S. S. Belyaev

Radioisotopic, Culture-Based, and Oligonucleotide Microchip Analyses of Thermophilic Microbial Communities in a Continental High-Temperature Petroleum Reservoir

ABSTRACT Activity measurements by radioisotopic methods and cultural and molecular approaches were used in parallel to investigate the microbial biodiversity and its physiological potential in formation waters of the Samotlor high-temperature oil reservoir (Western Siberia, Russia). Sulfate reduction with rates not exceeding 20 nmol of H2S liter−1 day−1 occurred at 60 and 80°C. In upper horizons (AB, A, and B), methanogenesis (lithotrophic and/or acetoclastic) was detected only in wells in which sulfate reduction did not occur. In some of the wells from deeper (J) horizons, high-temperature sulfate reduction and methanogenesis occurred simultaneously, the rate of lithotrophic methanogenesis exceeding 80 nmol of CH4 liter−1 day−1. Enrichment cultures indicated the presence of diverse physiological groups representing aerobic and anaerobic thermophiles and hyperthermophiles; fermentative organotrophs were predominant. Phylogenetic analyses of 15 isolates identified representatives of the genera Thermotoga, Thermoanaerobacter, Geobacillus, Petrotoga, Thermosipho, and Thermococcus, the latter four being represented by new species. Except for Thermosipho, the isolates were members of genera recovered earlier from similar habitats. DNA obtained from three samples was hybridized with a set of oligonucleotide probes targeting selected microbial groups encompassing key genera of thermophilic bacteria and archaea. Oligonucleotide microchip analyses confirmed the cultural data but also revealed the presence of several groups of microorganisms that escaped cultivation, among them representatives of the Aquificales/Desulfurobacterium-Thermovibrio cluster and of the genera Desulfurococcus and Thermus, up to now unknown in this habitat. The unexpected presence of these organisms suggests that their distribution may be much wider than suspected.

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang oil field (P. R. China)

The number of microorganisms of major metabolic groups and the rates of sulfate reduction and methanogenesis processes in the formation waters of the high-temperature horizons of Dagang oil field have been determined. Using cultural methods, it was shown that the microbial community contained aerobic bacteria oxidizing crude oil, anaerobic fermentative bacteria, sulfate-reducing bacteria, and methanogens. Using cultural methods, the possibility of methane production from a mixture of hydrogen and carbon dioxide (H2 + CO2) and from acetate was established, and this result was confirmed by radioisotope methods involving NaH14CO3 and 14CH3COONa. Analysis of enrichment cultures 16S rDNA of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not revealed. Phylotypes of the representatives of Thermococcus spp. were found among archaeal 16S rDNA. 16S rRNA genes of bacterial clones belong to the orders Thermoanaerobacteriales (Thermoanaerobacter, Thermovenabulum, Thermacetogenium, and Coprothermobacter spp.), Thermotogales, Nitrospirales (Thermodesulfovibrio sp.) and Planctomycetales. 16S rDNA of a bacterium capable of oxidizing acetate in the course of syntrophic growth with H2-utilizing methanogens was found in high-temperature petroleum reservoirs for the first time. These results provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs, indicating that syntrophic processes play an important part in acetate degradation accompanied by methane production.

Use of Microorganisms in the Biotechnology for the Enhancement of Oil Recovery

The existing methods of oil field exploitation give the opportunity to extract no more than half of the geological resources of oil; from carbonate oil collectors, only 20% of oil is extracted [1]. Furthermore, the portion of recovered oil is tending to decrease due to the development of deposits of viscous oils under difficult geological and physical conditions. Thus, new methods for enhancing oil recovery are needed to at least maintain oil extraction at a constant level. There are various chemical and physicochemical methods for enhancing oil recovery. The method of secondary flooding, developed by the Soviet academician A.P. Krylov in the 1930s, is widely used in many oilproducing countries. Surface water is pumped into the oil stratum through a system of water-injection wells, which results in an increase in the stratal pressure. This technology is highly efficient. Chemical methods, used in combination with secondary flooding, belong to the so-called tertiary methods of oil recovery enhancement. These include immiscible flooding (alkaline or polymeric) and miscible flooding with solvents and acids, as well as flooding with the use of surface-active compounds (surfactant flooding, or microemulsion flooding). The surfactants widely used in Europe and North America are chemically synthesized oil sulfonates. Gas injection is also applied, with CO 2 , N 2 , and hydrocarbon gases being mainly used. Thermal methods include injection of a heat carrier (hot water or vapor) or intrastratal combustion. Selective plugging of highly permeable layers for the improvement of flooding is little used in Europe and Siberia but is widely applied in North America, Canada in particular [2]. The development of methods for microbial enhancement of oil recovery started in the middle of the 20th century. They are highly efficient and diverse, environmentally safe, and relatively cheap, although science-intensive. Three substantial prerequisites exist that allow us to believe in the prospects for the development of new efficient biotechnologies for enhancing oil recovery based on the geochemical activity of microorganisms. First, oil fields exploited with water flooding are widely inhabited by aerobic and anaerobic microorganisms belonging to various physiological groups, which retain viability and biochemical activity in oil strata. Second, microorganisms are able to degrade oil hydrocarbons to smaller and more labile organic molecules and to synthesize such oil-releasing agents as ee 2 , ec 4 , fatty acids, alcohols, polysaccharides, surfactants, and other technologically active substances. Third, microorganisms are capable of in situ production of oil-releasing substances directly in zones and microzones containing residual oil. Present research that aims at the development of biotechnologies for oil recovery enhancement should be concerned with study of the regularities of microbial distribution and geochemical activity in oil fields under different geophysical conditions, as well as with advanced investigation of physiological and biochemical characteristics of microorganisms and development of methods for regulation of microbial processes in oil fields [3].

Production of Oil-Releasing Compounds by Microorganisms from the Daqing Oil Field, China

Twenty pure cultures isolated from formation waters of the Daqing oil field were studied with respect to their capacity to produce surface-active compounds in media with individual hydrocarbons, lower alcohols, and fatty acids. Aerobic saprotrophic bacteria belonging to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, Clavibacter, Pseudomonas, and Acinetobacter decreased the surface tension of cultivation media from 55–63 to 28–44 mN/m. Strains of Bacillus cereus, Rhodococcus ruber, andBacillus licheniformis produced biosurfactants most actively. Bacteria of the genera Rhodococcus, Dietzia, Kocuria, and Gordonia produced exopolysaccharides in media with hydrocarbons. Culture liquids of the strains of R. ruberand B. licheniformis exhibited an oil-releasing effect. Thus, the Daqing oil field is inhabited by aerobic bacteria capable of producing effective oil-releasing agents.

[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.

Phylogenetic Diversity of Aerobic Saprotrophic Bacteria Isolated from the Daqing Oil Field

A diverse and active microbial community in the stratal waters of the Daqing oil field (China), which is exploited with the use of water-flooding, was found to contain aerobic chemoheterotrophic bacteria (including hydrocarbon-oxidizing ones) and anaerobic fermentative, sulfate-reducing, and methanogenic bacteria. The aerobic bacteria were most abundant in the near-bottom zones of injection wells. Twenty pure cultures of aerobic saprotrophic bacteria were isolated from the stratal waters. Under laboratory conditions, they grew at temperatures, pH, and salinity values typical of the stratal water from which they were isolated. These isolates were found to be able to utilize crude oil and a wide range of hydrocarbons, fatty acids, and alcohols. Phylogenetic analysis carried out with the use of complete 16S rRNA sequences showed that the isolates could be divided into three major groups: gram-positive bacteria with a high and a low G+C content of DNA and gram-negative bacteria of the γ-subclass of the Proteobacteria. Gram-positive isolates belonged to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, and Clavibacter. Gram-negative isolates belonged to the genera Pseudomonas and Acinetobacter. In their 16S rRNA sequences, many isolates were similar to the known microbial species and some probably represented new species.

[The properties of hydrocarbon-oxidizing bacteria isolated from the oilfields of Tatarstan, Western Siberia, and Vietnam].

Eleven strains of hydrocarbon-oxidizing bacteria, isolated from oilfields and representing the genera Rhodococcus, Gordonia, Dietzia, and Pseudomonas, were characterized as mesophiles and neutrophiles. Rhodococci were halotolerant microorganisms growing in a media containing up to 15% NaCl. All the strains oxidized n-alkanes of crude oil. An influence of the cultivation temperatures (28 or 45°C) and organic supplements on the degradation of C12-C30n-alkanes in oxidized oil by two bacterial strains of the genus Pseudomonas was shown. The introduction of acetate, propionate, butyrate, ethanol, and sucrose led mainly to decreased oxidation of petroleum paraffins. At certain cultivation temperatures, the addition of volatile fatty acid salts increased the content of certain n-alkanes in oxidized oil as compared to crude oil.

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.

Microbiology of formation waters from the deep repository of liquid radioactive wastes Severnyi

The presence, diversity, and geochemical activity of microorganisms in the Severnyi repository of liquid radioactive wastes were studied. Cultivable anaerobic denitrifiers, fermenters, sulfate-reducers, and methanogens were found in water samples from a depth of 162-405 m below sea level. Subsurface microorganisms produced methane from [2-(14)C]acetate and [(14)C]CO(2), formed hydrogen sulfide from Na(2) (35)SO(4), and reduced nitrate to dinitrogen in medium with acetate. The cell numbers of all studied groups of microorganisms and rates of anaerobic processes were higher in the zone of dispersion of radioactive wastes. Microbial communities present in the repository were able to utilise a wide range of organic and inorganic compounds and components of waste (acetate, nitrate, and sulfate) both aerobically and anaerobically. Bacterial production of gases may result in a local increase of the pressure in the repository and consequent discharge of wastes onto the surface. Microorganisms can indirectly decrease the mobility of radionuclides due to consumption of oxygen and production of sulfide, which favours deposition of metals. These results show the necessity of long-term microbiological and radiochemical monitoring of the repository.

[Microbiological processes in a high-temperature oil field].

Thermophilic sulfate-reducing bacteria (SRB) oxidizing lactate, butyrate, and C12–C16n-alkanes of oil at a temperature of 90°C were isolated from samples of water and oil originating from oil reservoirs of the White Tiger high-temperature oil field (Vietnam). At the same time, no thermophiles were detected in the injected seawater, which contained mesophilic microorganisms and was the site of low-temperature processes of sulfate reduction and methanogenesis. Thermophilic SRB were also found in samples of liquid taken from various engineering reservoirs used for oil storage, treatment, and transportation. These samples also contained mesophilic SRB, methanogens, aerobic oil-oxidizing bacteria, and heterotrophs. Rates of bacterial production of hydrogen sulfide varied from 0.11 to 2069.63 at 30°C and from 1.18 to 173.86 at 70°C μg S/(l day); and those of methane production, varied from 58.4 to 100 629.8 nl CH4/(l day) (at 30°C). The sulfur isotopic compositions of sulfates contained in reservoir waters and of hydrogen sulfide of the accompanying gas indicate that bacterial sulfate reduction might be effective in the depth of the oil field.