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Oil exploration and production are considered to be the most important benchmark for the progress of any nation. Oil exploration faces much operational hindrances during the life time of the field. One of the major problems that the oil industry faces is oil field souring and souring mediated microbial induced corrosion (MIC). Oil field souring and MIC exerts additional burden on oil industries due maintenance and replacement of production units and controlling environmental hazards.
Assessment of corrosion causing microbial communities and their estimation is very important to design method to control these communities. Present study deals with the investigation of the microbial communities responsible for the souring mediated corrosion. To address the problem of souring mediated corrosion, production water samples from 5 different oil fields sites were collected and subjected to culturable and non-culturable studies using various microbiological and molecular biology techniques. Samples from 4 onshore sites: Kathloni and Dikom oil fields from north-eastern India and Kalol and Ghandhar oil fields from western part of India and one offshore oil field situated in Bombay High were collected.
Samples were enriched various selective media to harness maximum culturable communities. Since the samples were collected from oil water separators which were operated at 37 -42 °C, all the enrichments were done at these temperatures. The external oil production facilities installed in these oil fields are highly suffering from souring and MIC due to enormous increase in the microbial activities. These microbial communites which were inactive or less active in harsh reservoir conditions were exceptionally active in oil production facilities due to favourable temperature and availability of nutrients. The hydrogen sulfide concentration at Kathloni oil field production facilities was 35 mg.L-1 in well head sample which increased to 105 mg.L-1 separator tank and further increased to 210 mg.L-1 in storage tanks.
Phylogenetic identification of the microbial communities by 16S rRNA gene indicated that Lyngby medium favored the growth of several Firmicutes related to Clostridium thiosulfatireducens, Clostridium subterminale and Fusibacter paucivorans. Lyngby medium equally supported the growth of Enterobacteriaceae, similar to the genera Enterobacter, and Citrobacter. Modified API RP 38 and Baar’s medium favoured the growth of many spore forming and non spore forming SRB related to the genera Desulfotomaculum, Desulfomicrobium, Desulfobulbus, and Desulfovibrio. These phylotypes were well acclimatized to the production water environment causing severe problems in the production facilities.
The dominating species in the production water samples were Clostridium subterminale and Desulfovibrio vulgaris. Isolates belonging to both the species were isolated from all the production water samples. Many isolated species showed similarity with previously reported species from oil fields. Strains affiliated to Desulfotomaculum halophilum , Desulfovibrio alaskensis and Desulfovibrio longus were isolated from investigated oil fields. Earlier these species were reported as novel species isolated from different oil fields around the world. Isolated strains from same geographical location when subjected to the DGGE profiling revealed the interspecies and intraspecies diversity among the isolates. All the isolated strains from the same location were delineated in different genotypic clusters.
All isolates were able to produce hydrogen sulfide in the range of 55 to 250 mg.L-1. It was observed that hydrogen sulfide production was significantly higher in the microbes isolated from Lyngby medium. Most of the isolates have salinity tolerance range from 0 to 6% and optimal salinity between 0.5 to 2%. It was also noted that sulfide production by the isolates was decrease when they were grown at higher salinity. The optimum temperature ranges from 35- 40°C and maximum growth temperature varies from 40-45°C.
To explore the non-culturable diversity, bisulfite reductase gene was employed. Dissimilatory sulfite reductase gene (dsr) encode for dissimilatory sulfite reductase enzyme. This enzyme catalyses the formation of sulfide from sulfite and hence can be used to study diversity of the bacteria which produce hydrogen sulfide.
PCR amplification of the dsrB gene of pure SRB strains was carried out by using DSRp2060F and DSR4R primers sets. Semi-nested approach was used to make the amplicon useful for DGGE analysis. Semi-nested PCR overcome the drawback of poor amplification while using direct PCR with GC clamp attached primer to amplifying target sequence. Hence semi nested strategy where amplification product of the first PCR can be used as template for consecutive PCR with GC-clamp attached primers was successfully used.
When semi-nested PCR amplified dsrB gene products were subjected to DGGE, all the amplicons derived from pure SRB strains showed a single bright band that migrated at a particular position. Species belonging to same genera like Desulfobulbus proponicus (SRB8) and Desulfobulbus rhabdoformis (SRB2); Desulfomicrobium norvegicum (SRB10) and Desulfomicrobium escambiense (SRB6); Desulfovibrio termitidis (SRB9) and Desulfovibrio gigas (SRB7) showed distinctly separated and distantly migrated bands.
Denaturating Gradient Gel Electrophoresis of the bisulfite reductase (dsr) gene amplified from DNA isolated from production water samples showed very diverse banding pattern. This revealed highly diverse population of sulfidogenic communities in the production water samples collected from various geographical locations. The DGGE band affiliated to Desulfotomaculum aeronauticum like sequences was present in all the production water samples except production water collected from Kathloni oil fields. The DGGE pattern of K1 and D1 oil field were similar, probably because of similar in-situ oil field conditions. However, D1 oil field samples have two additional bands which were phylogenetically affiliated to the species Desulfotomaculum aeronauticum and Desulfitobacterium hafniense. SRB affiliated to Desulfotomaculum thermoacxetooxidans and Desulfovibrio vulgaris were present in production water of both Kathloni and Dikom oil fields from northeastern part of India.
Quantification of SRB in the production water samples is done by quantitative real-time PCR of the dsr gene. The quantitative real-time method to estimate dsr gene using Sybr-Green chemistry revealed lowest detection limit of 1o2 dsr gene copies. The optimized primer ratio was 400nM for each primer when used in qRT-PCR. The dsrB standard curve was linear from 4.5 × 107 to 4.5 × 102 copies per reaction with the slope of -3.59 (R2 value =0.994). The 16S rDNA standard curve was linear from 9 × 108 to 90 copies per reaction with a slope of -3.025 (R2 value =0.997). Production water samples from north-eastern and western part had approximately equal number of sulfate reducers in the range of 105 copies per ml of production water except G2 production water with approximately 106 dsrB gene copies per ml of production water. Indeed, highest dissolved sulfide was recorded in G2 production waters. The 16S rRNA gene copies were in the magnitude range of 106 -107 copies per ml of production water with highest copies in D2 production waters. D2 sample also showed lowest dsrB gene copies. This could be attributed to the fact that SRB have to compete with other bacteria present in the production water and hence, absolute number and proportion of SRB remain low in D2 production waters.
Primary screening of the biocides was focused on selection of the suitable biocide against SRB in the production water sample collected from oil-water separator. The quantification result revealed that dsrB gene copies of Desulfovibrio vulgaris culture decreased by 72% and 97.6% with 15 ppm and 20 ppm concentration of BNPD in 24 hours. This result showed that 20 ppm concentration of BNPD can control the growth of Desulfovibrio vulgaris effectively. The South Kadi production water enriched cultures showed decrease of 94.17%, 97.92% and 98.07% in dsrB gene copies with biocidal concentration of 10, 15 and 20 mg.L-1 in 24 hours in respect to control. The dsrB gene copy number decreased from 36925 (control, 0 mg.L-1 biocide) to 715 copies ml-1 in biocidal concentration of 20 mg.L-1.
The aim of the present investigation was to estimate the sulfidogenic communities dominating the oil fields. For this purpose, different media with different sulfur sources were used. To grow sulfate reducing bacteria, media with sodium lactate and a sulfate source was used, whereas fermentative TRB were grown in S7 medium supplemented with peptone and sodium thiosulfate. In order to find out the most suitable method to control sulfidogenic communities, research studies were focused on identifying these communities so treatments could be designed accordingly. Although most strains were previously found in oil fields, TERI GP7, showing 96% similarity with Clostridium thiosulfatireducens, has not yet been reported. TERI GP7 was found in the oil-water separator tanks and is highly sulfide producing. This phylotype could be a potential candidate for separation tank souring in the north-eastern fields. This initial study can pave the way for designing methods for the quick and early detection of sulfidogenic communities and the prophylaxis of reservoir souring. In conclusion, the dsrB gene based qRTPCR assay can be applied as a rapid, high throughput, and accurate tool to obtain an insight into community diversity and size of sulfate reducers in production water samples. This method may also prove useful in developing mRNA based reverse transcriptase real-time PCR assays to quantify bisulfate reductase gene expression.
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