Abstract
The goal of this work was to study the overall genomic diversity of microorganisms of the Dagang high-temperature oilfield (PRC) and to characterize the metabolically active fraction of these populations. At this water-flooded oilfield, the microbial community of formation water from the near-bottom zone of an injection well where the most active microbial processes of oil degradation occur was investigated using molecular, cultural, radiotracer, and physicochemical techniques. The samples of microbial DNA and RNA from back-flushed water were used to obtain the clone libraries for the 16S rRNA gene and cDNA of 16S rRNA, respectively. The DNA-derived clone libraries were found to contain bacterial and archaeal 16S rRNA genes and the alkB genes encoding alkane monooxygenases similar to those encoded by alkB-geo1 and alkB-geo6 of geobacilli. The 16S rRNA genes of methanogens (Methanomethylovorans, Methanoculleus, Methanolinea, Methanothrix, and Methanocalculus) were predominant in the DNA-derived library of Archaea cloned sequences; among the bacterial sequences, the 16S rRNA genes of members of the genus Geobacillus were the most numerous. The RNA-derived library contained only bacterial cDNA of the 16S rRNA sequences belonging to metabolically active aerobic organotrophic bacteria (Tepidimonas, Pseudomonas, Acinetobacter), as well as of denitrifying (Azoarcus, Tepidiphilus, Calditerrivibrio), fermenting (Bellilinea), iron-reducing (Geobacter), and sulfate- and sulfur-reducing bacteria (Desulfomicrobium, Desulfuromonas). The presence of the microorganisms of the main functional groups revealed by molecular techniques was confirmed by the results of cultural, radioisotope, and geochemical research. Functioning of the mesophilic and thermophilic branches was shown for the microbial food chain of the near-bottom zone of the injection well, which included the microorganisms of the carbon, sulfur, iron, and nitrogen cycles.
Highlights
All stages of DNA and RNA isolation and the reagents used were tested in a preliminary study of an enrichment culture of hydrocarbon-oxidizing bacteria isolated from the Kongdian bed (Shestakova et al, 2011b)
The retrieved sequences of the δ-Proteobacteria included the genes of a thermophilic sulfate-reducing bacterium Desulfomicrobium thermophilum (99% similarity) and of mesophilic sulfate reducers, of sulfurreducing and iron-reducing bacteria (Geobacter hydrogenophilus, 97%), as well as of an uncultured bacterium remotely related to a syntrophic bacterium Smithella sp
Back-flushed water was sampled during the application of a biotechnology for enhanced oil recovery based on injection of nitrogen and phosphorus salts together with a water-air mixture and/or a hydrogen peroxide solution as an oxygen source
Summary
Microbial communities of petroleum reservoirs have been extensively studied using molecular, radioisotope, and culturebased techniques (Belyaev et al, 1982; Nazina et al, 1985, 1995; Voordouw et al, 1996; Magot et al, 2000; BonchOsmolovskaya et al, 2003; Orphan et al, 2003). Aerobic bacteria detected in these environments are considered contaminants arriving into the reservoir from the surface during drilling or with injection water. Analysis of 12 metagenomes from hydrocarbon resource environments (An et al, 2013) revealed that anaerobic communities were common, cores from oil sands and coal beds had unexpectedly high proportions of aerobic hydrocarbon-degrading bacteria. Head et al (2014) evaluated several hypotheses that might explain the occurrence of organisms conventionally considered to be aerobic, in nominally anoxic petroleum reservoir habitats. These hypotheses are: “(1) Samples were exposed to oxygen during sampling, transport and storage allowing growth of aerobic organisms initially present at low abundance. These hypotheses are: “(1) Samples were exposed to oxygen during sampling, transport and storage allowing growth of aerobic organisms initially present at low abundance. (2) Oxygen was supplied externally to the reservoir by meteoric water. (3) A ‘cryptic’ aerobic community that uses in situ generated oxygen may be present in some petroleum systems. (4) The aerobes detected are capable of anaerobic metabolism.”
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