Microbial and Monosaccharide Composition of Biofilms Developing on Sandy Loams from an Aquifer Contaminated with Liquid Radioactive Waste.

The development of microbial biofilms increases the survival of microorganisms in the extreme conditions of ecosystems contaminated with components of liquid radioactive waste (LRW) and may contribute to the successful bioremediation of groundwater. The purpose of this work was to compare the composition of the microorganisms and the exopolysaccharide matrix of the biofilms formed on sandy loams collected at the aquifer from a clean zone and from a zone with nitrate and radionuclide contamination. The aquifer is polluted from the nearby surface repository for liquid radioactive waste (Russia). The phylogenetic diversity of prokaryotes forming biofilms on the sandy loams' surface was determined during 100 days using high-throughput sequencing of the V4 region of the 16S rRNA genes. Scanning electron microscopy was used to study the development of microbial biofilms on the sandy loams. The ratio of proteins and carbohydrates in the biofilms changed in the course of their development, and the diversity of monosaccharides decreased, depending on the contamination of the sites from which the rocks were selected. The presence of pollution affects biofilm formation and EPS composition along with the dominant taxa of microorganisms and their activity. Biofilms establish a concentration gradient of the pollutant and allow the microorganisms involved to effectively participate in the reduction of nitrate and sulfate; they decrease the risk of nitrite accumulation during denitrification and suppress the migration of radionuclides. These biofilms can serve as an important barrier in underground water sources, preventing the spread of pollution. Pure cultures of microorganisms capable of forming a polysaccharide matrix and reducing nitrate, chromate, uranyl, and pertechnetate ions were isolated from the biofilms, which confirmed the possibility of their participation in the bioremediation of the aquifer from nonradioactive waste components and the decrease in the radionuclides' migration.

Metagenomic and Culture-Based Analyses of Microbial Communities from Petroleum Reservoirs with High-Salinity Formation Water, and Their Biotechnological Potential.

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.

Genome Analysis and Potential Ecological Functions of Members of the Genus Ensifer from Subsurface Environments and Description of Ensifer oleiphilus sp. nov.

The current work deals with genomic analysis, possible ecological functions, and biotechnological potential of two bacterial strains, HO-A22T and SHC 2-14, isolated from unique subsurface environments, the Cheremukhovskoe oil field (Tatarstan, Russia) and nitrate- and radionuclide-contaminated groundwater (Tomsk region, Russia), respectively. New isolates were characterized using polyphasic taxonomy approaches and genomic analysis. The genomes of the strains HO-A22T and SHC 2-14 contain the genes involved in nitrate reduction, hydrocarbon degradation, extracellular polysaccharide synthesis, and heavy metal detoxification, confirming the potential for their application in various environmental biotechnologies. Genomic data were confirmed by cultivation studies. Both strains were found to be neutrophilic, chemoorganotrophic, facultatively anaerobic bacteria, growing at 15-33 °C and 0-1.6% NaCl (w/v). The 16S rRNA gene sequences of the strains were similar to those of the type strains of the genus Ensifer (99.0-100.0%). Nevertheless, genomic characteristics of strain HO-A22T were below the thresholds for species delineation: the calculated average nucleotide identity (ANI) values were 83.7-92.4% (<95%), and digital DNA-DNA hybridization (dDDH) values were within the range of 25.4-45.9% (<70%), which supported our conclusion that HO-A22T (=VKM B-3646T = KCTC 92427T) represented a novel species of the genus Ensifer, with the proposed name Ensifer oleiphilus sp. nov. Strain SHC 2-14 was assigned to the species 'Ensifer canadensis', which has not been validly published. This study expanded the knowledge about the phenotypic diversity among members of the genus Ensifer and its potential for the biotechnologies of oil recovery and radionuclide pollution treatment.

Ways of Long-Term Survival of Hydrocarbon-Oxidizing Bacteria in a New Biocomposite Material-Silanol-Humate Gel.

Immobilized bacterial cells are presently widely used in the development of bacterial preparations for the bioremediation of contaminated environmental objects. Oil hydrocarbons are among the most abundant pollutants. We have previously described a new biocomposite material containing hydrocarbon-oxidizing bacteria (HOB) embedded in silanol-humate gels (SHG) based on humates and aminopropyltriethoxysilane (APTES); high viable cell titer was maintained in this material for at least 12 months. The goal of the work was to describe the ways of long-term HOB survival in SHG and the relevant morphotypes using the techniques of microbiology, instrumental analytical chemistry and biochemistry, and electron microscopy. Bacteria surviving in SHG were characterized by: (1) capacity for rapid reactivation (growth and hydrocarbon oxidation) in fresh medium; (2) ability to synthesize surface-active compounds, which was not observed in the cultures stored without SHG); (3) elevated stress resistance (ability to grow at high Cu2+ and NaCl concentrations); (4) physiological heterogeneity of the populations, which contained the stationary hypometabolic cells, cystlike anabiotic dormant forms (DF), and ultrasmall cells; (5) occurrence of piles in many cells, which were probably used to exchange genetic material; (6) modification of the phase variants spectrum in the population growing after long-term storage in SHG; and (7) oxidation of ethanol and acetate by HOB populations stored in SHG. The combination of the physiological and cytomorphological properties of the cells surviving in SHG for long periods may indicate a new type of long-term bacterial survival, i.e., in a hypometabolic state.

Genome analysis of Pollutimonas subterranea gen. nov., sp. nov. and Pollutimonas nitritireducens sp. nov., isolated from nitrate- and radionuclide-contaminated groundwater, and transfer of several Pusillimonas species into three new genera Allopusillimonas, Neopusillimonas, and Mesopusillimonas.

Two facultatively anaerobic, chemoorganoheterotrophic bacterial strains, designated JR1/69-2-13T and JR1/69-3-13T, were isolated from nitrate- and radionuclide-contaminated groundwater (Ozyorsk town, South Urals, Russia). Both strains were found to be motile, Gram-stain negative rod-shaped neutrophilic, psychrotolerant bacteria that grow within the temperature range from 5-10 to 33 °C at 0-3 (0-5)% NaCl (w/v). The major cellular fatty acids were identified as C16:0, C16:1 ω7c, C18:1 ω7c and C17:0 cyclo. The major polar lipids were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol and unidentified aminophospholipids. The genomic G + C content of strains JR1/69-2-13T and JR1/69-3-13T was determined to be 57.2 and 57.9%, respectively. The 16S rRNA gene sequences of the strains showed high similarity between each other (98.6%) and to members of the genera Pusillimonas (96.8-98.4%) and Candidimonas (97.1-98.0%). The average nucleotide identity and digital DNA-DNA hybridization (dDDH) values among genomes of the new isolates and Pusillimonas and Candidimonas genomes were below 84.5 and 28.8%, respectively, i.e., below the thresholds for species delineation. Based on the phylogenomic, phenotypic and chemotaxonomic characterisation, we propose assignment of strains JR1/69-3-13T (= VKM B-3223T = KCTC 62615T) and JR1/69-2-13T (= VKM B-3222T = KCTC 62614T) to a new genus Pollutimonas as the type strains of two new species, Pollutimonas subterranea gen. nov., sp. nov. and Pollutimonas nitritireducens sp. nov., respectively. As a result of the taxonomic revision of the genus Pusillimonas, three novel genera, Allopusillimonas, Neopusillimonas, and Mesopusillimonas are also proposed; and Candidimonas bauzanensis is reclassified as Pollutimonas bauzanensis comb. nov. Genome analysis of the new isolates suggested molecular mechanisms of their adaptation to an environment highly polluted with nitrate and radionuclides.

Crude Oil Degradation in Temperatures Below the Freezing Point by Bacteria from Hydrocarbon-Contaminated Arctic Soils and the Genome Analysis of Sphingomonas sp. AR_OL41.

Intensive human activity in the Arctic region leads to hydrocarbon pollution of reservoirs and soils. Isolation of bacteria capable of growing at low temperatures and degrading oil and petroleum products is of scientific and practical value. The aim of this work was to study the physiology and growth in oil at temperatures below 0 °C of four strains of bacteria of the genera Pseudomonas, Rhodococcus, Arthrobacter, and Sphingomonas-previously isolated from diesel-contaminated soils of the Franz Josef Land archipelago-as well as genomic analysis of the Sphingomonas sp. AR_OL41 strain. The studied strains grew on hydrocarbons at temperatures from -1.5 °C to 35 °C in the presence of 0-8% NaCl (w/v). Growth at a negative temperature was accompanied by visual changes in the size of cells as well as a narrowing of the spectrum of utilized n-alkanes. The studied strains were psychrotolerant, degraded natural biopolymers (xylan, chitin) and n-alkanes of petroleum, and converted phosphates into a soluble form. The ability to degrade n-alkanes is rare in members of the genus Sphingomonas. To understand how the Sphingomonas sp. AR_OL41 strain has adapted to a cold, diesel-contaminated environment, its genome was sequenced and analyzed. The Illumina HiSeq 2500 platform was used for AR_OL41 genome strain sequencing. The genome analysis of the AR_OL41 strain showed the presence of genes encoding enzymes of n-alkane oxidation, pyruvate metabolism, desaturation of membrane lipids, and the formation of exopolysaccharides, confirming the adaptation of the strain to hydrocarbon pollution and low habitat temperature. Average nucleotide identity and digital DNA-DNA hybridization values for genomes of the AR_OL41 strain with that of the phylogenetically relative Sphingomonas alpine DSM 22537T strain were 81.9% and 20.9%, respectively, which allows the AR_OL41 strain to be assigned to a new species of the genus Sphingomonas. Phenomenological observations and genomic analysis indicate the possible participation of the studied strains in the self-purification of Arctic soils from hydrocarbons and their potential for biotechnological application in bioremediation of low-temperature environments.

Structure elucidation and gene cluster annotation of the O-antigen of Halomonas titanicae TAT1 containing three residues of 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid.

A halotolerant hydrocarbon-oxidizing bacterium Halomonas titanicae strain TAT1 was isolated from a petroleum reservoir. The O-polysaccharide (O-antigen) was isolated from the lipopolysaccharide of H. titanicae TAT1 and studied by component analyses and 1D and 2D NMR spectroscopy. The following structure of the repeating linear pentasaccharide O-unit, containing only aminosugars, was established: →4)-ß-d-GlcpNAc3NAcA-(1 â†’ 4)-ß-d-GlcpNAc3NAcA-(1 â†’ 6)-α-d-GlcpNAc-(1 â†’ 4)-ß-d-GlcpNAc3NAcA-(1 â†’ 6)-α-d-GlcpNAc-(→, where d-GlcNAc3NAcA indicates 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid. The O-antigen gene cluster was identified in the genome of H. titanicae TAT1 and compared with available database sequences. The genes revealed in the O-antigen gene cluster and the assigned functions of putative proteins were consistent with the established polysaccharide structure.

Microbial Communities of Seawater and Coastal Soil of Russian Arctic Region and Their Potential for Bioremediation from Hydrocarbon Pollutants.

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.

Correction: Semenova et al. Physiological and Genomic Characterization of Actinotalea subterranea sp. nov. from Oil-Degrading Methanogenic Enrichment and Reclassification of the Family Actinotaleaceae. Microorganisms 2022, 10, 378.

The authors wish to make the following correction to this paper [...].

Physiological and Genomic Characterization of Actinotalea subterranea sp. nov. from Oil-Degrading Methanogenic Enrichment and Reclassification of the Family Actinotaleaceae.

The goal of the present work was to determine the diversity of prokaryotes involved in anaerobic oil degradation in oil fields. The composition of the anaerobic oil-degrading methanogenic enrichment obtained from an oil reservoir was determined by 16S rRNA-based survey, and the facultatively anaerobic chemoorganotrophic bacterial strain HO-Ch2T was isolated and studied using polyphasic taxonomy approach and genome sequencing. The strain HO-Ch2T grew optimally at 28 °C, pH 8.0, and 1-2% (w/v) NaCl. The 16S rRNA gene sequence of the strain HO-Ch2T had 98.8% similarity with the sequence of Actinotalea ferrariae CF5-4T. The genomic DNA G + C content of strain HO-Ch2T was 73.4%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the genome of strain HO-Ch2T and Actinotalea genomes were 79.8-82.0% and 20.5-22.2%, respectively, i.e., below the thresholds for species delineation. Based on the phylogenomic, phenotypic, and chemotaxonomic characterization, we propose strain HO-Ch2T (= VKM Ac-2850T = KCTC 49656T) as the type strain of a new species within the genus Actinotalea, with the name Actinotalea subterranea sp. nov. Based on the phylogenomic analysis of 187 genomes of Actinobacteria we propose the taxonomic revision of the genera Actinotalea and Pseudactinotalea and of the family Actinotaleaceae. We also propose the reclassification of Cellulomonas carbonis as Actinotalea carbonis comb. nov., Cellulomonas bogoriensis as Actinotalea bogoriensis comb. nov., Actinotalea caeni as Pseudactinotalea caeni comb. nov., and the transfer of the genus Pseudactinotalea to the family Ruaniaceae of the order Ruaniales.

Sulfidogenic Microbial Communities of the Uzen High-Temperature Oil Field in Kazakhstan.

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.

Soehngenia longivitae sp. nov., a Fermenting Bacterium Isolated from a Petroleum Reservoir in Azerbaijan, and Emended Description of the Genus Soehngenia.

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 1933PT, 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 1933PT and the type strain of the most closely related species Soehngenia saccharolytica DSM 12858T 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 1933PT (VKM B-3382T = KCTC 15984T) 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.

Draft Genome Sequence of Halomonas titanicae Strain TAT1, a Hydrocarbon-Oxidizing Halophilic Bacterium Isolated from a Petroleum Reservoir in Russia.

The draft genome sequence of a moderately halophilic bacterium, Halomonas titanicae strain TAT1, isolated from production water of the Romashkinskoe oilfield (Russia) is presented. The genome is annotated for elucidation of the metabolic pathways involved in hydrocarbon degradation and nitrate reduction in petroleum-contaminated hypersaline environments.

Draft genome sequence data and analysis of Shinella sp. strain JR1-6 isolated from nitrate- and radionuclide-contaminated groundwater in Russia.

Shinella sp. strain JR1-6 is a Gram-negative, facultatively anaerobic, non-spore-forming, motile, rod-shaped bacterium isolated from radionuclide- and nitrate-contaminated groundwater. This bacterium reduces nitrate to N2. Strain JR1-6 has potential for removal of nitrate contamination, which is the main reason for the interest in sequencing its genome. Here, we present a set of features of Shinella sp. strain JR1-6, together with the description of its genomic sequencing and annotation. The draft genome of strain JR1-6 has a size of ∼7.09 Mb and contains 6,945 genes, including 62 RNA genes. In the genome of strain JR1-6, the genes were revealed encoding nitrate reduction to N2, as well as the genes associated with metal resistance, showing its adaptation to the conditions of the environment and possible role in nitrate removal from contaminated groundwater. The draft genome sequence of Shinella sp. strain JR1-6 is available at DDBJ/EMBL/GenBank under the accession no. SHMI00000000.

Draft Genome Sequence of a Fermenting Bacterium, Soehngenia sp. Strain 1933P, Isolated from a Petroleum Reservoir in Azerbaijan.

The draft genome sequence of a mesophilic fermenting bacterium, Soehngenia sp. strain 1933P, isolated from production water of the Binagady petroleum reservoir (Republic of Azerbaijan), is presented. The genome is annotated for elucidation of the metabolic potential and taxonomic position of strain 1933P.

Draft Genome Sequence of Geotoga petraea Strain HO-Geo1, Isolated from a Petroleum Reservoir in Russia.

The draft genome sequence of Geotoga petraea strain HO-Geo1, a bacterium isolated from production water of the Vostochno-Anzirskoe petroleum reservoir in Russia, is presented. The genome of strain HO-Geo1 is annotated for elucidation of the metabolic potential and its possible function in the subsurface microbial community and biotechnological application.

Geobacillus proteiniphilus sp. nov., a thermophilic bacterium isolated from a high-temperature heavy oil reservoir in China.

A rod-shaped, spore-forming, thermophilic, chemoorganotrophic, aerobic or facultatively anaerobic bacterial strain, 1017T, was isolated from production water sampled at the Dagang oilfield (PR China), and was characterized by using a polyphasic approach. The strain is capable of anaerobic glucose fermentation. Nitrate is reduced to nitrite. Optimal growth was observed at 60-65 °C, at pH between pH 7.0 and 7.5, and with 1-2 % (w/v) NaCl. The major cellular fatty acids were iso-C17 : 0, anteiso-C17 : 0, iso-C15 : 0, iso-C16 : 0 and C16 : 0. The predominant polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analysis based on the 16S rRNA, gyrB and parE gene sequences indicated that the isolate belonged to the genus Geobacillus and was most closely related to Geobacillus thermoleovorans KCTC 3570T (99.5, 96.1 and 97.9 % sequence similarity, respectively). Genome sequencing revealed a genome size of 3.57495 Mb and a DNA G+C content of 51.8 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain 1017T and G. thermoleovorans KCTC 3570T were 95.9 and 64.9 %, respectively. Results of phylogenomic metrics analysis of the genome and 1172 core genes of strain 1017T and its physiological and biochemical characteristics confirmed that strain 1017T represented a novel species of the genus Geobacillus, for which the name Geobacillusproteiniphilus sp. nov. is proposed. The type strain is 1017T (=VKM B-3132T=KCTC 33986T).

Draft Genome Sequence of a Fermenting Bacterium, "Sphaerochaeta halotolerans" 4-11T, from a Low-Temperature Petroleum Reservoir in Russia.

The draft genome sequence of an anaerobic fermenting bacterium, "Sphaerochaeta halotolerans" strain 4-11T, isolated from formation water of a low-temperature petroleum reservoir in Russia is presented. The genome is annotated to elucidate the taxonomic position of the strain 4-11T and to extend the public genome database.

Whole-genome sequence data and analysis of type strains 'Pusillimonas nitritireducens' and 'Pusillimonas subterraneus' isolated from nitrate- and radionuclide-contaminated groundwater in Russia.

Two strains, 'Pusillimonas nitritireducens' JR1/69-2-13T and 'Pusillimonas subterraneus' JR1/69-3-13T, of aerobic, motile, Gram-negative, non-spore-forming, organotrophic, psychrotolerant bacteria were isolated from a sample of nitrate- and radionuclide-contaminated groundwater in Russia. Here we describe the draft genomes of these strains. The sequenced and annotated genome of the strain JR1/69-2-13T contained 4.3 Mbp with 4108 protein-coding genes. The genome of the strain JR1/69-3-13T contained 4.5 Mbp with 4260 protein-coding genes. Genome analysis of both strains provides an insight into the genomic basis of their resistance to nitrate, heavy metals and metalloids. The draft genome sequences of strains 'Pusillimonas nitritireducens' JR1/69-2-13T and 'Pusillimonas subterraneus' JR1/69-3-13T are available at DDBJ/EMBL/GenBank under the accession nos. https://www.ncbi.nlm.nih.gov/nuccore/PDNV00000000 and https://www.ncbi.nlm.nih.gov/nuccore/PDNW00000000, respectively.

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.