T. L. Babich

[Microbiological investigations of high-temperature horizons of the Kongdian petroleum reservoir in connection with field trial of a biotechnology for enhancement of oil recovery].

The physicochemical conditions and microbiological characteristics of the formation waters of the Kongdian oilfield of the Dagang oilfield (China) were studied. It was demonstrated that this oilfield is a high-temperature ecosystem with formation waters characterized by low mineralization. The concentrations of nitrogen and phosphorus compounds, as well as of electron acceptors, are low. Oil and oil gas are the main organic matter sources. The oilfield is exploited with water-flooding. The oil stratum was inhabited mostly by anaerobic thermophilic microorganisms, including fermentative (102–105 cells/ml), sulfate-reducing (0–102 cells/ml), and methanogenic (0–103 cells/ml) microorganisms. Aerobic bacteria were detected mainly in the near-bottom zone of injection wells. The rate of sulfate reduction varied from 0.002 to 18.940 μg S2− l−1 day−1 and the rate of methanogenesis from 0.012 to 16.235 μg CH4 l−1 day−1. Microorganisms with great biotechnological potential inhabited the oilfield. Aerobic thermophilic bacteria were capable of oxidizing oil with formation of biomass, the products of partial oxidation of oil (volatile acids), and surfactants. During growth on the culture liquid of oil-oxidizing bacteria, methanogenic communities produced methane and carbon dioxide, which also had oil-releasing capabilities. Using various labeled tracers, the primary filtration flows of injected solutions at the test site were studied. Our comprehensive investigations allowed us to conclude that the method for microbial enhancement of oil recovery based on the activation of the stratal microflora can be applied in the Kongdian oilfield.

The Phylogenetic Diversity of Aerobic Organotrophic Bacteria from the Dagang High-Temperature Oil Field

The distribution and species diversity of aerobic organotrophic bacteria in the Dagang high-temperature oil field (China), which is exploited with water-flooding, have been studied. Twenty-two strains of the most characteristic thermophilic and mesophilic aerobic organotrophic bacteria have been isolated from the oil stratum. It has been found that, in a laboratory, the mesophilic and thermophilic isolates grow in the temperature, pH, and salinity ranges characteristic of the injection well near-bottom zones or of the oil stratum, respectively, and assimilate a wide range of hydrocarbons, fatty acids, lower alcohols, and crude oil, thus exhibiting adaptation to the environment. Using comparative phylogenetic 16S rRNA analysis, the taxonomic affiliation of the isolates has been established. The aerobic microbial community includes gram-positive bacteria with a high and low G+C content of DNA, and γ and β subclasses of Proteobacteria. The thermophilic bacteria belong to the genera Geobacillus and Thermoactinomyces, and the mesophilic strains belong to the genera Bacillus, Micrococcus, Cellulomonas, Pseudomonas, and Acinetobacter. The microbial community of the oil stratum is dominated by known species of the genus Geobacillus (G. subterraneus, G. stearothermophilus, and G. thermoglucosidasius) and a novel species “Geobacillus jurassicus.” A number of novel thermophilic oil-oxidizing bacilli have been isolated.

Microbiological and production characteristics of the high-temperature Kongdian petroleum reservoir revealed during field trial of biotechnology for the enhancement of oil recovery

Microbiological technology for the enhancement of oil recovery based on the activation of the stratal microflora was tested in the high-temperature horizons of the Kongdian bed (60°C) of the Dagang oil-field (China). This biotechnology consists in the pumping of a water-air mixture and nitrogen and phosphorus mineral salts into the oil stratum through injection wells in order to stimulate the activity of the stratal microflora which produce oil-releasing metabolites. Monitoring of the physicochemical, microbiological, and production characteristics of the trial site has revealed large changes in the ecosystem as a result of the application of biotechnology. The cell numbers of thermophilic hydrocarbon-oxidizing, fermentative, sulfate-reducing, and methanogenic microorganisms increased 10–10000-fold. The rates of methanogenesis and sulfate reduction increased in the near-bottom zone of the injection wells and of some production wells. The microbial oil transformation was accompanied by the accumulation of bicarbonate ions, volatile fatty acids, and biosurfactants in the formation waters, as well as of CH4 and CO2 both in the gas phase and in the oil. Microbial metabolites promoted the additional recovery of oil. As a result of the application of biotechnology, the water content in the production liquid from the trial site decreased, and the oil content increased. This allowed the recovery of more than 14000 tons of additional oil over 3.5 years.

Detection of n-alkane biodegradation genes alkB and ladA in thermophilic hydrocarbon-oxidizing bacteria of the genera Aeribacillus and Geobacillus

Ability to degrade crude oil n-alkanes was revealed in new strains of thermophilic bacilli isolated from petroleum reservoirs and a hot spring: Geobacillus toebii В-1024, Geobacillus sp. 1017, and Aeribacillus pallidus 8m3. The strains utilized С10–С30n-alkanes (В-1024), С10, C11, and С13–С19,22n-alkanes (1017), and C11–C29n-alkanes (8m3). In all three strains, PCR amplification with specific degenerate oligonucleotide primers revealed the alkB gene encoding rubredoxin-dependent alkane monooxygenase. In strains В-1024 and 1017, fragments of the genes homologous to the ladA gene determining flavin-dependent alkane monooxygenase were also amplified. Nucleotide sequences of these genes were practically identical to those of the genes ladAαB23, ladAβB23, and ladBB23, which were revealed previously in Geobacillus thermoleovorans strain B23. For the latter strain, activity of respective enzymes in the oxidation of long-chain n-alkanes has been shown. Thus, simultaneous presence of the alkB and ladA genes coding alkane monooxygenases responsible for oxidation of medium-chain and long-chain n-alkanes in thermophilic bacilli was revealed for the first time.

Identification of hydrocarbon-oxidizing Dietzia bacteria from petroleum reservoirs based on phenotypic properties and analysis of the 16S rRNA and gyrB genes

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol %, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002T, respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classification of strain 32d as a D. maris strain and strain 263 as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

Microorganisms of the Carbonate Petroleum Reservoir 302 of the Romashkinskoe Oilfield and Their Biotechnological Potential

Diversity, geochemical activity, and biotechnological potential of the microorganisms from oil bed 302 of the Romashkinskoe oilfield (Tatarstan, Russia) are reported. The microbial community contained almost no aerobic microorganisms. Sulfate-reducing (103−106 cells/mL) and fermentative bacteria (102−105 cells/mL) predominated in the oilfield. Sulfate reduction was the predominant process in formation water with the rates up to 26.6 μg S2−L/day. The number of methanogens and methanogenesis rate in formation water did not exceed 104 cells/mL and 8.19 μg CH4 L/day, respectively. Analysis of the 16S rRNA gene clone library revealed the sequences of denitrifying bacteria of the genera Sulfurimonas and Thauera. The oil recovery technique combining the stimulation of fermentative bacteria and suppression of sulfate reducers in the oilfield was proposed for development of the bed 302. Fermentative bacteria could be activated by the traditional method, i.e., injection of molasses and nitrogen and phosphorus mineral salts through the injection wells. Introduction of high concentrations of nitrate will activate the growth of denitrifying bacteria, suppress the growth of sulfidogenic bacteria, and result in decreased sulfide concentration in formation water. The proposed biotechnology is technologically simple and environmentally friendly.

Microorganisms of low-temperature heavy oil reservoirs (Russia) and their possible application for enhanced oil recovery

Physicochemical and microbiological characteristics of formation waters low-temperature heavy oil reservoirs (Russia) were investigated. The Chernoozerskoe, Yuzhno-Suncheleevskoe, and Severo-Bogemskoe oilfields, which were exploited without water-flooding, were shown to harbor scant microbial communities, while microbial numbers in the water-flooded strata of the Vostochno-Anzirskoe and Cheremukhovskoe oilfields was as high as 106 cells/mL. The rates of sulfate reduction and methanogenesis were low, not exceeding 1982 ng S2–/(L day) and 9045 nL СН4/(L day), respectively, in the samples from water-flooded strata. High-throughput sequencing of microbial 16S rRNA gene fragments in the community of injection water revealed the sequences of the Proteobacteria (74.7%), including Betaproteobacteria (40.2%), Alphaproteobacteria (20.7%), Gammaproteobacteria (10.1%), Deltaproteobacteria (2.0%), and Epsilonproteobacteria (1.6%), as well as Firmicutes (7.9%), Bacteroidetes (4.1%), and Archaea (0.2%). DGGE analysis of microbial mcrA genes in the community of injection water revealed methanogens of the genera Methanothrix, Methanospirillum, Methanobacterium, Methanoregula, Methanosarcina, and Methanoculleus, as well as unidentified Thermoplasmata. Pure cultures of bacteria of the genera Rhodococcus, Pseudomonas, Gordonia, Cellulomonas, etc., capable of biosurfactant production when grown on heavy oil, were isolated. Enrichment cultures of fermentative bacteria producing significant amounts of volatile organic acids (acetic, propionic, and butyric) from sacchariferous substrates were obtained. These acids dissolve the carbonates of oil-bearing rock efficiently. Selection of the efficient microbial technology for enhanced recovery of heavy oil from terrigenous and carbonate strata requires model experiments with microbial isolates and the cores of oil-bearing rocks.

Microbiological and production characteristics of the Dagang high-temperature heavy oil reservoir (block no. 1) during trials of the biotechnology for enhanced oil recovery

Microbiological and biogeochemical data on the Kongdian bed (block no. 1) of the Dagang high-temperature oilfield during trials of the biotechnology for enhanced oil recovery are reported. Oil-bearing horizons of block no. 1 are characterized by high temperature (56.9–58.4°C), complex geological conditions, and heavy oil (density 0.966–0.969 g/cm3). The biotechnology implied injecting oxygen as an air-water mixture or H2O2 together with aqueous solution of nitrogen and phosphorus mineral salts through injection wells in order to activate the oilfield microbial community. In the course of trials, an increase in abundance of aerobic and anaerobic microorganisms was revealed, as well as increased methanogenesis rate in formation water. Microbial oxidation of heavy oil resulted in increased concentration of mineral carbonates dissolved in formation water, changes in the stable carbon isotopic composition δ13C/Σ(CO2 + HCO3- + CO32-), formation of biosurfactants, and decreased interfacial tension of formation water. Application of the biotechnology at the Kongdian bed (block no. 1) resulted in additional recovery of 6331 t oil. Oil viscosity in the zone of production wells located at the North block of the Kongdian bed decreased by 11%. A total of 46152 t additional oil was recovered at three experimental sites of the Dagang oilfield (North block and block no. 1 of the Kongdian bed and the Gangxi bed), which is an indication of high efficiency of the technology for activation of the oilfield microflora for heavy oil replacement from high-temperature oilfields.

Phylogenetic Diversity of Microorganisms from the Sludge of a Biogas Reactor Processing Oil-Containing and Municipal Waste

High-throughput sequencing of the 16S rRNA gene fragments was used to determine the phylogenetic diversity of prokaryotes, including human pathogens, in the liquid phase of the sludge of a biogas reactor processing oil-containing and municipal waste. A unique microbial community was found to develop in the sludge, which comprised the microorganisms of municipal wastewater (bacteria of human feces) and specific groups of aerobic and anaerobic microorganisms, which possibly arrived with oil-containing water. In the 16S rRNA gene library, the sequences of representatives of Firmicutes prevailed (54.9%), which belonged to anaerobic bacteria of the genera Gelria (26.6%), Syntrophomonas (6.0%), Lutispora (2.0%), and uncultured Clostridia (group MBA03, 11.1%). The Proteobacteria sequences (20.7%) belonged mostly to the metabolically diverse members of the genus Pseudomonas (13.8%). The phylum Bacteroidetes (7%) was represented by uncultured bacteria (VadinBC27 wastewater-sludge group), while members of the phylum Cloacimonetes were mainly syntrophic bacteria Candidatus Cloacamonas (7.5%). The sequences of bacteria commonly occurring in oilfields (Clostridia, Anaerolinea, Bacteroidetes, sulfate-reducing Deltaproteobacteria, members of the family Syntrophaceae, and of the genera Thauera, Pseudomonas, Dechloromonas, and Petrimonas) were revealed. No sequences of bacteria known to be pathogenic to humans were found. The cultured microorganisms were aerobic organotrophic and anaerobic fermenting, denitrifying, and methanogenic prokaryotes. Fermenting and methanogenic enrichments grew on a broad range of organic substrates (sucrose, glycerol, starch), producing volatile fatty acids (acetate, n-butyrate, and propionate), gases (Н2, СО2, and CH4), and decreasing pH of the medium from 7.0 to 4.5–5.0. The possible application of the biogas reactor sludge as a source of fermenting and methanogenic anaerobic prokaryotes, as well as of aerobic hydrocarbonoxidizing bacteria for oilfield introduction and for production of new preparations for enhanced oil recovery and for bioremediation of oil contamination is discussed.

Microbial Community and in situ Bioremediation of Groundwater by Nitrate Removal in the Zone of a Radioactive Waste Surface Repository

The goal of the present work was to investigate the physicochemical and radiochemical conditions and the composition of the microbial community in the groundwater of a suspended surface repository for radioactive waste (Russia) and to determine the possibility of in situ groundwater bioremediation by removal of nitrate ions. Groundwater in the repository area (10-m depth) had elevated concentrations of strontium, tritium, nitrate, sulfate, and bicarbonate ions. High-throughput sequencing of the V3–V4/V4 region of the 16S rRNA gene revealed the presence of members of the phyla Proteobacteria (genera Acidovorax, Simplicispira, Thermomonas, Thiobacillus, Pseudomonas, Brevundimonas, and uncultured Oxalobacteraceae), Firmicutes (genera Bacillus and Paenibacillus), and Actinobacteria (Candidatus Planktophila, Gaiella). Canonical correspondence analysis suggested that major contaminant – nitrate, uranium, and sulfate shaped the composition of groundwater microbial community. Groundwater samples contained culturable aerobic organotrophic, as well as anaerobic fermenting, iron-reducing, and denitrifying bacteria. Pure cultures of 33 bacterial strains belonging to 15 genera were isolated. Members of the genera Pseudomonas, Rhizobium, Cupriavidus, Shewanella, Ensifer, and Thermomonas reduced nitrate to nitrite and/or dinitrogen. Application of specific primers revealed the nirS and nirK genes encoding nitrite reductases in bacteria of the genera Pseudomonas, Rhizobium, and Ensifer. Nitrate reduction by pure bacterial cultures resulted in decreased ambient Eh. Among the organic substrates tested, sodium acetate and milk whey were the best for stimulation of denitrification by the microcosms with groundwater microorganisms. Injection of these substrates into the subterranean horizon (single-well push-pull test) resulted in temporary removal of nitrate ions in the area of the suspended radioactive waste repository and confirmed the possibility for in situ application of this method for bioremediation.