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ENVIRONMENTAL BIOTECHNOLOGY

Molecular- and cultivation-based analyses of microbialcommunities in oil field water and in microcosms amendedwith nitrate to control H 2 S production

Raji Kumaraswamy & Sara Ebert & Murray R. Gray &

Phillip M. Fedorak & Julia M. Foght

Received: 28 June 2010 /Revised: 18 October 2010 /Accepted: 18 October 2010 /Published online: 6 November 2010# Springer-Verlag 2010

Abstract Nitrate injection into oil fields is an alternative to biocide addition for controlling sulfide production ( sour-ing ) caused by sulfate-reducing bacteria (SRB). This studyexamined the suitability of several cultivation-dependent and cultivation-independent methods to assess potentialmicrobial activities (sulfidogenesis and nitrate reduction)and the impact of nitrate amendment on oil field microbiota. Microcosms containing produced waters from twoWestern Canadian oil fields exhibited sulfidogenesis that was inhibited by nitrate amendment. Most probable number (MPN) and fluorescent in situ hybridization (FISH)analyses of uncultivated produced waters showed low cellnumbers ( 10 3 MPN/ml) dominated by SRB (>95%relative abundance). MPN analysis also detected nitrate-reducing sulfide-oxidizing bacteria (NRSOB) and hetero-trophic nitrate-reducing bacteria (HNRB) at numbers toolow to be detected by FISH or denaturing gradient gelelectrophoresis (DGGE). In microcosms containing pro-duced water fortified with sulfate, near-stoichiometricconcentrations of sulfide were produced. FISH analyses of the microcosms after 55 days of incubation revealed that Gammaproteobacteria increased from undetectable levels to5 20% abundance, resulting in a decreased proportion of Deltaproteobacteria (50 60% abundance). DGGE analysisconfirmed the presence of Delta- and Gammaproteobacteriaand also detected Bacteroidetes. When sulfate-fortified produced waters were amended with nitrate, sulfidogenesis

was inhibited and Deltaproteobacteria decreased to levelsundetectable by FISH, with a concomitant increase inGammaproteobacteria from below detection to 50 60%abundance. DGGE analysis of these microcosms yieldedsequences of Gamma- and Epsilonproteobacteria related to presumptive HNRB and NRSOB ( Halomonas , Marinobac-terium , Marinobacter , Pseudomonas and Arcobacter ), thussupporting chemical data indicating that nitrate-reducing bacteria out-compete SRB when nitrate is added.

Keywords Anaerobic microbes . Nitrate reduction .Sulfidogenesis . Reservoir souring . MPN . DGGE

Introduction

Oil fields can harbor a diversity of microbes that may beindigenous or introduced during water-flooding for second-ary recovery of oil (Magot 2005 ). Injection water that contains sulfate may stimulate the activity of sulfate-reducing bacteria (SRB) in the reservoir or surfacefacilities, resulting in production of noxious H 2 S i n a process known as souring . Additionally, sulfate-reducingArchaea such as Archaeoglobus have been found in hot (~80 C) oil fields (Gittel et al. 2009 ). Sulfide increasesmaintenance costs by causing precipitation of transitionmetals and corrosion of pumps and pipes, and alsoincreases processing costs by decreasing the quality of theoil (Vance and Thrasher 2005 ). Thus, souring is a phenomenon of great concern to oil producers, leading toimplementation of various methods for its control. Injectionof biocides into reservoirs to inhibit SRB is a commonsouring control method. However, this treatment is expen-sive, requiring frequent injection because it is effective onlyfor a short duration or is ineffective because microbial

R. Kumaraswamy : S. Ebert : P. M. Fedorak : J. M. Foght ( * )Biological Sciences, University of Alberta,Edmonton, AB T6G 2E9, Canadae-mail: [email protected]

M. R. GrayChemical and Materials Engineering, University of Alberta,Edmonton, AB T6G 2V4, Canada

Appl Microbiol Biotechnol (2011) 89:2027 2038DOI 10.1007/s00253-010-2974-8

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biofilms develop resistance to the biocides (Telang et al.1998 ). The biocides can also be hazardous to human healthand environment, therefore worker safety issues are also possible (Cord-Ruwisch et al. 1987 ).

Addition of nitrate to oil fields to control souring isconsidered to be a potentially cost-effective and less toxicalternative to biocide treatment, particularly in offshore oilfields where injection water is high in sulfate, and also incontinental oil fields (Reinsel et al. 1996 ; Telang et al.1997 ; Larsen et al. 2006 ; Eckford and Fedorak 2002 ). In principle, nitrate addition stimulates the growth of nitrate-reducing bacteria (NRB) and inhibits SRB activity andgrowth. Several mechanisms have been proposed, includ-ing: (a) heterotrophic nitrate-reducing bacteria (HNRB) out-compete SRB for electron donors in reservoirs, thusdecreasing sulfide production (Hubert and Voordouw2007 ; Gittel et al. 2009 ); (b) nitrate-reducing sulfide-oxidizing bacteria (NRSOB) carry out nitrate-dependent oxidation of sulfide to sulfate, thus decreasing existingsulfide concentrations (Telang et al. 1999 ); (c) nitrite, anintermediate in microbial nitrate reduction, accumulates andincreases the local redox potential, which in turn inhibitsmicrobial sulfate reduction (Greene et al. 2003 ); (d) nitritealso inhibits a key SRB enzyme (dissimilatory sulfitereductase; DsrAB) that catalyzes reduction of sulfite tosulfide, thus preventing sulfide production (Greene et al.2003 ); and (e) nitrate addition causes some SRB to shift their metabolism from sulfate reduction to nitrate reduction(Seitz and Cypionka 1986 ).

All these mechanisms have the potential to stop or evenreverse microbial sulfide production when nitrate is added tosouring reservoirs. However, detecting the presence andstimulating the activities of HNRB and NRSOB is of particular interest in the control of souring. Laboratoryenrichment cultures, microcosms and bioreactors have beenused to demonstrate that adding nitrate to the water samplesfrom souring oil fields successfully controls sulfide produc-tion (Dunsmore et al. 2006 ; Eckford and Fedorak 2002 ;Hubert et al. 2003 ). However, there also have been caseswhen nitrate addition failed to control sulfide formation(Kaster et al. 2007 ; Kjellerup et al. 2005 ). Thus, a better understanding of the microbiological and chemical compo-sition of oil field fluids is needed to predict the success of nitrate addition for oil field souring control. This outcomemight be achieved using a multipronged approach for microbial community characterization involving cultivationof microorganisms, polymerase chain reaction (PCR) analy-sis of 16 S rRNA genes using universal primers followed byDNA sequencing, and performing in situ hybridization of active microbes using group- or genus-specific 16 S rRNAgene probes. Although a few peer-reviewed studies haveutilized denaturing gradient gel electrophoresis (DGGE) toexamine oil field samples (e.g., Jurelevicius et al. 2008 ;

Bdtker et al. 2009 ), the combination of DGGE withfluorescent in situ hybridization (FISH) and most probablenumber (MPN) enumeration has not been reported. There-fore, in this study we measured microbial souring of produced waters from two continental oil fields previouslyexamined for souring control (Eckford and Fedorak 2002 ),and observed control of souring by nitrate amendment inmicrocosms. This was accompanied by MPN estimation of different functional groups (SRB, HNRB, NRSOB andfermentative microorganisms). To complement the chemicalanalysis of microbial activity and MPN estimation, we alsoanalyzed the effect of nitrate amendment on the microbialcommunity structure in the produced waters using culture-independent DGGE and FISH analyses. The study evaluatedhow well the activities predicted by MPN and molecular analysis of produced waters corresponded to communityactivity observed in microcosms.

Materials and methods

Oil fields, sampling and chemistry of produced water samples

Two oil fields in Alberta, Canada, previously shown inlaboratory studies to be amenable to nitrate control of souring(Eckford and Fedorak 2002 ), were sampled. Produced water samples were collected from free water knock out units,which are well-head treatment facilities for oil water separation. Ownership had changed since the last samplingin 2001, thus oil fields N and P in the 2001 studiescorrespond to oil fields D and H, respectively, in the current study. Biocide addition to oil field D was stopped 1 week prior to sampling in September 2005, and no biocides were being added to oil field H. Temperature, pH (using color pHast indicator strips; EM Science, Gibbstown, NJ) andsulfide concentrations in produced waters were measuredimmediately in the field as previously described (Eckfordand Fedorak 2002 ). Water samples were aseptically collectedin sterile 4-l wide mouth bottles and subsamples wereimmediately transferred to sterile, sealed, anaerobic 125-mlserum bottles as described by Eckford and Fedorak ( 2002 ).The samples in the serum bottles were transported to thelaboratory and stored at 4 C for a maximum of 24 h beforeanalysis. Sulfate, nitrate, and chloride concentrations weredetermined by ion chromatography (Eckford and Fedorak 2002 ). Nitrite was determined by a colorimetric assay(Griess-Romijn-van Eck 1966 ).

MPN enumerations

Selective liquid media were used to enumerate five physio-logical groups of microbes by the three-tube MPN technique.

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Each medium contained 20 g/l NaCl to approximate thesalinity of the produced waters. All media except HNRB andmodified Butlin s media (below) were prepared using strict anaerobic methods, dispensing9-ml portions of the media intoHungate tubes with various headspace gas compositions. SRBwere enumerated using two different media: modified Butlin smedium (Eckford and Fedorak 2002 ) with lactate as solecarbon source and modified Widdel Pfennig medium con-taining six carbon sources: lactate (2.7 mM), acetate(4.9 mM), propionate (4 mM), decanoate (0.58 mM), benzoate (0.7 mM), and ethanol (7.6 mM) (Hulecki et al.2009 ). Fermenters were grown in medium containing (per liter): 30 g tryptic soy broth (Becton Dickinson, Sparks,MD), 0.5 g cysteine and 1 mg resazurin, sealed with aheadspace of N 2 . Compositions of the media used toenumerate HNRB and NRSOB and the methods used for scoring have been described by Eckford and Fedorak ( 2002 ). NRSOB tubes had a headspace of 10% CO 2 , balance N 2whereas HNRB tubes were sealed in Hungate tubes under anaerobic headspace and monitored for N 2 O gas production.

Using syringes, the sealed tubes of sterile media wereinoculated with 1 ml of serial 10-fold dilutions of the produced water samples prepared in sterile, anaerobicsaline. Tubes were incubated for 8 weeks in the dark at room temperature (~22 C) before scoring. Statisticaldifferences ( p

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DNA extraction

Cell pellets for DNA extraction were obtained from 100-mlsamples of uncultivated produced water by centrifugation at 10,000 g for 20 min. Likewise, the pellets obtained from10 ml of microcosm samples or 9 ml of selected 10

1

dilution MPN tubes were used for DNA extraction usingthe Soil DNA Extraction Kit (MoBio Laboratories, USA)according to the procedure described by the manufacturer.The quality and yield of the extracted DNA was determined by electrophoresis in 1.5% agarose. The soi l DNAextraction kit was compared with another commercialDNA extraction kit and a laboratory DNA extraction protocol for DNA yield and DGGE patterns using the produced water samples. Results were comparable so thesoil kit was used for further DNA extractions.

PCR amplification of 16 S rRNA gene fragments

Approximately 500 bp of the bacterial 16 S rRNA genewere amplified for DGGE analysis using universal primer 341 F with a GC clamp and primer 907R (Schefer andMuyzer 2001 ). PCR amplifications were performed in25 l total volume containing 5 to 100 ng of templateDNA, 10 to 25 pmol of each primer and 12.5 l of PCR master mix (Promega, Madison, WI) containing 400 mM of each deoxynucleoside triphosphate, 50 units/ml of TaqDNA polymerase (Promega) and 3 mM MgCl 2 . For PCR with 341 F-GC and 906 R primers, an initial denaturationstep at 95 C for 2 min was used, followed by 35 cycles of denaturation at 94 C for 30 s, primer annealing at 55 Cfor 1 min, and primer extension at 72 C for 1 min. A finalextension step at 72 C for 10 min was used, followed by afinal holding step at 4 C.

PCR products were checked for correct size by electrophoresis in 1.5% (wt/vol) agarose gels, stained withethidium bromide. Whenever PCR reactions gave no product with DGGE primers (due to low concentrations of template DNA), nested PCR with primers PB36F andPB38R (Foght et al. 2004 ) was used to amplify nearly fulllength 16 S rRNA genes (~1,500 bp), and 1 l of the product was re-amplified using DGGE primers 341 F-GCand 907R. For PCR with PB36F and PB38R primers, aninitial denaturation step at 95 C for 4 min was used,followed by 30 cycles of denaturation at 94 C for 45 s, primer annealing at 54 C for 1 min, and primer extensionat 73 C for 2 min. A final extension step at 73 C for 10 min was used, followed by a final holding step at 4 C.

DGGE analysis of PCR products

DGGE was performed as described previously (Schefer andMuyzer 2001 ) using the D-Code System (Bio-Rad Labora-

tories, Ontario, Canada) with 1-mm-thick gels. PCR products(350 450 ng) were mixed with 5 loading dye, applieddirectly onto 6% polyacrylamide gels with denaturinggradients from 0% to 80% denaturant (where 100%denaturant contains 7 M urea and 40 vol% formamide), thendeveloped at 96 V for 16 h. After electrophoresis, the gelswere stained for 30 min in 50 ml of a 1:5 dilution of SYBR Gold (Molecular Probes, Eugene, OR) in nuclease-free water,rinsed in deionized water (Milli-Q system), and photo-graphed using a Fujifilm FLA-5000 scanner. The gel wasre-stained with ethidium bromide and individual bands wereexcised under UV illumination, suspended in 10 l of Milli-Q water and stored overnight at 4 C. The eluted DNA (3

5 l) was re-amplified using the original DGGE primer setsand an aliquot of each product was run on a denaturinggradient gel to ascertain quality. The PCR products were purified using Roche PCR purification kits (Roche Labora-tories, Canada), sequenced using reverse primer 907R andBigDye Terminator V3.1 mix (Applied Biosystems Inc.,USA) and resolved on an Applied Biosystems 3730 DNAanalyzer in the Molecular Biology Services Unit (BiologicalSciences Dept., University of Alberta). Typically, thesequences of excised DNA bands were 450 550 nt in length.All excised bands yielded single sequences, that is, cloningwas not required to separate co-migrating PCR products.

Comparative sequence analysis

Sequences were checked for chimeras using the Mallardchimera check program ( http://www.cardiff.ac.uk/biosi/ research/biosoft/ ), and trimmed using BioEdit softwareversion 7.0.9 ( http://www.mbio.ncsu.edu/BioEdit/bioedit.html ; Ibis Biosciences, Carlsbad, CA). Valid sequenceswere compared to the GenBank database using the BLASTalgorithm ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Thesequences determined in this study have been deposited inGenBank as accession numbers FJ535621 FJ535651.

FISH analysis

Pellets from 10-ml samples of produced waters and micro-cosms, or from 9 ml of 10

1 dilutions of MPN cultures werecollected by centrifugation as described above, washed oncewith 10 mM phosphate buffer (pH 7.2) containing 130 mM NaCl, and resuspended in 0.5 ml of the same buffer.Subsequently, the cells were fixed with 4% paraformalde-hyde (w/v) for 1 3 h and resuspended in 10 mM phosphate buffer and 60% (v/v) ice-cold absolute ethanol (final volume,0.5 ml). Sample volumes of 5 10 l were used for hybridization with Cy3- or Alexa-labeled probes (IntegratedDNA Technologies, USA). Hybridization was carried out inhybridization buffer with formamide concentrations appro- priate for each probe at 46 C for 1.5 h and then incubated in

http://www.cardiff.ac.uk/biosi/research/biosoft/http://www.cardiff.ac.uk/biosi/research/biosoft/http://www.mbio.ncsu.edu/BioEdit/bioedit.htmlhttp://www.mbio.ncsu.edu/BioEdit/bioedit.htmlhttp://blast.ncbi.nlm.nih.gov/Blast.cgihttp://blast.ncbi.nlm.nih.gov/Blast.cgihttp://www.mbio.ncsu.edu/BioEdit/bioedit.htmlhttp://www.mbio.ncsu.edu/BioEdit/bioedit.htmlhttp://www.cardiff.ac.uk/biosi/research/biosoft/http://www.cardiff.ac.uk/biosi/research/biosoft/

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Fig. 1 FISH analysis of cells harvested from uncultivated producedwater samples or from sulfate-fortified microcosms after 55 days of incubation. a , b Produced water from oil fields D and H, respectively, probed with SRB-Cy3 ( red ) and EUB338-Alexa ( green ). c, d Nitrate-unamended microcosms inoculated with water from oil fields D andH, respectively, probed with SRB-Alexa ( green ) and EUB338-Cy3

(red ). e, f Nitrate-amended microcosms inoculated with water from oilfields D and H, respectively, probed with GAM42A-Alexa ( green ) andEUB338-Cy3 ( red ). Cells targeted by both probes appear yellow in all panels. A mixture of all four SRB probes targeting different taxawithin the Deltaproteobacteria was used in a d

Table 2 FISH analysis of uncultivated produced water samples, selected MPN dilutions and microcosms with or without nitrate amendment

Oil field Sample type FISH analysis

Probes giving positive results Abundance relative to EUB338 signal (%)

D Produced water SRB probes a >95

H Produced water SRB probes a >95

D Microcosm unamended with nitrate SRB probesa

50

60GAM42A 10 20

D Microcosm amended with nitrate GAM42A 50 60

H Microcosm unamended with nitrate SRB probes a 50 60

GAM42A 5 10

H Microcosm amended with nitrate GAM42A 50 60

D MPN dilution with HNRB medium b GAM42A 60 70

D MPN dilution with fermenter medium b SRB probes a 40 50

H MPN dilution with fermenter medium b SRB probes a 40 50

a A mixture of four SRB probes was used for detection and quantification

bLowest dilution positive culture (10

1 dilution)


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tree construction (not shown) was used to infer the phylogenetic affiliation. The sequences from bands W2and W7 from the two oil fields (Fig. 2) were most similar (99% and 97%) to an acetate-oxidizing, sulfate-reducing Desulfobacter (Table 3); band W1 from oil field D wasmost similar (96%) to an uncultivated Bacteroidetes; and band W8 (oil field H) had 100% identity over 457 bases tothe fermentative homoacetogen Acetobacterium carbinoli-cum strain VNs25 isolated from a deep subsurface naturalgas storage aquifer (Basso et al. 2009 ).

Thus, the produced water samples harbored a communi-ty with low cell density (MPN) and low diversity, primarilycomposed of SRB (FISH) and some other genera (DGGE).The dominance of SRB suggested the potential for sulfido-genesis in the presence of sulfate, a hypothesis that wassubsequently tested in microcosm studies.

Microbial activity in sulfate-fortified microcosms

Water samples from both oil fields were sealed in anaerobicmicrocosms and fortified with ~3 mM sulfate, similar to

concentrations measured in produced water in previousstudies (Table 1). Notably, no exogenous electron donorswere added to the microcosms, so all activity detected inthe microcosms was due to endogenous electron sourcesthat also can be used for microbial reduction of sulfate andnitrate (Grigoryan et al. 2008 ; Vance and Brink 1994 ;Gauthier et al. 1992 ; Kleikemper et al. 2002 ). Nitrate wasadded to half the microcosms and the concentrations of sulfate, sulfide, nitrate and nitrite were monitored duringincubation (Fig. 3).

Duplicate microcosms prepared with both oil fieldwaters without nitrate amendment had comparable patternsof sulfate removal and near stoichiometric production of sulfide (Fig. 3a and c ). Microcosms from oil field H hadshorter lag times and faster rates of sulfate reduction thanoil field D microcosms, suggesting a more active or larger indigenous sulfate-reducing community, although there wasno statistically significant difference between the SRBMPN values in these two oil fields (Table 1; p

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was detected during incubation (Fig. 3b and d ), consistent with the previous study (Eckford and Fedorak 2002 ). Nitrate was nearly depleted within 10 days of incubation,and approximately 2 mM nitrite accumulated. No changesin sulfate, nitrate or sulfide were observed in the heat-killedcontrols (data not shown). Thus, the microcosms demon-strated the potential for the indigenous microbial commu-nities in both oil fields to produce sulfide in the absence of nitrate, and conversely to inhibit sulfidogenesis whennitrate was added.

FISH and DGGE analyses of microcosms

Duplicate microcosms were analyzed by FISH and DGGEafter 55 days of incubation to observe shifts in microbialcommunity composition in response to sulfate fortification, both with and without addition of nitrate.

FISH analysis of the microcosms from both oil fieldsincubated without nitrate showed that 50 60% of the totalcells hybridized to the mixture of SRB probes and 5 20%hybridized to the gammaproteobacterial probe (Table 2 and

Fig. 2 DGGE gel showing bacterial 16 S rRNA genefragments amplified fromuncultivated produced water samples or from sulfate-fortifiedmicrocosms after 55 days of incubation. Lanes 1 and 2:nested-PCR DGGE of uncultivated produced water

from oil fields D and H,respectively; lanes 3 6 : direct DGGE of oil field Hmicrocosms; lanes 7 10: direct DGGE of oil field Dmicrocosms. Lanes 3 , 4, 7 and8: microcosms not amendedwith nitrate; lanes 5 , 6 , 9 and10: microcosms amended withnitrate. Pairs of lanes represent duplicate microcosms. Numbersrefer to sequenced bandsidentified in Table 3

Fig. 3 Chemical analyses of representative microcosmscontaining produced water samples from oil fields D and H,fortified with sulfate. a Oil fieldD, unamended with nitrate; b oilfield D, nitrate-amended; c oilfield H, unamended with nitrate;d oil field H, nitrate-amended


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souring in oil field waters, to predict the potential for controlling souring by nitrate amendment, and to under-stand the effect of nitrate amendment on sulfidogenic oilfield microbial communities. Although the samples wecollected in 2005 did not have detectable levels of sulfide,the fields have a history of souring and their sulfidogeniccommunities previously responded to nitrate amendment (Eckford and Fedorak 2002 ). Therefore, it was reasonableto expect that the 2005 water samples would still harbor SRB and would behave in the same fashion if fortified withsulfate. Notably, when oil field H was subsequentlysampled in 2007, sulfide was again detected (Hulecki et al. 2010 ), supporting selection of these sites for study.

MPN enumeration and FISH analysis revealed domi-nance of SRB in the water samples and indicated the potential for sulfidogenesis that was corroborated inmicrocosm studies. However, in isolation, neither MPN,FISH nor DGGE analysis of the original water samplescould adequately predict control of souring by nitrateaddition. For example, MPN analysis of oil field D water revealed the presence of HNRB but detected only lownumbers of NRSOB and therefore could not unequivocally predict the potential for nitrate to prevent sulfide generationin microcosms. Although microcosms did demonstrate hownitrate amendment could control souring, the time requiredfor analysis (2 7 weeks of incubation) was a disadvantage.DGGE analysis of produced waters was inadequate for detecting microbes potentially involved in either souring or nitrate reduction, but DGGE analysis of nitrate-amendedmicrocosm samples identified putative heterotrophic nitratereducers such as Halomonas , Marinobacter and Pseudo-monas as well as epsilonproteobacterial sequences puta-tively associated with nitrate reduction coupled to sulfideoxidation.

The inability of the two molecular methods to detect thekey microbial players in the uncultivated water samples could be explained by PCR bias due to low numbers of cells in thewater samples, low proportions of the target phyla, and/or metabolic inactivity of groups such as HNRB and NRSOB inthe produced water samples (a bias inherent to FISH analysisis that metabolically active cells produce strong signalswhereas inactive or slowly metabolizing cells produce weak or no fluorescent signals). Improvements would includeanalyzing the microcosms using FISH and/or DGGE at intervals during microcosm incubation, and utilizing addi-tional FISH probes, particularly those targeting the NRSOBEpsilonproteobacterium Arcobacter (Watanabe et al. 2000 ),and Bacteroidetes probes to complement CF319a, whichoften underestimates Bacteroidetes abundance (O'Sullivan et al. 2002 ).

Regardless of the inherent shortcomings of the individ-ual analyses, the combination of methods revealed severalcorrelations and intriguing observations about sulfidogene-

sis and nitrate inhibition in these oil field waters. SRB weredetected in high numbers in the water samples by MPN anddominated the uncultivated microbiota according to FISH,even though sulfide and sulfate concentrations were belowdetection levels. This observation may be explained by thefact that some SRB ferment in the absence of sulfate(Oppenberg and Schink 1990 ; Muyzer and Stams 2008 ),and is supported by the correspondence of SRB andfermenter numbers using MPN enumeration. This correla-tion was subsequently confirmed by DGGE analysis of fermenter MPN cultures from both oil fields, detectingsequences related to putative SRB such as Desulfovibrioand Desulfotigum spp. FISH analysis of fermenter culturesfrom oil field H that showed dominance of SRB (40 50%relative abundance) further strengthened this observation.Water samples from the same oil fields sampled in 2007(Hulecki et al. 2010 ) had significant concentrations of substrates such as benzene, toluene, xylenes, propionateand acetate, which are substrates for SRB (Foght 2008 ;Muyzer and Stams 2008 ).

The dynamics of nitrate, nitrite, sulfide and sulfate production and removal in microcosms were qualitativelysimilar for both oil fields. However, the shorter lag time andfaster sulfate reduction rate in oil field H microcosmsunamended with nitrate did not correspond to detection of specific taxa by DGGE or FISH or abundance of metabolictypes by MPN. As a result of microbial nitrate reduction,~2 mM nitrite accumulated in the nitrate-amended micro-cosms from both oil fields. The logical inference is that HNRBwere stimulated and competed with SRB, and additionally theaccumulated nitrite likely inhibited sulfate reduction (Have-man et al. 2005 ; Kaster et al. 2007 ; Hubert and Voordouw2007 ), thus decreasing SRB abundance. This SRB decreasein nitrate-amended microcosms was also noted using FISH,in which Gammaproteobacteria dominated. DGGE analysisshowed a corresponding increase in putative P . stutzeri , Halomonas , Marinobacterium and Marinobacter , whichinclude hydrocarbon-associated HNRB species. For example, in the nitrate-amended microcosms, the best sequencematch for band N2F, Marinobacterium sp. JW3.2b, wasassociated with fuel hydrocarbons and another close match, Marinobacterium sp. IC961 (AB196257), was the dominant clone in libraries prepared from a mesothermic petroleumreservoir (Pham et al. 2009 ). Similarly, Marinobacter and Halomonas spp. have been grown with hydrocarbons under nitrate-reducing conditions (Bonin et al. 2002 ; Larsen et al.2006 ), and Marinobacter spp. have been isolated from oilfields including those undergoing nitrate treatment (Sette et al. 2007 ). In fact, Marinobacter sp. dominated biofilmsdeveloped from oil field produced water flow-throughsystems treated with nitrate (Dunsmore et al. 2006 ) andwere deemed to play a very important role in nitratetreatment to control souring. Some Marinobacter species


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such as M . aquaeolei effect incomplete reduction of nitrate tonitrite, which might account for accumulation of nitrite in themicrocosms (Huu et al. 1999 ). Therefore, detection of thesegenera in nitrate-amended microcosms using DGGE and theobservation of dominant Gammaproteobacteria in nitrate-amended microcosms and MPN cultures using FISH iscomplementary evidence that they contributed to heterotrophic nitrate reduction suppressing sulfidogenesis. Although NRSOB such as Arcobacter spp. were detected in themicrocosms and some of the members of this genusincompletely reduce nitrate to nitrite (Gevertz et al. 2000 ),this reaction may not have an important role in suppressingsulfate reduction in situ at these sites because no sulfide wasdetected in the produced water samples. Another consider-ation is that produced water samples represent only the planktonic component of oil field microbiota, whereas theattached community, which is technically very difficult toaccess, may have a different microbial composition andactivities in situ.

In sulfate-fortified microcosms incubated without nitrateamendment the SRB remained dominant according to FISHanalysis. However, no sequenced DGGE bands wereaffiliated with sulfate-reducing Deltaproteobacteria; thismay be due to our inability to excise and sequence all bands from the DGGE gel. In addition to SRB in thesemicrocosms, FISH detected Gammaproteobacteria (5 20%abundance) and DGGE yielded sequences affiliated with Marinobacterium , Halomonas and uncultivated Bacteroi-detes, indicating that fortification with sulfate allowedgrowth or activity of metabolic types in addition to SRB.

Thus, the combination of complementary culture-dependent and culture-independent methods predicted andsubsequently demonstrated the effect of nitrate amendment on sulfate reduction in the oil field water samples. Themolecular results were generally inadequate for analyzinglow cell density, uncultivated water samples. The potentialmechanism of sulfide suppression in these samples (HNRBoutcompeting SRB) was better understood when themolecular techniques were supported with cultivation- based microcosms and MPN analysis. Further insights andrefinements would result from expanding this study tocompare actively souring oil fields that respond and thosethat do not respond to nitrate injection.

Acknowledgements This work was funded by Schlumberger Cam- bridge Research Ltd. (UK) and NSERC (Canada). We thank DevonEnergy Corporation and Harvest Energy for access to their oil fields.

References

Amann RI, Stromley J, Devereux R, Key R, Stahl DA (1992) Molecular and microscopic identification of sulfate-reducing bacteria inmultispecies biofilms. Appl Environ Microbiol 58:614 623

Basso O, Lascourreges JF, Le Borgne F, Le Goff C, Magot M (2009)Characterization by culture and molecular analysis of themicrobial diversity of a deep subsurface gas storage aquifer.Res Microbiol 160:107 116

Bdtker G, Lysnes K, Torsvik T, Bjrnestad E, Sunde E (2009)Microbial analysis of backflowed injection water from a nitrate-treated North Sea oil reservoir. J Ind Microbiol Biotechnol36:439 450

Bonin PC, Michotey VD, Mouzdahir A, Rontani JF (2002) Anaerobic biodegradation of squalene: using DGGE to monitor the isolationof denitrifying bacteria taken from enrichment cultures. FEMSMicrobiol Ecol 42:37 49

Cochran WG (1950) Estimation of bacterial densities by means of most probable number . Biometrics 6:105 116

Cord-Ruwisch R, Kleinitz W, Widdel F (1987) Sulfate-reducing bacteria and their activities in oil production. J Pet Technol21:97 106

Davidova I, Hicks MS, Fedorak PM, Suflita JM (2001) The influenceof nitrate on microbial processes in oil industry productionwaters. J Ind Microbiol Biotechnol 27:80 86

Dunsmore B, Youldon J, Thrasher DR, Vance I (2006) Effects of nitrate treatment on a mixed species, oil field microbial biofilm. JInd Microbiol Biotechnol 33:454 462

Eckford RE, Fedorak PM (2002) Chemical and microbiologicalchanges in laboratory incubations of nitrate amendment "sour" produced waters from three western Canadian oil fields. J IndMicrobiol Biotechnol 29:243 254

Foght J (2008) Anaerobic biodegradation of aromatic hydrocarbons: pathways and prospects. J Mol Microbiol Biotechnol 15:93 120

Foght J, Aislabie J, Turner S, Brown CE, Ryburn J, Saul DJ, Lawson W(2004) Culturable bacteria in subglacial sediments and ice from twoSouthern Hemisphere glaciers. Microb Ecol 47:329 340

Gauthier MJ, Lafay B, Christen R, Fernandez L, Acquaviva M, BoninP, Bertrand JC (1992) Marinobacter hydrocarbonoclasticus gen.nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int J Syst Bacteriol 42:568 576

Gevertz D, Telang AJ, Voordouw G, Jenneman GE (2000) Isolationand characterization of strains CVO and FWKO B, two novel

nitrate-reducing, sulfide-oxidizing bacteria isolated from oil field brine. Appl Environ Microbiol 66:2491 2501Gittel A, Sorensen KB, Skovhus TL, Ingvorsen K, Schramm A (2009)

Prokaryotic community structure and sulfate reducer activity inwater from high-temperature oil reservoirs with and without nitrate treatment. Appl Environ Microbiol 75:7086 7096

Greene EA, Hubert C, Nemati M, Jenneman GE, Voordouw G (2003) Nitrite reductase activity of sulphate-reducing bacteria preventstheir inhibition by nitrate-reducing, sulphide-oxidizing bacteria.Environ Microbiol 5:607 617

Griess-Romijn-van Eck (1966) Physiological and chemical test for drinking water. NEN 1056, IY-2 Nederlandse NormalisatieInstituut Rijswijk, The Netherlands

Grigoryan AA, Cornish SL, Buziak B, Lin S, Cavallaro A, Arensdorf JJ, Voordouw G (2008) Competitive oxidation of volatile fatty

acids by sulfate- and nitrate-reducing bacteria from an oil field inArgentina. Appl Environ Microbiol 74:4324 4335Harmsen HJ, Akkermans AD, Stams AJ, de Vos WM (1996)

Population dynamics of propionate-oxidizing bacteria under methanogenic and sulfidogenic conditions in anaerobic granular sludge. Appl Environ Microbiol 62:2163 2168

Haveman SA, Greene EA, Voordouw G (2005) Gene expressionanalysis of the mechanism of inhibition of Desulfovibrio vulgarisHildenborough by nitrate-reducing, sulfide-oxidizing bacteria.Environ Microbiol 7:1461 1465

Hubert C, Nemati M, Jenneman G, Voordouw G (2003) Containment of biogenic sulfide production in continuous up-flow packed-bed bioreactors with nitrate or nitrite. Biotechnol Prog 19:338 345

Appl Microbiol Biotechnol (2011) 89:2027 2038 2037

8/13/2019 H2s 1

12/13

Hubert C, Voordouw G (2007) Oil field souring control by nitrate-reducing Sulfurospirillum spp. that outcompete sulfate-reducing bacteria for organic electron donors. Appl Environ Microbiol73:2644 2652

Hulecki JC, Foght JM, Fedorak PM (2010) Storage of oil field- produced waters alters their chemical and microbiologicalcharacteristics. J Ind Microbiol Biotechnol 37:471 481

Hulecki JC, Foght JM, Gray MR, Fedorak PM (2009) Sulfide persistence in oil field waters amended with nitrate and acetate.J Ind Microbiol Biotechnol 36:1499 1511

Huu NB, Denner EB, Ha DT, Wanner G, Stan-Lotter H (1999) Marinobacter aquaeolei sp. nov., a halophilic bacterium isolatedfrom a Vietnamese oil-producing well. Int J Syst Bacteriol 49(Pt 2):367 375

Jurelevicius D, von der Weid I, Korenblum E, Valoni E, Penna M,Seldin L (2008) Effect of nitrate injection on the bacterialcommunity in a water oil tank system analyzed by PCR-DGGE.J Ind Microbiol Biotechnol 35:251 255

Kaster KM, Grigoriyan A, Jenneman G, Voordouw G (2007) Effect of nitrate and nitrite on sulfide production by two thermophilic,sulfate-reducing enrichments from an oil field in the North Sea.Appl Microbiol Biotechnol 75:195 203

Kim HM, Hwang CY, Cho BC (2010) Arcobacter marinus sp. nov. Int J Syst Evol Microbiol 60:531 536

Kjellerup BV, Veeh RH, Sumithraratne P, Thomsen TR, Buckingham-Meyer K, Frolund B, Sturman P (2005) Monitoring of microbialsouring in chemically treated,produced-waterbiofilm systems usingmolecular techniques. J Ind Microbiol Biotechnol 32:163 170

Kleikemper J, Schroth MH, Sigler WV, Schmucki M, Bernasconi SM,Zeyer J (2002) Activity and diversity of sulfate-reducing bacteriain a petroleum hydrocarbon-contaminated aquifer. Appl EnvironMicrobiol 68:1516 1523

Kumaraswamy R, van Dongen U, Kuenen JG, Abma W, vanLoosdrecht MC, Muyzer G (2005) Characterization of microbialcommunities removing nitrogen oxides from flue gas: theBioDeNOx process. Appl Environ Microbiol 71:6345 6352

Larsen J, Skovhus TL, Agerbk M, Thomsen TR, Nielsen PH (2006)Bacterial diversity study applying novel molecular methods onHalfdan produced waters. NACE - International CorrosionConference Series, Houston, Paper 06668

Magot M (2005) Indigenous microbial communities in oil fields. In:Ollivier B, Magot M (eds) Petroleum microbiology. ASM Press,Washington, pp 21 33

Muyzer G, Stams AJM (2008) The ecology and biotechnology of sulphate-reducing bacteria. Nat Rev Microbiol 6:441 454

Oppenberg B, Schink B (1990) Anaerobic degradation of 1, 3- propanediol by sulfate-reducing and by fermenting bacteria.Antonie Leeuwenhoek 57:205 213

O'Sullivan LA, Weightman AJ, Fry JC (2002) New degenerateCytophaga-Flexibacter-Bacteroides-specific 16 S ribosomal

DNA-targeted oligonucleotide probes reveal high bacterialdiversity in River Taff epilithon. Appl Environ Microbiol68:201 210

Pernthaler J, Glckner FO, Schnhuber W, Amann R (2001)Fluorescence in situ hybridization (FISH) with rRNA-targetedoligonucleotide probes. In: Paul JH (ed) Methods in microbiol-ogy. Academic Press, San Diego, pp 207 226

Pham VD, Hnatow LL, Zhang S, Fallon RD, Jackson SC, Tomb JF,DeLong EF, Keeler SJ (2009) Characterizing microbial diversityin production water from an Alaskan mesothermic petroleumreservoir with two independent molecular methods. EnvironMicrobiol 11:176 187

Reinsel MA, Sears JT, Stewart PS, Mclnerney MJ (1996) Control of microbial souring by nitrate, nitrite or glutaraldehyde injection ina sandstone column. J Ind Microbiol 17:128 136

Schefer H, Muyzer G (2001) Denaturing gradient gel electrophoresisin marine microbial ecology. In: Paul JH (ed) Methods inmicrobiology. Academic Press, pp 425 468

Seitz, HJ, Cypionka H (1986) Chemolithotrophic growth of Desulfo-vibrio desulfuricans with hydrogen coupled to ammonification of nitrate or nitrite. Arch Microbiol 164:63 67

Sette LD, Simioni KC, Vasconcellos SP, Dussan LJ, Neto EV, OliveiraVM (2007) Analysis of the composition of bacterial communitiesin oil reservoirs from a southern offshore Brazilian basin.Antonie Leeuwenhoek 91:253 266

Sorokin DY, Teske A, Robertson LA, Kuenen JG (1999) Anaerobicoxidation of thiosulfate to tetrathionate by obligately heterotrophic bacteria, belonging to the Pseudomonas stutzeri group.FEMS Microbiol Ecol 30:113 123

Telang AJ, Ebert S, Foght JM, Westlake DWS, Jenneman GE, GevertzD, Voordouw G (1997) Effect of nitrate injection on themicrobial community in an oil field as monitored by reversesample genome probing. Appl Environ Microbiol 63:1785 1793

Telang AJ, Ebert S, Foght JM, Westlake DWS, Voordouw G (1998)Effects of two diamine biocides on the microbial communityfrom an oil field. Can J Microbiol 44:1046 1065

Telang AJ, Jenneman GE, Voordouw G (1999) Sulfur cycling inmixed cultures of sulfide-oxidizing and sulfate- or sulfur-reducing oil field bacteria. Can J Microbiol 45:905 913

Vance I, Brink DE (1994) Propionate-driven sulphate-reduction by oil-field bacteria in a pressurised porous rock bioreactor. ApplMicrobiol Biotechnol 40:920 925

Vance I, Thrasher DR (2005) Reservoir souring: mechanisms and prevention. In: Ollivier B, Magot M (eds) Petroleum microbiol-ogy. ASM Press, Washington, pp 123 142

Watanabe K, Watanabe K, Kodama Y, Syutsubo K, Harayama S(2000) Molecular characterization of bacterial populations in petroleum-contaminated groundwater discharged from under-ground crude oil storage cavities. Appl Environ Microbiol66:4803 4809


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