ENVIRONMENTAL BIOTECHNOLOGY

Functional genes (dsr) approach reveals similar sulphidogenicprokaryotes diversity but different structure in saline watersfrom corroding high temperature petroleum reservoirs

Jing Guan & Bing-Liang Zhang &

Serge Maurice Mbadinga &

Jin-Feng Liu & Ji-Dong Gu & Bo-Zhong Mu

Received: 24 April 2013 /Revised: 25 July 2013 /Accepted: 25 July 2013 /Published online: 15 August 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Oil reservoirs and production facilities are general-ly contaminated with H2S resulting from the activity ofsulphidogenic prokaryotes (SRP). Sulphidogenesis plays amajor role in reservoir souring and microbial influenced cor-rosion in oil production systems. In the present study,sulphidogenic microbial diversity and composition in salineproduction fluids retrieved from three blocks of corrodinghigh temperature (79~95 °C) oil reservoirs with high sulfateconcentrations were investigated by phylogenetic analyses ofgene fragments of the dissimilatory sulfite reductase (dsr).Analysis of dsr gene fragments revealed the presence of sev-eral clusters of sulphidogenic prokaryotes that cover the ordersDesulfovibrionales (Desulfovibrio, Desulfomicrobiumthermophilum), Desulfobacterales (Desulfobacterium,Desul fosarcina , Desul fococcus , Desul fo t ignum ,Desulfobotulus, Desulfobulbus), Syntrophobacterales(Desulfacinum, Thermodesulforhabdus, Desulforhabdus),Clostridiales (Desulfotomaculum) and Archaeoglobales(Archaeoglobus); among which sequences affiliated to mem-bers of Desulfomicrobium, Desulfotomaculum andDesulfovibrio appeared to be the most encountered generawithin the three blocks. Collectively, phylogenetic and non-

metric multidimensional scaling analyses indicated similar butstructurally different sulphidogenic prokaryotes communitieswithin the waters retrieved from the three Blocks. This studyshow the diversity and composition of sulphidogenic prokary-otes that may play a role in the souring mediated corrosion ofthe oilfield and also provides a fundamental basis for furtherinvestigation to control oil reservoir souring and corrosion ofpipelines and topside installations.

Keywords Corrosion .dsrgenes .Highsulfate content .Hightemperature oil reservoir . Saline productionwater .

Sulphidogenic prokaryotes

Introduction

Sulphidogenic prokaryotes (SRP) constitute a diverse groupof anaerobic microorganisms that share the exclusive ability toproduce sulfide while oxidizing various electron donorssources. Dissimilatory sulfate reduction and production ofH2S has a central role in the global sulfur cycle and representsone of the most significant organic matter mineralizationprocesses in oil field habitats. During the process of secondaryoil recovery, most often water is injected into the reservoir toincrease pressure and enhance recovery. The supply of sulfateas an electron acceptor and the presence of oil organics andtheir degradation products as electron donors facilitate theenrichment and growth of sulphidogenic prokaryotes in thereservoir, as well as in piping and topside installations (Sundeand Torsvik 2005; Vance and Thrasher 2005).

The activity of SRP causes severe economic problems dueto the reactivity and toxicity of the produced hydrogen sulfide(H2S). In addition to microbiologically influenced corrosionand reservoir souring, the efficiency of oil production is

Electronic supplementary material The online version of this article(doi:10.1007/s00253-013-5152-y) contains supplementary material,which is available to authorized users.

J. Guan :B.<L. Zhang : S. M. Mbadinga : J.<F. Liu :B.<Z. Mu (*)State Key Laboratory of Bioreactor Engineering and Institute ofApplied Chemistry, East China University of Science andTechnology, Shanghai 200237, People’s Republic of Chinae-mail: bzmu@ecust.edu.cn

J.<D. GuSchool of Biological Sciences, The University of Hong Kong,Pokfulam Road, Hong Kong SAR, People’s Republic of China

Appl Microbiol Biotechnol (2014) 98:1871–1882DOI 10.1007/s00253-013-5152-y

decreased due to plugging by SRP biomass and precipitatedmetal sulfide (Davidova et al. 2001; Nemati et al. 2001a).

Awide range of corrosion inhibition researches have beenimplemented to study the problems associated with oil and gasproduction and, at the same time, to protect the environment(Bodtker et al. 2008; Gardner and Stewart 2002; Gieg et al.2011; Hubert 2010; Hubert and Voordouw 2007; Lin et al.2009; Myhr et al. 2002; Nazina et al. 2002; Nemati et al.2001b; Zuo et al. 2004). However, information on the diver-sity and distribution of SRP in oil reservoirs when temperatureis beyond 75 °C is still very limited.

A wide range of SRP community that include several dis-tinct bacterial phyla such as δ-Proteobacteria (Desulfovibrio,Desulfomicrobium, Desulfovermiculus, Desulfanium,Thermonsulforhabdus, Desulfobacter, Desulfobulbus,Desulfotignum, and Desulfobacerium), Firmicutes(Desulfotomaculum), Nitrospira (Thermodesulfovibrio), andThermodesulfobacterium (Thermodesulfobacter) andEuryarchaeota (Archaeoglobus) within the Archaea have beenreported to occur in oil reservoirs ecosystems (Gieg et al. 2011;Youssef et al. 2009). However, there is no single 16S rRNAgene-targeting probe or PCR primer enabling the detection ofall SRP simultaneously since this group of microorganismswasclassified based on their sulphidogenic capabilities. The dsrgene, which encodes for the key enzyme in the anaerobicsulfate respiration pathway, is present in all known SRP. Al-though the dsrAB approach allows a better illustration of thediversity of SRP, and also the discovery of novel dsrAB genesnot yet identified by culturing method (Savage et al. 2010), itmay varywith each individual environmental samples. Becauseof the high specificity of the functional genes, not all complexenvironmental samples can show an abundant diversity byusing the dsrAB method. The 16S rRNA gene using universalprimers for almost all of the bacteria clone libraries showedthat, only one SRP related to Syntrophus sp. was detectable inoil reservoirs sample (Guan et al. 2013)

Identification of oil reservoir microorganisms has so farmost frequently been assessed by cultivation-dependentmethods, and cultivation-independent methods have onlybeen introduced into the field of reservoir microbiology inthe past decade. Information currently available on the micro-bial communities and especially on the abundance ofsulphidogenic prokaryotes associated with high temperaturein oil reservoirs or production systems is sparse.

The use of 16S rRNA gene special groups’ method candisplay an abundant diversity when close attention to thedistribution of some familiar phyla are given in oil field.However, oil reservoir breed abundant microorganisms notjust limited to the six known groups of SRP but far greatestnumber of SRP includingmembers affiliatedwith theArchaea.

The goal of this study was to investigate the diversity andcomposition of SRP in saline waters from corroding hightemperature oil reservoirs using a combination of nested

PCR phylogenetic analyses of dsrAB gene and PCR-DGGEassays. The three analyzed blocks (hereafter refer to as BlockC, T and H) share similar sulphidogenic prokaryotes diversitybut different structure.

Materials and methods

Description of the sampling site

Production water samples were obtained from three differentoil exploration/exploitation blocks in Jiangsu oilfield (Blocksmean large areas of land awarded to oil drilling and explora-tion companies by a country's government). The oilfield wasselected due its reservoir souring and pipeline corrosion back-ground. From an initial average of 510 days for the mainte-nance of the pipelines, souring and corrosion have dramati-cally increased in recent years hence reducing the mainte-nance of the oilfield facilities to just three to four months (timespan for corrosion failure). High souring and high corrosioncause a loss of approximately 85 million RMB (~13.86 mil-lion USD by June 2013) annually to the entire oilfield. Thecharacteristics of the petroleum reservoir water samples areshown in Table 1. Five liters of production water sample fromeach production oil well were collected directly from theproduction valve of the pipeline at the well head into sterilebottles after initial flushing out for 10 to 15 min. The bottleswere completely filled with oil/water mixture, tightly sealedand immediately transported back to laboratory for treatment.Filtration, after separation between oil and water phases, wasused to concentrate the microbial biomass for DNA-basedphylogenetic analyses.

Nucleic acid extraction

The residual oil in samples was removed by heating to 50 °Cfor 15 min and then phase separation was accomplished in 2 Lsterile separatory funnel. The obtained samples were filteredthrough 0.22 μm polycarbonate membranes (25 mm diameter;Millipore, Bedford, USA). The polycarbonate membranes con-taining the cells from water were placed in a centrifuge tube.Genomic DNAs were then extracted from the concentratedbiomass by using the AxyPrep™ Bacterial Genomic DNAMaxiprep Kit (Axygen Biosciences, Inc.) following the proce-dure provide by the manufacturer.The yield and quality ofDNAs were analyzed by agarose gel electrophoresis on 1.0 %(w/v). Extracted DNAs were stored at −20 °C until further use.

PCR amplification of dsrAB gene

A two-step nested PCR amplification reaction was performedto obtain dsrAB gene fragments suitable for further analysis(Giloteaux et al. 2010). dsrAB gene was initially amplified

1872 Appl Microbiol Biotechnol (2014) 98:1871–1882

using the primer set DSR1F/4R primer and then subsequentlynested with dsr619AF/dsr1905BR for the second run. PCR am-plifications were performed with 0.2 mM each deoxynucleosidetriphosphate (dNTP), 1.5 μL 50 mM MgCl2,5 μL 10×iTaqMix,0.5U Taq (TaKaRa Biotechnology Co., Ltd, Dalian, China),0.25 μM each primer, 2 μL DNA template in a final volume of50μL.DNAamplificationwas performedwith aminicycler PTC200 (MJ Research) starting with 5 min at 95 °C, followed by35 cycles consisting of denaturation (45 s at 94 °C), annealing for45 s at either 55 °C for DSR1F/4R (Wagner et al. 1998) or 54 °Cfor dsr619AF/dsr1905BR (Giloteaux et al. 2010), extension (90 sat 72 °C), and a final extension at 72 °C for 10 min. AmplifieddsrAB fragments were resolved by electrophoresis in a 1% (w/v)agarose gel in 1×TAE buffer to confirm the expected size of theproducts (~ 1.2 kb).

Cloning, sequencing and analysis of drsAB sequences

An approximately 1.2 kb fragment the dsrAB genes thatwere nested amplified using the primer set dsr619AF/dsr1905BR were directly ligated into the pMD®19-T Simplevector (TaKaRa Biotechnology Co., Ltd, Dalian, China)and transformed into competent Escherichia coli cells(Shanghai Lifefeng Biotech Co., Ltd) according to theinstructions of the manufacturer. Correct inserts werechecked by PCR using the vector specific primers RV-M(5´-GAGCGGATAACAATTTCACACAGG-3´) and M13-47 (5´-CGCCAGGGTTTTCCCAGTCACGAC-3´). ThePCR reactions were carried out as follows: 5-min initial de-naturation of DNA at 95 °C, followed by 20 cycles of 1 mindenaturation at 94 °C, 1 min primer annealing at 52 °C, 1 min

extension at 72 °C and a final extension step at 72 °C for10min. Positive clones in each library were randomly selectedfor sequencing on an automated ABI Prism 377 DNA analyz-er by using the RV-M sequencing primers. Primers and vectorswere manually removed from the obtained DNA sequences.

PCR amplification of dsrB and DGGE analysis

A dsrB fragment of approximately 350-bp was amplified fromextracted DNA obtained from samples in three different blocksof Jiangsu oilfield using the primers pair DSRp2060F-GC(Geets et al. 2006) and DSR4R (Wagner et al. 1998).ThePCR amplification was carried out in a 25 μL reaction mixturecontaining approximately 20 ng template, 12.5 μL Premix ExTaq (TaKaRa Biotechnology Co., Ltd), 0.5 μM concentrationsof each primer, 2 μL DNA template and dd H2O. The cyclingconditions were as follows: hot-start at 95 °C for 5 min, follow-ed by 35 cycles of denaturation at 94 °C for 40s, annealing at55 °C for 45 s, and elongation at 72 °C for 1 min. The cyclingwas completed by a final extension step at 72 °C for 10 min toreduce the occurrence of doublet artifacts bands during DGGEanalyses. A gradient of 30 - 60 % (w/v) denaturant (the 100 %(w/v) denaturant solution contains 7 M urea and 40 % (v/v)formamide) was constructed in a 1.5 mm thick 8 % (w/v) pol-yacrylamide gel. Gels were at a constant voltage of 160 V for4.5 h using a Protean II system (Bio-Rad, USA). Gels wereincubated in a 1× TAE buffer at a stable temperature of 60 °C.Randomly selected bands of interest were isolated from the gelusing a sterile tip and the DNA containing acryl amide frag-ments were incubated overnight at room temperature in sterilePCR water to allow DNA diffusion out of the polyacrylamide

Table 1 Characterization of the samples from Jiangsu oil reservoirs

Block C Block T Block H

CH 2-9 CH 2-36 CH 2-42 CH 2-43 CH 2-48 T83-8 T83-1 T83-6 T83-7 T83-10 H 88-11 H 88-39

Depth (m) 2580.4 2622.3 2206.9 2072.5 2071.6 2514.2 2475.2 2375.3 2497.5 2522.0 2521.5 2525.4

Temp (°C) 94 95 83 79 79 92 91 88 91 92 90 90

Water flooding(year)

14 14 14 14 14 6 6 6 6 6 3 3

pH 6.93 7.07 6.67 6.86 6.55 6.79 6.87 7.00 6.98 6.45 6.36 6.44

Water type NaHCO3 NaHCO3 NaHCO3 NaHCO3 NaHCO3 NaHCO3 NaHCO3 NaHCO3 NaHCO3 NaHCO3 NaHCO3 NaHCO3

Mineralization(g/l)

21.43 23.04 21.13 18.68 19.63 8.78 6.76 131.66 11.20 10.32 10.79 14.42

Cl- (g/l) 11.89 13.18 12.06 10.40 13.04 5.72 2.59 124.53 4.77 4.41 6.28 4.33

SO42- (mg/l) 457.67 82.31 nd nd 86.40 193.00 469.20 462.00 856.07 436.26 571.00 3273.63

PO43- (mg/l) 5.10 4.28 2.75 4.46 0.70 8.25 21.30 53.00 29.56 53.06 10.45 11.85

NO3- (mg/l) 19.54 23.46 12.63 12.48 0.44 0.66 5.27 0.02 12.42 15.04 14.13 21.54

K++Na+ (g/l) 8.13 8.75 7.94 7.01 6.41 2.82 2.87 6.44 4.23 3.75 3.90 5.15

Ca2++Mg2+

(mg/l)96.11 136.37 182.29 129.20 95.00 40.60 77.30 179.00 19.22 13.86 20.30 31.68

H2S (mg/l) 30.56 40.08 19.53 34.11 nd nd 1.25 nd 8.19 54.53 97.10 78.80

* nd: not detectable

Appl Microbiol Biotechnol (2014) 98:1871–1882 1873

matrix. The solution was directly used for further amplifica-tions. Excised bands were re-amplified using the cycling pre-viously described (primer set DSRp2060F-GC/DSR4R, 20 cy-cles), and re-run on DGGE to confirm their identity and purityprior to purification and direct sequencing with primer setDSRp2060F/DSR4R.

Phylogenetic analysis of dsr gene fragments

Recovered partial dsr gene sequences were initially compared toGenBank database for preliminary identification using theBLASTN and BLASTX routines (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Gene fragments were translated into proteinsequences with Expasy (http://web.expasy.org/translate/).MEGA 5 software was used to align the protein sequencesand generate phylogenetic trees. Bootstrap of 1000 replicateswas adopted to assign confidence levels at the nodes of the trees.Comparison of dsr gene clone libraries constructed from thethree Blocks was based on relative abundance data for differentOTUs identified. Non-metric multidimensional scaling (NMDS)was used to determine the similarity between the Blockswith the

community analysis software PRIMER 6 (Clarke and Gorley2006). The NMDS plot is a representation of how different theSRP communities are from each other (at the genus level) basedon clustering of like samples. Bray-Curtis similarities werecalculated on the OTU relative abundance matrices.

Nucleotide sequence accession numbers

Dsr gene fragments obtained in this study were deposited inGenBank andwere assigned the accession numbers KC285781to KC285837 for clones’ libraries (see Table S1) andKC480059 to KC480065 for randomly selected DGGE bands.

Results

Physicochemical characteristics of oil reservoirs productionfluids

The characteristics of the investigated oil reservoirs produc-tion fluids are summarized in Table 1. Fluids were collected

Clone JSO_H11b29 (KC285834)Clone JSO_C36b22 (KC285831)

Desulfomicrobium orale dsrA (AY083030)

Desulfomicrobium norvegicum dsrA (AB061532)

Desulfomicrobium apsheronum dsrA (AF482459)

Desulfonatronovibrio hydrogenovorans dsrA (AF418197)Desulfobacterium anilini dsrA (AF482455)High-temperature oil production water dsrA (FN376512)

Clone JSO_T8b32 (KC285784)Desulfobacterium oleovorans dsrA (AF418201)

Desulfobacter latus dsrA (U58124)

Desulfotignum balticum clone DSM7044-1dsrA (AF420284)

Desulfobacter vibrioformis dsrA (AJ250472)

Anaerobic hydrocarbon-degrading consortia dsrA (AF327309)

Desulfocella halophila dsrA (AF418200)

Desulfobulbus propionicus dsrA (AF218452)

Desulfobulbus rhabdoformis dsrA (AJ250473)

Desulfobacterium vacuolatum dsrA (AF418203)

Desulfobotulus sapovorans dsrA (U58120)

Clone JSO_T10b16 (KC285783)Mixture of the water from Japanese oil field dsrA (AB493909)

Clone JSO_C42b33 (KC285782)Desulfococcus multivorans dsrA (U58126)

Desulfovibrio fructosovorans dsrA (AB061538)

Reed rhizosphere Desulfovibrio sp. LVS-21 dsrA (AM494497)

Desulfovibrio desulfuricans dsrA (AF273034)

Clone JSO_H11b09 (KC285787)

Clone JSO_C48b06 (KC285806)Desulforhabdus amnigena dsrA (AF337901)

Desulfacinum infernum dsrA (AF418194)

Clone JSO_H39b63(KC285786)

Deltaproteoba

cteria

NitrospiraThermodesulfovibrio yellowstonii dsrA (U58122)

99

98

92

64

90

81

72

71

60

60

5%Fig. 1 Phylogenetic tree ofenvironmental bacterial dsrAgene retrieved from tree blocks ofoilfields showing the distributionsof the OTUs related to sulfur-reducing bacteria and closelyrelated sequences from GenBankdatabase. Alignments to relatedsequences were performed withMEGA 5 software. The topologyof the tree was obtained with theneighbor-joining method. Thevalues at the nodes are bootstrapvalues (n=1000 replicates) of ≥75 % are reported. The scale barrepresents 5 % sequencedivergence. Sampling locationsare named as in Table 1 and thesequences retrieved from thecorresponding injected waterwere also shown

1874 Appl Microbiol Biotechnol (2014) 98:1871–1882

Desulfosarcina variabilis str. Montpellier dsrB (AF191907) Desulfosarcina variabilis dsrB (AJ310429)

Salt marsh clone PIMOD08 dsrB (AY741567) East Sea gas hydrate-bearing sediment dsrB (JN798935)

Desulfococcus oleovorans Hxd3 dsrB (AF418201) Desulfobacterium oleovorans DSM 6200 dsrB (AF418201) Clone JSO_H11b39 (KC285826)

Marine clone DB_dsr12 dsrB (EU350969) Clone JSO_C48b67 (KC285825)

Desulfococcus multivorans dsrB (U58126) Desulfobacter vibrioformis dsrB (AJ250472)

Desulfobacter latus dsrB (U58124) Desulfobacterium vacuolatum DSM 3385 dsrB (AF418203)

Clone JSO_C48b47 (KC285824) Desulfotignum balticum dsrB (AF482463) Marine Desulfotignum balticum dsrB (AF420284)

Desulfoba

cteraceae

Desulfonatronovibrio hydrogenovorans DSM 9292 dsrB (AF418197) Clone JSO_T1b38 (KC285827)

Desulfomicrobium orale dsrB (AY083030) Desulfomicrobium norvegicum dsrB (AB061532) Desulfomicrobium apsheronum dsrB (AF482459)

Desulfomicrobiaceae

Desulfobacterium anilini dsrB (AF482455) Desulfotomaculum thermobenzoicum dsrB (AF273030)

Clone JSO_C48b17 (KC285811) Clone JSO_T10b08 (KC285818)

Desulfotomaculum acetoxidans dsrB (AF271768) uncultured bacterium dsrB (FJ948562)

Desulfotomaculum thermosapovorans dsrB (AF271769) Desulfotomaculum sp. Eth-2 dsrB (JQ304755) Sea sediments clone B17 dsrB (JQ304784)

Clone JSO_C9b23 (KC285812) Desulfotomaculum sp. Srb55 dsrB (AB260069)

Desulfotomaculum geothermicum dsrB (AF273029) Desulfotomaculum bacterium dsrB (JQ520153) Clone JSO_T10b04 (KC285817) 2km Deep environment dsrB (FJ948568)

Clone JSO_H11b10 (KC285821)

Desulfotomaculum

Desulfovibrio fructosovorans dsrB (AB061538) Clone JSO_C42b28 (KC285805)

Desulfovibrio desulfuricans dsrB (AF273034) Desulfovibrio

Desulfobulbus propionicus dsrB (AF218452) Desulfobulbus rhabdoformis dsrB (AJ250473) Clone JSO_C36b20 (KC285790) Clone JSO_T10b06 (KC285791)

Desulfobacteraceae

Thermodesulforhabdus norvegica dsrB (AJ277293) Clone JSO_H11b02 (KC285792)

High-temperature oil production water dsrB (FN376544) Desulfacinum infernum DSM 9756 dsrB (AF418194)

Syntrophobacteraceae

Deep-sea hydrothermal environments dsrB (AY354081) Clone JSO_T6a05 (KC285794) )/JSO_H39b43 (KC285795)

Clone JSO_C9a22 (KC285796) Archaeoglobus profundus dsrB (AF071499)

Archaeoglobus veneficus dsrB (BAF64852) Archaeoglobus veneficus SNP6 dsrB (YP 004342656)

Archaeoglobus infectus DSM 18877 dsrB (BAF64849) Archaeoglobus fulgidus DSM 4304 dsrB (NP 069260) Archaeoglobus fulgidus dsrB (M95624) Archaeoglobus fulgidus dsrB (JQ045863)

High-temperature oil production water dsrB (FN376487)

Archa

eoglobus

Nitrospira Thermodesulfovibrio yellowstonii dsrB (U58122)

99

99

97

99

98

100

99

99

99

99

78

99

98

97

96

95

96

96

96

95

94

88

88

68

65

95

5%

Fig. 2 Phylogenetic tree ofenvironmental bacterial dsrBgene retrieved from tree blocks ofoilfields showing the distributionsof the OTUs related to SRP andclosely related sequences fromGenBank database. Alignmentsto related sequences wereperformed with MEGA 5software. The topology of the treewas obtained with the neighbor-joining method. The values at thenodes are bootstrap values(n=1000 replicates) of ≥ 75 % arereported. The scale bar represents5 % sequence divergence.Sampling locations are named asin Table 1 and the sequencesretrieved from the correspondinginjected water were also shown

Appl Microbiol Biotechnol (2014) 98:1871–1882 1875

from twelve oil-producing wells located in three differentBlocks of the Jiangsu oilfield. The fluids were characterizedwith pH values from slightly acidic (pH ~6.36) to neutral (pH~7.07). Mineralization varied from 6.76 g/l to 131.65 g/l, withhigh chloride (2.6 g/l −124.52 g/l) and high sulfate contents

(0.082 g/l–3.27 g/l). The amount of Na++K+ was ranged from2.82 g/l to 8.75 g/l, and Ca2++Mg2+ was between 13.86 mg/land 182.29 mg/l. The ratio (Na++K+)/( Ca2++Mg2+) in thetwelve oil reservoirs varied from 48 to 203.5; indicating thatmonovalent cations dominate over divalent cations. H2S

Desulfotignum balticum clone DSM7044-1 dsrB (AF420284)

Desulfotignum balticum dsrB (AF482463)

Desulfobacter vibrioformis dsrB (AJ250472)

Desulfobacter latus dsrB (U58124)

Desulfobacterium vacuolatum DSM 3385 dsrB (AF418203)

East Sea gas hydrate-bearing sediment dsrB (JN798935)

Marine clone DB_dsr12 dsrB (EU350969)

Desulfosarcina variabilis str. Montpellier dsrB(AF191907)

Desulfosarcina variabilis dsrB (AJ310429)

Desulfococcus multivorans dsrB (U58126)

Desulfobacterium oleovorans DSM 6200 dsrB (AF418201)

Desulfococcus oleovorans Hxd3 dsrB (AF418201)

Desulfoba

cteraceae

JSO DGGE band C1, C5, C10, C13, TH9, T14

Desulfovibrio desulfuricans dsrB (AF273034) Desulfovibrionaceae

Desulfomicrobium orale dsrB (AY083030)

Desulfomicrobium norvegicum dsrB (AB061532)

Desulfomicrobium apsheronum dsrB (AF482459)

Desulfomicrobiaceae

2km Deep environment dsrB (FJ948562)

JSO DGGE band C11, TH11

High-temperature oil production water dsrB (JQ520153)

2km Deep environment dsrB (FJ948568)

Desulfotomaculum sp. Srb55 dsrB (AB260069)

Desulfotomaculum geothermicum dsrB (AF273029)

Desulfotomaculum acetoxidans dsrB (AF271768)

Desulfotomaculum thermobenzoicum dsrB (AF273030)

Desulfotomaculum thermosapovorans dsrB (AF271769)

Sea sediments clone B17 dsrB(JQ304784)

Desulfotomaculum sp. Eth-2 dsrB (JQ304755)

Desulfotomaculum

Desulfobulbus propionicus dsrB (AF218452)

JSO DGGE band T12

Desulfobulbus rhabdoformis dsrB (AJ250473)

Desulfobulbaceae

JSO DGGE band TH10

Desulfarculus baarsii DSM 2075 dsrB (NC 014365)

Thermodesulforhabdus norvegica dsrB (AJ277293)

JSO DGGE band T5, C6, H6

Desulfacinum infernum DSM 9756 dsrB (AF418194)

JSO DGGE band T4

High-temperature oil production water dsrB (FN376553) Syntroph

obacteraceae

Archaeoglobus fulgidus dsrB (M95624)

High-temperature oil production water dsrB (FN376487)

Archaeoglobus fulgidus dsrB (JQ045863)

Archaeoglobus fulgidus DSM 4304 dsrB (NP 069260)

JSO DGGE band C2, C4,C4, TH3, TH13 Archaeoglobus profundus dsrB (AF071499)

Archaeoglobus profundus DSM 5631 dsrB ()

Deep-sea hydrothermal environments dsrB (AY354081)

Archaeoglobus infectus DSM 18877 dsrB (BAF64849)

Archaeoglobus veneficus dsrB (BAF64852)

Archaeoglobus veneficus SNP6 dsrB (YP 004342656)

Archa

eoglobus

Nitrospira Thermodesulfovibrio yellowstonii dsrB (U58122)

75 100

67

100

100

61 100

99

99

99

92 99

92 98

98

98

80

71

65

64

76

99

5% Fig. 3 Phylogenetic tree ofenvironmental bacterial dsrBgene retrieved from PCR-DGGEband (tree blocks of oilfields)showing the distributions of theOTUs related to SRP and closelyrelated sequences from GenBankdatabase. Alignments to relatedsequences were performed withMEGA 5 software. The topologyof the tree was obtained with theneighbor-joining method. Thevalues at the nodes are bootstrapvalues (n=1000 replicates) of ≥75 % are reported. The scale barrepresents 5 % sequencedivergence. Sampling locationsare named as in Table 1 and thesequences retrieved from thecorresponding injected waterwere also shown

1876 Appl Microbiol Biotechnol (2014) 98:1871–1882

varied from none detectable amounts to as high as 97.10 mg/l.Nitrate (NO3

-) and phosphate (PO43-) were also detectable but

in relatively minor proportions.

Sulphidogenic prokaryotes composition in production fluidsfrom three distinct Blocks

In this work, SRP communities in tree blocks of oilfields wereanalyzed using phylogenetic analyses of dsrAB gene se-quences. Five producing wells from Block C (C9, C36, C42,C43 and C48), five producing wells from Block T (T8, T1,T6, T7 and T10) and two producing wells from Block H (H11and H39) were sampled. A total of 168, 179, and 77 cloneswere randomly selected from the libraries established fromproduction water samples collected respectively from BlockC, Block T, and Block H. After screening all the sequences,there were 10, 7 and 8 operational taxonomy units (OTUs)obtained from Block C, Block T, and Block H respectivelyand they were separately classified into 13 different phyloge-netic groups. The phylogenetic affiliation of the dsrAB genesequences is presented in Fig. 1 and Fig. 2. The compositionof sulphidogenic prokaryotes in each block is given in Fig. 3.

Block C

The ten OTUs from Block C were affiliated with the genusArchaeoglobus (1.2 % of total clones), Desulfomicrobium(31.0 % of total clones),Desulfovibrio (31.0 % of total clones),Desulfotomaculum (20.2 % of total clones), Thermo-desulforhabdus (2.4 % of total clones), Desulforhabdus

(1.2 % of total clones), Desulfotignum (6.0 % of total clones),Desulfosarcina (2.4 % of total clones),Desulfobulbus (4.2 % oftotal clones) and Desulfobotulus (0.6 % of total clones).

Block T

The seven OTUs from Block T were affiliated with the genusArchaeoglobus (1.7 % of total clones), Desulfomicrobium(27.9 % of total clones),Desulfovibrio (25.7% of total clones),Desulfotomaculum (29.6 % of total clones), Desulfobacterium(1.7 % of total clones), Desulfobulbus (11.7 % of total clones)and Desulfobotulus (1.7 % of total clones).

Block H

The eight OTUs from Block H were affiliated with the genusArchaeoglobus (7.8 % of total clones), Desulfomicrobium(50.6 % of total clones), Desulfovibrio (2.6 % of total clo-nes),Desulfotomaculum (24.7 % of total clones), Thermo-desulforhabdus (1.3 % of total clones), Desulfacinum (7.8 %of total clones), Desulfococcus (2.6 % of total clones),Desulfosarcina (2.6 % of total clones).

Phylogenetic analyses of dsrA gene sequences

The phylogenetic affiliation of the dsrA gene sequences ispresented in Fig. 1. Six OTUs were affiliated with the genusDesulfomicrobium, Desulfobacterium, Desulfobotulus,Desulfovibrio, Desulforhabdus, Desulfacinum. For instance,OTUs Block H clone H11b29 and OTUs Block C clone

Fig. 4 Denaturing gradient gel electrophoresis of dsrB PCR products.Sequenced bands are indicated on the Figure. The lanes from left to rightare samples in block T (T3, T1, T6, T7, T8, T10), blocks H (H7, H10,

H11, H21, H31, H39) and block C (C3, C2, C9, C36, C43, C48, C42).Only representative samples were analyzed. DGGE bands labeled THmeans these bands were detected both in block T and block H

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C36b22 were closely related to Desulfomicrobium orale dsrA(AY083030) (Loy et al. 2002), OTUs Block T clone T8b32 wasclosely related to Desulfobacterium oleovorans dsrA(AF418201) (Friedrich 2002), OTUs Block C clone C42b33and OTUs Block T clone T10b16 were closely related toDesulfobotulus sapovorans dsrA (U58120) (Michael Wagner

1998), OTUs Block H clone H11b09 was closely related toDesulfovibrio desulfuricans dsrA (AF273034) (Laue et al.2001), OTUs Block C clone C48b06 was closely related toDesulforhabdus amnigena dsrA (AF337901) (Klein et al.2001), OTUs Block H clone H39b63 was closely related toDesulfacinum infernum dsrA (AF418194) (Friedrich 2002).

Fig. 5 Non-metric MDS analysis of the SRP communities present inJiangsu oilfield based on OTU frequencies in dsrAB gene clone libraries.The MDS plot is a representation of how different the SRP communities

are from each other (at the genus level) based on clustering of likesamples. Similarity contour lines from cluster analyses are superimposedon to the MDS plot

1878 Appl Microbiol Biotechnol (2014) 98:1871–1882

Phylogenetic analyses of dsrB gene sequences

The phylogenetic affiliation of the dsrB gene sequences ispresented in Fig. 2. Ten OTUs were affiliated with the genusArchaeoglobus (clone T6a05, clone C9a22, clone H39b43),Desulfacinum (clone H11b02), Desulfobulbus (clone C36b20,cloneT10b06), Desulfovibrio (cloneC42b28), Desulfotomaculum(clone T10b04, cloneH11b10, cloneC9b23, clone C48b17, cloneT10b08), Desulfomicrobium (clone T1b38), Desulfotignum (cloneC48b47), Desulfosarcina (clone C48b67), Desulfococcus (cloneH11b39).

Phylogenetic characterization of randomly selecteddsrB-DGGE

The phylogenetic affiliation of the dsrB-DGGE gene sequences ispresented in Fig. 3. The dsrB sequences corresponding to fivebands clustered with representatives of the family‘Archaeoglobaceae’. A total of five Archaeoglobus fulgidus-relat-ed sequenceswere found amongDGGEbands (C2, C3, C4, TH3,and TH13). A total of six Desulfovibrio desulfuricans-relatedsequences were found among DGGE bands (C1, C5, C10, C13,TH9, and T14). OneDGGE bandwas relatedwithDesulfobulbuspropionicus (T12), and twowithDesulfotomaculum (C11, TH11).Four bands clustered with representatives of the family‘Syntrophobacteraceae’. One DGGE band was related withThermodesulforhabdus norvegica (T4), and three withDesulfacinum infernum (T5, C6, and H6). DGGE band TH10showed similarities withDesulfarculus baarsii. Denaturing gradi-ent gel electrophoresis of dsrB PCR products is showed in Fig. 4.

Similarities and differences of in the SRP communities

Non-metric MDS analysis of the SRP communities present inJiangsu oilfield based on OTU frequencies in dsrAB geneclone libraries. Similarity contour lines from cluster analysesare superimposed on to the MDS plot (Fig. 5). In general, allthe samples are at least 30% similar; samples in Block Tare atleast 40 % similar whereas samples in Block H are at least60 % similar. Samples T6 and H11, T1 and T8, C9 and C39,C43 and T7 are at least 70 % similar.

Discussion

This present study aimed to characterize the sulphidogenicprokaryotes population in twelve oil reservoirs productionfluids collected at three Blocks from a highly souring andcorroding high temperature (79~95 °C) oilfield in China inorder to bring insights into the diversity and structural compo-sition of the SRP communities associated with these ecologicalsites. The investigated oil reservoirs encompass a wide range ofphysico-chemical conditions, including high mineralization as

high as 131.65 g/l, slightly acidic pH, high sulphate (up to3.27 g/l), high chloride (up to 124.53 g/l) content and a highratio of monovalent cations over divalent ones. The presence ofSRP communities in such extreme environments is rarelyreported (Gieg et al. 2011). Nevertheless, phylogenetic analy-ses revealed the identification of several SRPwithin all samplesresembling to those observed in other investigation of variouspetroleum reservoirs (Duncan et al. 2009; Gieg et al. 2011;Youssef et al. 2009). We observed a relatively high diversity ofSRP among the three Blocks though some SRP were onlydetected in minor proportions. The most encountered thermo-philic SRP include members affiliated with the generaArchaeoglobus, Desulfotomaculum, Desulfomicrobium,Desulfacinum and Thermodesulforhabdus. Growth tempera-tures varying from 40 °C to 90 °C have been documented forknown closest relatives (Magot et al. 2000; von Jan et al. 2010)with a maximum optimum temperature of 82 °C reported forArchaeoglobus profundus (von Jan et al. 2010). These temper-atures are close to those reported in the present study (Table 1).It is generally accepted that metals corrosion is stimulated byanaerobic microbes, especially sulphidogenic prokaryotes viaformation of corrosive H2S (Nakagawa et al. 2006). Indeed, ashigh as 97.10 mg/L of H2S were detected in the samplesdescribed in the present investigation. Such high concentrationsof H2S are indicative of high activity of sulphidogenic prokary-otes. However, though microbial induced corrosion at hightemperature is not commonly investigated; some relatives ofthermophilic SRP retrieved from our samples collected atJiangsu oilfield have nevertheless been reported to as metals

Table 2 PCR-DGGE analyses (bands) and relative abundance of differ-ent SRP phylogenetic groups as revealed by the clone library (clones)

SRP affiliation Relative abundance (%) PCR-DGGE

dsrA geneClones

dsrB geneClones

Bands Detected

Archaeoglobus 0 2.6 C2, C4, C4, TH3,TH13

Desulfomicrobium 31.4 1.9 -

Desulfovibrio 5.4 18.2 C1, C5, C10, C13,TH9,T14

Desulfotomaculum 0 25.0 C11, TH11

Thermodesulforhabdus 0.2 0.9 T4

Desulforhabdus 0.5 0 -

Desulfacinum 0 1.4 T5, C6, H6

Desulfococcus 0 0.5 -

Desulfotignum 0 2.4 -

Desulfosarcina 0 0.9 -

Desulfarculus 0 0 TH10

Desulfobulbus 0 6.6 T12

Desulfobacterium 1.2 0 -

Desulfobotulus 0.9 0 -

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corroding microbes under thermophilic conditions(Anandkumar et al. 2009a; Anandkumar et al. 2009b; Duncanet al. 2009; Islam and Karr 2013; Lapaglia and Hartzell 1997).Although mesophiles SRP such as members of the genus

Desulfovibrio have been reported from high temperature oilreservoirs (Dahle et al. 2008; Mbadinga et al. 2012), theywould have found their niche in the cooler part of the oilreservoirs investigated herein.

Fig. 6 Frequency distribution of dsrAB (classified into major phyloge-netic groups) recovered in clone libraries from the three Blocks of Jiangsuoilfield. Values shown above the error bars represent the percentage of

samples in which different genera of SRP were identified. The error barsrepresent 1×standard error

1880 Appl Microbiol Biotechnol (2014) 98:1871–1882

Phylogenetic analyses of dsrAB genes clone sequences

Phylogenetic analysis based on dsrAB gene sequences by themethod of nested-PCR was applied for detecting the majorphylogenetic groups of SRP in the three blocks (Giloteauxet al. 2010). Phylogenetic analysis of dsrA gene sequencesrevealed a lower diversity than phylogenetic analysis of dsrBgene sequences of SRP in this study. Only Deltaproteobacteriawere detected in dsrA gene sequences. Six OTUswere affiliatedwith the genera Desulfomicrobium, Desulfobacterium,Desulfobotulus, Desulfovibrio, Desulforhabdus. Nevertheless,not merelyDeltaproteobacteria but alsoClostridia (Firmicutes)and Archaeoglobi (Euryarchaeota) were detected in phylogenet-ic analyses of dsrB gene clone sequences. Ten OTUs wereaffiliated with members of the genera Archaeoglobus,Desulfotomaculum, Desulfomicrobium, Desulfococcus,Desulfovibrio, Desulfacinum, Thermodesulforhabdus,Desulfotignum, Desulfosarcina, and Desulfobulbus.

Phylogenetic analyses of dsrB genes DGGE sequences

Since the phylogenetic analysis of dsrB gene resulted in amuch high diversity of SRP communities, a PCR-DGGEbased on dsrB was implemented. It is displayed that clonedsrB sequence analysis of clone libraries established from theproduction water samples in the three Blocks (C, T, and H)agrees with the PCR-DGGE based approach; resulting in theoccurrence of several DGGE bands indicating the presence ofa much higher diversity in dsrB (Fig. 3). However, six generaof SRP were not detectable in DGGE band sequences. GeneraDesulforhabdus, Desulfobacterium and Desulfobotulus wereonly detected in dsrA-clone sequences, whereas generaDesulfococcus, Desulfosarcina and Desulfotignum were onlydetected in dsrB-clone sequences. Desulfomicrobium-like se-quences were also found in both dsrA-Clone and dsrB-clonelibraries. Table 2 summarizes the relative abundance of dif-ferent SRP phylogenetic groups as revealed by the dsrA-clonelibrary and the dsrB-clone library as well as those detected viaPCR-DGGE approach.

Frequency distribution of sulphidogenic prokaryotes

In the present study, the frequency distribution of dsrAB generecovered in clone libraries from constructed from the threeblocks of Jiangsu oilfield is shown in Fig. 6. Five oil producingwells were investigated in Block C and Block T respectivelyand two oil wells belonged to Block H. Thirteen types of SRPwere analyzed in total; there were 10, 7 and 8 types in Block C,Block T and Block H respectively. For Block C and Block T,Desulfovibrio, Desulfotomaculum and Desulfomicrobiumwere the most frequently encountered phylogenetic group;while Desulfomicrobium, Desulfotomaculum, ArchaeoglobusandDesulfacinumwere most frequently recovered in Block H.

NMDS analysis and frequency distribution of SRP within thethree Blocks indicated that the diversity of SRP communitiesare similar among different Blocks but different in structurecomposition.

The present investigation which aimed to provide a pictureof the diversity and composition of sulphidogenic prokaryotesthat would have played a role in the souring mediated corro-sion of the oilfield provides some fundamental basis to controloil reservoir souring and corrosion of pipelines and topsideinstallations via nitrate/nitrite injection.

Acknowledgments This work was supported by the National NaturalScience Foundation of China (Grant No. 41073055) and the NSFC/RGCJoint Research Fund (No. 41161160560).

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