Anaerobic, fermentative, halotolerant bacteria, strains 4-11^Tand 585, were isolated from production water of two low-temperature petroleum reservoirs (Russia) and were characterized by using a polyphasic approach. Cells of the strains were spherical, non-motile and 0.30–2.5 µm in diameter. Strain 4-11^Tgrew optimally at 35 °C, pH 6.0 and 1.0–2.0% (w/v) NaCl. Both strains grew chemoorganotrophically with mono-, di- and trisaccharides. The major cellular fatty acids of both strains were C_14:0, C_16:0, C_16:1ω9 and C_18:03-OH. Major polar lipids were glycolipids and phospholipids. The 16S rRNA gene sequences of the strains 4-11^Tand 585 had 99.9% similarity and were most closely related to the sequence ofSphaerochaeta associataGLS2^T(96.9, and 97.0% similarity, respectively). The G+C content of the genomic DNA of strains 4-11^Tand 585 were 46.8 and 46.9%, respectively. The average nucleotide identity and digital DNA–DNA hybridization values between the genomes of strain 4-11^TandS. associataGLS2^Twere 73.0 and 16.9%, respectively. Results of phylogenomic metrics analysis of the genomes and 120 core proteins of strains 4-11^Tand 585 and their physiological and biochemical characteristics confirmed that the strains represented a novel species of the genusSphaerochaeta, for which the nameSphaerochaeta halotoleranssp. nov. is proposed, with the type strain 4-11^T(=VKM B-3269^T=KCTC 15833^T). Based on the results of phylogenetic analysis,Sphaerochaeta coccoideswas reclassified as member of a new genusParasphaerochaetagen. nov.,Parasphaerochaeta coccoidescomb. nov. The generaSphaerochaetaandParasphaerochaetaform a separate clade, for which a novel family,Sphaerochaetaceaefam. nov., is proposed.

A microbial enhanced oil recovery (MEOR) technique was tested at low-temperature heavy oil reservoirs (Russia). The bioaugmentation approach used is based on the introduction of hydrocarbon-oxidizing bacteria into the oilfield in combination with an injection of oxygen as a H₂O₂ solution in order to initiate the first stage of hydrocarbon oxidation and of (NH₄)₂HPO₄ as a source of biogenic elements. Before the pilot trials, the microorganisms of petroleum reservoirs were investigated by high-throughput sequencing, as well as by culture-base and radioisotope techniques. Molecular studies revealed the differences in microbial composition of the carbonate and terrigenous oil reservoirs and the communities of injection and formation water. Aerobic bacteria Rhodococcus erythropolis HO-KS22 and Gordonia amicalis 6-1 isolated from oilfields oxidized oil and produced biosurfactants. Fermentative enrichment and pure cultures produced considerable amounts of low fatty acids and alcohols from sacchariferous substrates. In core-flooding tests, 43.0–53.5% of additional heavy oil was displaced by aerobic bacteria, producing biosurfactants, and 13.4–45.5% of oil was displaced by fermentative bacteria, producing low fatty acids, alcohols, and gas. A total of 1250 t additional oil was recovered as a result of the application of an MEOR technique at the Cheremukhovskoe heavy oil reservoir and Vostochno-Anzirskoe reservoir with light conventional oil.

The development of Arctic regions leads to pollution of marine and coastal environments with oil and petroleum products. The purpose of this work was to determine the diversity of microbial communities in seawater, as well as in littoral and coastal soil, and the potential ability of their members to degrade hydrocarbons degradation and to isolate oil-degrading bacteria. Using high-throughput sequencing of the V4 region of the 16S rRNA gene, the dominance of bacteria in polar communities was shown, the proportion of archaea did not exceed 2% (of the total number of sequences in the libraries). Archaea inhabiting the seawater belonged to the genera Nitrosopumilus and Nitrosoarchaeum and to the Nitrososphaeraceae family. In the polluted samples, members of the Gammaproteobacteria, Alphaproteobacteria, and Actinomycetes classes predominated; bacteria of the classes Bacteroidia, Clostridia, Acidimicrobiia, Planctomycetia, and Deltaproteobacteria were less represented. Using the iVikodak program and KEGG database, the potential functional characteristics of the studied prokaryotic communities were predicted. Bacteria were potentially involved in nitrogen and sulfur cycles, in degradation of benzoate, terephthalate, fatty acids, and alkanes. A total of 19 strains of bacteria of the genera Pseudomonas, Aeromonas, Oceanisphaera, Shewanella, Paeniglutamicibacter, and Rhodococcus were isolated from the studied samples. Among them were psychrotolerant and psychrophilic bacteria growing in seawater and utilizing crude oil, diesel fuel, and motor oils. The data obtained suggest that the studied microbial communities could participate in the removal of hydrocarbons from arctic seawater and coastal soils and suggested the possibility of the application of the isolates for the bioaugmentation of oil-contaminated polar environments.

A methanogenic enrichment growing on a medium with methanol was obtained from a petroleum reservoir (Republic of Azerbaijan) and stored for 33 years without transfers to fresh medium. High-throughput sequencing of the V4 region of the 16S rRNA gene revealed members of the genera Desulfovibrio, Soehngenia, Thermovirga, Petrimonas, Methanosarcina, and Methanomethylovorans. A novel gram-positive, rod-shaped, anaerobic fermentative bacterium, strain 1933P^T, was isolated from this enrichment and characterized. The strain grew at 13–55 °C (optimum 35 °C), with 0–3.0% (w/v) NaCl (optimum 0–2.0%) and in the pH range of 6.7–8.0 (optimum pH 7.0). The 16S rRNA gene sequence similarity, the average nucleotide identity (ANI) and in silico DNA–DNA hybridization (dDDH) values between strain 1933P^T and the type strain of the most closely related species Soehngenia saccharolytica DSM 12858^T were 98.5%, 70.5%, and 22.6%, respectively, and were below the threshold accepted for species demarcation. Genome-based phylogenomic analysis and physiological and biochemical characterization of the strain 1933P^T (VKM B-3382^T = KCTC 15984^T) confirmed its affiliation to a novel species of the genus Soehngenia, for which the name Soehngenia longivitae sp. nov. is proposed. Genome analysis suggests that the new strain has potential in the degradation of proteinaceous components.

The reserves of light conditional oil in reservoirs with low-salinity formation water are decreasing worldwide, necessitating the extraction of heavy oil from petroleum reservoirs with high-salinity formation water. As the first stage of defining the microbial-enhanced oil recovery (MEOR) strategies for depleted petroleum reservoirs, microbial community composition was studied for petroleum reservoirs with high-salinity formation water located in Tatarstan (Russia) using metagenomic and culture-based approaches. Bacteria of the phyla Desulfobacterota, Halanaerobiaeota, Sinergistota, Pseudomonadota, and Bacillota were revealed using 16S rRNA-based high-throughput sequencing in halophilic microbial communities. Sulfidogenic bacteria predominated in the studied oil fields. The 75 metagenome-assembled genomes (MAGs) of prokaryotes reconstructed from water samples were assigned to 16 bacterial phyla, including Desulfobacterota, Bacillota, Pseudomonadota, Thermotogota, Actinobacteriota, Spirochaetota, and Patescibacteria, and to archaea of the phylum Halobacteriota (genus Methanohalophilus). Results of metagenomic analyses were supported by the isolation of 20 pure cultures of the genera Desulfoplanes, Halanaerobium, Geotoga, Sphaerochaeta, Tangfeifania, and Bacillus. The isolated halophilic fermentative bacteria produced oil-displacing metabolites (lower fatty acids, alcohols, and gases) from sugar-containing and proteinaceous substrates, which testify their potential for MEOR. However, organic substrates stimulated the growth of sulfidogenic bacteria, in addition to fermenters. Methods for enhanced oil recovery should therefore be developed, combining the production of oil-displacing compounds with fermentative bacteria and the suppression of sulfidogenesis.

Application of seawater for secondary oil recovery stimulates the development of sulfidogenic bacteria in the oil field leading to microbially influenced corrosion of steel equipment, oil souring, and environmental issues. The aim of this work was to investigate potential sulfide producers in the high-temperature Uzen oil field (Republic of Kazakhstan) exploited with seawater flooding and the possibility of suppressing growth of sulfidogens in both planktonic and biofilm forms. Approaches used in the study included 16S rRNA and dsrAB gene sequencing, scanning electron microscopy, and culture-based techniques. Thermophilic hydrogenotrophic methanogens of the genus Methanothermococcus (phylum Euryarchaeota) predominated in water from the zone not affected by seawater flooding. Methanogens were accompanied by fermentative bacteria of the genera Thermovirga, Defliviitoga, Geotoga, and Thermosipho (phylum Thermotogae), which are potential thiosulfate- or/and sulfur-reducers. In the sulfate- and sulfide-rich formation water, the share of Desulfonauticus sulfate-reducing bacteria (SRB) increased. Thermodesulforhabdus, Thermodesulfobacterium, Desulfotomaculum, Desulfovibrio, and Desulfoglaeba were also detected. Mesophilic denitrifying bacteria of the genera Marinobacter, Halomonas, and Pelobacter inhabited the near-bottom zone of injection wells. Nitrate did not suppress sulfidogenesis in mesophilic enrichments because denitrifiers reduced nitrate to dinitrogen; however, thermophilic denitrifiers produced nitrite, an inhibitor of SRB. Enrichments and a pure culture Desulfovibrio alaskensis Kaz19 formed biofilms highly resistant to biocides. Our results suggest that seawater injection and temperature of the environment determine the composition and functional activity of prokaryotes in the Uzen oil field.