Identification and Phylogenetic Analysis of Thermophilic Sulfate-Reducing Bacteria in Oil Field Samples by 16S rDNA Gene Cloning and Sequencing

Anaerobe Ž1998. 4, 165]174 Article No. an980156

ENVIRONMENTAL MICROBIOLOGY

Identification and Phylogenetic Analysis of Thermophilic Sulfate-Reducing Bacteria in Oil Field Samples by 16S rDNA Gene Cloning and Sequencing Jyh-Yih Leu1, Caroline P. McGovern-Traa 2, Andrew J. R. Porter 2, William J. Harris 2 and W. Allan Hamilton 2 1

Environmental Biology Division, Development Center for Biotechnology, 81 Chang-Hsing Street, Taipei, Taiwan. R.O.C. 2 Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK (Received 15 October 1997, accepted 30 March 1998 Key Words: Desulfotomaculum, hydrocarbon reservoir souring, sulfidogenesis, thermophilic sulfate-reducing bacteria, thermophilic anaerobe

Thermophilic sulfate-reducing bacteria ŽSRB. have been recognized as an important source of hydrogen sulfide ŽH 2 S. in hydrocarbon reservoirs and in production systems. Four thermophilic SRB enrichment cultures from three different oil field samples Žsandstone core, drilling mud, and production water. were investigated using 16S rDNA sequence comparative analysis. In total, 15 different clones were identified. We found spore-forming, low GqC content, thermophilic, sulfate-reducing Desulfotomaculum-related sequences present in all oil field samples, and additionally a clone originating from sandstone core which was assigned to the mesophilic Desulfomicrobium group. Furthermore, three clones related to Gram-positive, non-sulfate-reducing Thermoanaerobacter species and four clones close to Clostridium thermocopriae were found in enrichment cultures from sandstone core and from production water, respectively. In addition, the deeply rooted lineage of two of the clones suggested previously undescribed, Gram-positive, low GqC content, thermophilic, obligately anaerobic bacteria present in production water. Such thermophilic, non-sulfate-reducing microorganisms may play an important ecological role alongside SRB in oil field environments.

Introduction Sulfate-reducing bacteria ŽSRB. are strictly anaerobic microorganisms responsible for the terminal mineralization of organic material in anoxic environments. The growth of SRB in oil-bearing reservoirs has been Address correspondence to: Dr Jyh-Yih Leu. Phone: Ž02. 27325123 ext. 4202. Fax: Ž02. 27334693. E-mail: [email protected] or Professor W. Allan Hamilton: Phone: Ž01224. 273100. E-mail: [email protected]

1075-9964r98r030165q 10 $30.00r0

Q 1998 Academic Press

shown to be responsible for oil formation souring w 1,2x . So far, the mechanisms of the process are not fully understood, and consequently reservoir souring associated with sulfide-producing microorganisms is still seen as a major research topic. The majority of studies on SRB in oil field environments have concentrated on the ecology and physiology of mesophilic microorganisms, which grow optimally between 208C and 408C. However, most oilbearing reservoirs, especially in North Sea fields, Q 1998 Academic Press

166

J.-Y. Leu et al.

exist in deep geological strata, with temperatures above 608C w 3x . Hot oil field environments constitute ecological niches in which thermophilic microorganisms growing above 508C are likely to be responsible for the mineralization of organic material. It has been suggested therefore, that thermophilic sulfate reducers, which have been recently isolated from or detected in such environments using cultivation methods w 1,4]10x , play an important role in the biogenic production of H 2 S in oil reservoirs and in production systems. Therefore, the identification and characterization of thermophilic SRB present in oil field environments is an essential requirement in order to understand the mechanism of reservoir souring. To date, growing thermophilic SRB in pure culture has remained a challenging task due to both their slow growth rate and the limitation of existing cultivation techniques. Their close association with other thermophilic fermentative bacteria also contributes to the difficulty in obtaining pure SRB cultures. The incomplete description of thermophilic SRB species in natural environments has profoundly hindered the understanding of their bacterial diversity and ecological role. With sensitive and specific molecular techniques, SRB can now be identified independent of their growth in pure culture. Reverse sample genome probing ŽRSGP. has been developed by Voordouw and his colleagues w 11,12x to analyse the diversity of SRB in samples obtained from oil fields after liquid culture enrichment. Amann et al. w 13x have successfully identified specific SRB populations within developing and established biofilms using general and specific hybridization probes. Combined with polymerase chain reaction ŽPCR., gene cloning and sequencing, the use of molecular biological techniques, especially those that take advantage of the 16S rRNA molecule, has become a powerful tool for studying the diversity and structure of natural microbial communities. The presence of previously unknown microorganisms has been discovered in microbial communities of open sea, hot spring, thermal mats, and soil through comparison of cloned 16S rRNA genes w 14]18x . The direct amplification of partial 16S rRNA sequences, from a marine sediment, using PCR has demonstrated the existence of novel SRB species that have not yet been obtained in pure culture w 19x . In the present study, the comparative analysis of cloned 16S rDNA sequences from four thermophilic SRB enrichment cultures obtained from samples taken from different oil field locations was used to identify sulfate-reducing microorganisms. We have found thermophilic, sulfate-reducing Desulfotomaculumrelated sequences present in all oil field samples, and a clone prepared from one of the samples was found to belong to the Desulfomicrobium group. In addition, clones related to Gram-positive, low GqC content, thermophilic, non-sulfate-reducing, anaerobic bacte-

Table 1. Description of thermophilic sulfate-reducing bacterial cultures used in this study Enrichment culture Sample

Depth Žfeet. below the sea floor.

12FL

13FA

Sandstone core Žinner material.

Sandstone core Žinner. material.

3504

3504

FP5F Drilling mud

11500

OB5M Production water

—

Location

North Sea

North Sea

North Sea

Oman

Carbon substrate

Lactate

Acetate

Propionate

Butyrate

Salinity Ž% .

0.2

0.2

0.2

2

Temperature Ž8C .

55

55

55

55

ria were also found.

Materials and Methods SRB enrichment cultures Four SRB thermophilic enrichment cultures Ž12FL, 13FA, FP5F, and OB5M., which were grown at 558C from three different oil field samples Žsandstone core, drilling mud, and production water. and described in Table 1, were analysed by 16S rDNA comparative analysis. Sandstone core was taken from a vertical depth of 3504 feet under the North Sea floor by Corex ŽU.K.. Ltd, Aberdeen. The core samples were transported aseptically and kept in anaerobic boxes with argon gas to minimize O 2 exposure. The inner material of a 5-day-old core was aseptically separated from the outer area, which was likely to be invaded by drilling mud, and was used for enrichment of SRB cultures. Water-based drilling mud was sampled from another field at 11500 feet under the North Sea floor. Production water was collected from oil production systems in Oman. Mud and production water samples were taken in sterilized plastic bottles pre-flushed with N2 , filled to the top and sealed to prevent oxidation. SRB enrichment medium was modified from Widdel and Bak w 20x . Medium contained Žper liter distilled water. NaCl 20 g Žsaltwater. or 2 g Žfreshwater., MgCl 2 ? 6H 2 O 3 g Žsaltwater. or 0.4 g Žfreshwater., CaCl 2 ? 2H 2 O 0.15 g Žsaltwater. or 0.1 g Žfreshwater., Na 2 SO4 4 g, NH 4 Cl 0.25 g, KH 2 PO4 0.2 g, KCl 0.5 g, yeast extract 1 g and resazurin Ž0.1%, wrv., adjusted to pH 7.4]7.6. In addition, 1 mL of trace element mixture, 1 mL of selenite solution, 30 mL of NaHCO3 solution, 7.5 mL of sulfide solution w 20x were added to each liter of medium, along with a carbon source. The carbon substrates used for cultures 12FL, 13FA, FP5F, and OB5M were lactate Ž20

Identification of Thermophilic Sulfate-reducing Bacteria in Oil Field Samples mM., acetate Ž20 mM., propionate Ž16 mM., and butyrate Ž10 mM., respectively. Media were inoculated with 4]5% inoculum and incubated at 558C. The growth of sulfide-producing microorganisms including SRB was indicated by the turbidity of culture medium and the detection of H 2 S w 21x . Observation of cells in the light microscope was also routinely used to determine the growth of sulfide-producing microorganisms. With the exception of primary enrichments, defined SRB medium Žwithout yeast extract. supplemented with vitamin mixture Ž1 mLrL. Žcontaining distilled water 100 mL, p-aminobenzoic acid 5 mg, biotin 1 mg, nicotinic acid 10 mg, pantothenic acid 5 mg, pyridoxal 15 mg, thiamine 10 mg, vitamin B 12 5 mg, and riboflavin 5 mg. was used for the growth and maintenance of SRB cultures.

167

16.8 mL of dATP Ž1.25 mM., 16.8 mL of dCTP Ž1.25 mM., 16.8 mL of dGTP Ž1.25 mM., 16.8 mL of dTTP Ž1.25 mM., and 16 mL of pure sterilized water. The reaction mixture was overlaid with 100 mL of mineral oil. Amplification was performed using a Techne PHC-2 thermal cycler ŽScotlab, Aberdeen, Scotland, U.K.. and the following parameters: 30 cycles of denaturation at 958C for 1 min, primer annealing at 558C for 1 min, and chain extension at 728C for 1 min. This was followed by 7 min at 728C to allow the extension of all molecules to be completed. 16S rDNA PCR products were separated in 1.4% agarose electrophoresis gels stained with ethidium bromide Ž1 mgrmL. and visualized by UV excitation. Amplified 16S rDNA gene products were excised from agarose gel and purified by using QIAEX Gel Extraction Kit ŽQIAGEN, Germany..

DNA isolation A 20 mL sample of fully grown cell culture was pelletted by centrifugation at 10 000 = g at 48C for 10 min. The cell pellet was resuspended in 4 mL SE buffer Ž150 mM NaCl, 100 mM EDTA; pH 8.0. and then centrifuged at 10 000 = g, 48C for 10 min. After removing the supernatant, the pellet was resuspended in 2.5 mL SE buffer and 55 mL fresh lysozyme Ž50 mgrmL. was added at room temperature for 20 min to lyze cells. The suspension was then mixed with 220 mL sodium dodecyl sulfate ŽSDS. Ž25%, wrv. and incubated at 608C for 20 min. Total genomic DNA was isolated from lyzed bacterial cells by treatment with proteinase, prior to extraction with phenolrchloroformrisoamyl alcohol and precipitation with ethanol w 22x . Amplification of 16S rRNA genes The polymerase chain reaction ŽPCR. w 23x was used to amplify 16S rRNA genes from purified genomic DNA. Eubacterial universal primers compared to the Escherichia coli 16S rRNA genes w 24x were used to selectively amplify eubacterial rDNA. The forward primer D9 Ž 59-GGggatccAGAGTTTGATCCrATGGCTCAG. w 25x corresponded to base pair Žbp. positions 8 to 27 in E. coli 16S rRNA and the reverse primer A Ž 59-GGggatccGTATTACCGCGrTGCTGCTGG. w 25x corresponded to the bp complement of positions 536 to 518 in E. coli 16S rRNA. The lower case letters indicate the BamH I restriction site of PCR primers. The PCR reaction contained in 100 mL: 2 mL of genomic DNA Ž50 ngrmL., 10.4 mL of 10 = PCR reaction buffer, 0.4 mL of Taq DNA polymerase, 2 mL of forward primer Ž25 mM., 2 mL of reverse primer Ž25 mM.,

Construction of 16S rDNA clone library PCR performed using forward primer D9 and reverse primer A resulted in an approximate 500 bp product as determined by gel electrophoresis. Amplification products were cleaned as above, digested with BamH I, ligated into BamH I cut pUC 18 ŽBoehringer Mannheim, U.K.. in both directions and used to transform competent E. coli strain XL1-Blue ŽStratagene, La Jolla, CA.. using standard methods w22x.

16S rDNA sequencing and analysis Library clones were sequenced after purification with QIAprep Spin Plasmid Kit-250 ŽQIAGEN, Germany.. Initially, a ddT-terminated extension reaction ŽT Track. using the universal reverse primer Ž59-CAGGAAACAGCTATGAC. was performed to distinguish different clones. Complete sequence of unique clones was obtained using the universal forward primer Ž59-CGTTTTCCCAGTCACGAC. and reverse primer Ž59-CAGGAAACAGCTATGAC. w 26x . The 16S rDNA sequence data obtained was subjected to comparison analysis in the GenBank database ŽDaresbury Laboratory, U.K.. and then manually aligned to, on the basis of primary and secondary structural consideration, a 16S rRNA database selected from the Ribosomal Database Project ŽRDP. version 2.2, or the GenBank database using genetic data environment ŽGDE. multiple sequence editor w 27x . Pairwise genetic distances were computed using the method of Jukes and Cantor w 28x and phylogenetic trees were constructed from genetic distances using the neighbor-joining method in the PHYLIP package version 3.4 w 29x . Tree topology was examined using 100 bootstrapped data sets. For the

168

J.-Y. Leu et al.

bootstrapping, we used the following sequence of events during the analysis: SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE. All programs are available as part of the PHYLIP package in Daresbury, U.K.

Results SRB enrichment cultures Four thermophilic SRB enrichment cultures ŽTable 1. were obtained: 12FL and 13FA grown on 0.2% NaCl medium with lactate and acetate, respectively, were enriched from sandstone core, FP5F oxidizing propionate at 0.2% NaCl was from drilling mud taken from a different, deeper and hotter reservoir, and OB5M enriched on 2% NaCl medium with butyrate was from production water from a third reservoir in a quite different geographical location. These 558C growth enrichment cultures were subsequently transferred from primary enrichments at least three times in defined SRB medium with an appropriate substrate as carbon and energy source. After subculturing three times, cultures were found to grow at significantly reduced rates compared with the original enrichments, and so yeast extract Ž200 mgrL. was added to stimulate growth. Total genomic DNA extracted from each of the four thermophilic SRB enrichment cultures was subject to PCR amplification of 16S rRNA genes. 16S rDNA gene cloning and sequencing A total of 22, 18, 17, and 12 recombinant plasmids were prepared for cloning of 12FL, 13FA, FP5F, and OB5M, respectively. Consequently, T track analysis of 19, 13, 9, and 10 representative clones, all of which were identified as containing approximately a 500 bp rDNA insert in agarose gel electrophoresis after digestion with BamH I, was carried out for the cultures 12FL, 13FA, FP5F, and OB5M, respectively. For the purpose of this study, the same T tracking sequence pattern was regarded as indicative of the same sequence. Totals of 6, 6, 2, and 8 unique clone patterns were found for the cultures 12FL, 13FA, FP5F, and OB5M, respectively, and these patterns were named and distinguished by their sources, and their sequences determined. Sequence analysis All 16S rDNA sequences obtained were analysed using the GenBank database at Daresbury in the U.K.. Upon examination of nucleotide sequences, three pairs of clones Ž12FL-3 and 12FL-4, 13FA-5 and

13FA-6, FP5F-1 and FP5F-2. were demonstrated as having identical sequences. For each pair of identical sequences in the same culture, only one was used for comparative analysis. A total of 19 clones, therefore, were used to construct phylogenetic trees. For each clone, approximately 500 nucleotides were unambiguously aligned with known species. The majority of clones were linked to thermophilic anaerobes, with the exception of 12FL-1 which was grouped with mesophilic bacteria. The phylogenetic tree shows that all clones sequenced fall into three major groups: clones 12FL-2, 12FL-3, 13FA-2, 13FA-5, OB5M-2, OB5M-3, OB5M-4, OB5M-5, OB5M-6, and OB5M-8 were members of group I ŽGram-positive, low GqC content, non-sulfate-reducing, thermophilic, obligately anaerobic bacteria., clones 12FL-5, 12FL-6, 13FA-1, 13FA-3, 13FA-4, FP5F-1, OB5M-1, and OB5M-7 were assigned to group II ŽGram-positive, spore-forming, low GqC content, sulfate-reducing, thermophilic, obligately anaerobic bacteria., and only one clone, 12FL-1, clustered with Gram-negative, mesophilic, SRB Žgroup III. in the d-subdivision of the Proteobacteria. Group I contains four phylogenetic clusters: a lineage branch consisting of OB5M-2 and OB5M-8, and clusters A, B, and C ŽFigure 1.. The identical names of known thermophilic microorganisms are also found in the clusters A, B, and C, respectively, as defined by Rainey et al. w 30x . Eight sequenced clones from this study were grouped into cluster A: the four clones 12FL-2, 12FL-3, 13FA-2, and 13FA-5 originally from a sandstone core were most closely related to Thermoanaerobacter species; the four clones OB5M-3, OB5M-4, OB5M-5, and OB5M-6 originally from production water had Clostridium thermocopriae w 31x as their closest described relative. Strong support was given for grouping of these clones in cluster A Ž99% relatedness in 100 bootstrap data sets.. An unusually long branch length was found with clone 13FA-2. The CHECK_CHIMERA program within the Ribosomal Database Project ŽRDP. w 32x was used for clone 13FA-2 and did support the presence of chimeric artifacts. The sequences of clone 13FA-2 has a 185 bp segment from the sequence beginning at the 59-end which is highly similar to that of 13FA-3 and closely related to the sequences of Desulfotomaculum thermobenzoicum, whereas the remaining sequences are highly similar to those of Thermoanaerobacter finnii. Sequence analysis showed that the lineage represented by clones OB5M-2 and OB5M-8 was deeply rooted in cluster A. The branching of this lineage with cluster A was found to be strong by bootstrap analysis Ž89% in 100 trees tested.. Because of the novelty and deep-branching of these two cloned sequences, the CHECK_CHIMERA program was also used to test whether they might be PCR-generated chimeric sequences. However, the results suggested

Identification of Thermophilic Sulfate-reducing Bacteria in Oil Field Samples

169

Figure 1. An unrooted phylogenetic tree showing the relationships of all clones found in four thermophilic, sulphate-reducing bacterial cultures with other bacteria. The tree was constructed using 479 unambiguous positions of 16S rDNA genes. 12FL, 13FA, FP5F, and OB5M refer to cultures 12FL, 13FA, FP5F, and OB5M, respectively. Scale bar indicates 10 nucleotide changes per 100 sequence positions.

that they are unlikely to be chimeras. The relationship of OB5M-2 and OB5M-8 to other bacteria is, however, not certain. In addition, two 100% identical clones Ž12FL-3 and 13FA-5. from different carbon enrichment cultures, but originally from the same

sample, were found within this group, and other clones in group I also showed nearly identical sequences. Comparison of the sequences of clone OB5M-2 with that of clone OB5M-8, and also the sequences of clone OB5M-3 with that of clone OB5M-4

170

J.-Y. Leu et al.

showed single base differences at positions 346 and 176, respectively, corresponding to the 16S rRNA sequence of E. coli w 24x . These positions are situated in conserved regions, suggesting that these mismatches are probably PCR-generated errors. Similarly, comparison of the sequences of clone 12FL-2 with that of clone 12FL-3, and also the sequences of clone OB5M-5 with that of clone OB5M-6 showed a single base difference at position 89, and three base differences at positions 325 Žin conserved regions., 465, and 479, respectively, corresponding to 16S rRNA sequence of E. coli. These positions are in more variable regions and although possibly attributable to PCR-generated errors, they may be real differences. From the phylogenetic tree, group II is significantly separated from group I. Group II is represented by members of the spore-forming, thermophilic, SRB genus Desulfotomaculum w 33x . Clones in this group were found from all thermophilic SRB cultures examined in this study. A total of eight clones strongly clustered with thermophilic Desulfotomaculum species. The association of clones 12FL-5, 12FL-6, 13FA-1, 13FA-3, 13FA-4, OB5M-1, and OB5M-7 with Desulfotomaculum thermobenzoicum w 34,35x and Desulfotomaculum australicum w 36x is strongly supported by a bootstrap value of 92% in 100 resampling trees. It should be noted that clone OB5M-7 has an unusually short branch length. The use of the CHECK_CHIMERA program proved that OB5M-7 is a chimeric clone, consisting of a 56 bp sequence segment from the sequence beginning at the 59-end which is highly similar to that of clone OB5M-3 and related to that of Clostridium thermocopriae. The remaining sequences Ž406 bp. of OB5M-7, however, are closely related to Desulfotomaculum thermobenzoicum. Clone FP5F-1 was closely related to Desulfotomaculum geothermicum w 37x , sharing 87.3% sequence similarity. Within this group, three pairs of 100% identical clones were found: 12FL-5 and 13FA-3 as well as 12FL-6 and 13FA-1 were obtained from different cultures, but from the same sample: 13FA-4 and OB5M-1, on the other hand, were from different cultures obtained from different samples widely separated geographically. One clone, 12FL-1, although from the thermophilic culture 12FL surprisingly clustered with species of mesophilic SRB Žgroup III. and this clone was most closely related to Desulfomicrobium species.

Discussion Thermophilic sulfate reducers have been recognized as an important source of H 2 S in hot oil reservoirs and in their associated production systems. Little attention, however, has been paid to identification and characterization of these microorganisms from such environments as growing, isolating, and identi-

fying thermophilic species using classical cultural methods is difficult and time-consuming. In this study, 16S rRNA gene analysis was directly applied to identify thermophilic SRB present in four thermophilic SRB cultures 12FL, 13FA, FP5F, and OB5M ŽTable 1.. Although comparative analysis of cloned rDNA sequences provides a method for assessing the diversity of thermophilic species in samples without pure culture isolation, the introduction of potential error or bias from the requisite methodology including cell lysis w 38x , DNA extraction and purification w 39x , genomic properties Žthe size and number of genome within a cell as well as the organization and number of rRNA genes. w 40x , PCR amplification w 41x , and cloning w 42x remains to be noted. In this study, chimeric sequences were effectively detected in clones 13FA-2 and OB5M-7 using the CHECK_CHIMERA program through the RDP w 32x . Each of these two clones comprised an rDNA segment identical to that from the other clone. However, they were still affiliated to the group with which the remaining dominant segment clustered. The generation of chimeric rDNA sequences during PCR amplification procedures has recently been documented with mixtures of pure culture isolates w 16x and with environmentally-derived, uncultivated bacteria w 42,43x . Although rDNA-based molecular biological techniques can potentially involve biases, they have been used to evaluate effectively the structure and diversity of a microbial community from environmentally derived mixed populations w 15,44x . Universal eubacterial PCR primers were used to amplify a region of 16S rRNA genes in order to obtain as many sequences as possible from the cultures. A total of 51 clones were obtained from four thermophilic SRB cultures. On the basis of sequence analysis, all clones, except one clone from culture 12FL which clustered with Desulfomicrobium species, were related to either thermophilic, non-sulfatereducing anaerobes defined in cluster A by Rainey et al. w 30x , or thermophilic, sulfate-reducing Desulfotomaculum species ŽFigure 1.. Clones related to thermophilic microorganisms in cluster A, or to Desulfotomaculum strains can be found in all four cultures, with the single exception of FP5F from which only a Desulfotomaculum-related species was obtained. These clones, however, may represent only a minority of the total population that might be expected from three environmental samples. The observation of only a few bacterial groups may result from the bias of the molecular techniques employed. A more likely explanation for this low bacterial diversity is that the cultures were initially selectively enriched using SRB media with specific carbon substrates at 558C, conditions under which only few bacterial groups can grow.

Identification of Thermophilic Sulfate-reducing Bacteria in Oil Field Samples The initial aim of this study was to identify thermophilic SRB present in four SRB cultures growing at 558C. However, the sequence analysis found that more than half of the clones clustered with Grampositive, non-sulfate-reducing, thermophilic, obligately anaerobic bacteria Žgroup I. ŽFigure 1.. These clones were found in three of the cultures, but not in culture FP5F. The discovery of thermophilic, nonsulfate-reducing organisms in SRB cultures is somewhat surprising. A reasonable explanation is that such organisms might be capable of oxidizing carbon substrate such as lactate, acetate, butyrate, or yeast extract in the growth medium. Although no report has shown that these known thermophilic, fermentative bacteria in group I have the ability to oxidize lactate, acetate, or butyrate, most of these organisms are capable of using yeast extract. In addition, this group of bacteria have recently been discovered to oxidize carbon substrates previously not known to be used. For example, some Thermoanaerobacter species have been reported to oxidize amino acids w 45x . The sequenced thermophilic, non-sulfate-reducing bacteria in this study, therefore, may be able to grow on lactate or short chain fatty acids. In group I, most clones were affiliated into cluster A previously defined by Rainey et al. w 30x . Members of cluster A were described with unique 16S rDNA signature nucleotides and the high intracluster similarity Ž95.2% to 99.5%.; however, they were phenotypically incoherent with respect to spore formation, polysaccharolytic activity, and DNA base composition Ž31 to 38 mol% GqC.. The affiliation of bacterial clones in group I demonstrated that distinct bacterial groups were present in different samples. In cluster A, the clones 12FL-2, 12FL-3, 13FA-2, and 13FA-5 originally from sandstone core from under the North Sea floor strongly clustered with Thermoanaerobacter species. Thermoanaerobacter species have recently been classified as thermophilic, carbohydrate-fermenting anaerobes with the ability to reduce thiosulfate to sulfide w 46x . They oxidize carbohydrates to acetate, lactate, ethanol, H 2 and CO2 with thiosulfate reduction, but not sulfate reduction, to produce sulfide. More recently, a Thermoanaerobacter strain has also been found in an oil-producing well w 47x , using thiosulfate, but not sulfate, as electron acceptor. This H 2 S from thiosulfate reduction has been proposed to increase the risk of corrosion and reservoir souring w 47]48x . The clones OB5M-3, OB5M-4, OB5M-5, and OB5M-6 from hot production water in Oman were most closely related to Clostridium thermocopriae. Clostridium thermocopriae is a fermentative thermophile capable of cellulose degradation and isolated from animal feces, compost, soil, and a hot spring w 31x . The lineage of clones OB5M-2 and OB5M-8 was deeply rooted in cluster A Ž89% relatedness in 100 bootstrap data

171

sets.. It is possible that this linkage represents a novel bacterial genus. Full length sequences would be required in order to resolve this deep phylogenetic relationship. When considering the many previous studies of 16S rRNA comparative analysis, the detection of previously unknown organisms in natural microbial communities is no longer considered novel or surprising. In the phylogenetic tree produced from this study ŽFigure 1., the rooted lineage of clones OB5M-2 and OB5M-8 indicates that a further previously undescribed group of bacteria may exist in these cultures. These microorganisms may have the ability to reduce sulfate to sulfide. The only thermophilic SRB obtained in this study are members of the genus Desulfotomaculum Žgroup II. ŽFigure 1.. Thermophilic SRB are found within the eubacterial genera Desulfotomaculum, Thermodesulfobacterium, Thermodesulfovibrio w 49,50x , and Thermodesulforhabdus w 51x , and the archaebacterial genus Archaeoglobus w 52]53x . Moderately thermophilic Desulfotomaculum species are spore-forming, Grampositive anaerobes with a temperature optimum of 558C to 658C. The genus exhibits a great nutritional versatility; lactate, fatty acids, alcohols, and H 2 are their common substrates for growth. In group II, two Ž12FL-5, 12FL-6., three Ž13FA-1, 13FA-3, 13FA-4., and two ŽOB5M-1, OB5M-7. representative clones were related to both Desulfotomaculum thermobenzoicum and Desulfotomaculum australicum, sharing 84.2% to 94.1% sequence similarity. The clone FP5F-1 from culture FP5F was more closely related to Desulfotomaculum geothermicum, sharing 87.3% sequence identity. These sequences may be derived from previously undescribed Desulfotomaculum strains. Phylogenetic grouping, therefore, revealed that Desulfotomaculumlike species were found in all four thermophilic SRB cultures, and different strains appeared to be present in different samples. The cultures used were originally enriched from different samples taken from different oil field locations ŽTable 1.. Therefore, the results suggest that Desulfotomaculum species appear to be common and widely distributed over oil field environments. Interestingly, clones 13FA-4 and OB5M-1 obtained from two different oil fields, North Sea and Oman, respectively, had identical sequences ŽFigure 1., which indicated that this type of Desulfotomaculum strain is probably widespread through distant oil fields. The common presence of thermophilic Desulfotomaculum species in oil field environments has also been demonstrated in previous studies using cultivation methods w 5,8x or the immunomagnetic recovery methods w 54x . Stetter and his colleagues w 55x have also suggested that hyperthermophilic sulfatereducing archaebacteria, Archaeoglobus species, with growth temperature range from 648C to 928C and an optimum of approximately 838C, may be responsible

172

J.-Y. Leu et al.

for the generation of sulfide in hot oil fields. In our previous study, an Archaeoglobus strain has been isolated from production water in North Sea, growing at the temperatures between 658C and 908C w 56x . Therefore, by integrating many studies on thermophilic sulfate reducers, it can be postulated that both Archaeoglobus species and Desulfotomaculum species may play important roles in the biogenic production of H 2 S in hot oil field environments, with hyperthermophilic Archaeoglobus species more likely being the major contributor in environments with temperatures above 658C, and Desulfotomaculum species being responsible for sulfide production in environments with temperatures between 458C and 708C. Of 51 clones, only one clone, 12FL-1 from the thermophilic culture 12FL surprisingly grouped with mesophilic, SRB Žgroup III. in the d-subdivision of the Proteobacteria. This clone was most closely related to Desulfomicrobium species which are mesophilic, straight short rod, lactate-utilizing SRB. In theory, mesophilic Desulfomicrobium species should not grow in a 558C environment; however, Parkes and his colleagues w 57x have found that a mesophilic SRB isolate, strain 80, which was obtained from deep sediment below the Japan Sea floor and had 88% sequence similarity Žover 785 base pairs. with Desulfovibrio salexigens, demonstrated a very broad and high range of growth temperatures Ž15 to 658C, with an optimum at 258C.. Therefore, the possibility of the Desulfomicrobium-like species present in culture 12FL having originated from the inner section of sandstone core I under the North Sea floor cannot be ruled out. In this study, cultures 12FL and 13FA are the most relevant to the understanding of the SRB origins, since both were enriched from the inner section of the same sandstone core derived from an unproduced oil field in the North Sea, and without invasion of drilling mud. 16S rDNA analysis indicates that Desulfotomaculum-like strains are present in cultures 12FL and 13FA, and may be the indigenous SRB in these oil reservoirs. These sulfate reducers may therefore be particularly important in reservoir souring. To understand the role SRB play in natural oil field environments, the comparative analysis of cloned 16S rDNA sequences should be used to characterize additional oil field samples without prior cultivation. Subsequently, SRB rDNA sequences retrieved from environmental samples by cloned sequencing could also be used for designing group- or species-specific hybridization probes to identify specific SRB, and also to determine their abundance and distribution. Recently, phylogenetic analysis has been successfully used to assess the divergence of 16S rRNA sequences from SRB in a sandy marine sediment and the developed hybridization probes have been able to evaluate efficiently the 16S rRNA of phylogenetically defined

SRB groups w 19x . The present study is the first application of cloned 16S rDNA sequence analysis to examine the SRB cultures from oil field environments, and also to identify thermophilic sulfate-reducing microorganisms in cultures from different oil field samples in a range of locations. Phylogenetic analysis revealed that the thermophilic sulfate reducers, at least Desulfotomaculum-related species, may be common and widespread over oil fields and play an important role in the production of sulfide. Further exploration with a Desulfotomaculum group-specific probe in oil field samples will enable the confirmation and localization of Desulfotomaculum species. In addition to thermophilic SRB, the thermophilic, non-sulfatereducing bacteria discovered in this study may also have an important ecological role, either directly or alongside SRB, in hydrocarbon reservoir souring.

References 1. Cord-Ruwisch R., Kleinitz W. and Widdel F. Ž1987. Sulfatereducing bacteria and their activities in oil production. J Pet Technol Jan.: 97]106 2. Schapira S. F.D. and Sanders P.F. Ž1990. Present status of souring in the North Sea. Proceeding Seminar Souring Reservoirs, Pet Sci Technol Inst, Aberdeen 3. Barth T. and Riis M. Ž1992. Interactions between organic acid anions in formation waters and reservoir mineral phases. Org Geochem 19: 455]482 4. Beeder J., Nilsen R.K., Rosnes J.T., Torsvik T. and Lien T. Ž1994. Archaeoglobus fulgidus isolated from Hot North Sea oil field waters. Appl Environ Microbiol 60: 1227]1231 5. Bernard F.P., Connan J. and Magot M. Ž1992. Indigenous microorganism in connate water of many oil fields: a new tool in exploration and production techniques. Soc Pet Engineer Paper 24811: 467]476 6. Burger E.D., Addington D.V. and Crews A.B. Ž1991. Reservoir souring: bacterial growth and transport. Proc. 4th Internat. IGT Symp. on Gas, Oil and Environ. Biotechnol. Colorado Springs, Dec. 1991 7. Cochrane W.J., Jones P.S., Sanders P.F., Holt D. M. and Moseley M. J. Ž1988. Studies on the thermophilic sulfatereducing bacteria from a souring North Sea oil field. Soc Pet Engineer Paper 18368: 301]316 8. Rosnes J.T., Torsvik T. and Lien T. Ž1991. Spore-forming thermophilic sulfate-reducing bacteria isolated from North Sea oil field waters. Appl Environ Microbiol 57: 2302]2307 9. Rueter P., Rabus R., Wilkes H., Aeckersberg F., Rainey F.A., Jannasch H.W. and Widdel F. Ž1994. Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria. Nature ŽLondon. 372: 455]458 10. Stetter K.O., Huber R., Blochl E., Kurr M., Eden R.D., Flelder M., Cash H. and Vance I. Ž1993. Hyperthermophilic archaea are thriving in deep North Sea and Alaskan oil reservoirs. Nature ŽLondon. 365: 743]745 11. Voordouw G., Voordouw J.K., Jack T.R., Foght J., Fedorak P.M. and Westlake D.W. S. Ž1992. Identification of distinct communities of sulfate-reducing bacteria in oil fields by reverse sample genome probing. Appl Environ Microbiol 58: 3542]3552 12. Voordouw G., Voordouw J.K., Karkhoff-Schweizer R.R., Fedorak P.M. and Westlake D.W.S. Ž1991. Reverse sample genome probing, a new technique for identification of bacteria in environmental samples by DNA hybridization, and its application to identification of sulfate-reducing bacteria in oil field samples. Appl Environ Microbiol 57: 3070]3078

Identification of Thermophilic Sulfate-reducing Bacteria in Oil Field Samples 13. Amann R.I., Stromley J., Devereux R., Key R. and Stahl D.A. Ž1992. Molecular and microscopic identification of sulfatereducing bacteria in multispecies biofilms. Appl Environ Microbiol 58: 614]623 14. Barns S.M., Fundyga R.E., Jeffries M. W. and Pace N.R. Ž1994. Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment. Proc Natl Acad Sci USA 91: 1609]1613 15. Fuhrman J.A., McCallum K. and Davis A.D. Ž1993. Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific oceans. Appl Environ Microbiol 59: 1294]1302 16. Liesack W. and Stackebrandt E. Ž1992. Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J Bacterial 174: 5072]5078 17. Schmidt T.M., DeLong E.F. and Pace N.R. Ž1991. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J Bacteriol 173: 4371]4378 18. Ward D.M., Weller R. and Bateson M.M. Ž1990. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature ŽLondon. 345: 63]65 19. Devereux R. and Mundfrom G.W. Ž1994. A phylogenetic tree of 16S rRNA sequences from sulfate-reducing bacteria in a sandy marine sediment. Appl Environ Microbiol 60: 3437]3439 20. Widdel F. and Bak F. Ž1992. Gram-negative mesophilic sulfate-reducing bacteria. In Balows A., Truper H.G., Dworkin M., Harder W. and Schleifer K.H. Žeds. The prokaryotes. 2nd Edn, pp 3352]3378. Springer-Verlag, New York 21. Cline D.J. Ž1969. Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol Oceanogr 14: 454]458 22. Sambrook J., Fritsch E.F. and Maniatis T. Ž1989. Molecular cloning: a laboratory manual. 2nd Edn Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 23. Saiki R.K., Gelfand D.H., Stoffel S., Scharf S.J., Higuchl R., Horn G.T., Mullis K.B. and Erlich H.A. Ž1988. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487]491 24. Brosius J., Palmer M.L., Kennedy P.J. and Noller H.F. Ž1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci USA 75: 4801]4805 25. Lane D.J. Ž1991. 16Sr23S rRNA sequencing. In Stackebrandt E. and Goodfellow M. Žeds. Nucleic acid techniques in bacterial systematics, pp 115]175. John Wiley and Sons, Chichester, U.K. 26. Sanger F., Nicklen S. and Coulson A.R. Ž1977. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463]5467 27. Larsen N., Olsen G.J., Maiddak B.L., McCaughey M.J., Overbeek R., Macke T.J., Marsh T.L. and Woese C.R. Ž1993. The ribosomal database project. Nucleic Acids Res 21: 3021]3023 28. Jukes T.H. and Cantor C.R. Ž1969. Evolution of protein molecules. In Munro H. N. Žed. Mammalian protein metabolism, pp 21]132. Academic Press, New York 29. Felsenstein J. Ž1993. PHYLIP Žphylogenetic inference package., version 3.5lc. Department of Genetics, University of Washington, Seattle, USA 30. Rainey F.A., Ward N.L., Morgan H.W., Toalster R. and Stackebrandt E. Ž1993. Phylogenetic analysis of anaerobic thermophilic bacteria: aid for their reclassification. J Bacteriol 175: 4772]4779 31. Jin F., Yamasato K. and Toda K. Ž1988. Clostridium thermocopriae sp. nov., a cellulolytic thermophile from animal feces, compost, soil and a hot spring in Japan. Int J Syst Bacteriol 38: 277]281 32. Maidak B.L., Larsen N., McCaughey M.J., Overbeek R., Olsen G.J., Fogel K., Blandy J. and Woese C.R. Ž1994. The ribosomal database project. Nucleic Acids Res 22: 3485]3487 33. Widdel F. Ž1992a. The genus Desulfotomaculum. In Balows A., Truper H.G., Dworkin M., Harder W. and Schleifer K.H. Žeds. The prokaryotes. 2nd Edn, pp 1792]1799. Springer-Verlag, New York 34. Redburn A. C. and Patel B.K.C. Ž1993. Phylogenetic analysis of Desulfotomaculum thermobenzoicum using polymerase chain reaction-amplified 16S rRNA-specific DNA. FEMS Microbiol Lett 113: 81]86 35. Tasaki M., Kamagata Y., Nakamura K. and Mikama E. Ž1991. Isolation and characterization of a thermophilic benzoate-

36.

37.

38.

39.

40.

41. 42.

43.

44. 45.

46.

47.

48.

49.

50.

51.

173

degrading, sulfate-reducing bacterium, Desulfotomaculum thermobenzoicum sp. nov. Arch Microbiol 155: 348]352 Love C.A., Patel B.K. C., Nichols P.D. and Stackebrandt E. Ž1993. Desulfotomaculum australicum, sp. nov., a thermophilic sulfate-reducing bacterium from the Great Artesian Basin of Australia. System Appl Microbiol 16: 244]251 Daumas S., Cord-Ruwisch R. and Garcia J.L. Ž1988. Desulfotomaculum geothericum sp. nov., a thermophilic, fatty aciddegrading, sulfate-reducing bacterium isolated with H2 from geothermal ground water. Antonie van Leeuwenhoek 54: 165]178 Picard C., Ponsonnet C., Paget E., Nesme X. and Simonet P. Ž1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol 58: 2717]2722 Liesack W., Weyland H. and Stackebrandt E. Ž1991. Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria. Microb Ecol 21: 191]198 Farrelly V., Rainey F.A. and Stackebrandt E. Ž1995. Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species. Appl Environ Microbiol 61: 2798]2801 Reysenbach A., Giver L.J., Wickham G.S. and Pace N.R. Ž1992. Differential amplification of rRNA genes by polymerase chain reaction. Appl Environ Microbiol 58: 3417]3418 Moyer C.L., Dobbs F. C. and Karl D.M. Ž1994. Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii. Appl Environ Microbiol 60: 871]879 Kopczynski E.D., Bateson M.M. and Ward D.M. Ž1994. Recognition of chimeric samll-subunit ribosomal DNAs composed of g en es from u n cu ltiv ated m icroorg a n ism s. A ppl Environ Microbiol 60: 746]748. DeLong E.F., Franks D.G. and Alldredge A.L. Ž1993. Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages. Limnol Oceanogr 38: 924]934 Faudon C., Fardeau M.L., Heim J., Patel B., Magot M. and Olliver B. Ž1995. Peptide and amino acid oxidation in the presence of thiosulfate by members of the genus Thermoanaerobacter. Curr Microbiol 31: 152]157 Lee Y.E., Jain M.K., Lee C., Lowe S.E. and Zeikus J.G. Ž1993. Taxonomic distinction of saccharolytic thermophilic anaerobes: description of Thermoanaerobacterium xylanolyticum gen. nov., sp. nov., and Thermoanaerobacterium saccharolyticum gen. nov., sp. nov.; reclassification of Thermoanaerobium brockii, Clostridium thermosulfurogenes, and Clostridium thermohydrosulfuricum E100-69 as Thermoanaerobacter brockii comb. nov., Thermoanaerobacterium thermosulfurigenes comb. nov., and Thermoanaerobacter thermohydrosulfuricus comb. nov., respectively; and transfer of Clostridium thermohydrosulfuricum 39E to Thermoanaerobacter ethanolicus. Int J Syst Bacteriol 43: 41]51 Fardeau M.L., Cayol J.L., Magot M. and Ollivier B. Ž1993. H 2 oxidation in the presence of thiosulfate, by a Thermoanaerobacter strain isolated from an oil-producing well. FEMS Microbiol Lett 113: 327]332 Ravot G., Magot M., Fardeau M.L., Patel B.K.C., Prensier G., Egan A., Garcia J.L. and Ollivier B. Ž1995. Thermotoga elfii sp. nov., a novel thermophilic bacterium from an African oilproducing well. Int J Syst Bacteriol 45: 308]314 Henry E.A., Devereux R., Maki J.S., Gilmour C.C., Woese C.R., Mandelco L., Schauder R., Remsen C.C. and Mitchell R. Ž1994. Characterization of a new thermophilic sulfate-reducing bacterium Thermodesulfovibrio yellowstonii, gen. nov. and sp. nov.: its phylogenetic relationship to Thermodesulfobacterium commune and their origins deep within the bacterial domain. Arch Microbiol 161: 62]69 Widdel F. Ž1992b. The genus Thermodesulfobacterium. In Eds Balows A., Truper H.G., Dworkin M., Harder W. and Schleifer K.H. The Prokaryotes. 2nd Edn, pp 3390]3392. Springer-Verlag, New York Beeder J., Torsvik, T. and Lien T. Ž1995. Thermodesulforhabdus norvegicus gen. nov., sp. nov., a novel thermophilic sulfatereducing bacterium from oil field water. Arch Microbiol 164: 331]336

174

J.-Y. Leu et al.

52. Burggraf S., Jannasch H.W., Nicolaus B. and Stetter K.O. Ž1990. Archaeoglobus profundus sp. nov., represents a new species within the sulfate-reducing archaebacteria. Syst Appl Microbiol 13: 24]28 53. Stetter K.O. Ž1988. Archaeoglobus fulgidus gen. nov., sp. nov.: a new taxon of extremely thermophilic archaebacteria. Syst Appl Microbiol 10: 172]173 54. Christensen B., Torsvik T. and Lien T. Ž1992. Immunomagnetically captured thermophilic sulfate-reducing bacteria from North Sea oil field waters. Appl Environ Microbiol 58: 1244]1248

55. Stetter K.O., Laurer G., Thomm M. and Neuner A. Ž1987. Isolation of extremely thermophilic sulfate reducers: evidence for a novel branch of archaebacteria. Science 236: 822]824 56. Leu J.Y. Ž1996. PhD thesis: Isolation, characterisation, and identification of sulphate-reducing bacteria from oil field environments. University of Aberdeen, U.K. 57. Parkes R.J., Cragg B.A., Bale S.J., Getliff J.M., Goodman K., Rochelle P.A., Fry J.C., Weightman A.J. and Harvey S.M. Ž1994. Deep bacterial biosphere in Pacific Ocean sediments. Nature ŽLondon. 371: 410]413