic hydrocarbonmetabolism

The oilfields studied are located about 50 km from eachother on the North Slope of Alaska. Some evidence ofanaerobic oil biodegradation has been previouslyreported for some oilfields in this region (Masterson et al.,2001; Holba et al., 2004). Production water samples fromboth fields contained numerous metabolites indicative ofanaerobic hydrocarbon biodegradation (Table 1). C1- toC3- alkylsuccinates as putative signature metabolites ofanaerobic methane, ethane, and propane decompositionwere detected, respectively, as were a variety of fattyacids that might represent the downstream metabolites ofthese alkanes. Benzylsuccinate, a metabolite of anaero-bic toluene decay, and a variety of monoaromatic acidsand alcohols associated with anaerobic or aerobic hydro-carbon metabolism were also identified (Table 1). Thedetection of (methyl) naphthoic acids and the naphthalenering reduction metabolite, 5, 6, 7, 8-tetrahydro-2-naphthoic acid, indicated anaerobic polycyclic aromatic

Table 1. Putative hydrocarbon metabolites and their concentrations(in mM) detected in production water samples taken from two hotAlaska North Slope oilfields.

Identified metabolite Field A Field B

Anaerobic alkylbenzene metaboliteBenzylsuccinate ND 0.033

Benzoate and associated anaerobic metabolitesBenzoate 2.9 1.6Cyclohexane carboxylate 3.5 1.1Pimelate ND 0.9Glutarate 2.1 2.2

Other putative monoaromatic metabolitesToluate (o-, m-, p-) 0.23–1.6 0.26–0.44Phthalate (m-, p-) 0.22–0.84 0.71–2.3Tolylacetate (o-, m-, p-) 0.27–0.37 0.12–0.19Hydroxybenzoate (m-, p-, CH3-) 0.003–0.22 0.035–0.15Phenol 8.5 2.6Cresol (o-, m-, p-) 2.1–2.3 0.62–0.89

Naphthalene and methylnaphthalene metabolites1-Naphthoate ND 0.0212-Naphthoate 0.11 0.155,6,7,8-Tetrahydronaphthoate ND 0.017Methylnaphthoates DET 0.097Dimethylnaphthoates ND DET

Anaerobic alkane metabolitesMethylsuccinate 6.1 5.0Ethylsuccinate 8.1 7.2Propylsuccinate (two peaks) 2.4 1.3

Alkanoic acidsPentanoate 15.0 0.83Hexanoate 6.6 0.085Heptanoate 2.4 0.04Octanoate 0.57 ND

ND, not detected; DET, detected but peak too small to quantify usingintegration software.

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hydrocarbon (PAH) metabolism (Aitken et al., 2004;Meckenstock et al., 2004). These results provided fieldevidence that the microbial populations associated withthe oilfield operations were capable of anaerobic hydro-carbon decay.

Rates of in situ sulfate reduction

Sulfate reduction rates (SRR) were determined on avariety of production well and processing facility samplesfrom the two fields. The radiotracer assay did not substan-tially alter the ambient sulfate pool, so the measured SRRreflects the in situ rate (Ulrich et al., 1997; Davidova et al.,2001). With notable exception, the SRR measured in pro-duction well and processing facility samples from Field A(ranging from 6 to 807 mmol S l-1 day-1) were substantiallyhigher than the comparable values in Field B samples(0.13–10 mmol S l-1 day-1) (Table S1). These results cor-relate with the higher concentrations of sulfate found inField A (2 mM) compared with Field B (0.3 mM), suggest-ing that sulfate reduction is a more prevalent electron-accepting process in Field A than in the Field B.

Laboratory evidence for anaerobic hydrocarbonbiodegradation

The SRR measurements also correlated well with resultsfrom biodegradation assays using production waters from

the two oilfields as inocula in the presence of a varietyof hydrocarbon substrates under thermophilic (55°C)sulfate-reducing and methanogenic conditions. Enhancedlevels of sulfate consumption were observed when Field Aproduction water was incubated in the presence of C6–C12

n-alkanes, field paraffins or crude oil relative to substrate-free controls (Fig. 1A). In contrast, no significant amountsof sulfate were consumed in comparable sulfate-amended incubations established from Field B productionwater (Fig. 1B). Rather, methanogenesis appeared to bean important electron-accepting process in Field B, assignificantly enhanced levels of methane were producedin sulfate-free cultures in the presence of a C6–C12

n-alkane mixture or crude oil relative to substrate-unamended controls (Fig. 1D). Correspondingly, relativelylow amounts of methane were produced when the Field Asample was incubated under methanogenic conditionswith the hydrocarbon substrates (Fig. 1C). Coupled withthe SRR results, these data pinpoint sulfate reduction asan important electron-accepting process at Field A,whereas methanogenesis predominates in Field B.

Transfers of primary enrichments and methanogeniccrude oil biodegradation

Positive enrichments exhibiting enhanced methanogen-esis or sulfate reduction with hydrocarbons relative to

Fig. 1. Measured sulfate consumption (A andB) or methane production (C and D) in oilfieldproduction water samples from Field A (A andC) or Field B (B and D) incubated in theabsence and the presence of a variety ofhydrocarbons at 55°C. Results are after a24-week incubation period.

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the substrate-free controls (Fig. 1) were transferred to afresh medium and incubated at 55°C. After approxi-mately 6 months of incubation, only Field B enrichmentreplicates amended with crude oil (1 ml) showedincreased levels of methane relative to the oil-free inocu-lated controls over the 90 days following transfer(Fig. 2). The transfers on other substrates did not showmethane production or sulfate reduction relative to thecontrols (including Field A transfers) (data not shown).Although the crude oil in the Field B methanogenic incu-bations was added in excess, analysis of the oil layerafter 90 days of incubation showed some depletionof the lighter hydrocarbon fractions (e.g. C10 to C16

n-alkanes) relative to sterile control incubations(Table S2). These active methanogenic enrichmentswere transferred again to fresh medium containing alower amount of crude oil (0.1 ml) in the presence orabsence of glass beads and were incubated at 55°Cfor a prolonged period without disturbance. Afterapproximately 6 months of incubation, methane concen-trations were determined in the cultures, and replicateincubations were used for oil component and metaboliteanalysis.

Hydrocarbon analysis by gas chromatography-massspectrometry (GC-MS) showed > 50% depletion (with anerror of �12.9% based on the recovery of the extractionstandard squalane) of C9 to C31 n-alkanes in the activeenrichments relative to an uninoculated sterile control(Fig. 3A). Some losses of the branched alkanes pristaneand phytane were also observed. Analysis of mono- andpolycyclic aromatic hydrocarbons also showed depletionof these hydrocarbons in the oils extracted from inocu-lated cultures relative to the sterile control (Fig. 3B and C).Substantially higher amounts of CH4 were measured

in transferred incubations containing glass beads(91 � 7.6 mmol, n = 3) relative to those that did not(15.6 � 5.1 mmol, n = 3), which was nearer to the amountof methane produced in the oil-unamended incubations(9.1 � 5.3 mmol, n = 3). In order to determine whether theamount of CH4 produced in the inoculated, oil-amendedincubations correlated with the amount of hydrocarbonconsumed, a mass balance calculation was performed(described in Experimental procedures). Based on theamount of C9 to C31 n-alkanes consumed by the activeoil-degrading enrichments, 117 mmol of CH4 was theoreti-cally expected (Table S3). The actual CH4 formed in theoil-amended cultures averaged 82 mmol (after normalizingfor CH4 formed in the oil-free controls). Thus, about 70%of the predicted amount of CH4 could be accounted forexperimentally.

In the oil-degrading enrichments, the aqueous phasewas extracted and analysed for the presence of signatureanaerobic hydrocarbon metabolites after the 6-monthincubation period. However, no chromatographic peakswere evident in the oil-degrading enrichments that werenot also present in the oil-free or oil-containing sterileincubations. The lack of anaerobic hydrocarbon metabo-lites may be due to the detection limits of the assay(Duncan et al., 2009) as only relatively small volumes ofculture fluid (40 ml) were analysed.

Molecular analysis of the Field B oil-degradingenrichment culture

One archaeal 16S rRNA gene sequence OTU wasdetected in the crude oil-degrading enrichment fromField B. It was most similar (> 99%, Fig. 4) to variousuncultured archaeon clones closely related to Methano-thermobacter species and to sequences previouslydetected in samples collected in 2006 from the samefield (Duncan et al., 2009). A single mcrA OTU was alsoobtained from the enrichment. Its deduced amino acidsequence was most similar (> 99%) to that of thededuced amino acid sequence of the most abundantmcrA OTU from a mcrA clone library constructed fromthe 2006 sample (Fig. S1).

Five bacterial 16S rRNA gene sequence OTUs (98%similarity level), affiliating with four different orders(Thermotogales, Synergistales, Defferibacterales andThermoanaerobacterales), were also obtained from theoil-degrading enrichment (Fig. 5; Table S4). For four ofthe OTUs, the most similar sequences include those fromthe 2006 clone libraries (Duncan et al., 2009) or fromAnaerobaculum hydrogeniformans strain OS1 (16S rRNAgene sequence Accession No. FJ862996) that was previ-ously isolated from Field B (Maune and Tanner, 2008).The fifth OTU forms a clade together with unculturedSynergistales clones that is distinct from the genus

Fig. 2. Methane production from Field B enrichments transferredfrom cultures shown in Fig 1D in the presence (circles) or absence(squares) of crude oil at 55°C. The data shown are from theaverage of four crude oil-amended incubations and two oil-freeincubations. The mmol values were calculated from culturescontaining 20 ml headspace volumes.

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Anaerobaculum (Fig. 5, Table S4). It should be noted thatbacterial sequences similar to those in the enrichmentswere not dominant in the 2006 clone libraries whereineach comprised less than 2% of the 2006 samples(Duncan et al., 2009).

Discussion

This study provides evidence that thermophilic anaerobicpopulations associated with hot oil reservoirs can biode-grade crude oil components under the prevailing condi-tions. Analysis of hot produced water samples from twoAlaska North Slope oilfields revealed a suite of metabo-lites indicative of the anaerobic biotransformation of ali-phatic and aromatic hydrocarbons (Table 1). Detecting

known, signature metabolites of anaerobic hydrocarbonbiotransformation provides unequivocal evidence that thisprocess occurs in situ and is an approach that has beenwidely used to show that such metabolism occurs inhydrocarbon-contaminated terrestrial aquifers (e.g. Beller,2000; Griebler et al., 2004; Gieg and Suflita, 2005). Thismethod also offered metabolic proof of in situ anaerobicoil biodegradation in reservoirs (Aitken et al., 2004). In ourstudy, we found that several metabolites were present atconcentrations greater than 1 mM (Table 1), substantiallyhigher than the levels typically measured in hydrocarbon-impacted groundwater systems (Gieg and Suflita, 2005;Ledin et al., 2005). In other oilfield fluids sampled fromAlaska’s North Slope, we also detected relatively highlevels of C1 to C4-alkylsuccinates and downstream fatty

Fig. 3. Hydrocarbon (HC)-to-squalane peak area ratios for (A) n-alkanes ranging from C9 to C31, (B) select polycyclic aromatic hydrocarbonsand (C) select monoaromatic hydrocarbons. The open bars represent the results from a sterile incubation amended with crude oil, whereasthe black and grey bars represent duplicate inoculated oil-amended Field B enrichment cultures. Results are after a 6-month incubationperiod. PR, pristane; PH, phytane; Naph, naphthalene; MeNaph, methylnaphthalene; Phenanth, phenanthrene; Xyl, xylene; EtBz,ethylbenzene; 135TMB, 1,3,5-trimethylbenzene; EtTol, ethyltoluene.

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acids suggesting the in situ anaerobic metabolism of C1 toC4 n-alkanes (Duncan et al., 2009). Geological evidencehas long shown that such gaseous hydrocarbons arebiodegraded in petroleum reservoirs (Huang and Larter,2005). The detection of metabolites indicative of anaero-bic hydrocarbon biodegradation in both oilfield samplesoffers strong in situ evidence that petroleum componentscan serve as substrates for the anaerobic microbial popu-lations inhabiting Field A and B reservoirs.

Sulfate-reducing bacteria have been widely cultivatedfrom oilfields and are notorious for their pernicious activi-ties that lead to souring and/or corrosion (Vance andThrasher, 2005). Sulfate in oilfields can originate frommarine waters used for secondary oil recovery practices(waterflooding). On Alaska’s North Slope, for example,seawater is used for waterflooding, but the practice differsdepending on the particular field. In Field B operations,reservoir fluids are typically recovered after oil removaland reinjected to maintain formation pressures with sea-water used only intermittently as needed. In contrast,seawater is routinely used for waterflooding in the Field A

facility (our personal communication with operators).These operational practices likely reflect the overallhigher sulfate concentrations measured in Field A (2 mM)and lower concentrations in Field B (0.3 mM) and offer areasonable explanation for the differences in microbialactivities we measured in these fields. In Field A, forexample, the SRR were substantially higher (up to fiveorders of magnitude) than those measured in Field Bsamples (Table S1). Interestingly, the SRR in Field B weresimilar to those measured in other oilfields (0.02–6.75 mMS day-1) using a similar radiotracer assay (Davidova et al.,2001; Bonch-Osmolovskaya et al., 2003; Nazina et al.,2006).

The differing SRR results in the two oilfield samplescorrelated well with our primary enrichment resultsshowing that the Field A populations were able to reducesulfate in response to the added hydrocarbons (Fig. 1A)while the Field B microbial communities did not (Fig. 1B).Accordingly, we expected to successfully enrich a sulfate-reducing population able to utilize a variety of hydrocar-bons from Field A. However, the desired activity was lost

Fig. 4. Phylogenetic relationships of archaeal clones from the Field B enrichment with respect to related sequences. The tree is constructedfrom approximately 560 bp 16S rRNA gene sequences using the neighbour-joining algorithm. One thousand bootstrap replications wereperformed; only values greater than 700 are shown. The sequence shown in bold (BARCe03) and followed by an asterisk is from theenrichment. It is representative of 46 sequences having > 99% similarity. Sequences in bold beginning with ‘PS’, ‘2P’ and ‘CO’ are fromsamples taken from the same field in 2006 (Duncan et al., 2009).

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upon repeated transfer for reasons that remain unclear. Itis possible that the microorganisms in the Field A enrich-ments preferentially oxidized the water-soluble organiccompounds present in the production water samples(Table 1), rather than the added hydrocarbon substrates,and that this effect was more pronounced upon transfer ofthe initial incubations.

In contrast, the Field B enrichment efforts resulted inthe successful cultivation of a methanogenic consortiumable to consume crude oil components at 55°C. We foundthat providing increased surface area in the form of glassbeads to the oil-amended enrichments resulted in moreextensive methane production than in the absence ofextra surface area. We previously observed that a fargreater amount of methane was produced from crude oilwhen a methanogenic consortium was incubated in thepresence of reservoir core material (Gieg et al., 2008). Ina petroleum reservoir, microbial cells likely adhere to rocksurfaces and exist as biofilms (Sanders and Sturman,2005), so the inclusion of an inert solid surface appearedto be a fruitful strategy for cultivating oil-degradingreservoir-associated anaerobes.

There has been much debate in the literature aboutwhether the microbes identified in oilfield systems aretruly indigenous to the petroleum reservoir or whetherthey have been introduced through reservoir processingvia drilling or waterflooding (Magot, 2005). For example,many mesophilic species have been cultured or identi-fied by phylogenetic analysis from thermophilic reservoirsamples, thus suggesting that they may not be indig-enous (Magot, 2005; Li et al., 2007a; Dahle et al., 2008).One of the key problems in studying microbial commu-nities indigenous to oil reservoirs is that it is extremelydifficult and costly to obtain uncontaminated petrolifer-ous rock samples that have not been exposed to pro-duction fluids. Thus, production well head samplesremain as the most reasonable option but some uncer-tainty always remains as to whether the populationsfound in production fluids truly reflect the indigenous res-ervoir population (Magot, 2005; Dahle et al., 2008).Given this caveat, though, the microbial species identi-fied in the production well-derived methanogenic consor-tium do show a strong association with thermophilicoilfield systems as they are similar to sequences

Fig. 5. Phylogenetic relationships of bacterial clones from the Field B enrichment with respect to related sequences. The tree is constructedfrom approximately 1250 bp 16S rRNA gene sequences using the neighbour-joining algorithm. One thousand bootstrap replications wereperformed; only values greater than 700 are shown. The five sequences shown in bold (e.g. BBACe04, etc.) and followed by an asterisk arefrom the enrichment. They are representative OTUs of sequences with > 98% similarity. The number in parentheses following the accessionnumber indicates the total of clones represented by the OTU. Sequences in bold beginning with ‘PS’, ‘2P’, ‘CO’ and Anaerobaculumhydrogeniformans strain OS1 are from samples taken from the same field in 2006 (Duncan et al., 2009).

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retrieved from other high-temperature reservoirs(Orphan et al., 2000; Bonch-Osmolovskaya et al., 2003;Nazina et al., 2006; Li et al., 2007a; Gittel et al., 2009)and to samples previously collected from other locationsin the Field B facility (Duncan et al., 2009). Also, thesequences in the culture were very different from thosepreviously detected in a sample from a seawater lineleading to Field B where genera such as Pseudomonas,Sulfurimonas and Arcobacter were identified (Duncanet al., 2009). It is worth noting that the predominant bac-terial sequences identified in the oil-degrading enrich-ment (affiliating mainly with uncultivated clones in theOrders Thermotogales, Synergistales, Defferibacteralesand Thermoanaerobacterales) were minor componentsof the bacterial clone libraries prepared directly fromother Field B facility samples in which bacterial generasuch as Thermovirga and Desulfomicrobium were moreabundant (Duncan et al., 2009). No sequences closelyrelated (99%) to BBACe11 (29% in the enrichment) werepresent in the 2006 clone libraries, but strain A. hydro-geniformans OS1 was obtained from one 2006 sample.A sequence similar to BBACe04 (24% in the enrichment)was found in one 2006 library (0.3% of total) and onesequence similar to BBACe02 (20% in the enrichment,0.2% in the 2006 clone library). Sequences closelyrelated to BBACe03 (20% in the enrichment) were foundin three of the 2006 clone libraries, with the greatestrepresentation at 1.7%. No sequences closely related toBBACEe10 (7% in the enrichment) were obtained fromthe 2006 clone libraries. These observations demon-strate an enrichment of the identified phylotypes as aresult of laboratory cultivation efforts.

The closest cultivated relatives of the bacterial phylo-types identified in the oil-utilizing consortium (Fig. 5,Table S4) have known fermentative or syntrophiccapabilities (Ravot et al., 1995; Hattori et al., 2000;Orphan et al., 2000; 2003; Balk et al., 2002; Bonch-Osmolovskaya et al., 2003; Li et al., 2007a; Maune andTanner, 2008) but are not known to metabolize hydro-carbons. However, only a handful of studies even reportthe testing of crude oil as a substrate during the culti-vation of microbes from reservoir fluids (Grabowskiet al., 2005; Magot, 2005; Gray et al., 2009) so little isknown about the species in reservoirs able to enzymati-cally attack hydrocarbons. The bioconversion of hydro-carbons to methane is a syntrophic process involvingthe cooperation between organisms (such as syntrophsor fermentors) that can carry out step-wise hydrocarbonmetabolism to produce simple substrates (CO2, H2

and/or acetate) that the methanogens can convert tomethane (Gieg et al., 2008; Jones et al., 2008). Severalpossible routes for this metabolism have been recog-nized, wherein the incomplete oxidation of hydrocarbonscoupled with syntrophic acetate oxidation and methano-

genic CO2 reduction (with H2) is thought to be a keyprocess in reservoirs (Dolfing et al., 2007; Jones et al.,2008). Indeed, the most closely related cultivatedspecies to the predominant methanogen OTU identifiedin the Field B enrichment are H2/CO2-using Methano-thermobacter species that are prevalent in manythermophilic reservoirs (Orphan et al., 2000; 2003;Bonch-Osmolovskaya et al., 2003; Nazina et al., 2006;Mochimaru et al., 2007; Li et al., 2007a; Gray et al.,2009). Thus, the predominant methanogen phylotype islikely playing a key role in the syntrophic decompositionof crude oil components in the consortium and inanaerobic oilfield systems. We also hypothesize that theidentified bacterial members of the consortium play keyroles as syntrophs or fermenters in the methanogenicdecomposition of crude oil components in the Field Breservoir environment as they emerged as the dominantspecies under controlled enrichment conditions wherecrude oil components were consumed (Fig. 3). However,testing such a hypothesis is beyond the scope of thisstudy.

Here, we have provided evidence for in situ anaerobichydrocarbon metabolism and microbial activity from twodistinct thermophilic oilfields on Alaska’s North Slope.We have also successfully cultivated a microbial consor-tium from petroleum reservoir fluids capable of metabo-lizing crude oil components to methane and haveidentified consortial members that are likely to beplaying key roles in this process. Ongoing studies withthis consortium will help us to better understand howsuch anaerobes function in the petroliferous subsurfacebiosphere.

Experimental procedures

Sample collection

Samples from two Alaska North Slope oilfields (designatedFields A and B) were collected in May 2007 and in January2008. General geological and biogeochemical characteristicsof these fields have been previously described (Mastersonet al., 2001; Houseknecht and Bird, 2006). In May 2007, two1 l samples from the hot (> 50°C) oilfield facilities were col-lected at production well heads into sterile Nalgene bottles,closed without a headspace, shipped overnight, and imme-diately placed in an anaerobic glove bag (~5% H2 in N2) uponreceipt in the laboratory. Anaerobicity of the samples wasconfirmed following the addition of resazurin, a redox indica-tor, which remained clear. These samples were used toestablish anaerobic hydrocarbon-utilizing enrichment cul-tures and for metabolite profiling. Multiple samples were sub-sequently collected in January 2008 from the same oilfields atmultiple production well heads, injection wells, and fromcentral processing facilities into sterile glass bottles that wereflushed with N2 prior to shipment to the laboratory. Uponreceipt, these latter samples were used to assess rates of insitu sulfate reduction.

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Establishment of hydrocarbon-utilizing enrichments

Fluids from the oilfields collected in May 2007 were used toestablish thermophilic enrichments under sulfate-reducingand methanogenic conditions. Analysis by ion chromatogra-phy (below) showed that the Field A and B fluids initiallycontained 2 mM and 0.3 mM sulfate, respectively, while bothsamples contained ~0.5 M chloride. Incubations were pre-pared in the anaerobic glove bag by mixing the given produc-tion water sample with an equal volume of a sulfide-reduced,bicarbonate-buffered marine mineral medium (Widdel andBak, 1992). Sulfate-reducing enrichments were establishedby adding sulfate from a sterile anoxic stock solution to yieldan initial concentration of 15 mM, whereas no sulfate wasadded to the enrichments established under methanogenicconditions. Triplicate incubations were amended with one ofthe following hydrocarbon substrate preparations: (i) an equalmixture (v/v) of C6H14, C8H18, C10H22 and C12H26 (278 mmoltotal hydrocarbon), (ii) an equal mixture of C14H30, C16H34 andC18H38 (130 mmol total hydrocarbon), (iii) 50 mmol C28H58 (dis-solved in the inert hydrocarbon carrier heptamethylnonane(HMN), (iv) crude oil from Alaska’s North Slope (1 ml), or (v)0.05 g of field paraffins, deposited and scraped from oilfieldequipment in Oklahoma (dissolved in HMN). Sterile, inocu-lated substrate-free and uninoculated incubations containingsubstrate served as negative controls. All incubations werestatically incubated at 55°C in the dark. Methane and sulfate(and initial chloride) concentrations were measured over timevia gas and ion chromatography respectively (Gieg et al.,2008).

Transfers of initial enrichments

Initial incubations showing a loss of sulfate or methane pro-duction above that of the substrate-free inoculated controlswere transferred to fresh media with the appropriate sub-strate and monitored for additional activity (first transfers).Multiple replicates were established with crude oil (asdescribed above) as the substrate in the absence of addedelectron acceptors. Successful incubations were againtransferred and amended with a lesser amount of oil(0.1 ml, second transfers). Some replicates, both oil-amended and oil-free, were provided with washed, sterileglass beads (1 mm diameter, BioSpec Products, Bartles-ville, OK) to adjust available surface area for microbial colo-nization. Previous work showed an increased rate andextent of methanogenesis from crude oil in the presence ofsolid surfaces (Gieg et al., 2008). These latter transferswere used to determine crude oil composition changes andfor microbial community analysis by 16S rRNA genesequencing (below).

Hydrocarbon metabolite analysis of oilfield samples

To look for putative anaerobic hydrocarbon metabolites pro-duced in oilfields, a portion of each sample collected in May2007 (250 ml) was acidified in the anaerobic glove bag, thenanalysed by GC-MS as previously described (Gieg andSuflita, 2005). Metabolite identification was based on com-parison of GC-MS profiles with authentic standards. Quanti-

fications were made by comparison of metabolite peak areaswith those of authentic standards.

Sulfate reduction assay

Samples from both oilfields collected in January 2008 wereevaluated for the rates of in situ sulfate reduction using aradiotracer technique (Ulrich et al., 1997). Briefly, samples(10 ml) were dispensed into sterile anoxic serum bottlesflushed with nitrogen and supplemented with 2 mCi Na2

35SO4

(698 mCi ml-1, MP Biomedicals, Irvine, CA) per bottle from ananoxic sterile stock solution. The 35S-sulfate amendment didnot measurably change the background levels of sulfate inthe samples. The bottles were incubated at temperaturesclose to the respective ambient temperatures before the poolof total reduced inorganic sulfur was extracted by a chromiumreduction technique and quantified as previously described(Ulrich et al., 1997). Filter-sterilized water samples (0.22 mm)were used as negative controls.

Oil and metabolite analysis in enrichment cultures

Active oil-amended methanogenic enrichments (first trans-fers) were extracted with methylene chloride and processedfor GC-MS analysis as previously described (Gieg et al.,2008). The integrated response of the linear alkanes versusthat of the branched alkanes pristane and phytane withineach sample were compared for an initial quantification ofhydrocarbon loss relative to controls. For subsequent incu-bations (second transfers), cultures were acidified to pH < 2,the aqueous phase was separated from the oil layer andextracted with ethyl acetate prior to metabolite analysis (asabove) after 6 months of incubation. The oil layer wasextracted with methylene chloride following the addition ofsqualane as an extraction standard (0.5 ml of a 5 ml/10 mlstock = 0.25 ml = 0.48 mmol). Substrate-free and sterile incu-bations were treated in the same fashion. Both oil andmetabolite analyses were carried out according to theGC-MS method previously described (Gieg and Suflita, 2005;Gieg et al., 2008). Quantification of oil components wasmade by determining the detector response peak area ratiosof n-alkanes (C9 to C31) and select aromatic hydrocarbonsrelative to that of the added squalane extraction standard.Using this method of quantification, the assumption wasmade that squalane extraction efficiency was 100%.However, we determined the squalane response error byadding a known amount of squalane to water, extracting withmethylene chloride and analysing the extract by GC-MS (asabove). The recovery of squalane was then compared with ananalysis of the same amount of a non-extracted squalanestandard. By this method, the squalane response error was12.9% (n = 6). The amount of hydrocarbon in each samplewas estimated in order to perform mass balance calculationsfor the bioconversion of oil components to CH4. To do this, weassumed equivalent GC response factors for the squalanestandard and the other hydrocarbons of interest. The amount(mmol) of each of the n-C9–C31 alkanes was then determinedand normalized by the amount of the same hydrocarbon inthe sterile controls. This allowed us to calculate the amount ofn-alkane consumed by the cultures (Table S3). This value

Methanogenic crude oil biodegradation in hot oilfields 9

© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

was used to predict the amount of CH4 formed from eachn-alkane based on the stoichiometry established by Symonsand Buswell (1933). Summing these values facilitated thecomparison between actual and predicted amounts of CH4

(Table S3).

Community analysis by 16S rRNA gene sequencing

DNA was extracted from the active Field B methanogenicenrichment using a commercially available DNA kit (MO BIOPowerSoil DNA isolation kit, MO BIO Laboratories, Carlsbad,CA) that combined bead-beating and chemical steps to lysecells. Amplification of the eubacterial and archaeal 16S rRNAgenes used the primers 27F, 1391R (Woyke and Smith,2007) and ARC33F, 958R (Gieg et al., 2008), respectively,according to the protocols described by Woyke and Smith(2007) and Gieg and colleagues (2008). The mcrA library wascreated with the ME1 and ME2 primers and conditions (Haleset al., 1996) that produce approximately 760 bp fragments.All three libraries were created using the TOPO TA CloningKit (Invitrogen Corp., Carlsbad, CA). Transformed colonieswere transferred into 96-well microtitre plates containingtryptone-yeast-extract glycerol broth with ampicillin (Elsha-hed et al., 2003), grown overnight at 37°C, and stored at-75°C until sequencing. Plasmid DNA was purified from thetransformed cells and sequencing performed on an ABImodel 3730 capillary sequencer using the M13 flankingregions as sequencing primer sites (Microgen: The Labora-tory for Genomics and Bioinformatics, Oklahoma City, OK).Sequences in the 16S rRNA gene sequence clone librarieswere aligned using the greengenes NAST-aligner (DeSantiset al., 2006a), screened for chimeric sequences using Belle-rophon (Huber et al., 2004) and chimeras removed. The 16SrRNA bacterial gene sequence library contained 41sequences, the corresponding archaeal library contained 46sequences and the mcrA library contained 23 sequences (all> 99% nucleotide sequence similarity to each other, e.g. 1OTU). Distance matrices (DeSantis et al., 2006b) werecreated from the 16S rRNA archaeal and bacterial genesequence library and used by the DOTUR program (Schlossand Handelsman, 2005) to identify groups of sequencessharing a given level of similarity. The archaeal 16S rRNAgene sequences had > 99% similarity to each (1 OTU),DOTUR produced 5 OTUs at the 98% level of similarity fromthe bacterial 16S rRNA gene sequence library. Each bacterialOTU contained three or more sequences (Fig. 5, Table S4).One sequence was chosen from each OTU to serve as arepresentative sequence. For all libraries, similarities of therepresentative sequences to existing sequences wereobtained following BLASTN searches (Basic Local AlignmentSearch Tool, Altschul et al., 1990). BLASTX searches wereperformed on translated mcrA sequences. Sequences thatmost closely matched the representative clone sequencesand selected outgroup sequences were aligned using CLUST-ALX (version 1.81) (Thompson et al., 1997). The neighbour-joining method of Saitou and Nei (1987) was used todemonstrate the phylogenetic relationships among selectedsequences, as implemented in CLUSTALX (Thompson et al.,1997). Distances were calculated as % divergence betweenall pairs of sequences from the multiple alignment, alignmentpositions with gaps were excluded from the analysis and the

Kimura two-parameter distance correction for multiple substi-tutions was applied (Kimura, 1980). The support for the treebranches was estimated from 1000 bootstrap replicates(Felsenstein, 1985). Representative sequences weredeposited in GenBank under Accession No. GU357463–GU357474.

Acknowledgements

We thank Victoria Parisi for help in collecting samples fromFields A and B in January 2008 and Laura Christian for fieldparaffin scrapings. This study was supported by grantsfrom the Integrated Petroleum Environmental Consortium,National Science Foundation, ConocoPhillips, and start-up funds to L.G. from Faculty of Science, University ofCalgary. Access to the Field A and B facilities is gratefullyacknowledged.

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Fig. S1. Phylogenetic relationship of deduced methyl-coenzyme M reductase subunit A (mcrA) amino acidsequences from the Field B enrichment with respect torelated sequences. The tree is constructed from approxi-mately 219 amino acid positions (deduced from the mcrAnucleotide sequences) using the neighbour-joining algorithm.One thousand bootstrap replications were performed; onlyvalues greater than 700 are shown. The sequence shown inbold (BME09g02) and followed by an asterisk is from theenrichment. It is representative of 23 sequences > 99% simi-larity. Sequences in bold beginning with ‘PS’ are fromsamples taken from the same field in 2006 (Duncan et al.,2009).Table S1. In situ sulfate reduction rates (SRR) determinedfrom a variety of production well and processing facilitysamples from two oilfields on Alaska’s North Slope.Table S2. Alkane-to-pristane or alkane-to-phytane peak arearatios for crude oil incubated in the presence (inoculated) orabsence (sterile) of a methanogenic population enriched froma hot oilfield production well from Field B on Alaska’s North

12 L. M. Gieg, I. A. Davidova, K. E. Duncan and J. M. Suflita

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Slope. Bolded numbers show a decrease of the ratio in inocu-lated incubations relative to sterile incubations indicating thatbiodegradation has occurred.Table S3. Amounts (mmol) of n-alkanes present in sterile andinoculated crude oil-amended incubations enriched from pro-duction well fluids from Field B on Alaska’s North Slope andexpected amounts (mmol) of methane based on predictedstoichiometric reactions.

Table S4. Summary of OTU in hydrocarbon-degradingmethanogenic culture enriched from Field B.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials suppliedby the authors. Any queries (other than missing material)should be directed to the corresponding author for thearticle.

Methanogenic crude oil biodegradation in hot oilfields 13

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