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Methanogenic Microbial Community Composition ofOily Sludge and Its Enrichment Amended with AlkanesIncubated for Over 500 DaysLi-Ying Wang a , Wei Li a , Serge Maurice Mbadinga a , Jin-Feng Liu a , Ji-Dong Gu b & Bo-Zhong Mu aa State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry , EastChina University of Science and Technology , Shanghai , Chinab School of Biological Sciences , The University of Hong Kong , Hong Kong SAR , ChinaAccepted author version posted online: 27 Mar 2012.Published online: 12 Jul 2012.

To cite this article: Li-Ying Wang , Wei Li , Serge Maurice Mbadinga , Jin-Feng Liu , Ji-Dong Gu & Bo-Zhong Mu (2012)Methanogenic Microbial Community Composition of Oily Sludge and Its Enrichment Amended with Alkanes Incubated for Over500 Days, Geomicrobiology Journal, 29:8, 716-726, DOI: 10.1080/01490451.2011.619634

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Geomicrobiology Journal, 29:716–726, 2012Copyright © Taylor & Francis Group, LLCISSN: 0149-0451 print / 1521-0529 onlineDOI: 10.1080/01490451.2011.619634

Methanogenic Microbial Community Composition of OilySludge and Its Enrichment Amended with AlkanesIncubated for Over 500 Days

Li-Ying Wang,1 Wei Li,1 Serge Maurice Mbadinga,1 Jin-Feng Liu,1 Ji-Dong Gu,2

and Bo-Zhong Mu1

1State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East ChinaUniversity of Science and Technology, Shanghai, China2School of Biological Sciences, The University of Hong Kong, Hong Kong SAR, China

Methanogenic microbial community is responsive to the avail-ability of hydrocarbons and such information is critical for theassessment of hydrocarbon degradation in remediation and alsoin biologically enhanced recovery of energy from non-producingoil reserves. In this study, methanogenic enrichment cultures fromoily sludge amended with n-alkanes (C15-C20) showed a develop-ment of active methanogenic alkanes-degrading consortium forover a total of 1000 days of incubation at 37◦C. Total genomicDNAs were extracted from three types of samples, the originaloily sludge (OS), the sludge after incubation for 500 days un-der methanogenic condition without any external carbon addi-tion (EC), and the enrichment culture from the EC amendedwith n-alkanes (ET) incubated for another 500 days. The phy-logenetic diversities of microbial communities of the three sampleswere analyzed by PCR amplification of partial 16S rRNA genes.The catabolic genes encoding benzylsuccinate synthase (bssA)and alkylsuccinate synthase (assA) were also examined byPCR amplification. These results provide important evidencein that microbial populations in an oily sludge shifted frommethanogenic aromatic compounds degrading communities to po-tential methanogenic alkane-degrading communities when the en-richment was supplemented with n-alkanes and incubated underanaerobic conditions.

Keywords microbial community, methanogenesis, n-alkanes, oilysludge

Received 13 May 2011; accepted 30 August 2011.This work was supported by the National Natural Science Founda-

tion of China (Grant No. 41073055) and the 863 Program (Grant No.2009AA063503).

Address correspondence to Bo-Zhong Mu, East China Universityof Science and Technology, Shanghai, 200237, China. E-mail: bzmu@ecust.edu.cn

INTRODUCTIONThe modern civilization dependent heavily on fossils fuels,

but petroleum supplies are becoming increasingly limited. Whenoil reservoirs are closed after drilling and pumping, there are stillmore than 50% of the total crude oil remaining as residues ofnon-recoverable fraction due to tight binding to subsurface ma-trices. Recovery of energy as methane gas that is biologicallyproduced as the result of methanogenic oil biodegradation inpetroleum reservoir is becoming economic feasible. Studyingthe organisms of methanogenic oil biodegradation has becomevery important to enhance the recovery of energy assets as bio-genic methane from residual oils stranded in petroleum ecosys-tems.

Oily sludge originating from oil refineries is a complex mix-ture with high content of petroleum hydrocarbons and otherorganics, would represent a special habitat for metabolicallyspecific microorganisms both aerobic and anaerobic. Methanecan be generated by microbial breaking down of hydrocarbons(Zengler et al. 1999; Gieg et al. 2008; Jones et al. 2008; Wanget al. 2011). Methanogenesis can also play an important rolein remediation of hydrocarbons contaminated sites in anoxicenvironments (Gray et al. 2010). Crude oil in some petroleumreservoirs could be converted to methane only by the addition ofan exogenous microbial community, as such, the use of exoge-nous microorganisms (inoculating oil reservoirs with enrichedmethanogenic hydrocarbon-degrading consortium) to stimulatemethane production has also been proposed (Gieg et al. 2008).

Laboratory investigation of methanogenic hydrocarbon-degrading microbial communities is one of the most importantsteps for possible application of methanogenic hydrocarbon-degradation in remediation of crude oil contamination or en-hancing the recovery from non-producing oil reserves. Theobjectives of this study were to enrich alkane-degradingmethanogenic culture and to elucidate the microbial commu-nity shift during acclimation and enrichment over a long timeof incubation.

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METHANOGENIC ALKANES DEGRADATION 717

METHODS

Sample Collection and Preparationof Enrichment Cultures

The oily sludge was collected from the bottom layer of awaste oil treatment tank at Shanghai Oil Refinery. A sterile 5 Lglass bottle was filled completely, immediately sealed with cap,transported back to the laboratory then stored at room temper-ature (22◦ C) shortly before use. The enrichment culture wasinitiated by inoculating 10 mL (20%) of the oily sludge into anautoclaved serum bottle containing 40 mL of a previously ster-ilized basal medium, sealed with butyl rubber stopper (BellcoGlass, NY, Vineland, USA) and aluminum seal, and incubatedat 37◦C in the dark.

The medium was comprised of namely A, B, and C threeparts. Part A contained (g/L): NaCl, 0.20; MgCl2·6H2O, 1.20;CaCl2·2H2O, 0.10; NH4Cl, 0.25; KH2PO4, 0.20; and KCl, 1.30;rezasurin, 0.0001. Part B was a solution of NaHCO3 (2.5 g/L)alone. Part C was made of 1.0 mL of trace elements and 1.0mL of vitamin stock solution. The trace elements stock solutioncontains (g/L): CoCl2·6H2O, 0.50; CuCl2, 0.10; FeCl2·4H2O,7.50; H3BO3, 1.00; MnCl2·4H2O, 0.50; Na2MoO4·2H2O, 0.10;NiCl2·6H2O, 0.10; ZnCl2, 0.50. The vitamin stock solutioncontains (g/L): vitamin B12, 0.0001; D(+)-biotin, 0.02; folicacid, 0.02; nicotinic acid, 0.05; p-aminobenzoic acid, 0.05; Ca-D(+)-pantothenate, 0.05; pyridoxine-HCl, 0.1; riboflavin, 0.05;thiamine-HCl, 0.05; thioctic acid, 0.05.

To prepare the basal medium before use, Part A was auto-claved at 121◦C for 20 min, supplemented with Parts B and C,which were sterilized by filtration with 0.2 µm-pore-size mem-brane filter (Millipore). The final medium under a stream ofpure nitrogen gas was reduced with Na2S·9H2O (0.50 g/L). Inorder to deplete the indigenous carbon and energy source, e.g.,aromatic hydrocarbons, alkanes, organic acids etc. in the initialoily sludge collected, no extra substrates were amended to theserum bottles containing basal medium and the sludge was usedas inoculums in EC. The prepared basal medium (40 mL) with afinal pH 7.2 was dispensed anaerobically into each serum bottle(120 mL) that was sealed with butyl rubber stopper and crimpseal, and the head space of serum bottle was filled with pure N2

(99.99%) after passing through heated copper filings.ET for methanogenic alkanes-degradation was established

by transferring 5 mL of the EC after 500 days and incubatedinto each 120 mL serum bottle containing 45 mL of the mediumdescribed above, with n-alkanes mixture (C15-C20). The mix-ture of n-alkanes (C15-C20) was prepared according to Wanget al. (2011) and 100 µmol of the total n-alkane mixture wastransferred into each ET serum bottle. Controls inoculated withenrichment culture (EC) but without n-alkanes were also pre-pared to account for the background methanogenesis from theinoculant. All incubations were kept at 37◦C in the dark.

For the proportion of the total n-alkane mixture, n-penta-decane (C15; ≥99%), n-hexadecane (C16; 99%), n-heptadecane(C17; 99%), n-octadecane (C18; 99%), n-nonadecane (C19;

99%), and n-eicosane (C20; 99%) were purchased from Sigma-Aldrich (Milwaukee, WI). All the above media were preparedaccording to the Hungate technique as described by Bryant(1972).

Chemical AnalysesMethane and hydrogen produced in the headspace of serum

bottles were measured using a gas chromatograph equippedwith a column of 5 Å carbon molecular sieves, a flameionization detector (FID) and a thermal conductivity detector(TCD). The injector and the detectors temperatures were heldat 200◦C, the column temperature was initially held at 50◦C for5 min, then increased at a rate of 15◦C/min to 200◦C and holdat 200◦C for 15 min.

Methane concentrations were determined by using a workingcurve in which the coefficient of linear regression between CH4

concentrations and detection response was 0.994 (n = 6). Thealkanes in the serum bottles at the end of the incubation weredetermined on an Agilent 6890 model GC. A surrogate stan-dard (Cetyl chloride, 50.0 µl) was added to the bottles prior tothe extraction of alkanes from the microcosms four individualtimes. Pooled organic layers were transferred to a new vial andconcentrated under a stream of N2. The extracts were injectedonto GC to separate alkane components on an HP-5MS capillarycolumn (30 m × 0.25 mm × 0.25 µm film thickness, AgilentTechnologies, Inc.) by initially holding the oven at 60◦C for2 min, increased at 10◦C per min to 280◦C and held for 25 min.An Agilent 5975 inert mass selective detector (MSD) was usedto acquire data in the scanning mode. EI was operated at 70 eV,and ion source temperature was held at 230◦C. To quantify theresidual alkanes in extracts, the GC peak areas of alkanes rang-ing from C15 to C20 were integrated and then divided by the GCpeak area of the added cetyl chloride to obtain an n-alkane-to-standard ratio.

In order to test for the presence of (putative) intermediatemetabolites in the alkane-degrading methanogenic enrichmentcultures, a 5 mL sample was treated with ammonia water to∼pH 9.0 and then dry at 105◦C. Then after 10% butanol/sulfatesolution (0.5 ml) was added to the sample and esterified at 90◦Cfor 60 min. The resulting butyl esterified sample was then sub-jected to extraction. Volatile fatty acids (VFAs) were extractedwith n-dodecane whereas long chain fatty acids (LCFA) wereobtained by extraction with n-hexane. Sample extracts were an-alyzed with an Agilent 6890 GC equipped with an HP-5MScapillary column (30 m × 0.25 mm × 0.25 µm) and mass de-tector (MSD 5975). The oven temperature was held at 60◦C for2 min, then increased at 10◦C per min to 280◦C, and held for25 min. An Agilent 5975 inert mass selective detector (MSD)acquired the data in the scan mode. EI was operated at 70 eV,and ion source temperature was held at 230◦C.

DNA Extraction and PCR AmplificationGenomic DNAs were extracted as previously described

(Wang et al. 2011). Partial 16S rRNA genes were amplified

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718 L. Y. WANG ET AL.

using the primers 27F and 1492R for bacteria (Weisburg et al.1991; Brunk et al. 1996; Nazina et al. 2006), and the primers21F (DeLong, 1992) and 1041R (Kolganova et al. 2002) forarchaea. In addition, the assA and bssA genes were PCR am-plified from genomic DNAs using primers 1432F and ass/bssR, and primers 1213F and 1987R, respectively (Callaghan et al.2010). The PCR condition for amplification of the 16S rRNAgene was as follows: initial denaturation at 94◦C for 3 min, 25cycles of 94◦C for 30 sec, annealing at 55◦C for 30 sec, and72◦C for 90 sec, and a final elongation step of 72◦C for 7 min.The assA and bssA gene PCR conditions were as follows: initialdenaturation at 95◦C for 5 min, 45 cycles of 95◦C for 45 sec,annealing at 60◦C for 1 min, and 72◦C for 2 min, and a finalelongation step of 72◦C for 20 min.

Construction of 16S rRNA Gene and assA/bssA GeneClone Libraries

The amplified and then purified PCR products were directlycloned into E. coli using a pGEM R©-T Easy cloning vector(Promega, USA) following the manufacturer’s instructions. Theobtained white colonies were randomly picked and inoculatedinto 1 mL Luria Broth (LB) medium supplemented with ampi-cillin (50 mg/mL) and incubated overnight at 37◦C. The obtainedactive grown cultures were used as DNA template for PCR re-action with M13F/M13R primer set. Correct inserts were de-termined with automated ABI 377 sequencer (Dye TerminatorCycle Sequencing Ready Reaction FS Kit; PE Applied Biosys-tems) using M13F universal sequencing primer. Obtained DNAsequences were checked for vector by VecScreen Widget 1.0software before further analysis.

Phylogenetic Analysis16S rRNA gene sequences from each respective library were

aligned to a core set of sequences using the NAST alignmentalgorithm (DeSantis et al. 2006a) on the Greengenes website(http://greengenes.lbl.gov). Chimeric sequences were then iden-tified using Bellerophon, version 3 (Huber et al. 2004; DeSantiset al. 2006b). The remaining sequences were grouped into oper-ational taxonomic units (OTUs) by further-neighbor algorithmusing MOTHUR software (Schloss et al. 2009).

A similarity cut-off of 98% was used to consider distinctOTUs and one sequence in each OTU was chosen to representthe OTU for phylogenetic tree construction. For 16S rRNA genelibraries, the phylogenetic placement of each OTU was exam-ined using the Classifier program from the Ribosomal DatabaseProject II (Wang et al. 2007). The nearest relatives of each OTUwere identified using the BLASTN network service (Altschulet al. 1997).

Types of assA/bssA OTUs were first detected by ORFfinder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) to find allopen reading frames, and ExPASY translate tool (http://expasy.org/tools/dna.html) were used to allow the translationof a nucleotide sequence to the corresponding amino acids se-quence and then aligned with several known assA/bssA gene

products. Phylogenetic trees were constructed based on theneighbor-joining algorithm (Saitou and Nei 1987) using theMEGA4 software (Tamura et al. 2007). Bootstrap analysis with1000 replicates was applied to assign confidence levels to thenodes in the trees.

Nucleotide Sequence Accession NumbersDNA sequences obtained in this study have been submitted

to the GenBank databases under accession numbers JF754491-JF754549.

RESULTS

Methane Production and n-alkanes Biodegradationfrom Cultured Oily Sludge

The oily sludge was inoculated in freshly prepared culturemedium and incubated under strictly anaerobic conditions. Asubstantial increase in the amount of methane production wasobserved with no obvious lag period. Methane accumulatedin the headspace of the bottles to reach 720 µmol on day-200, but the amounts of methane stayed almost the same forthe remaining incubation period (Figure 1A). However, in theenrichment transfer (ET) cultures amended with a mixture ofn-alkanes, the methane production was detected after an initiallag period of about 100 days, and 18 µmol of net methane wasproduced above the controls without n-alkane-amendment overthe 500 days incubation in ET (Figure 1B). The biodegradationpattern for the n-alkanes was evident from GC analysis after500 days incubation in ET, all six n-alkanes (C15, C16, C17,C18, C19, and C20) were reduced non-discriminatively in theactive methanogenic cultures with a loss of approximately 10%compared to sterilized controls.

The concentrations of residual n-alkanes recovered on day500 of incubation in the enrichment transfer (ET) cultures fromthe sterile control and active methanogenic incubations wereused to calculate the loss of individual alkanes in the cultureand the methane observed to the theoretical production was19.7% (Table 1). In addition, hydrogen at concentrations be-tween 2 to 13 µM, octadecanoic acid (0.07 µM), hexadecanoicacid (0.31 µM), tetradecanoic acid (0.69 µM), dodecanoic acid(1.32 µM), acetate (33.32 µM) and formate (10.91 µM) werealso detected on day 500 in ET cultures.

Archaeal CommunitiesFor the members in the domain Archaea, 35, 47 and 48

clones were randomly selected from OS, EC and ET for se-quencing. Of the total sequences screened by MOTHUR, 4, 7and 5 operational taxonomy units (OTUs) were obtained fromOS, EC and ET formed seven distinct phylogenetic groups(Figure 2).

The archaeal community composition of OS sample wasless diverse: OTU A1-22 (22.9% of total archaeal clones) wasclosely related to the H2-and-formate utilizing genus Methano-linea tarda, while A1-09 (34.3% of total archaeal clones) was

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METHANOGENIC ALKANES DEGRADATION 719

FIG. 1. Methane production in the methanogenic enrichment cultures(EC): (A) The first enrichment culture from oily sludge without addition of carbon or energysource; (B) Methanogenic enrichment transfer (ET) cultures amended with a mixture of n-alkanes (�), as compared to the unamended controls (•) and the data ofduplicate experiments values are shown.

closely related to methyl (o-methyl and/or methoxy) com-pounds utilizing genera Methanomethylovorans thermophilaand Methanomethylovorans hollandica, and the remaining ar-chaeal phylotypes were actually assigned to the unclassifiedEuryarchaeota without cultivated representatives.

Most of the sequences (68.1% of total archaeal clones) re-trieved from the EC were affiliated with H2 and formate uti-lizing methanogens. For instance, OTU A2-05 was closelyrelated to Methanobacterium subterraneum and Methanobac-terium formicicum, OTU A2-44 was affiliated with the genusMethanolinea, OTU A2-38 was closely related to Methanofol-lis tationis and Methanofollis liminatans, and OTUs A2-24 and

A2-07 were with the genus Methanocalculus. OTU A2-17 rep-resenting approximately 17% of total archaeal clones was af-filiated with the acetoclastic Methanoseata. The remaining ar-chaeal phylotypes were related to unclassified Euryarchaeota.

The archaeal community in ET was characterized by the oc-currence of acetoclastic methanogens (64.6% of total archaealclones) represented by OTUs A3-30 and A3-44 and closely re-lated to the genus Methanoseata as well as H2 and formateutilizing methanogens (31.3% of total archaeal clones) repre-sented by A3-23 and A3-22 affiliated with Methanolinea. Veryfew of Euryarchaeota members (OTU A3-48) without cultivatedrepresentatives were also retrieved from the ET.

TABLE 1Predicted and measured methane production in microcosms amended with n-alkanes and incubated at 37◦C for 500 days in the

enrichment transfer (ET) culture

Reactions

Substrateconsumed

(mmol)Theoretical CH4

production (mmol)

Measured CH4

production(mmol)

Percent oftheoreticalproduction

C15H32 + 7H2O → 3.5CO2 + 11.5CH4 C15 = 0.0008 0.009C16H34 + 7.5H2O → 3.75CO2 + 12.25CH4 C16 = 0.0013 0.0159C17H36 + 8H2O → 4CO2 + 13CH4 C17 = 0.0019 0.0247C18H38 + 8.5H2O → 4.25CO2 + 13.75CH4 C18 = 0.0012 0.0165C19H40 + 9H2O → 4.5CO2 + 14.5CH4 C19 = 0.0010 0.0145C20H42 + 9.5H2O → 4.75CO2 + 15.25CH4 C20 = 0.0007 0.0107Sum 0.0913 0.018 19.7%

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720 L. Y. WANG ET AL.

Methanocalculus pumilus (AB008853)EC clone A2-07 (1/47)Methanocalculus halotolerans (T) (AF033672)

Methanocalculus taiwanensis (AF172443)Methanocalculus

100 Methanocalculus taiwanensis (AF172443)EC clone A2-24 (10/47)

Methanocalculus chunghsingensis (AF321115)Methanofollis formosanus (AY186542)Methanofollis aquaemaris (AF262035)

EC clone A2-38 (16/47) Methanofollis

100

91

99 EC clone A2 38 (16/47)Methanofollis tationis (AF095272)Methanofollis liminatans (AY196677)

f

Uncultured Methanolinea sp.clone SMS-sludge-8 (AB479399)OS clone A1-22 (8/35)Methanolinea tarda (T) clone NOBI-1 (AB162774)

90

99

95

Methanolinea tarda (T) clone NOBI 1 (AB162774)Uncultured Methanolinea sp.clone AnDHS-A2 (AB434764)

Uncultured archaeon TA02 (AF229775)Uncultured euryarchaeote clone ARC19 (AY693813)

ET clone A3-23 (8/48)EC clone A2-44 (2/47)

Methanolinea

99

86100

81EC clone A2 44 (2/47)Uncultured Methanolinea sp.clone SMS-T-Pro-3 ( AB479407)

ET clone A3-22 (7/48)Uncultured Methanomethylovorans sp.clone KuA6 (AB077216)

Methanomethylovorans hollandica (AY260433)Methanomethylovorans thermophila (T) clone L2FAW (AY672821)

Methanomethylovorans100Methanomethylovorans thermophila (T) clone L2FAW (AY672821)OS clone A1-09 (12/35)ET clone A3-30 (19/48)

EC clone A2-17 (8/47)Toluene-degrading methanogenic consortium archaeon clone M2 (AF423188 )Uncultured Methanosaeta sp clone SMS-sludge-1 (AB479392) Methanoseata

89

100

100

Uncultured Methanosaeta sp.clone SMS sludge 1 (AB479392)Uncultured archaeon clone PL-35A3 (AY570656)

Uncultured Methanosaeta sp. (EU888812)ET clone A3-44 (12/48)

Methanobacterium aarhusense (AY386124)Methanobacterium beijingense (AY350742)

9388

100

Methanobacterium beijingense (AY350742)EC clone A2-05 (3/47)

Methanobacterium subterraneum (X99044)Methanobacterium formicicum clone FCam (AF028689)

Methanobacterium

Uncultured euryarchaeote clone GNA01G09 (EU731048)Uncultured archaeon clone Thp A 75 (EF444633)

100

100

Uncultured archaeon clone Thp A 75 (EF444633)EC clone A2-06 (7/47)OS clone A1-35 (10/35)

OS clone A1-41 (5/35)ET clone A3-48 (2/48)

Uncultured Euryarchaeal Clade100

93

100

2%

FIG. 2. Phylogenetic tree of the methanogens 16S rRNA gene phylotypes (shown in bold) retrieved from the oily sludge and methanogenic enrichment cultures(EC) without addition of carbon or energy sources and enrichment transfer (ET) cultures. Type sequences retrieved from GenBank database are shown with theiraccession number. Putative divisions are listed on the tree. Alignments to related sequences were performed with MEGA 4 software. The topology of the tree wasobtained by the neighbor-joining method. Bootstrap values (n = 1000 replicates) of ≥75% are reported. Scale bar = nucleotide changes per site.

Bacterial CommunitiesWithin the domain bacteria, 74, 82 and 52 clones were re-

trieved respectively from OS, EC and ET and 16, 11 and 8 OTUswere resulted, respectively. These 35 OTUs were phylogenet-ically affiliated with 13 different taxonomic clades (Figure 3).For OS, 10 of the 16 OTUs (60.8% of total clones) obtained werewithin the Proteobacteria, two (28.4%) with green non-sulfurbacteria (Chloroflexi), one (2.7%) with Nitrospira, one (5.4%)with OP11 and two (2.7%) with OP8.

From EC, one of the 11 OTUs was affiliated with theFirmicutes (47.6% of total clones), two with Chloroflexi(23.2%), two with Synergistetes (8.5%), three with theδ-Proteobacteria (13.4%), one with Actinobacteria (2.4%),one with Spirochaetes (2.4%) and one with OP11 (2.4%).Within the Firmicutes group, OTU B2–45 was closely re-lated to Clostridium sp. (Accession number AY554416), which

was identified in the production water from Gangxi oil field,China.

Among members of the Chloroflexi group, OTU B2-01 wasclosely related to clone MP14 retrieved from a mesophilicphenol-degrading methanogenic community (Chen et al. 2008),while OTU B2-08 was related to clone SJA-131 from an anaer-obic trichlorobenzene-transforming microbial consortium (vonWintzingerode et al. 1999). The Synergistetes constituents repre-sented by OTU B2-56 and OTU B2-30 were found being relatedto oil bed bacterium 11br (Accession number GU129077) andto clone 39 previously found in UASB reactor treating brewerywastewater methanogenic community (Diaz et al. 2006).

Within the δ-Proteobacteria OTU B2-17 and OTU B2-16 affiliate with Syntrophus spp. closely related to thoseidentified in hexadecane-degrading methanogenic community(Zengler et al. 1999); OTU B2-02, however, was an uncultured

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METHANOGENIC ALKANES DEGRADATION 721

member of the family Geobacteraceae obtained from a ura-nium bioremediation site (Holmes et al. 2007). The Actinobac-teria group is represented by OTU B2-05 which was closelyrelated to clone Nap1-8C (Accession number EU522663) ob-tained from hydrocarbon-degrading methanogenic community.OTU B2-80 was closely related to Spirochaetes clone ObedB-2A retrieved from a methanogenic coal enrichment culture com-munity (Penner et al. 2010). Members of the candidate division

OP11 were represented by OTU B2-22 closely related to cloneEub 2 retrieved from a toluene-degrading methanogenic com-munity (Ficker et al. 1999).

From ET, four of the eight OTUs were affiliated with theδ-Proteobacteria (38.5% of total clones), one with Firmicutes(30.8%), one with Thermotogae (15.4%) and two with Chlo-roflexi (15.4%). In the δ-Proteobacteria, OTUs B3-16 and B3-32 were affiliated to Syntrophus sp. clone B2, while B3-07 was

OS clone B1-03 (2/74)Brevundimonas sp. Dsp-7 (DQ676936)Brevundimonas vesicularis clone HU2 (EU862355)

Uncultured alpha proteobacterium (EF032743)OS l B1 19 (1/74)

α-Proteobacteria99

83 OS clone B1-19 (1/74)Brachymonas petroleovorans CHX (AY275432)

OS clone B1-08 (11/74)Uncultured Cupriavidus sp. (FJ978099)OS clone B1-49 (3/74)

Uncultured beta proteobacterium (AY118150)99

99

83

Uncultured bacterium (AY945911)OS clone B1-30 (1/74)Uncultured bacterium( DQ975219 )Uncultured beta proteobacterium (EU266800)OS clone B1-43 (3/74)

Uncultured bacterium (EU704617)

β-Proteobacteria

99

94

88

( )OS clone B1-05 (1/74)

Uncultured bacterium (AY945886)Uncultured bacterium (GU083550)OS clone B1-20 (8/74)Alcaligenes sp. Q-6 (AB246810)

OS clone B1-42 (8/74)

99

99

88

OS c o e (8/7 )OS clone B1-38 (7/74)Citrobacter amalonaticus (GU185859)Enterobacteriaceae bacterium KVD-unk-44 (DQ490332)Citrobacter sp. TAF (DQ083975)

γ-Proteobacteria

Uncultured Geobacteraceae bacterium clone: M21_1032 (EF668143)EC clone B2-02 (6/82)

88

100

EC clone B2-02 (6/82)Mesophilic phenol-degrading methanogenic consortia:MP91 (EF198033)

Syntrophus buswellii ( X85131)EC clone:B2-17 (2/82)ET clone:B3-17 (1/52)

EC clone:B2-16(3/82)Hexdecane degrading methanogenic consortia clone:B1 (AJ133794)

δ-Proteobacteria84

91

91

88

Hexdecane-degrading methanogenic consortia clone:B1 (AJ133794)ET clone:B3-07 (11/52)

Hexdecane-degrading methanogenic consortia clone:B2 (AJ133795)ET clone:B3-16 (2/52)

ET clone:B3-32 (6/52)

84

2%

FIG. 3. (A) Phylogenetic tree of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria 16S rRNA gene phylotypes ofsequence types (shown in bold) retrieved from the oily sludge and methanogenic enrichment cultures (EC) without addition of carbon or energy sources andenrichment transfer (ET) cultures. Type sequences retrieved from GenBank database are shown with their accession number. Putative divisions are listed on thetree. Alignments to related sequences were performed with MEGA 4 software. The topology of the tree was obtained by the neighbor-joining method. Bootstrapvalues (n = 1000 replicates) of ≥75% are reported. Scale bar = nucleotide changes per site.

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722 L. Y. WANG ET AL.

Mesophilic phenol-degrading methanogenic consortia:MP14 (EF198025)EC clone B2-01 (9/82)

Toluene-degrading methanogenic consortium bacterium clone:Eub 4 (AF423184)ET clone B3-23 (6/52)

Residual oil degrading methanogenic consortium clone: lg1e04 ( EU037975)

80

95

g g g g ( )Anaerobic TCB transforming microbial consortium clone: SJA-131 (AJ009492)EC clone B2-08 (10/82)

Anaerobic dechlorinating consortia clone: SHA-53 (AJ249111)Anaerobic TCB transforming microbial consortium clone:SJA-61 (AJ009469)ET clone B3-35 (2/52)

Longilinea arvoryzae (AB243673)Anaerobic dechlorinating consortia clone:SHA-105 ( AJ249113)

Chlorofexi

100

99

97

97

99

100

93

g ( )OS clone B1-74 (11/74)

Anaerolinea thermophila (AB046413)OS clone B1-13 (10/74)

Petroleum reservoir clone:11bR (GU129077)EC clone B2-56 (5/82)Anaerobic terephthalate-degrading consortium clone:TA19 (AF229792)Uncultured Synergistes sp. (EU721788)

Syn

ergiste

84

100

99

100

8399

Methanogenic UASB reactor treating brewery wastewater consortium clone:39 (DQ478741)EC clone B2-30 (2/82)

etes

Uncultured Thermodesulfovibrio sp.clone:B19_otu15 (DQ097680)Thermodesulfovibrio islandicus (T) clone R1ha3 (X96726)

Thermophilic phenol-degrading clone:TPD-27 (AY862525)OS clone B1-07 (2/74)

Nitrospira

Canadian low-temperature biodegraded oil reservoir clone:PL-35B7 (AY570625)

100

99

100

93 ET clone B3-43 (16/52)Residual oil degrading methanogenic consortium clone:lg1a03 (EU037969)

Residual oil degrading methanogenic consortium clone:lg1g03 (EU037978)Clostridium sp. 9B4 (AY554416)EC clone B2-45 (39/82)

Thermophilic phenol degrading methanogenic consortia: TP34(EF198045)

Firmicutes

Iron-reducing bacterium enrichment culture clone HN-HFO4 (FJ269084)

99

93

88

98

10077

Uncultured Coriobacteriales bacterium clone AMBC4 (AM935730)Hydrocarbon-degrading methanogenic consortia clone:Nap1-8C (EU522663)EC clone B2-05 (2/82)

Actinobacteria

EC clone B2-80 (2/82)Methanogenic coal enrichment culture consortia clone:ObedB-2A (EU073764))

Mesophilic phenol-degrading methanogenic consortia clone:MP16 (EF198027)

Spiroch

aetes

Thermotogales bacterium MesG1Ag4.2.16S.B (HM003109)

100

99

100

83

100

Dechlorinating microbial communities clone:Er-LLAYS-110 (EU542543)ET clone B3-01 (8/52)Alaskan mesothermic oil reservoir clone D004023B03 (EU721797)

Thermotogae

Thermophilic phenol-degrading methanogenic consortia clone: TP11 (EF198038)Candidate division OP8 clone: OPB95 (AF027060)OS clone B1-04 (1/74)Geothermal spring mat consortium clone:DTM39 (EF205501)

OP8

99

100

100

OS clone B1-72 (1/74)EC clone B2-77 (2/82)

Toluene-degrading methanogenic consortium bacterium clone:Eub 2 (AF423182)Uncultured eubacterium WCHB1-56 (AF050605)

OS clone B1-46 (4/74)Uncultured candidate division OP11 bacterium ( FJ82192)

OP11

100

100

100

99

2%

FIG. 3. (B) Phylogenetic tree of the Chloroflexi, Firmicutes, Actinobacteria, Thermotogae, Synergistetes, Nitrospira, Spirochaetes, OP8 and OP11 16S rRNAgene phylotypes of sequence types (shown in bold) retrieved from the oily sludge and methanogenic enrichment cultures (EC) without addition of carbon or energysources and enrichment transfer (ET) cultures. Type sequences retrieved from GenBank database are shown with their accession number. Putative divisions arelisted on the tree. Alignments to related sequences were performed with MEGA 4 software. The topology of the tree was obtained by the neighbor-joining method.Bootstrap values (n = 1000 replicates) of ≥75% are reported. Scale bar = nucleotide changes per site.

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METHANOGENIC ALKANES DEGRADATION 723

related to Syntrophus sp. clone B1, all of them were closely re-lated to Syntrophus spp. retrieved from a hexadecane-degradingmethanogenic community (Zengler et al. 1999). Firmicutes rep-resented by OTU B3-43, which was closely related to the clonePL-35B7 obtained from Canadian low-temperature biodegradedoil reservoir community (Grabowski et al. 2005).

Within the Thermotogae group, OTU B3-01 was closelyrelated to Thermotogales spp. obtained from temperatehydrocarbon-impacted sites as well as from mesothermic oilreservoirs (Nesbo et al. 2010). Members of the Chloroflexi rep-resented by OTU B3-23 and OTU B3-35 were closely relatedto clone Eub 4 obtained from toluene-degrading methanogeniccommunity (Ficker et al. 1999) and to clone SJA-61 re-trieved from an anaerobic trichlorobenzene-transforming mi-crobial consortium (von Wintzingerode et al. 1999).

To provide additional evidences on the microbial communi-ties capable of anaerobic degradation of petroleum hydrocar-bons, functional genes associated with the anaerobic degrada-tion of petroleum hydrocarbons (assA and bssA) were assessedin oily sludge and two methanogenic hydrocarbon-degradingcultures (Fig. 4). The results showed that bssA genes were onlyidentified in the EC whereas genes encoding alkylsuccinate syn-thase (assA) could only be detected in the ET amended with n-alkanes. Three bssA genotypes were identified from sequences

of the 52 clones, of which one genotype was most similar tothat of Thauera aromatica and the two other genotypes be-long to novel clades. Five assA genotypes were delineated fromthe sequences of the 79 clones, of which one genotype wasmost similar to that of Desulfatibacillum alkenivorans AK-01(Callaghan et al. 2008) and the other four genotypes belong tonovel clades.

DISCUSSIONMethanogenic degradation of petroleum hydrocarbons in

subsurface environments as well as in laboratory enrichmentcultures is a very slow process (Zengler et al. 1999; Jones et al.2008). An active alkanes-degrading methanogenic consortiumderived from a methanogenic oily sludge that produced methaneover a period of 500 days was successfully established in theenrichment transfer (ET) cultures amended with a mixture of n-alkanes (C15-C20), although the methanogenesis from n-alkanes(C15-C20) showed comparatively small amounts of methanecompared to theoretically predicted. Noteworthy, methanogen-esis was still in the phase of fast increasing after 500 days ofincubation in ET (Figure 1B), indicating that enough electrondonors were still present to support the growth and activity ofthe methanogenic community.

Oil contaminated aquifer sediment clone Fort Lupton OTU2 assA (ADJ51092)Oil contaminated aquifer sediment clone Fort Lupton OTU3 assA (ADJ51093)

97

76 Oil contaminated aquifer sediment clone Fort Lupton OTU3 assA (ADJ51093)Methanogenic degradation of Paraffin clone SDB OTU4 assA (ADJ51091)

Methanogenic degradation of Paraffin clone SDB OTU1 assA (ADJ51090)ET clone assA 55 (2/79)ET clone assA 42 (2/79)

100

99

ET clone assA 42 (2/79)ET clone assA 65 (73/79)ET clone assA 57 (1/79)

Oil contaminated aquifer sediment clone Fort Lupton OTU1 assA (ADJ51073)Methanogenic degradation of Paraffin clone SDB OTU2 assA (ADJ51074)

assA

89

100

100

99 Methanogenic degradation of Paraffin clone SDB OTU2 assA (ADJ51074)Oil contaminated river sediment clone Arthur Kill OTU3 assA (ADJ51072)Oil contaminated river sediment clone Arthur Kill OTU2 assA (ADJ51071)Desulfoglaeba alkanexedens assA (ADJ51097)Methanogenic degradation of Paraffin clone SDB OTU5 assA (ADJ51087)

10099

99

Methanogenic degradation of Paraffin clone SDB OTU5 assA (ADJ51087)Desulfatibacillum alkenivorans (strain AK-01) assA1 (ABH11460)ET clone assA 36 (1/79)

EC clone bssA40 (2/52)Thauera aromatica (AF113168)88 Thauera aromatica (AF113168)

Azoarcus sp. DN11 (AB285034)EC clone bssA16 (1/52)

EC clone bssA12 (49/52)Geobacter toluenoxydans bssA (EF123666)

bssA99 Geobacter toluenoxydans bssA (EF123666)

Geobacter grbiciae bssA (EF123664)SIP bssA clone LA07bs07 (GU133298)SIP bssA clone LA07bs24 (GU133309)SIP bssA clone LA07bs12 (GU133300)

100

99100

SIP bssA clone LA07bs12 (GU133300)

5%

FIG. 4. Neighbor-joining dendrogram of deduced amino acid sequences of assA/bssA genes from the methanogenic hydrocarbon-degrading enrichment cultures(EC) and enrichment transfer (ET) culture. Alignments to related sequences (shown with accession number) were performed with MEGA 4 software. The topologyof the tree was obtained with the neighbor-joining method. Bootstrap values (n = 1000 replicates) of ≥75% are reported. Scale bar = amino acid changes per site.

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724 L. Y. WANG ET AL.

This observation is supported by the detection of (putative)metabolite intermediates (long-chain fatty acids) as well asmethanogenic precursors (H2, formate and acetate) in ET, sug-gesting that 500-days was not longer enough for stoichiometricconversion of majority of the alkanes to methane. Due to theavailability of carbon sources, microbial community structuresdisplayed some differences between EC and ET.

Bacterial Community Structure Shift Duringthe Methanogenesis

Bacterial population displayed some differences in culturewith different interventions. Bacterial DNA sequences were an-alyzed based on the percentage representation of major phylumin clone libraries generated from the three different stages (Fig-ure 5A). The EC was characterized by the dominance of mem-bers of the Firmicutes and Chloroflexi, which mostly consists ofclone sequences from methanogenic oil/aromatic/alkanes degra-dation (Figure 3B). These typical fermentative bacteria (Firmi-cutes and Chloroflexi) were also identified in ET but exhibiteddifferent degradation activities for different substrates.

The order Clostridiales, the most commonly encounteredbacterial taxon in petroleum reservoir (Gray et al. 2010), withinFirmicutes has recently been speculated to be able to oxidizehydrocarbon or probably utilize various hydrocarbon intermedi-ates to generate methanogenic precursors (Kunapuli et al. 2007;Gieg et al. 2008); Chloroflexi spp. are recognized as obligateanaerobic group and also present at high frequency in manyhydrocarbon-impacted environments, and such organisms havebeen speculated to be responsible for the activation and primaryfermentation of toluene (Ficker et al. 1999; Gray et al. 2010).

In addition, this group of strictly anaerobic fermentative or-ganisms is able to use a variety of short-chain fatty acids to pro-

duce hydrogen/acetate/carbon dioxide and was found to formsyntrophic relationship with hydrogenotrophic methanogens(Sekiguchi et al. 2001). Thermotogales, however, have alwaysbeen recognized as thermophiles or hyperthermophiles, butmesophilic Thermotogales commonly known as “Mesotogas”have been found in temperate hydrocarbon-impacted sites aswell as in mesothermic oil reservoirs and also identified in ET,suggesting that these bacteria may also be metabolically activein the anaerobic degradation of oil hydrocarbons (Nesbo et al.2010).

Microorganisms phylogenetically related to the genus of Syn-trophus were suggested to initiate the activation of alkanesto produce lower molecular weight fatty acids and hydrogen(Zengler et al. 1999; Jones et al. 2008). Interestingly, Syntro-phus spp. appeared to be the most abundant bacteria in ET andthus we speculate that the initial attack of the alkanes could beby clones related to this group. Candidate division OP11 waspresent in OS and EC bacterial clone libraries, but it has nocultured representatives. This clade has a wide environmentaldistribution in surface, shallow, and deep subsurface environ-ments (Harris et al. 2004) and has been identified in a range ofhydrocarbon-impacted environments (Dojka et al. 1998; Phamet al. 2009), including toluene-degrading methanogenic con-sortium (Ficker et al. 1999). However, the role of OP11 in ourmethanogenic microcosms remains inconclusive at the moment.

Archaeal Community Structure Shift Duringthe Methanogenesis

Obviously, archaea displayed some differences in its com-munity structures in three samples analyzed in this study. Basedon the archaeal amplicons, analysis was made based on thepercentage representation of major lineage in clone libraries

FIG. 5. Relative contributions of different phylogenetic groups to microbial communities. (A) Relative proportion of archaeal lineages from 16S rRNA genesequences clone libraries constructed from DNA extracted from three different stages. (B) Relative proportion of bacterial lineages from 16S rRNA gene sequenceclone libraries constructed from DNA extracted from three different stages.

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METHANOGENIC ALKANES DEGRADATION 725

generated from the three samples and was shown in Figure 5B.Archaeal OTUs recovered from the OS were closely relatedto methyl compounds utilizer (Methanomethylovorans) and hy-drogenotrophic methanogens (Methanolinea) while in the ECand ET the archaea were characterized by the diverse mem-bers of hydrogenotrophic methanogens (Methanobacterium,Methanolinea, Methanofollis and Methanocalculus) and as wellas acetotrophic methanogens (Methanoseata).

A wide range of substrates at different concentrations mighthave been available in the original oily sludge to supportseveral types of methanogens. It is worth noting that OTUs(68.1%) related to hydrogenotrophic methanogens were pre-dominant in EC while OTUs (64.5%) affiliated with acetotrophicmethanogens dominant in ET through the further incubation andthen addition of n-alkanes. The emergence of Methanosaeta-liketogether with diverse hydrogenotrophic members suggests thatthese methanogens play key role in methanogenic degradationof oily sludge and alkanes.

Interestingly, Methanosaeta-like accounted for approxi-mately 2/3 in the ET. This is in line with the stoichiometryof alkane conversion to acetate and hydrogen suggested byZengler et al. (1999) in which near 2/3 of the methane is gener-ated from acetate and 1/3 from hydrogen, this matches the ratioof acetoclastic vs obligate CO2- reducing methanogens in theET archaeal clone library.

However, there are also data suggesting that this 2/3 to 1/3split in methanogenic pathways has been observed with purealkanes as substrate, but methanogenic crude oil degradation israther dominated by CO2-reduction (Jones et al. 2008). This hasimplications for the differences observed in methanogenic hy-drocarbon degradation in oily sludge relative to the enrichmenton pure alkanes.

Catabolic Genes Shift During the MethanogenicIncubations

Addition of fumarate onto the parent substrate (referred toas fumarate addition) plays a particularly dominant role inthe degradation of both saturated and aromatic hydrocarbons(Heider, 2007). Alkylsuccinate synthase (ass) as a key enzymeof anaerobic alkane oxidation has recently been proven to be avaluable functional biomarker gene in hydrocarbon-rich envi-ronments and enrichment cultures (Callaghan et al. 2010). Theinability to detect assA in EC does not mean that assA is notpresent; rather it may indicate that the population of anaerobicalkane’s degraders may be too low compared to the other mem-bers in the community. Similarly, this might also be true for thelack of bssA in the ET.

The presence of bssA in EC indicates that methanogenicaromatic hydrocarbon degradation was a dominant process fromthe hydrocarbons available in the oily sludge and the microbialcommunity had been shifted after the cultures were transferredto ET amended with n-alkanes. This assumption is reflected notonly by the dominance of organisms that have been implicated

in the initial activation of n-alkanes but also by the presence ofassA-like genes in ET.

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