microbial diversity, community composition and metabolic potential in hydrocarbon contaminated oily...

RESEARCH ARTICLE

Microbial diversity, community composition and metabolicpotential in hydrocarbon contaminated oily sludge: prospectsfor in situ bioremediation

Ranjit Das & Sufia K. Kazy

Received: 26 September 2013 /Accepted: 10 February 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Microbial community composition and metabolicpotential have been explored in petroleum-hydrocarbon-contaminated sludge of an oil storage facility. Culture-independent clone library-based 16S rRNA gene analysesrevealed that the bacterial community within the sludge wasdominated by the members of β-Proteobacteria (35 %),followed by Firmicutes (13 %), δ-Proteobacteria (11 %),Bacteroidetes (10 %), Acidobacter ia (6 %), α -Proteobacteria (3 %), Lentisphaerae (2 %), Spirochaetes(2 %), and unclassified bacteria (5 %), whereas the archaealcommunity was composed of Thermoprotei (54 %),Methanocellales (33 %), Methanosarcinales/Methanosaeta(8 %) andMethanoculleus (1 %) members. Methyl coenzymeM reductase A (mcrA) gene (a functional biomarker) analysesalso revealed predominance of hydrogenotrophic, methano-genic Archaea (Methanocellales, Methanobacteriales andMethanoculleus members) over acetoclastic methanogens(Methanosarcinales members). In order to explore the culti-vable bacterial population, a total of 28 resident strains wereidentified and characterized in terms of their physiological andmetabolic capabilities. Most of these could be taxonomicallyaffiliated to the members of the genera Bacillus ,Paenibacillus, Micrococcus, Brachybacterium, Aerococcus,and Zimmermannella, while two strains were identified asPseudomonas and Pseudoxanthomonas. Metabolic profilingexhibited that majority of these isolates were capable of grow-ing in presence of a variety of petroleum hydrocarbons as solesource of carbon, tolerating different heavy metals at higherconcentrations (≥1 mM) and producing biosurfactant during

growth. Many strains could grow under a wide range of pH,temperature, or salinity as well as under anaerobic conditionsin the presence of different electron acceptors and donors inthe growth medium. Correlation between the isolates and theirmetabolic properties was estimated by the unweighted pairgroup method with arithmetic mean (UPGMA) analysis.Overall observation indicated the presence of diverse groupsof microorganisms including hydrocarbonoclastic, nitrate re-ducing, sulphate reducing, fermentative, syntrophic, methan-ogenic and methane-oxidizing bacteria and Archaea withinthe sludge community, which can be exploited for in situbioremediation of the oily sludge.

Keywords Microbial community . Oily sludge .

Hydrocarbon .Bioremediation .16SrRNAgene .mcrAgene .

Diversity

Introduction

Management of huge quantities of oily sludge generated bypetroleum industries is a critical problem worldwide as im-proper handling of sludge may lead to severe environmentalpollution caused by its constituents, which include complexmixture of aliphatics, aromatics, nitrogen, sulphur, oxygen-containing compounds, asphaltenes, and metals (Militon et al.2010; Reddy et al. 2011). Various physicochemical methodsare available for the treatment of oily sludge, although manyof these technologies are costly, energy intensive, inefficientand not eco-friendly. Bioremediation technology based onnatural microbial population of contaminated sites has beenrecognized as a sustainable, economic, environmentallyfriendly and versatile alternative clean-up strategy(Cerqueira et al. 2011; Gillespie and Philp 2013). The prevail-ing physicochemical factors in the contaminated sites deter-mine the microbial population that can survive and flourish in

Responsible editor: Gerald Thouand

R. Das : S. K. Kazy (*)Department of Biotechnology, National Institute of Technology,Durgapur, Mahatma Gandhi Avenue, Durgapur 713 209, WestBengal, Indiae-mail: [email protected]

Environ Sci Pollut ResDOI 10.1007/s11356-014-2640-2

such harsh environments utilizing the contaminants, andtherefore, understanding of the inhabitant microbial activitiesin polluted ecosystems has been recognized as essential for themanagement of such environments (Kleinsteuber et al. 2012;Zhang et al. 2012; Gillespie and Philp 2013). Microbial com-munity profiling and identifying microorganisms playing keyrole in contaminant degradation could be useful in definingbioremediation strategies. Promoting the activity of the indig-enous microbial community (biostimulation) or introducingisolated microorganisms (from exogenous or endogenoussources, bioaugmentation) with appropriate degradation ca-pacity into the contaminated environment could acceleratebioremediation efficiency (Cerqueira et al. 2011; Almeidaet al. 2013).

Powerful culture-independent molecular techniques havebeen applied for characterizing complex microbial assem-blages and their metabolic potentials, which have yieldedamazing insights into the microbial community compositionand function in a variety of environments including oil-contaminated sites (de Vasconcellos et al. 2009; Chouariet al. 2010; Militon et al. 2010; van der Kraan et al. 2010;Mayumi et al. 2011; Hazen et al. 2013; Daffonchio et al.2013). Investigators have reported diverse groups of nitrate-reducing, sulphate-reducing, fermentative, iron-reducing,syntrophic and methanogenic bacteria from oil-associatedenvironments (Liu et al. 2009; van der Kraan et al. 2010;Ren et al. 2011; Kobayashi et al. 2012). In recent years,Archaea have gained intensive focus due to their abundancein almost every ecosystem, and investigators have demonstrat-ed that archaeal community structure and function are morecomplex than expected before. Archaea have been reportedfrom oil-containing environments including petroleum reser-voirs, oil fields, underground crude oil storage cavities andhydrocarbon-polluted aquifers and have been found to beinvolved in mineralization of petroleum hydrocarbons, espe-cially through methanogenesis under anaerobic conditions(Kleikemper et al. 2005; van der Kraan et al. 2010; Mayumiet al. 2011; Kobayashi et al. 2012; Zhang et al. 2012).Methanogenic Archaea has been known to express methylcoenzyme M reductase (MCR), which catalyzes the terminalstep in biogenic methane production. The methyl coenzymeM reductase A (mcrA) gene, encoding the alpha subunit ofMCR, has been recommended as a functional biomarker ofmethanogenesis and methanogenic Archaea (Luton et al.2002; Dhillon et al. 2005; Buriánková et al. 2013).

Conventional culture-dependent approaches have alsobeen extensively used for isolating different microorganismsfrom contaminated habitats and for assessing their physiolog-ical and metabolic potentials (Kostka et al. 2011; Ferrera-Rodríguez et al. 2013). The exploitation of inhabiting micro-organisms with proven degradation potentials and survivabil-ity in the contaminated environment is crucial for a successfulbioaugmentation-based bioremediation strategy (Almeida

et al. 2013). In this study, we have gained an insight on thecomposition and activity of archaeal and bacterial communi-ties based on 16S ribosomal RNA (rRNA) and mcrA geneclone library analyses in petroleum-hydrocarbon-contaminated sludge obtained from an oil storage facilitylocated at Durgapur, West Bengal, India. Culturable bacterialmembers were identified and characterized in terms of theirpotentials to grow in the presence of different hydrocarbons/compounds as sole source of carbon during growth, to growunder anaerobic conditions utilizing different electron accep-tors and over a wide range of temperature and pH, to producebiosurfactant and to tolerate various heavy metals duringgrowth. Our observations provide valuable insights on archae-al and bacterial communities in hydrocarbon-contaminatedoily sludge and on their metabolic potentials, which can beexploited for in situ bioremediation of the sludge.

Materials and methods

Sample collection, physicochemical analysis and enumerationof microorganisms

Oil sludge was collected in sterile screw-capped glass amberbottles (1-L capacity) from sludge storage tank of an oilreservoir facility of the Bharat Petroleum CorporationLimited, located at Rajbandh, Durgapur (23° 48′ N, 87° 32′E), India, and immediately stored on ice. Physicochemicalparameters including pH, temperature, oxidation reductionpotential (ORP), conductivity and salinity of the sample weremeasured on site using an Orion Star 140TM series meter(Thermo Electron Corporation, Beverly, MA). Immediatelyafter reaching laboratory, the samples were kept at 4 °C, andaliquots were used for DNA extraction and microbial countanalysis. Further chemical analyses (total petroleum hydrocar-bon (TPH), aliphatic component, poly aromatic hydrocarbons,anions, metals, etc.) of the sludge were done by S KMitra Pvt.Ltd., Kolkata, India. TPH, aliphatic component and aromatichydrocarbons were analyzed following the method describedby Mishra et al. (2001). Gas chromatographic analysis wasdone using a Varian CP 3800 gas chromatograph (Varian,USA) equipped with flame ionization detector. Authenticstandard (Supelco, Sigma-Aldrich, USA) was used for identi-fication of individual component. Metals and heavy metalswere analyzed following the American Public HealthAssociation APHAmethods (methods 3500 and 3111B) usingatomic absorption spectrometer or titration as applicable.Sulphate, phosphate, nitrate and bicarbonate were estimatedusing spectrophotometric or titrimetric method (as applicable)following the APHA methods (methods 4500 and 2320B).

Enumeration of bacteria was done onReasoner’s 2A (R2A)and mineral salts agar media under aerobic condition. TheR2A medium was comprised of the following ingredients

Environ Sci Pollut Res

(gram per litre of deionized water): yeast extract, 0.5; peptone,0.5; casein acid hydrolysate, 0.5; glucose, 0.5; soluble starch,0.5; Na pyruvate, 0.3; K2HPO4, 0.3; MgSO4·7H2O, 0.05; andagar powder, 15. The composition of mineral salt medium(MSM) was as follows (gram per litre of deionized water):K2HPO4, 0.348; KH2PO4, 0.272; NaCl, 4.68; NH4Cl, 1.07;KCl, 1.49; Na2SO4, 0.43; MgCl2·2H2O, 0.2; CaCl2·2H2O,0.03; yeast extract, 5.0; agar powder, 15; and 2.5 mL of traceelement stock solution that was composed of the followingcompounds (gram per litre of deionized water): EDTA, 0.05;MgSO4·7H2O, 3; MnSO4·H2O, 0.5; NaCl, 1; FeSO4·H2O,0.1; anhydrous CaCl2, 0.1; Al (SO4)2, 0.01; H3BO3, 0.01;Na2MoO4·2H2O, 0.01; and Na2 SeO3, 0.001. Initial pH valuesof the media were adjusted to 7.0–7.2 with NaOH priorsterilization (121 °C and 15 lb seq−1 inch for 15 min). Filter-sterilized trace element solution was added to the sterileMSM. Inoculated agar plates were incubated at 30 °C andcolony-forming units (CFUs) were determined after 72 h.

Total community DNA extraction and PCR amplificationof 16S rRNA and mcrA genes

Total community DNA was extracted from the sludge(100 mL) using the Epicenter Water DNA extraction kit(Epicenter, Madison, WI, USA) according to the manufac-turer’s instructions. Bacterial and archaeal 16S rRNA geneswere amplified by polymerase chain reaction (PCR) using thecombination of respective universal primer pairs 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -TACGGYTACCTTGTTACGACTT-3 ) for bacteria (Lane 1991)and Arch21F (5 -TTCCGGTTGATCCYGCCGGA-3 ) andArch958R (5 -YCCGGCGTTGAMTCCAATT-3 ) forArchaea (DeLong 1992). The 50-μL PCR reaction mixturecontained 2 μL of template DNA (50 ng μL−1), 10 pmol ofeach of the oligonucleotide primer, 2.5 mMMgCl2, 5 μL 10×PCR reaction buffer (200 mM Tris–HCl, 100 mM KCl, pH8.4), 0.2 mM each dNTP and 1.5 U Taq DNA polymerase(Fermentas, USA). For archaeal amplification, PCR mastermix contained additional 2-μL BSA (20 mg mL−1). Thermalcycling condition for bacterial primers was as follows: aninitial denaturation at 95 °C for 5 min, followed by 35 cyclesof 94 °C for 30 s, annealing at 58 °C for 45 s and extension at72 °C for 90 s followed by a 10-min final extension at 72 °C.Archaeal 16S rRNA gene was amplified with the followingcondition: an initial denaturation at 95 °C for 3 min followedby 35 cycles of denaturation at 94 °C for 45 s, annealing at55 °C for 50 s and elongation at 72 °C for 60 s. The finalelongation step was extended to 10 min at 72 °C and incuba-tion at 4 °C until further processing. Fragments of mcrA genewere amplified using the specific primer pair ME1 (5 -GCMATGCARATHGGWATGTC-3 ) and ME2 (5 -TCATKGCRTAGTTDGGRTAGT-3 ) (Hales et al. 1996).The reaction condition was as follows: an initial denaturation

at 94 °C for 3 min, followed by 35 cycles of 94 °C for 60 s,annealing at 48 °C for 50 s and extension at 68 °C for 60 sfollowed by a 7-min final extension at 68 °C and incubated at4 °C. Amplified PCR products were analyzed on 1 % agarose(Sigma-Aldrich, USA) gel running in 1× TAE buffer andstained with ethidium bromide for visualization under UVtransilluminator. PCR products (1.5 kb for 16S rRNA geneand 0.76 kb formcrA gene) were cut out of the gel and purifiedwith QIAquick gel extraction kit (QIAGEN Ltd., Hilden,Germany), according to the manufacturer’s instructions andresuspended in nuclease-free water.

Clone library construction and analysis of 16S rRNAand mcrA genes

Gel-purified 16S rRNA and mcrA gene products were clonedinto the pGEM-T Easy vector (Promega, Madison, WI) andtransformed into Escherichia coli JM109 competent cells fol-lowing the manufacturer’s instructions. After overnight incu-bation of LB plates containing 40 μg mL−1 of X-gal,24 μg mL−1 of IPTG and 100 μg mL−1 of ampicillin, the whitecolonies were randomly selected as positive clones. Two clonelibraries oil water bacteria (OWB) for Bacteria and archaea oilwater (AOW) for Archaea were constructed using a total of 192and 112 positive clones of Bacteria and Archaea, respectively.A total of 58 positive clones were selected formcrA gene clonelibrary (MCR) construction. For screening of the clone librar-ies, reamplification of PCR products of 16S rRNA and mcrAgene fragments was performed using vector specific primersSP6 (5′-ATT TAG GTG ACA CTATAG-3′) and T7 (5′-TAATAC GAC TCA CTA TAG GG-3′). Positive amplified PCRproducts of 16S rRNA gene were restriction digested by theenzymes HaeIII and MspI for Archaea and MspI and RsaI forBacteria. The mcrA gene PCR products were digested byMspIrestriction enzyme. Each 16-μL reaction volume contained8-μL PCR products, 2 μL 10× buffer, 1.5 U enzyme(Fermentas, USA) and 5.5-μL DEPC-treated water (Sigma-Aldrich, USA). Digestion process was carried out for about14–16 h in 37 °C water bath. All digested products wereanalyzed by 2.5 % agarose (Sigma-Aldrich, USA) gel electro-phoresis. Similar restriction patterns were grouped, and eachgroup was referred as an operational taxonomic unit (OTU)/ribotype or restriction fragment length polymorphism (RFLP)group. At least one representative clone from each dominantOTU/ribotype or RFLP group was selected for sequencing ofthe 16S rRNA or mcrA gene insert. All the clones were storedin 15 % glycerol at −80 °C for future use.

DNA sequencing and phylogenetic analysis

The plasmid DNAwas isolated from selected clones of differ-ent OTUs using HiPura Plasmid DNA extraction kit(Himedia, India) according to the manufacturer’s instructions.


Partial sequences of the 16S rRNA and mcrA gene fragmentswere determined on an automated ABI 3730XL sequencerusing T7/SP6 universal vector specific primers. The 16SrRNA gene s equence s we r e examined by theCHECK_CHIMERA program of the Ribosomal DatabaseProject (RDP-II) and compared with 16S rRNA gene se-quences deposited in GenBank database of the NationalCenter for Biotechnology Information (NCBI) using theBLAST service to determine their approximate phylogeneticaffiliations. Initial classification was made using classifierprogramme in Ribosomal Database Project and Greengenes(http://greengenes.lbl.gov/cgi-bin/nph-index.cgi). The re-trieved 16S rRNA gene sequences from the GenBank werealigned with our sequences by using ClustalW, and phyloge-netic analysis was done by MEGA 4.0 (Tamura et al. 2007)following neighbour-joining methods incorporating Jukes-Cantor distance correction (Jukes and Cantor 1969).Methanospirillum hungatei (M60880) was selected as out-group. Bootstrap analysis with 100 resamplings was per-formed to assign confidence levels to the nodes in the trees.Sequences of mcrA gene were analyzed by BLAST to searchfor the similarity with other relevant sequences available in theGenBank database. The mcrA gene sequences were translatedinto amino acids at Expert Protein Analysis System (SIBExPASy) browsing with website (http://web.expasy.org/translate) and aligned by ClustalW. Phylogenetic tree ofmcrAwas based on comparison of 210–230 amino acids withdistance correlation using parsimony method by MEGA 5.03software. The evolutionary distance was accomplished with1,000 bootstrap analyses used to assign the confidence levelsto the nodes in the tree (Li et al. 2012; Buriánková et al. 2013).

Isolation and identification of culturable bacterial strains

Thirteen bacterial strains were isolated from the oily sludgeusing R2A agar plate following serial dilution technique anddesignated as bharat petroleum durgapur (BPD) strains.Fifteen strains were isolated through enrichment on MSMplate (without yeast extract) supplemented with(100 mg L−1) benzene, toluene, ethylbenzene and xylene(BTEX) mixture or naphthalene or Na benzoate as sole carbonsource and designated as BT, NP and NB strains, respectively.Inoculated agar plates were incubated at 30 °C for 7 daysunder aerobic condition. Colonies were isolated, purified byrepeated subculturing and stored in 15 % glycerol at −80 °Cfor long time preservation of the strains. Genomic DNAwasisolated from the bacterial strains using standard phenol-chloroform extraction procedure (Sambrook et al. 1990).PCR amplification of bacterial 16S rRNA gene fragmentswas performed, and phylogenetic affiliations of the strainswere ascertained by analyzing 16S rRNA gene sequence (first450–500 base) of each isolate as described above.

Metabolic characterization of bacterial isolates

The potential of the isolates to grow in the presence of differ-ent hydrocarbons/compounds as sole source of carbon wasevaluated in MSM. Each strain was incubated at 30 °C for7 days in a liquid medium supplemented with 100 mg L−1 ofthe following hydrocarbons/compounds: benzene (B), toluene(T), ethyl-benzene (E), xylene (X), BTEX mixture, Na ben-zoate, naphthalene, phenol, m-cresol, p-cresol, anthracene,fluorene, phenanthrene, and pyrene. Growth was measuredby recording the optical density of each culture at 600 nm.Heavy metal sensitivity of the isolates was investigated fol-lowing growth at 30 °C for 7 days on agar plates of Tris-minimal medium supplemented with different heavy metalsalts (Cd(NO3)2, Pb(NO3)2, K2Cr2O7, ZnCl2, NiCl2 andCu(NO3)2) at various concentrations ranging from 0.1 to10 mM (Kazy et al. 1999). Growth potential of the isolateswas evaluated in liquid MSM at different pH (3–12), temper-ature (20–40 °C) and NaCl concentrations (0–10 %, w/v).Surfactant production ability of each isolate was examinedusing kerosene with culture supernatant as described byCerqueira et al. (2011). Anaerobic growth potential of thestrains was tested in agar plates of anaerobic medium(HiMedia, India). Multiple electron acceptors utilizing theability of the strains were investigated following the strains'growth at different anaerobic conditions (nitrate-reducing,sulphate-reducing, selenate-reducing and iron-reducing) inanaerobic agar plates of MSM supplemented with differentelectron acceptors (sodium nitrate, sodium sulphate, sodiumthiosulphate, sodium sulphite, sodium selenate and ferricchloride; 20 mM of each) and donors (glucose, 0.05 % orpyrene, 10 mg L−1) (Yan et al. 2012). The pH of the mediumwas adjusted to 7.2 after adding 1 g L−1 of L-cysteine HCl as areducing reagent. All experiments were conducted in an an-aerobic glove box (Coy Laboratories, model 1025 S/N) filledwith 85 % N2, 10 % H2, and 5 % CO2 gases.

Statistical analysis

The estimated sequence coverage of the constructed 16SrRNA gene libraries of both bacteria and Archaea was calcu-lated from the equation C=[1−(n1/N)]×100, where n1 is thenumber of phylotypes appearing only once in the library andN is the total number of clones examined (Good 1953). TheShannon-Weaver index (H') was estimated from the formulaH'=−∑s

i=1 pi ln pi, where pi is the proportion of each phylo-type and i relative to the total number of phylotypes. Therarefaction curves were produced using the AnalyticRarefaction 1.3 program of the Stratigraphy Lab, Universityof Georgia (http://www.uga.edu/~strata/software/index.html).Correlation between the strains with metabolic and physio-logical properties was estimated by unweighted pair groupmethod with arithmetic mean (UPGMA) analysis, and


resemblance dendrogram was drawn using the percentageidentity matrix. The UPGMA analysis and dendrogram gen-eration were done using the Multivariate Statistical Package(MVSP, version 3.1).

Nucleotide sequence accession numbers

The sequences of bacterial and archaeal 16S rRNA genesobtained from this study have been deposited in theGenBank database under the following accession numbersJF331637-JF331653, JF773284-JF773307, JN022447-JN022469, JN377807-JN377824, JN225425, HQ263257-HQ263265 and JQ420131.

Results and discussion

Physicochemical properties of sludge and enumerationof microorganisms

Physicochemical characteristics of the sludge were analyzedto assess the prevailing environmental conditions available tothe inhabitant microbial community (Table 1). The pH (6.3)and temperature (30.2 °C) of the sludge were within the rangeat which most of the microorganisms grow well with effectivecatabolic activities. Negative ORP value (−189 mV) indicatedthe prevalence of reducing the environment within the sludgepit. Presence of sulphate (116.53 mg L−1), nitrate(1.45 mg L–1) and bicarbonate (78 mg L−1) could be correlat-ed with the available electron-accepting regimes.Conductivity value (920 μS cm−1) indicated the abundanceof cations and anions within the sludge, which is also sup-ported by the presence of different metals and anions withinthe sludge. However, the salinity of the sample was low(0.451 g L−1). TPH content was around 54 % of the sludgevolume, wherein aliphatic component was measured at around35 %. Among the poly aromatic hydrocarbons (PAHs), naph-thalene, fluorine, phenanthrene, anthracene and pyrene weredetected in less amounts. Overall physicochemical parametersindicated that the sludge sample is slightly acidic with lowsalinity, moderate temperature and different reducing regimes,which have been correlated earlier with methanogenic envi-ronment (Kobayashi et al. 2012). Cultivable aerobic, hetero-trophic bacteria were enumerated by CFU counts on differentmedia (R2A and MSM). The R2A medium yielded higherCFU counts (4.10±0.424×106 mL−1) compared with thecounts obtained using MSM (1.25±0.212×106 mL−1).

Molecular analysis of microbial diversity

In order to investigate the diversity of bacterial and archaealpopulations in the sludge, community DNA was extracted,nearly full-length 16S rRNA genes were amplified using either

bacterial or archaeal specific primer set, and two clone libraries(OWB, for bacteria and AOW, for Archaea) were constructed.Bacterial clone library was prepared using 192 clones andarchaeal library was made with 112 clones. Based on amplifiedribosomal DNA restriction analysis (ARDRA), clones of bac-terial library were grouped into 67 distinct OTUs (or ribotypes),whereas archaeal clones were grouped into 21 unique OTUs(Table 2). Rarefaction analysis using the ARDRA data showednear saturation of OTU numbers indicating satisfactory cover-age of these libraries (Fig. 1). High Shannon diversity index (H)and equitability (E) for bacterial library (H=3.819; E=0.908)indicated high diversity of bacterial populations compared withthat of the archaeal members in the sludge (Table 2).

Phylogenetic analysis

Phylogenetic diversity and affiliation of microbial groupswere ascertained using partial sequence of 16S rRNA gene

Table 1 Physicochemical characteristics of the oily sludge

Parameters Results

Temperature 30.2 °C

pH 6.3

ORP (mV) −189Conductivity (μS cm−1) 920

Salinity (g L−1) 0.451

Total N (mg L−1) 598

Total P (mg L−1) 41

Total S (mg L−1) 300

Total organic carbon (%, v/v) 3.6

SO42− (mg L−1) 116.53

PO43− (mg L−1) 5.02

NO32− (mg L−1) 1.45

HCO3− (mg L−1) 78

Fe (mg L−1) 49

Na (mg L−1) 167

Mg (mg L−1) 21

K (mg L−1) 31

Pb (mg L−1) 0.6

Cd (mg L−1) 0.3

Mn (mg L−1) 1.22

Total petroleum hydrocarbons (%, w/v) 54.82

Poly aromatic hydrocarbons (mg L−1)

Naphthalene 0.76

Fluorene 0.0036

Phenanthrene 0.002

Anthracene 0.002

Aliphatic components (%, w/v) 34.64

Microbial counts

R2A agar medium (CFU mL−1) (4.10±0.424)×106

MS agar medium (CFU mL−1) (1.25±0.212)×106


of one or more representative clone(s) from nearly allribotypes (OTUs) of both the clone libraries (bacterial andarcheal). From bacterial library (OWB), 47 OTUs, coveringalmost 90 % of the library were identified (Table 3), while 16ribotypes were identified from archaeal library covering al-most 96 % of the library (Table 4). Phylogenetic trees wereconstructed incorporating sequences retrieved from our sam-ple as well as from various hydrocarbon-containing sites(Figs. 2a, b and 3a). Proteobacteria members were predomi-nant (almost 50%) in the bacterial community, and within thisphylum, β-Proteobacteria was the most dominant (35.1 %)followed by δ-Proteobacteria (11.4 %) and α-Proteobacteria(3.1 %). However, a representative of γ-Proteobacteria wasnot detected within this sample, which could also be due totheir poor abundance in the sludge microbiota. The absence(or poor abundance) of γ-Proteobacteria might be correlatedwith the low salinity, nutrient limitation and presence ofhigher concentrations of toxic hydrocarbons within the sludgeas suggested by previous investigators (LaMontagne et al.2004; Gerdes et al. 2005; Hernandez-Raquet et al. 2006;Popp et al. 2006). Among the other phyla, Firmicutes(12.8 %), Bacteroidetes (10.3 %), Acidobacteria (6.2 %),Lentisphaerae (2.5 %), Spirochaetes (2.1 %), Chloroflexi(0.5 %) and unclassified members (5 %) were detected(Fig. 4a). The archaeal library (AOW) was constituted by themembers ofCrenarchaeota (53.4%) andEuryarchaeota (42%).Phylum Crenarchaeota was found to be solely represented byThermoprotei members, while phylum Euryarchaeota was rep-resented by the members of Methanocellales (33.1 %),

Methanosarcinales /Methanosaetaceae (8 %) andMethanomicrobiales/Methanoculleus (1 %) (Fig. 4b).

Proteobacteria

Four most abundant (OWB-18, -186, -132 and -99) and fewless abundant ribotypes (OWB-138, -26, -33, -176, -71, -47, -107, -4 and -80) were affiliated to β-Proteobacteria (Fig. 2a).Among these ribotypes, several (OWB-186, -176, -99, -80, -71, -47 and -18) showed strong lineage with unculturedRhodocyclaceae member (forming a distinct clade with100 % bootstrap support) as well as with well-knownhydrocarbon-metabolizing bacterium Thauera aromatica(X77118) of Rhodocyclaceae family. Rhodocyclaceae mem-bers have been known to possess versatile metabolic capabil-ities including ability to grow under oligotrophic conditionsand also found to be abundant in petroleum-associated envi-ronments (Ren et al. 2011). Particularly, Thauera andAzoarcus genera under this family were found to degrade alarge number of aromatic compounds under both aerobic andanaerobic conditions using different sets of enzymes.Rhodocyclaceae members have also been known to utilizedifferent substances like nitrate, perchlorate, Fe(III) and othermetals as electron acceptors during biodegradation of aromat-ic hydrocarbons under anaerobic conditions (Ding et al. 2008;Mao et al. 2010). The third most abundant ribotype (OWB-132) and two minor ones (OWB-138 and -107) showed stronglineage with Janthinobacterium andHerbaspirillum genera ofβ-Proteobacteria, which have previously been reported ascarbazole (N-heterocyclic aromatic compound derived fromcoal tar and shale oil)-degrading organisms or associated withchlorophenol-contaminated soil sediment, respectively (Imet al. 2004; Inoue et al. 2005). Three OTUs (OWB-26,OWB-33 and OWB-4, together with 3.7 % library coverage)showed s t rong l ineage wi th (99 % boo t s t r ap )Comamonadaceae family members including Brachymonaspetroleovorans. Previous investigators have demonstratedabundance of a romat ic hydrocarbon degrad ingB. petroleovorans strains in petroleum-contaminated sedi-ments and refinerywastewater. This organismwas also knownto grow and utilize a range of light hydrocarbons (C5–C10),aromatic compounds (toluene, m-cresol) or gasoline, whichindicated the role of this bacterium in environmental degrada-tion of hydrocarbons (Rouviere and Chen 2003; Allen et al.2007; Lors et al. 2010). Sequences representing two lessabundant ribotypes (OWB-150 and -78) could be affiliatedto α-Proteobacteria with close lineage to Rhodovarius andRhodobacter spp. Previous investigators have reported theassociation of Rhodobacter-type organisms with PAH andcrude oil degradation in marine environments. AlsoRhodobacteraceae members were found as the key playersfor oil degradation in the Gulf of Mexico beach sands impact-ed by deepwater horizon oil spill (Melcher et al. 2002; Kostka

Table 2 Diversity parameters of bacterial and archaeal clone libraries

Diversity estimate Bacteria Archaea

Total number of clones 192 112

Number of distinct OTUs 67 21

Sequence coverage (%) 90 96

Shannon diversity index (H) 3.819 2.299

Simpson's index (1/D) 3.278 6.024

Equitability (E) 0.908 0.755

0 50 100 150 2000

10

20

30

40

50

60

70

Num

ber

of O

TU

s

Number of clones

BacteriaArchaebacteria

Fig. 1 Rarefaction analysis of 16S rRNA gene clones for different OTUsof bacterial and archaeal library retrieved from oily sludge sample


Table3

Phylogeneticaffiliatio

nsof

OTUsin

thebacterialclone

library

retrievedfrom

oily

sludge

No.of

OTUs

No.of

clones

(%coverage)

Closestphylogeneticaffiliatio

n(A

ccession

no.)

Identity(%

)Ph

ylum

Source

ofclosestaffiliation

645

(23.1)

UnculturedRhodocyclaceaeclone(EU266828)

100

β-Proteobacteria

Tar-oil-contam

inated

aquifer

11(0.5)

Thaueraarom

atica(X

77118)

93β-Proteobacteria

Isolateundernitrate-reducing

condition

110

(5.2)

Janthinobacteriumlividum

(EU275366)

99β-Proteobacteria

Rhizosphere

soilof

Acaciamacracantha

13(1.6)

Herbaspirillum

sp.(AM989099)

100

β-Proteobacteria

Treated

drinking

water

12(1)

Herminiim

onas

arsenicoxydans

(AY728038)

99β-Proteobacteria

Arsenic-richenvironm

ents

37(3.7)

Brachym

onas

petroleovorans

CHX(AY275432)

97–99

β-Proteobacteria

Industrialwastewater

sludge

15(2.6)

Rhodovarius

lipocyclicus

(NR_025629)

99α-Proteobacteria

Industrialhygienecontrolsource

11(0.5)

Rhodobacter

sp.(AM888193)

96α-Proteobacteria

Sedimentsam

ple

24(2)

Syntrophorhabdus

sp.(JN

627945)

99δ-Proteobacteria

Productio

nwater

ofpetroleum

reservoir

11(0.5)

UnculturedTh

ermodesulfobacteriumsp.(GU120562)

100

δ-Proteobacteria

Leaking

oilw

ellclose

toPitchLake

28(4.2)

Syntrophus

sp.(AJ133795)

96–97

δ-Proteobacteria

Anaerobicenrichment

17(3.7)

Smith

ella

sp.(HQ133035)

96δ-Proteobacteria

Crude

oil-contam

inated

soilof

Shenglio

ilfields

12(1)

UnculturedSyntrophaceaebacterium

(FJ898339)

98δ-Proteobacteria

Oilfield

form

ationwater

29(4.6)

UnculturedAcetivibriosp.(EF6

13402)

99Firmicutes

Benzene-m

ineralizingconsortiu

m

12(1)

UnculturedPeptococcaceaebacterium

(EU016416)

90Firmicutes

Anaerobicbenzene-degradingenrichment

12(1)

UnculturedVeillonellaceae

bacterium

(JQ087052)

99Firmicutes

Sedimento

fahydrocarbon-contam

inated

aquifer

28(4.2)

UnculturedFirmicutes

bacterium

(JQ919709)

96Firmicutes

Gasoline-pollu

tedsoil

12(1)

Unculturedbacterium

clone(G

U390116)

97Firmicutes

Anaerobicdigester

treatin

gfeedstock

11(0.5)

UnculturedClostridiabacterium

BTEX(EU522653)

96Firmicutes

Oilsand

tailingssettlingbasin

11(0.5)

Unculturedbacterium

clone(FM206190)

88Firmicutes

Chlorinated

ethene-contaminated

groundwater

419

(9.8)

Bacterium

enrichmentculture

clone(G

U196214)

99–100

Bacteroidetes

Anaerobicdigester

11(0.5)

UnculturedBacteroides

sp.(AY780552)

96Bacteroidetes

Chlorinated

ethene-degrading

cultu

re

312

(6.2)

UnculturedAcidobacteria

bacterium

(AJ617856)

98–99

Acidobacteria

Oxic-anoxicinterphase

offloodedsoil

24(2.1)

Spirochaetaceaebacterium

clone(H

Q133202)

93–95

Spirochaetes

Crude

oil-contam

inated

soilof

Shenglio

ilfields

11(0.5)

UnculturedChloroflexibacterium

(DQ463707)

83Chloroflexi

LakeTanganyika

anoxichypolim

nion

25(2.5)

UnculturedLentisphaeraebacterium

(FM206077)

96Lentisphaerae

Chlorinated

ethane-contaminated

groundwater

12(1)

Unculturedbacterium

clone(CT573834)

98Unclassifiedbacteria

Every

municipalwastewater

treatm

entp

lant

14(2)

Unculturedbacterium

clone(FN643499)


Ricefieldsoilundermethanogeniccondition

14(2)

Unculturedbacterium

cloneKB-1

2(AY780556)


Chlorinated

ethene-degrading

cultu

re


et al. 2011). Eight ribotypes were affiliated to δ-Proteobacteria and among these two (OWB-166 and OWB-182) showed strong lineages with thermophilic anaerobicbacterium clone (AY168740) retrieved from terephthalate-degrading community and with thermophilic, anaerobichydrocarbon-metabolizing bacterium Syntrophorhabdus sp.Another OTU (OWB-116) showed strong lineage (100 %bootstrap support) with thermophilic sulphate-reducing anaer-obic organism Thermodesulfobacterium sp. (GU120562) re-ported from leaking oil well (Jeanthon et al. 2002). Ouranalysis revealed that Syntrophaceae family members areabundant (12 %) in this sludge sample. Several ribotypes(OWB-175, -153, -48, -7 and -96) were found to be stronglyrelated with the members of this family, particularly withSyntrophus and Smithella spp. Similar sequences have beenpreviously retrieved from methanogenic, anaerobichydrocarbon-containing environments (Kasai et al. 2005;Gray et al. 2011). Investigators have provided collective evi-dence for direct role of Syntrophaceaemembers in the activa-tion and oxidation of crude oil alkanes via long-chain fattyacid to acetate and hydrogen in methanogenic environments

(Gray et al. 2011; Mayumi et al. 2011). Members of the genusSyntrophus were known to convert anaerobic hydrocarbondegradation products (propionate, acetate, butyrate, etc.) intoCO2, H2, and formate, which could be utilized byhydrogenotrophic organisms, such as methanogens (Allenet al. 2007; Gan et al. 2012). Another Syntrophaceae genusSmithella has been reported to be directly involved in theanaerobic hydrocarbon degradation in methanogenic environ-ments (Gray et al. 2011). The presence of such syntrophicorganisms indicated that methanogenesis could be a predom-inant process in our sample (Allen et al. 2007).

Table 4 Phylogenetic affiliations of OTUs/RFLP groups in archaeal clone libraries retrieved from oily sludge

Archaealdomain

OTUs/RFLPgroups

No. of clones(% coverage)

Closest affiliation (accession no.) Identity(%)

Putative groups Isolation source of closestaffiliation

16S rRNA gene library

3 6 (5.6) Uncultured Archaeon clone (AB161333) 96 Crenarchaeota Petroleum-contaminated soil

1 2 (2) Uncultured Crenarchaeote clone(GU135499)

96 Crenarchaeota Pavin lake sediment

2 40 (35.8) Uncultured Crenarchaeote clone(EF420184)

99 Crenarchaeota Oil sand tailings pond

2 11 (10) Uncultured Leachate sediment clone(HQ141848)

99 Crenarchaeota Leachate sediment

1 9 (8) UnculturedMethanosarcinales (GU120495) 100 Euryarchaeota Leaking oil well close to Pitch Lake

3 26 (23.2) Uncultured Euryarchaeote clone(AY487177)

94–97 Euryarchaeota Food soil of Cubitermes fungifaber

1 4 (3.6) Uncultured Archaeon clone(GU453446)

97 Euryarchaeota Hydrocarbon-contaminated aquifer

2 7 (6.3) Ammonia-oxidizing Archaeon(FN691494)

86–94 Euryarchaeota Neuston of Lake Llebreta

1 1 (1) Uncultured Methanoculleus sp.(JF808024)

99 Euryarchaeota Oil reservoir from Yabase oilfield

mcrA gene library

2 15 (24.1) Uncultured Methanobacterium sp.(AFD09541)

98 Methanobacteriales Sediment of Lake Monoun

2 18 (30.9) Methanocella conradii HZ254(YP005380187)

94–96 Methanocellales Rice field soil from China

3 8 (13.7) Uncultured Methanocella sp. (AFD09519) 94 Methanocellales Sediment of Lake Monoun

1 3 (5.1) Uncultured methanogenic Archaeon(AER57881)

95 Methanomicrobiales Oil field subsurface soil

1 7 (12.1) Methanoculleus bourgensisMS2(AAL29285)

97 Methanomicrobiales Landfill waste materials

1 4 (6.9) Methanosarcina mazei Gö1 (AAM30936) 99 Methanosarcinales Unknown

�Fig. 2 Phylogenetic tree based on bacterial 16S rRNA gene sequences ofaα-, β-, and δ-Proteobacteria ribotypes and b Firmicutes, Bacteroidetes,Acidobacteria, Spirochaetes, Chloroflexi, Lentisphaerae and unclassifiedbacteria ribotypes retrieved from oily sludge. Trees were constructedusing the neighbour-joining method incorporating Jukes-Cantor distancecorrections. Sequence ofMethanospirillum hungateiwas used as the out-group. Sequences were designated as OWB. One thousand bootstrapanalyses were conducted, and bootstrap values >50 % were indicated atthe nodes. Scale bar=0.05 change per nucleotide position


Herbaspirillum sp. (AM989099)Anthracene-degrading bacterium SBANT22 (HM596196)OWB-138 (JN022451) 1.6%

Janthinobacterium sp. (FJ889626)Janthinobacterium lividum (EU275366)OWB-132 (JF773297) 5.2%

Herminiimonas arsenicoxydans (AY728038)OWB-107 (JN225425) 1%

Pseudorhodoferax soli (EU825700 )Aquaspirillum giesbergeri (AB074522)

Petroleum contaminated aquifer clone (DQ663909)OWB-26 (JF773296) 1.6%Hydrocarbon utilizing clone (AB274848)

OWB-33 (JF773287) 1.6%OWB-4 (JN022469) 0.5%Uncultured Comamonadaceae bacterium clone BS (FJ495224)Brachymonas petroleovorans strain CHX (AY275432)Oil contaminated soil clone FD_17 (DQ984534)

Thauera sp. (GQ215095)Thauera aromatica (X77118)

OWB-80 (JN022461) 0.5%OWB-18 (JF773284) 7.3%OWB-99 (JF773305) 4.2%OWB-176 (JF773300) 1.6%Uncultured Rhodocyclaceae clone D12_22 (EU266828.1 )OWB-71 (JF773303) 1.6%

Dagang oilfield clone (AY770938)OWB-186 (JF773286) 6.8%

Uncultured beta proteobacterium (GU120572)OWB-47 (JN022447) 1.6%

β-proteoba

cteria

Rhodovarius lipocyclicus (NR 025629)OWB-150 (JF773295) 2.6%

Hydrocarbon contaminated aquifer clone WCHB1-87 (AF050533)Rhodobacter sp. (AM888193)

OWB-78 (JN022458) 0.5%Arsenite-oxidizing biofilm clone 30 (AY168740)

α-proteoba

cteria

Thermophilic anaerobic bacterium clone TTA_B25 (AY297973)OWB-166 (JN022454) 1%OWB-182 (JF773299) 1%

Syntrophorhabdus sp. (AB611035)Uncultured bacterium clone BB-B2 (GQ844327)

OWB-116 (JN022456) 0.5%Uncultured Thermodesulfobacterium sp. (GU120562)

OWB-175 (JN022455) 1%Dagang oilfield clone (AY770973)Uncultured Syntrophaceae bacterium clone BNB-526 (FJ898339)

OWB-153 (JN022453) 1.6%Alkane-degrading methanogenic clone L9B-5 (FN646542)

Syntrophus buswellii (X85131)OWB-48 (JF773294) 1.6%

Production water oil reservoir clone L9B-6 (FN646543)Syntrophus sp. (AF126282)

OWB-7 (JF773293) 3.7%Anaerobic sludge digester clone (CT574012)

Smithella sp. (HQ133035)OWB-96 (JN022465) 0.5%Syntrophus sp. (AJ133794)

δ-proteoba

cteria

Methanospirillum hungatei (M60880)

83100

100

100

100

98

70100

96

65

52

99

99

72

100

90

78100

67

90

75

100

82

51

85

99

75

7463

99

88

90

99

81

100

100

65

0.05

a


Firmicutes

Nine ribotypes could be affiliated to phylum Firmicutes cov-ering 12.8 % of the bacterial population. Several OTUs

(OWB-112, OWB-126, OWB-51, OWB-189 and OWB-101) showed strong lineages with Clostridia, which werepreviously reported from oil-associated environments (vander Kraan et al. 2010) as well as from hydrocarbon-

Unc

lass

ifie

d ba

cter

iaAnaerobic sludge digester clone (CT573834)Uncultured MarinegpA bacterium (CU924245)

Leachate sediment clone (HQ184024)OWB-145 (JN022452) 1%

Uncultured bacterium clone KB-1 2 (AY780556)OWB-22 (JF773291) 2%

OWB-103 (JF773306) 2%Uncultured bacterium clone (FN643499)

Victivallis vadensis (NR 027565)Uncultured Victivallaceae bacterium clone (FJ788663)

OWB-15 (JF773289) 2%OWB-59 (JN022463) 0.5%Uncultured bacterium clone (AF296205)

Uncultured Lentisphaerae bacterium clone (FM206077) Len

tisph

aeria

OWB-156 (JN022467) 0.5%Uncultured Chloroflexi bacterium clone (DQ463707)

Sulfate-reducing prokaryotes (EF999396)

Chloroflexi

Spirochaeta smaragdinae (U80597)Treponema sp. (AF320287)

Spirochaetaceae bacterium clone (HQ133201)Uncultured bacterium clone (EF515401)OWB-8 (JF773298) 1.6%OWB-42 (JN022457) 0.5% Sp

irocha

etes

Allisonella histaminiformans (AF548373)Uncultured Veillonellaceae bacterium clone (JQ087052)

Benzene-contaminated groundwater clone (AY214180)OWB-163 (JF773304) 1%

Uncultured Acetivibrio sp. (EF613402)Uncultured bacterium clone (AY214179)OWB-189 (JN377824) 2% OWB-101 (JF773292) 2.6%

Acetivibrio cellulolyticus (L35516)Clostridium clariflavum (AB186359)

Uncultured Peptococcaceae bacterium clone (EU016416)OWB-51 (JN022462) 1%

Lutispora thermophila (AB186360)Clostridium from anoxic bulk soil (AJ229251)

OWB-66 (JN022464) 1.6%OWB-134 (JF773290) 2.6%

Propionate-oxidizing consortia clone (FM956337)Uncultured Bacillus sp. (EU250948)OWB-112 (JN022448) 1%

Clostridium sp. (FJ808611)Desulfotomaculum salinum (AY918122)

Uncultured Pelotomaculum sp. (AY607142)Uncultured Clostridia bacterium clone (EU522653)

OWB-126 (JN022450) 0.5%

Firmicutes

AY571457 Uncultured bacteriumOWB-167 (JN022468) 0.5%

Chlorinated ethenes treated groundwater clone(FM206190)Uncultured Bacteroides sp. (AY780552)

Anaerobic sludge digester clone (CU918036)OWB-183 (JN022459) 0.5%Iron-reducing enrichment culture (DQ676996)

Butyricimonas virosa (AB443949)OWB-54 (JF773301) 5.2%Anaerobic sludge digester clone (CU922674)OWB-144 (JF773302) 3.1%Bacterium enrichment culture (GU196214)

OWB-110 (JN022466) 0.5%OWB-125 (JN022449) 1%Uncultured Bacteroidetes bacterium clone (CU924584 )

Bacteroidetes

Uncultured Acidobacteria bacterium clone (EF457371)Aliphatic hydrocarbon-contaminated soil clone (AM935271)

OWB-168 (JF773307) 1.6%OWB-152 (JF773288) 4.1%OWB-170 (JN022460) 0.5%

Oxic-anoxic interphase of flooded soil clone (AJ617856)Uncultured bacterium clone (FM956893) A

cido

bacteria

Methanospirillum hungatei (M60880)

95100

100

9896

100

70

66

99

69

100

9865

100

99

9377

91

82

100

92

100

99

100

100

99

98

95

90

100

100

100

100

100

100

99

99

82

95

85100

90

100

98

90

96

5096

94

83

66

67

58

60

0.05

b

Fig. 2 (continued)


degrading methanogenic consortia from oil sand tailings set-tling basin of Canada (EU522653). The fermenting Clostridiamembers could hydrolyze and ferment complex substrates toproduce longer chain fatty acids, acetate, CO2, H2, NH4

+ andHS−, and many of these products could be utilized by themethanogenic organisms present within the community (Liuet al. 2011). Sequences of the minor ribotype (OWB-126) alsoshowed lineage with Pelotomaculum, which was found assyntrophic organisms closely associated with hydrogen-consuming organisms known to be involved in interspecieshydrogen transfer (Kleinsteuber et al. 2008). Nearly 5% of thebacterial population (represented by OWB-189 and OWB-101) showed strong affinity with the anaerobic generaAcetivibrio, which were also recovered previously fromhydrocarbon-contaminated groundwater or from anoxic oilrefinery sludge (Kleinsteuber et al. 2008). Ribotype OWB-51 showed identity with uncultured Peptococcaceae bacteri-um clone (EU016416). Peptococcaceaemembers were foundto be involved in anaerobic benzene degradation under iron-reducing or nitrate-reducing condition (Kunapuli et al. 2007;van der Zaan et al. 2012). Two ribotypes (OWB-66 andOWB-134, together representing 4.2 % of the bacterial li-brary) showed close relation with propionate-oxidizing con-sortium member involved in syntrophic oxidation of propio-nate (the major intermediary product in the anaerobic decom-position of hydrocarbons) under anaerobic methanogenicconditions.

Bacteroidetes, Acidobacteria, Lentisphaerae, Spirochaetes,Chloroflexi, and unclassified bacteria

Five sequences (represented by OWB-54, OWB-110, OWB-125, OWB-144 and OWB-183) showed high identity withuncultured Bacteroidetes. Bacteroidetes members have beenreported from sulphate-reducing anaerobic environments andknown to be involved in benzene, toluene and other hydro-carbon mineralization under such conditions (Liu et al. 2009;Müller et al. 2006). Phylum Acidobacteriawas represented bythree ribotypes (OWB-152, -168, and -170) covering morethan 6 % of the bacterial library. Acidobacteriamembers werealso known to be highly adaptable, metabolically flexible andwidely distributed in hydrocarbon-contaminated environ-ments (George et al. 2009; Militon et al. 2010). PhylumLentisphaerae was represented by two ribotypes (OWB-15and -59) that shared close relation among them as well as withanaerobic microorganism Victivallis vadensis and otherLentisphaerae bacterium clone, which have been reportedfrom dichloroethane-contaminated groundwater (Imfeldet al. 2010). Ribotypes OWB-8 and OWB-42 (representingmore than 2 % of the bacterial clone library) showed closerelation to strictly anaerobic Spirochaetaceae members previ-ously reported from hexadecane-degrading methanogeniccommunity of crude oil-contaminated soil of Sangli oil field.

Phylum Chloroflexi was represented by only one ribotype(OWB-156) that is also a close affinity with sulphate-reducing bacteria. Few ribotypes (OWB-103, OWB-22 andOWB-145, together 5 % clone library coverage) were relatedto unclassified bacteria involved in chlorinated ethene-degrading community or syntrophic acetate oxidation undermethanogenic conditions (Imfeld et al. 2010; Gan et al. 2012).

Archaeal community composition

The archaebacterial members were grouped into two differentclusters under phyla Crenarchaeota and Euryarchaeota.OTUs (ribotypes) belonging to Euryarchaeota could be affil-iated to the members under three different orders such asMe t h a n om i c ro b i a l e s , Me t h a n o c e l l a l e s a n dMethanosarcinales. Majority of archaebacterial OTUs(AOW-4, -13, -38, -44, -65, and -92, representing more than30 % of the archaeal library) showed lineage withhydrogenotrophic, methanogenic archaeon as well as withsimilar microorganism under Methanocellales. RibotypeAOW-7, representing 8 % of the archaeal library, showed arelation with uncultured acetoclastic methanogenic organismsunder Methanosarcinales and Methanosaetaceae. RibotypeAOW-15, representing 1 % of the archaeal library, could beaffiliated to methanogenic Methanomicrobiales organism un-der the genus Methanoculleus. Previous investigators havealso shown the abundance of methanogenic Archaea likeMethanosaeta, Methanoculleus, as well as other members ofMethanosarcinales and Methanomicrobiales in various oil-contaminated environments (Kasai et al. 2005; Liu et al.2009; Pham et al. 2009; Liu et al. 2011; Mayumi et al. 2011;Kobayashi et al. 2012). Methanogenesis is a common processin many anaerobic environments playing an important role forthe degradation of hydrocarbons in polluted soils, aquifers andoil reservoirs, thus contributing considerably to the minerali-zation of petroleum hydrocarbons (Kleikemper et al. 2005;Pham et al. 2009; Kobayashi et al. 2012). Methanogenicorganisms have been known to be catabolically dependenton few simple compounds such as CO2, H2, acetate, methanol,formate, etc., which may be produced by the activity offermenting, sulphate-reducing and syntrophic bacteria presentw i t h i n the commun i t y (Mayumi e t a l . 2011 ) .Hydrogenotrophic methanogens (H2 and CO2 utilizers) havebeen known to contribute indirectly to petroleum hydrocarbondegradation by keeping H2 concentrations low, so that fer-mentation of hydrocarbons becomes exergonic and thatfermenting organism can grow (Garcia et al. 2000;Kleikemper et al. 2005). Acetoclastic methanogens (acetateor methanol utilizers) have been known to contribute directlyto petroleum hydrocarbon degradation by removing the endproducts of fermentation (Kleikemper et al. 2005; Gray et al.2011). Our data on archaebacterial community revealed thatamong the methanogenic organisms, hydrogenotrophic


Methanobacterium thermoautotrophicum (B69022)

Methanobacterium formicicum (BAI67099.1)

Uncultured Methanobacterium sp. (AFD09541.1)

MCR-10MCR-6

Acidic peat bog clone (ABN68964.1)

MCR-15MCR-50MCR-5

Methanocella conradii HZ254 (YP005380187.1)MCR-36

MCR-8Methanocella paludicola SANAE (YP003355571.1)

Uncultured Methanocella sp. (AFD09519.1)

Uncultured methanogenic Archaeon (AER57881.1)

Uncultured methanogenic Archaeon (FN565473.1)MCR-21Methanoculleus thermophilus (AAK16834.1)

Methanoculleus bourgensis MS2 (AAL29285)

MCR-7Uncultured Archaeon (ACY42716.1)

Methanosarcina barkeri (E29525)Methanosarcina mazei Go1 (AAM30936.1)MCR-31

Methanopyrus kandleri (U57340.1 )

7083

64

66

68

74

62

7168

92

97

56

92

6299

82

52

97

63

0.5

Metha

nobacteriales

Metha

nocella

les

Metha

nomicrobiales

Metha

nosarcinales

Toluene contaminated wastewater clone (DQ399812)Petroleum contaminated soil Archaeon (AB161337)

Uncultured Archaeon clone (AB161333)AOW109 (JF331652), 1%AOW74 (JF331651), 1%AOW55 (JF331646), 3.6%

Uncultured Crenarchaeote (EU683335)AOW24 (JF331643), 2%

Pavin lake sediment clone (GU135499)Aeropyrum pernix (D83259)

Thermofiliaceae Archaeon (AF255605)Uncultured Thermoprotei Archaeon (HM041917)

Anaerobic Archaeon clone (HM749826)AOW56 (JF331647), 1%AOW72 (JF331650), 9%

Leachate sediment clone Ar3102 (HQ141848)Uncultured Archaeon clone AS-P (HM749826)

Oil sands tailings pond clone (EF420184)AOW6 (JF331638), 1%AOW12 (JF331640), 34.8%

Therm

oprotei

Toluene-degrading methanogenic Archaeon (AF423188)Uncultured Methanosarcinales (GU120495)AOW7(JF331639), 8%

Uncultured Methanosaetaceae (FJ156087)Methanothrix soehngenii (X16932)

Metha

nosarcinales

Hydrogenotrophic methanogenic Archaeon (EU155967)Uncultured Euryarchaeote (AY487177)AOW65 (JF331649), 4.5%

AOW4 (JF331637), 16%AOW92 (JF331653), 2.7%

Methanocella paludicola (NR028164)AOW13 (JF331641), 3.6%Hydrocarbon contaminated aquifer clone (GU453456)

AOW44 (JF331645), 5.3%Ammonia oxidizing Archaeon (FN691494)

Subseafloor sediment Archaeon (EU385654)AOW38 (JF331644), 1%

Metha

nocella

les

AOW15 (JF331642), 1%Uncultured Methanoculleus sp. (JF808024)

Metha

nomicrobiales

Anabaena circinalis (AF247588)

90100

99

95

92

98

71

60

54

94

100

87

95

100

80

51

99

79

91

100

57

52

99

0.05

Crena

rcha

eota

Euryarcha

eota

a

b


methanogens (Methanocellales and Methanomicrobialesmembers Methanocella and Methanoculleus, respectively)were dominant in the oily sludge followed by the presenceof acetoclastic Methanosarcinales and Methanosaetaceaemembers . Hydrogenot rophic Methanoce l la andMethanoculleus organisms have been known to produceme t h an e , u t i l i z i n g H2 and f o rma t e , wh e r e a sMethanosarcinales/Methanosaetaceae members can utilizeacetates, methyl amines and methanol for methane production(Sakai et al. 2008; Pham et al. 2009; Chouari et al. 2010). Gane t a l . ( 2012 ) have r epo r t ed t h a t a c e t o c l a s t i cMethanosarcinales/Methanosaetaceae and hydrogenotrophicMethanomicrobiales andMethanocellalesmembers can act asmethanogenic partners at around 30 °C. In general,methanogens exist in methane-producing consortia that in-clude H2-producing, sulphate-reducing and syntrophic organ-isms, which produce substrates as described above for thesurvival of methanogens (Mayumi et al. 2011). Therefore,our observation on the coexistence of diverse microbialgroups including syntrophic, H2-producing, and sulphate-

reducing organisms in association with methanogenic micro-organisms is reasonable.

Within Crenarchaeota, eight ribotypes (AOW-6, -12, -24, -55, -56, -72, -74 and -109) showed their affiliation withThermoprotei members, which were accommodated in twodistinct clades but could not be affiliated to any known orderunder this phylum. Among these, four ribotypes (AOW-6, -12, -56, and -72) representing almost 46 % of thearchaebacterial library showed their affinity with unculturedanaerobic Thermoprotei sequences retrieved from oil leachatesediment and oil sand tailings pond harbouring diverse me-thanogenic community (Penner and Foght 2010). SeveralOTUs (AOW-109, AOW-74 and AOW-55) showed strongrelation among them and with uncultured archaeon retrievedfrom unsaturated petroleum-contaminated soil (Kasai et al.2005). Crenarchaeota members have been described eitheras chemolithotrophs using ammonia/other reduced inorganiccompounds as energy sources or as chemoorganotrophs usingsimple/complex organic compounds (Lliro´s et al. 2008).However, their huge cosmopolitan distribution in a wide rangeof biogeochemically distinct environments and their complexphylogeny suggested their larger metabolic diversity and eco-physiological flexibility (Borrell et al. 2012). Current evi-dences have also suggested that some Crenarchaeota mem-bers may obtain energy from the anaerobic oxidation of meth-ane using a “dissimilatory” methane-oxidizing process(Biddle et al. 2006; Borrell et al. 2012). Abundance ofCrenarchaeota members in our sludge sample might be indi-cating their role in oxidation of methane produced by associ-ated methanogenic community, which may contribute signif-icantly in the mineralization of hydrocarbon constituents of oilsludge.

Analysis of methyl coenzyme M reductase A (mcrA) gene

Methanogenic community in the oily sludge was further char-acterized by the analysis of a functional marker mcrA geneencoding alpha subunit of the enzyme involved in the finalmethane-forming step during methanogenesis (Luton et al.2002; Buriánková et al. 2013). Restriction pattern analysisof cloned mcrA genes (58 clones) revealed the presence of13 RFLP groups within MCR library. Based on sequenceanalysis, almost 45 % of the mcrA clones were related to theMethanocellales members, 24 % to the Methanobacteriales,17 % to the Methanomicrobiales and 6.9 % to theMethanosarcinales members (Table 4). Such observation isin agreement with the archaeal 16S rRNA gene clone librarydata on sludge community, except that Methanobacterialesmembers were not detected by 16S rRNA gene analysis.Phylogenetic analysis (Fig. 3b) revealed the lineages ofmcrA sequences with the hydrogenotrophic methanogensMethanocella and Methanoculleus under Methanocellalesand Methanomicrobiales, respectively. Methanosarcinales-

Methanomicrobiales (1.0%)

Methanosarcinales/Methanosaetaceae(8.0%)

Thermoprotei (54.0%)

Methanocellales (33.0%)

Bacteroidetes (10.3%)

β-Proteobacteria (35.1%)

α-Proteobacteria (3.1%) δ- Proteobacteria (11.4%)

Firmicutes (12.8%)

Lantisphaeria (2.5%)Spirochaetes (2.1%)Chloroflexi (0.5%)

Unclassified-bacteria (5.0%)

Acidobacteria (6.2%)

a

b

Fig. 4 Relative abundance of a bacterial and b archaeal groups withinrespective clone libraries

�Fig. 3 a Phylogenetic tree of archaebacterial 16S rRNA gene sequencesretrieved from oily sludge. Tree was constructed using the neighbour-joining method incorporating Jukes-Cantor distance corrections. Se-quence of Anabaena circinalis was used as the out-group. Archaealsequences of this studywere designated asAOW.One thousand bootstrapanalyses were conducted, and bootstrap values >50 % were indicated atthe nodes. Scale bar=0.05 change per nucleotide position. b Phylogenetictree of archaebacterial mcrA gene based on translated, partial amino acidsequences. Tree was constructed using the maximum parsimony method.Sequences of this study were designated as MCR. One thousand boot-strap analyses were conducted, and bootstrap values >50 % were indicat-ed at the nodes. Scale bar=0.5 change per amino acid position


related mcrA sequences showed lineages with acetoclasticMethanosarcina, whereas Methanobacteriales-related mcrAsequences showed lineages with hydrogenotrophicMethanobacterium. The abundance of hydrogenotrophicmethanogens in both archaeal 16S rRNA and mcrA geneclone libraries suggested that these microorganisms might beinvolved in hydrogenotrophic methanogenesis within sludgeenvironment.

Characterization of culturable bacteria

Twenty-eight chemoorganotrophic bacterial strains were iso-lated from the oil sludge sample, and their phylogenetic affil-iation was ascertained through 16S rRNA gene sequence

analysis (Table 5; Fig. 5). Majority (more than 46 %) of thebacterial isolates were affiliated to the genus Bacillus. Sevenstrains (BT1, NB3, NB7, NP2, NP3, NP4, and NP5) could beidentified as Bacillus spp., strains NB1 and NB4 as Bacilluscereus, strains NP7 and BPD-15 as Bacillus subtilis and twostrains as Bacillus pumilus (BPD-14) and Bacillus tequilensis(BPD-18). Strains BT3, BPD-11 and BPD-12 (togetherrepresenting ~11 % of the culturable isolates) were identifiedas Paenibacillus spp. Four strains (BPD-1, -3, -6, and BPD-10), together representing more than 14 % of the culturableisolates, were affiliated to Aerococcus spp. of which BPD-10showed 100 % identity with Aerococcus viridians. StrainsBPD-4 and BPD-7 showed 98 % sequence identity withMicrobacteriaceae family member Zimmermannella faecalis.

Table 5 Phylogenetic affiliations of the bacterial strains isolated from oily sludge

StrainID

Accessionno.

Closest BLAST match (accession no.) Identity(%)

Putative groups Source of closest match

BT3 HQ263258 Paenibacillus lautus (GQ284372) 99 Firmicutes Sediment sample of natural spring

BPD-11 JN377814 Paenibacillus sp. DiSca6 (EF195092) 98 Firmicutes Heavy metal contaminated soil

BPD-12 JN377815 Paenibacillus sp. enrichment clone 9 (FJ930068) 98 Firmicutes Fermented composting materials

NP5 JN377823 Bacillus sp.G2DM-51 (DQ416786 ) 99 Firmicutes Landfill site of chromium-contaminated soil

NB3 HQ263262 Bacillus sp. ( DQ299199) 99 Firmicutes Soil sediments around a petroleum filling rig

NP2 HQ263260 Bacillus sp. 3LF 24T (FN666869) 99 Firmicutes Landfill 3-ft-depth soil

NB7 JN377822 Bacillus sp. RCT4 (FJ755945) 99 Firmicutes Effluent treatment plant for tannery wastewater

BPD-15 JQ420131 Bacillus subtilis strain: 318 ( AB513731) 100 Firmicutes Mangrove sediment

NP7 JN377819 Bacillus subtilis strain DX-3 (JN399219) 99 Firmicutes Zhanjiang Dongxing Refinery sewage

BPD-18 JN377818 Bacillus tequilensis SCSGAB0139 (JX315319) 99 Firmicutes Gorgonian coral collected in South China Sea

BPD-14 JN377816 Bacillus pumilus strain NB-1 (EU311215) 99 Firmicutes Solid waste from pesticide factory

NP3 HQ263265 Bacillus sp. MDS01 (EU236670 ) 99 Firmicutes Soil of iron mineral area

BT1 HQ263259 Bacillus cibi strain JG-30 ( AY550276 ) 99 Firmicutes Korean fermented seafood

NP4 HQ263261 Bacillus sp. LS23 (FJ937886) 100 Firmicutes Coastal waters of the South China Sea

NB1 JN377820 Bacillus cereus strain WJL-063 (FJ527559) 100 Firmicutes Soil polluted by chlorpyrifos

NB4 JN377821 Bacillus cereus strain ASK16 (KF256131) 100 Firmicutes Tannery effluent contaminated soil

BPD-1 JN377807 Aerococcus sp. SBL (HM582941) 99 Firmicutes Organic solvent compost soil

BPD-6 JN377811 Aerococcus sp. P3-2 (EU376006 ) 99 Firmicutes Sparasoma viridae in Grenada

BPD-10 JN377813 Aerococcus viridans (DQ386999 ) 100 Firmicutes Marine water

BPD-3 JN377808 Aerococcus sp. SMCC G885 (EU446131) 99 Firmicutes Deep terrestrial subsurface sediment

BPD-4 JN377809 Zimmermannella faecalis IFO15706(AB012591)

98 Actinobacteria Cow faeces

BPD-7 JN377812 Zimmermannella faecalis A11 (AM284993) 98 Actinobacteria Arsenic-contaminated soil

BT4 HQ263257 Micrococcus luteus strain 2PR58-3 (EU440972) 99 Actinobacteria Indian Ocean deep sea water

BPD-5 JN377810 Micrococcus lylae strain DSM20315(NR_026200)

99 Actinobacteria Human skin

NB6 HQ263264 Brachybacterium faecium DSM 4810(NR_074655)

99 Actinobacteria Poultry deep litter

BT2 HQ263256 Brachybacterium sp. PB10 (DQ643203) 98 Actinobacteria Deep-sea sediments

NB2 HQ263263 Pseudoxanthomonas spadix BD-a59(NR_074956)

99 γ-Proteobacteria BTEX-contaminated soil

BPD-16 JN377817 Pseudomonas fluorescens PGPR1 (HQ876462) 100 γ-Proteobacteria Agricultural field with semi-arid conditions


To our knowledge, it is the first report on Z. faecalis associatedwith hydrocarbon-contaminated environments. Strains NB6and BT2 were closely related to the genus Brachybacterium(with 98–99 % maximum identity). Two strains (BT4 and

BPD-5) could be related toMicrococcus luteus (99% identity)and Micrococcus lylae (100 % identity), respectively. Onlytwo gram-negative strains (BPD-16 and NB2) were recoveredfrom the sludge sample and identified as Pseudomonas

Paenibacillus lautus strain TSWCS3 (GQ284372)BT3 (HQ263258)BPD-11 (JN377814)Paenibacillus sp. DiSca6 (EF195092.1)Paenibacillus sp. PRE17 (EU880530)

Paenibacillus sp. SK-60 (FN666506)BPD-12 (JN377815)Paenibacillus barengoltzii strain THWCS9 (GQ284356) P

aeniba

cilla

ceae

NP5 (JN377823)Bacillus sp. G2DM-51 (DQ416786)Bacillus sp. HPC935(DQ299199)NB3 (HQ263262)Bacillus marisflavi strain (NR 025240)NP2 (HQ263260)NB7 (JN377822)

Bacillus licheniformis (DQ345289)BPD-15 (JQ420131)NP7 (JN377819)Bacillus subtilis strain DX-3 (JN399219.1)Bacillus tequilensis strain SCSGAB0139 (JX315319.1)BPD-18 (JN377818)

Bacillus pumilus strain 7-5 (EU912555)Bacillus pumilus strain ST267 (EU167543)BPD-14 (JN377816)

Bacillus sp.MDS01(EU236670)NP3 (HQ263265)Bacillus cibi strain JG-30(AY550276)BT1 (HQ263259)

NP4 (HQ263261)NB1 (JN377820)NB4 (JN377821)Bacillus thuringiensis strain Ss3(JF833089)Bacillus cereus strain ASK16 (KF256131)

Bacillaceae

Aerococcus sp. P3-2 (EU376006)BPD-1 (JN377807)BPD-6 (JN377811)BPD-10 (JN377813)BPD-3 (JN377808)Aerococcus sp. SBL (HM582941)Aerococcus viridans F00565 (DQ386999) A

erococcaceae

BPD-4 (JN377809.2)BPD-7 (JN377812)Zimmermannella faecalis (AB012591)Zimmermannella faecalis A11 (AM284993)

Microba

cteriaceae

BT4 (HQ263257)Micrococcus luteus strain 2PR58-3 (EU440972)BPD-5 (JN377810)Micrococcus lylae strain DSM 20315 (NR_026200)

Micrococcaceae

NB6 (HQ263264)BT2 (HQ263256)Brachybacterium faecium DSM 4810 (FN689567)

Brachybacterium sp. PB10 (DQ643203)Brachybacterium phenoliresistens (DQ822566) Dermab

acteraceae

Pseudoxanthomonas spadix AFH-5 (AM418384)Pseudoxanthomonas sp. BZ39 (HQ588836 )Pseudoxanthomonas spadix BD-a59(NR074956)NB2 (HQ263263) X

anthom

onad

aceae

Pseudomonas aeruginosa strain MW3AC(GQ180118)Pseudomonas putida strain MPR 4 (GQ900590)

BPD-16 (JN377817)Pseudomonas fluorescens PGPR1 (HQ876462)

Pseud

omon

adaceae

Methanospirillum hungatei (M60880.1)

100

8996

100

100

7679

100

100

92

98

99

97

78

100

100

89

82

99

98

83

86

70

100

99

90

9198

70

98

7388

94

99

0.05

Fig. 5 Phylogenetic tree based on 16S rRNA gene sequences of thebacterial isolates retrieved from oily sludge. Tree was constructed usingthe neighbour-joining method incorporating Jukes-Cantor distance cor-rections. Sequence of Methanospirillum hungatei was used as the out-

group. Bacterial isolates were designated as BT, NB, NP and BPD. Onethousand bootstrap analyses were conducted, and bootstrap values >70%were indicated at the nodes. Scale bar=0.05 change per nucleotideposition


fluorescens (BPD-16, 99% identity) and Pseudoxanthomonasspadix (NB2, 99 % identity). The majority of the microbialgroups detected by our culture-independent analysis could notbe recovered by cultivation; however, members of thegenus Bacillus were detected using both the culture-independent and culture-dependent techniques. Otherisolated genera, including Aerococcus, Paenibacillus,Pseudomonas, Pseudoxanthomonas, Zimmermannella,Micrococcus, and Brachybacterium, were not detected byclone library analysis, which could be due to their poorabundance within sludge microbiota or difficulties in DNAextraction or PCR biases as also suggested by Silva et al.(2013). A wide variety of aerobic, facultative anaerobic andanaerobic bacteria (as observed in the present study) have alsobeen recovered by previous investigators from hydrocarbon-contaminated environments worldwide. Such coexistencecould be possible due to the in situ oxygen production throughanaerobic bacterial metabolism or through possible influx ofwater (Silva et al. 2013). Abundance of gram-positive bacteriain the oily sludge could be attributed to their morphological,physiological and metabolic properties that allow them togrow in harsh environments. Such organisms are primarilychemoorganotrophs and can produce spores to survive underinhospitable environments including harsh chemical andphysical conditions (Narancic et al. 2012). Members of thegenus Bacillus have been found as predominant organisms inoil-contaminated environments and also have been known tobe more tolerant to high levels of hydrocarbons due to theirresistant endospores (Cerqueira et al. 2011).

All the isolates were further characterized in terms of theirability to grow in the presence of different hydrocarbons/compounds as sole carbon source in the medium, ability toproduce biosurfactant, heavy metal tolerance and growth re-sponses under various conditions (Fig. 6). Hydrocarbon utili-zation profile revealed that among the isolates, eight strains(B. pumilus BPD-14, B. tequilensis BPD-18, B. subtilis NP7,Z. faecalis BPD-7, Z. faecalis BPD-4, M. lylae BPD-5,M. luteus BT4, and Bacillus sp. BT1) were superior in utiliz-ing all the test compounds as the sole source of carbon andenergy during growth. However, other strains includingP. fluorescens BPD-16, B. subtilis BPD-15, Bacillus sp. NP3,Aerococcus sp. BPD-6, Aerococcus viridans BPD-10,Bacillus sp. NP4, Bacillus cereus NB4, Brachybacterium sp.NB6, Brachybacterium sp. BT2, Paenibacillus sp. BPD-12,Bacillus cereus NB1 and Paenibacillus lautus BT3 were alsocapable to grow in the presence of most of the carbon sourcestested. It was also important to note that all the isolates wereable to grow in the presence of different heavy metals (Cd, Pb,Cr, Zn, Ni, and Cu) at around 1-mM concentration in thegrowth medium, and among them, more than 50 % couldtolerate higher concentrations of almost all the heavy metalstested. Growth pattern at different pH showed that most of theisolated strains confluently grew at neutral to alkaline pH

range, while few (five) of them could grow well at acidic toneutral pH range, and interestingly, two strains (Z. faecalisBPD-7 and Z. faecalis BPD-4) showed good growth through-out the pH range (3–12) tested. Temperature sensitivity testrevealed that most of the strains could grow optimally at 20–30 °C, while few isolates (seven) also exhibited good growthat elevated temperature range (30–40 °C). Salinity of thegrowth medium did not exert negative effect on the growthof the isolates as almost all the strains showed good growtheven above 3 % NaCl concentration in the medium. It wasalso important to note that although all the strains were isolat-ed and grown aerobically, many of them (15 strains), particu-larly members of the genera Bacillus, Paenibacillus andAerococcus, were able to grow at anaerobic condition and toutilize multiple electron acceptors during anaerobic growth.Surfactant production was observed in the culture supernatantof most of the isola tes except Aerococcus andPseudoxanthomonas strains. Microbial surfactant productionhas been found as important for its ability to reduce theviscosity of oil and to facilitate emulsification, thereby en-hancing hydrocarbon biodegradability (Wolicka et al. 2010).All these properties might have facilitated the survival of suchorganisms within the sludge environment. In order to establishthe relationship between the isolated bacterial strains accord-ing to their different metabolic abilities such as hydrocarbonutilization potential, ability to grow under oxic/anoxic condi-tion, to utilize multiple electron acceptors, to grow at differentpH and temperature, to produce biosurfactant and to toleratevariety of heavy metals during growth, a UPGMA-basedstatistical analysis was performed (Fig. 6). All the parametershave clustered the isolates and described their non-taxonomicrelationship. The dendogram based on the above-mentionedproperties demonstrated diversification of isolates with re-spect to these selected traits. The dendogram revealed theformation of only small distinguishable subgroups (25 num-bers), with no apparent large clusters. It was further noted thatthe strains affiliated to different taxonomic groups sharedcommon traits. The observed majority of Bacillus spp. cor-roborated very well with previous reports on the widespreaddistribution of the members of this genus in diversepetroleum/PAH-contaminated environments (petroleum oilsludge, petroleum refinery and oil exploration sites, andpetroleum-contaminated soils) and have been extensively re-lated to petroleum biodegradation under both aerobic andanaerobic conditions (de Vasconcellos et al. 2009; Cerqueiraet al. 2011; Zhang et al. 2012; Banerjee and Ghoshal 2010;Reddy et al. 2011; Mukherjee and Bordoloi 2012). Moreover,Bacillus spp. have been known to be capable of denitrificationunder hydrocarbon-containing, nitrate-reducing environmentresulting in the production of bicarbonate (Wolicka et al.2010), which corroborated very well with the chemical pa-rameters of our sludge sample having nitrate-reducing condi-tion and substantial amount of bicarbonate. Abundance of


Paenibacillus strains in our sample corroborated well with theprevious reports on the same genus isolated from differenthydrocarbon-contaminated habitats ranging from petroleum-contaminated sediment, oil well and wastewater treatmentplant (Daane et al. 2001; Najafi et al. 2011). Paenibacillusspp. have been reported as efficient hydrocarbon-mineralizingorganisms, which could utilize naphthalene or phenanthreneas sole carbon source and could also degrade different PAHs(Daane et al. 2001; Sakai et al. 2005). Aerococcus andMicrococcus spp. have also been previously reported fromoil-associated environments and found as efficient hydrocar-bon degraders (de Vasconcellos et al. 2009; Lors et al. 2010;Mulla et al. 2011). Wang et al. (2010) have shown the pres-ence of alkane hydroxylase (key enzyme for bacterial alkaneoxidation in oil-contaminated environments) genes inBachybacterium spp. Pseudomonas spp., have been reportedfrom various oil-contaminated sites including aquifers, land-fills, mangrove forests, storage reservoirs, etc. and have beenextensively characterized for their well-known hydrocarbon-metabolizing ability (Bugg et al. 2000; Wang et al. 2010; Renet al. 2011; Kleinsteuber et al. 2012). Mukherjee and Bordoloi(2012) have reported the biodegradation of benzene, toluene,

xylene and pyrene compounds by a consortium comprising ofthree hydrocarbon-degrading bacterial strains, Bacillussubtilis DM-04, Pseudomonas aeruginosa M and NM isolat-ed from a petroleum-contaminated soil sample of north-eastIndia. The isolated strain NB2 was phylogenetically affiliatedto P. spadix, which has been associated with oil-contaminatedsites (Kim et al. 2008). Previous investigators have also ex-plored genomic properties and different petroleum-hydrocarbon-degrading versatility in Pseudoxanthomonasstrains (Patel et al. 2012; Lee et al. 2012; Choi et al. 2013).

Proposed hypothesis on in situ hydrocarbon degradationprocesses

Microbial community composition of the oily sludgeindicated the possibility of hydrocarbon mineralizationthrough the concerted effort of major metabolic groupsof microorganisms found in the sludge community. Inreducing environment, mineralization of organic matter ismore complex that it requires the cooperation of differentgroups of inhabitant microorganisms (Kleinsteuber et al.2012). In this sludge community, we have found several

BT

EX

Ben

zene

Tol

uene

Eth

ylbe

nzen

e

Xyl

ene

Na -

benz

oate

Nap

htha

lene

Phen

ol

m-C

reso

l

p-C

reso

l

Ant

hrac

ene

Fluo

rene

Phen

anth

rene

Pyre

ne

Cad

miu

m (C

d)

Lea

d (P

b)

Chr

omiu

m (

Cr)

Zin

c (Z

n)

Nic

kel (

Ni)

Cop

per (

Cu)

Gro

wth

at p

H (

2 -6)

Gro

wth

at p

H (

8-12

)

Gro

wth

att

emp

(20 -

25C

)

Hig

h at

tem

p ab

ove

40C

Gro

wth

at a

bove

3%

NaC

l

Aer

obic

Gro

wth

Ana

erob

ic g

row

th

Mul

tipl

e el

ectr

on a

ccep

tor

Surf

acta

nt p

rodu

ctio

n

Paenibacillus lautus BPD-11Pseudomonas fluorescens BPD-16Bacillus subtilis BPD-15Bacillus pumilus BPD-14Bacillus tequilensis BPD-18Bacillus sp. NP3Bacillus sp. NP5Pseudoxanthomonas spadix NB2Aerococcus sp. BPD-6Aerococcus sp. BPD-3Aerococcus sp. BPD-1 Aerococcus viridans BPD-10Bacillus sp. NP4Bacillus subtilis NP7Bacillus cereus NB4Zimmermannella faecalis BPD-7Zimmermannella faecalis BPD-4Brachybacterium sp. NB6Micrococcus lylae BPD-5Micrococcus luteus BT4Brachybacterium sp. BT2Bacillus sp. NB7Bacillus sp. NP2Bacillus sp. NB3Paenibacillus sp. BPD-12Bacillus cereus NB1Paenibacillus lautus BT3Bacillus sp. BT1

UPGMA clusters

Different hydrocarbons utilization

Heavy metals tolerance

Growth responses

Growth in presence of different hydrocarbons Growth above 5mM cadmium Growth above 5 mM lead Growth above 5mMchromium Growth above 5mM zinc Growth above 5mM nickel Growth above 5mM copper Growth at pH range 3-6Growth at pH range 8-12 Growth at temperature 20-25 C Growth at temperature above 40 C Growth above 3% NaClGrowth at aerobic condition Growth at anaerobic condition Utilization of multiple electron acceptors Surfactant productionof the isolates

3.6 3.0 2.4 1.8 1.2 0.6 0

Bacterial strains

Fig. 6 UPGMA analysis of physiological and metabolic properties of bacterial isolates retrieved from oily sludge. Filled cells indicated positiveresponse and blank cells indicated no response to different factors


16S rRNA gene sequences related to primary fermenters(Clostridium, Bacillus, Pelotomaculum, Peptococcaceaemembers), secondary fermenters (Acidobacteria ,Chloroflexi, Bacteroidetes, Spirochaetes), syntrophic(Syntrophus, Smithella, Pelotomaculum) and sulphate-reducing (Thermodesulfobacterium, Bacteroidetes,Peptococcaceae) organisms. In addition to these, se-quences related to hydrogenotrophic (Methanocellalesand Methanomicrobiales members) and acetoclastic(Methanosarcinales members) methanogenic Archaeahave been found to be predominated within the sludge.The syntrophic association between these microbial groups hasbeen well reported in methanogenic oil-associated environ-ments, wherein the fermentative, sulphate-reducing andsyntrophic organisms metabolize hydrocarbons through pro-duction of simple organic compounds, CO2, H2, etc., whichhave been known to be further metabolized by methanogenicmicroorganisms for their survival and activity resulting inmethane production (Garcia et al. 2000; Mayumi et al. 2011;Kleinsteuber et al. 2012). A similar kind of concerted metabolicinterplay might be going on within the sludge microbial com-munity to mineralize complex hydrocarbons throughmethanogenesis. Methanogenic archaeal diversity andmethanogenesis within the sludge environment was furthersupported by the abundance of functional biomarkermcrA geneof similar methanogens.Within the sludge community, we havealso found several sequences related to nitrate-reducing(Rhodocyclaceae members, Thauera, Acidobacteria), iron-reducing (Chloroflexi, Clostridiales members) andhydrocarbonoclastic (Comamonadaceae, Rhodobacteraceae,and several β-Proteobacteria members) organisms. Inhydrocarbon-contaminated and reducing environment, theavailability of alternative electron acceptors is a critical factorfor determining community composition. Presence of nitrate inthe sludge could serve as an easily available electron acceptorfor hydrocarbon degradation by facultative anaerobic,denitrifying Rhodocyclaceae members and Bacillus spp., andpossibly, these organisms have been found as predominatedwithin the sludge community. Other hydrocarbon-degradingmicroorganisms as found in the sludge might be thriving onthe degradation products or directly involved in complex hy-drocarbon degradation. Abundance of sequences related toCrenarchaeota members indicated the possible role of suchorganisms in anaerobic “dissimilatory”methane oxidation con-tributing in methane cycling within sludge environment(Biddle et al. 2006). The phylogenetic and metabolic diversityof the sludge microbial community suggests that a complexmicroflora might be involved in several geochemical cycles insitu resulting in possible oily sludge biodegradation(Grabowski et al. 2005). The hypothesis on community func-tions within sludge environment can be tested on a global scaleby metagenomics and metaproteomics approaches. Also, inorder to understand the function, interaction and ecological

significance of the inhabitant microorganisms further culture-dependent studies based on individual isolate as well asenriched consortia are required. Novel cultivation techniquescan be developed based on information obtained from themolecular studies, which might enable the cultivation of notyet cultured microorganisms. This would help to clarify thedirect/indirect role of different microorganisms in hydrocarbonmetabolism (Grabowski et al. 2005; Kleinsteuber et al. 2012).

Conclusion

Culture-independent clone library-based analyses revealedthat microbial community within the oily sludge was com-posed of diverse groups of microorganisms includinghydrocarbonoclastic, nitrate-reducing, sulphate-reducing, fer-mentative, syntrophic, methanogenic (both hydrogenotrophicand acetoclastic) and methane-oxidizing bacteria andArchaea. Cultivable bacterial community was predominatedby the members of the genus Bacillus, and almost all thebacterial isolates have the potential relevance to biodegrada-tion of hydrocarbons. Microbial community composition in-dicated the possibility of hydrocarbon degradation throughmetabolic interplay within syntrophic association betweenthe organisms, which could be exploited for in situ bioreme-diation of the oily sludge.

Acknowledgments This work was financially supported by the De-partment of Science and Technology, Government of India under FastTrack Project for Young Scientist scheme (SR/FT//LS-078/2008). Theauthors gratefully acknowledge the support for obtaining oily sludgesample from Bharat Petroleum Corporation Limited, Rajbandh (TOP),Durgapur, West Bengal, India. The authors are thankful to Dr. Pinaki Sar,Department of Biotechnology, Indian Institute of Technology Kharagpur,for his critical comments on the manuscript.

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