Distribution And Interaction Of Sulfate-Reducing Prokaryotes And Nitrate-Reducing Prokaryotes In Petroleum Reservoirs

Petroleum Reservoir is an extreme environment developed luxuriant microorganism, which play important roles in biogeochemical cycles. Sulfate-reducing prokaryotes (SRP) play a major role in microbial corrosion and reservoir souring in oil production systems. Nitrate-reducing bacteria (NRB) can inhibit the generation of hydrogen sulfide, thus reducing or mitigating the problems caused by SRP. Oil reservoir is also developed abundant NRB. Currently, the distribution of SRP and NRB in the reservoir environment and the interaction between them are still research hotspots. In this paper the composition and distribution of SRP and NRB in different temperature oil reservoirs and the community respons of SRP and NRB during nitrate-treatment were studied by using molecular biology methods. A strain of NRB which can effectively inhibit SRP was separated from oil reservoir produced fluid. And combined with chemical and biological analysis method to evaluate the effect of suppressing of sulfate-reducing bacteria by this strain. The results of this study have enriched the awareness on diversity of SRP and NRB in oil reservoir environment, and expanded the application of functional genes on studing NRB community in oil reservoir environment. This study provided a theoretical basis to ecological control SRP thus solving the problem of oil reservoir souring and equipment corrosion.The community structure and composition of SRP in different temperature oil fields (21 ℃ to 95 ℃) were investigated in this study by using dissimilatory sulfate reductase (dsrAB) genes for phylogenetic analyses. Use of 16S rRNA special groups’ method can display an abundant diversity than the use of 16S rRNA universal primers and dsrAB genes method. However, the dsrAB approach can reveal all-sided microbial community structure.16S rRNA gene library and dsrAB functional gene library analysis found that low temperature reservoir contain Desulfotomaculum, Desulfovibrio and Desulfomicrobium; 13 diverse genera of SRP belong to 5 orders(Desulfovibrionales, Desulfobacterales, Syntrophobacterales, Clostridiales and Archaeoglobales) be detected in high temperature oil reserviors, Desulfovibrio, Desulfomicrobium and Desulfotomaculum were dominate.This research using nitrate reductase functional genes (narG and napA) and nitrite reductase functional genes(nirS, nirK and nrfA) library analysis to study diversity of NRB in 25 different temperature (21 ℃ to 95 ℃) reservoirs from five oil fields:Daqing, Huabei, Shengli, Xinjiang and Jiangsu. The study found that the reservoir environment contain 38 different kinds of NRB, including 5 phyla:Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria and Cyanobacteria. NapA functional gene had detected 4 types of nitrate-reducing bacteria including:a-proteobacteria, β-proteobacteria, y-proteobacteria, ε-proteobacteria; the narG functional gene had detected a wide range of NRB types, including:a-proteobacteria, β-proteobacteria, y-proteobacteria, ε-proteobacteria, δ-proteobacteria, Bacilli, Actinobacteria and also had archaea:Methanomicrobia and Archaeoglobi; the nirK and nirS function gene had detected 5 types of NRB:a-proteobacteria, β-proteobacteria, y-proteobacteria, Cyanobacteria and Bacteroidia; nrfA functional gene only had detected Bacteroidia. The results show that most microorganisms containing nitrate reductase functional genes belonging to Proteobacteria; richer type of microorganism containing membrane-bound nitrate reductase functional genes belonging to Proteobacteria, Firmicutes and Euryarchaeota; the different types of nitrate/nitrite-reducing bacteria contained in the denitrification functional genes. Canonical correspondence analysis (CCA) show that the small molecule acid, NO3-, Cl-, pH values and other environmental factors in the reservoir environment have a considerable influence on the distribution of the different types of NRB.The use of nitrate to treat souring has been applied in Xinjiang oilfield operations. The method of functional gene library analysis was used to study the changes in the microbial community of the SRP and NRB in nitrate treatment reservoir. The dsrAB function of clone library analysis results show that the nitrate addition inhibits the alienation sulfite reductase activity. Baseline sample of sul fate-reducing bacteria including:Desulfotignum, Archaeoglobus, Desulfatitalea, and Desulfomicrobium, which was dominanted by Desulfotignum (70% of sequences, the same below);then nothing was detected in the treated samples, indicating that the growth and metabolism of the SRP was inhibited. The napA function gene clone library analysis showed that NRB are detected in the baseline sample including Rhodopseudomonas, Pseudomonas and Stappia, Rhodopseudomonas was the dominant bacteria (50%); after continuous injection nitrate for 6 months, Rhodopseudomonas was detected in the production fluid sample; after stop nitrate injection for a month, NRB community becomes Sulfurimonas, Sulfur ospirillum and Pseudomonas, Sulfurimonas and Sulfur ospirillium were the dominant bacteria (44%). NarG functional gene clone library analysis showed that the baseline samples was rich in NRB flora, including:Castellaniella. Pseudomonas, Comamonas. Thiobacillus and Geobacter, Pseudomonas was dominant(57%); after injecting nitrate for six months, narG functional gene diversity becomes relatively simple, only Denitromonas was detected:Pseudomonas and Denitromonas were detected in sample from after stop injection of nitrate for 1 month and Denitromonas (70%) was the dominant bacteria. Results of nrfA function gene clone library analysis show that the NRB composition Parabacteroides (100%) from the baseline sample into Thauera (100%), and finally only Geobacter was detected in the sample. Detected in the samples collected in a month after the stop injectting nitrate foud NR-SOB:Sulfurimonas and Sulfurospirillum.It indicated that nitrate treatment directly impacts the population of SRP and NRB, especially to NRB.In this study, an nitrate-reducing bacteria IB1 was isolated from oilfield production fluids,which could effectively inhibit SRP, by using metabolite detection and PCR-DGGE analysis. Inhibition rate of hydrogen sulfide is 99.06%;IB1 can reduce 71.5% of the corrosion rate of the steel; DGGE analysis showed that the specific bands of SRP had been weaken or even disappeared.