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.


Introduction
Oil field exploitation results in the exhaustion of oil reserves, oil biodegradation, and a decrease in oil quality. Microbial anaerobic oil degradation in oil fields is considered among the processes of transformation of certified light oil to heavy oil and then into bitumen [1]. As a rule, oil reservoirs do not contain free oxygen. Under these conditions, the possible electron acceptors for microorganisms are CO 2 , sulfate, sulfur and other oxidized sulfur compounds, or iron hydroxides [2,3]. Nitrate and other nitrogen oxides do not occur in formation water. In this ecosystem, oil is the main source of organic matter.
Anaerobic production of methane from oil was originally reported by S.I. Kuznetsov [4]. A wide range of works is devoted to the study of the composition of methanogenic syntrophic associations degrading oil [5][6][7][8][9][10]. The participation of bacteria of the Syntrophaceae family in the syntrophic degradation of alkanes together with methanogens has been shown [11][12][13][14][15]. Although the composition of the associations differed depending on the composition of the oil, the temperature and salinity of the habitat from which they were isolated, the main functional components were oil-degrading Deltaproteobacteria or/and Firmicutes and hydrogenotrophic and acetoclastic methanogens [16][17][18]. Using metagenomic analysis in syntrophic oil-degrading communities, in addition to the known species, new uncultured bacteria such as 'Atribacteria' or archaea 'Candidatus Methanoliparia', 'Candidatus Argoarchaeum', and 'Candidatus Syntrophoarchaeum' were revealed [19][20][21]. Moreover, recently non-syntrophic hydrocarbon degradation by the methanogenic archaeon 'Candidatus Methanoliparum' was shown, which essentially changes our view on anaerobic oil transformation in petroleum reservoirs [22].
Members of Actinobacteria were identified in various methanogenic crude oil-degrading consortia [14,23]. In the syntrophic thermophilic methanogenic community growing on crude oil, actinobacteria 'Candidatus Syntraliphaticia' were found, probably belonging to a new class of the phylum Actinobacteria [23]. Metatranscriptomic analysis indicated their ability to carry out anaerobic alkane degradation by activation with fumarate and subsequent oxidation to CO 2 , H 2 /formate, and acetate, which were then utilized by methanogens. Aerobic hydrocarbon-oxidizing actinobacteria of the genera Dietzia, Gordonia, Rhodococcus, and Mycobacterium have been repeatedly detected in oil fields exploited with water flooding in order to maintain formational pressure [2,24]. Based on metagenomics analysis, An and co-workers [25] have shown the existence of aerobic taxa and the genes for aerobic hydrocarbon degradation in anaerobic environments containing hydrocarbons (oil fields, oil sands, and coal beds). Although the metagenomic approach makes it possible to characterize the potential activity of a microbial community in general, isolation of components of the community and determination of their physiological and genomic properties is required to elucidate their ecological role in the environment. In the current study, a methanogenic oil-degrading enrichment culture was obtained from production water of the low-temperature oil reservoir. The microbial community composition determined by sequencing of the V4 fragments of the 16S rRNA gene revealed bacteria of the phyla Bacillota, Actinomycetota, and Pseudomonadota and of methanogenic archaea of the phylum Euryarchaeota. The key fermentative bacterial strains isolated from the enrichment belonged to the genus Actinotalea. These data indicate the need for further research of anaerobic oil-degrading microbial communities for elucidation of their impact in oil degradation in petroleum reservoirs.
The aim of this study was elucidation of the taxonomic position of actinobacterial strains and their role in the oil degradation. Using a polyphase taxonomy approach and genome sequencing, the fermentative strain HO-Ch2 T isolated from the methanogenic enrichment was described as a new species Actinotalea subterranea sp. nov., and its potential ecophysiological function in the oil field was discussed. Comparative analysis of 187 genomes of the class Actinomycetia resulted in the taxonomic revision of the family Actinotaleaceae. Bacteria Cellulomonas carbonis and Cellulomonas bogoriensis were reclassified as member of a genus Actinotalea as Actinotalea carbonis comb. nov. and Actinotalea bogoriensis comb. nov., respectively. Species Actinotalea caeni was assigned to a genus Pseudactinotalea as Pseudactinotalea caeni comb. nov., and the genus Pseudactinotalea was transferred from the family Actinotaleaceae of the order Cellulomonadales to the family Ruaniaceae of the order Ruaniales.

Development of Methanogenic Enrichment Growing on Crude Oil
The water sample obtained from the Cheremukhovskoe heavy oil reservoir (Ritek, Nurlat, Russia) was used for isolation of an anaerobic methanogenic enrichment growing on crude oil. The oil-bearing horizons located at the depth of 890-920 m had a temperature of 20.2-21.3 • C. Oil density of surface sample was 0.932 g/cm 3 (at 25 • C). The Cheremukhovskoe oilfield is exploited with water-flooding; production water remaining after oil separation was used for injection into the oilfield after mixing with fresh surface water. The sample of production water re-injected into the reservoir (PWRI) was collected at the well head of injection well 5600. This water sample had low redox value (Eh -28.9 mV) and total salinity 0.621 g·L −1 ; pH 7.9. Hydrocarbonate, chloride, and sulfate were the major anions, and K + + Na + were the major cations. Physicochemical and microbiological parameters of formation water have been reported previously [26]. The HO-5600 (HO, heavy oil) anaerobic enrichment was obtained by inoculation of the mineral salt medium (MS) containing the following (per liter distilled water): 3.0 g MgCl 2 ·6H 2 O, 0.15 g CaCl 2 ·2H 2 O, 0.25 g NH 4 Cl, 0.2 g KH 2 PO 4 , 0.5 g KCl, 5.0 g NaCl, 2.5 g NaHCO 3 , 0.001 g resazurin, 0.5 g Na 2 S·9H 2 O; 1 mL·l −1 trace elements [27]; pH 7.0-7.2; MS medium was amended with 0.5 g yeast extract and heavy oil (0.5% v/v). Argon was used as the gas phase. Cultivation was carried out in 100-mL glass vials hermetically sealed with rubber stoppers and metal caps under stationary conditions in the dark at 28 • C. In parallel, from the 5600 injection water sample an aerobic organotrophic enrichment was obtained in the medium containing the following (per liter distilled water): 5.0 g Bacto tryptone, 2.5 g yeast extract, 1.0 g glucose, and 5.0 g NaCl; pH 7.0-7.2.

DNA Isolation from the Oil-Degrading Enrichment, 16S rRNA Gene Amplification, and Sequencing
The methanogenic enrichment HO-5600 (20 mL culture from one glass vial) incubated for 36 weeks was filtered through membrane filters with 0.22 µm pores (Millipore, Merck, Darmstadt, Germany). After treatment with the solution containing 0.15 M NaCl and 0.1 M Na 2 EDTA (pH 8.0), the filters with biomass were used for DNA isolation. DNA was extracted from the HO-5600 enrichment using the Pure Link Microbiome DNA Purification KIT (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommendations. The V4 hypervariable region of the 16S rRNA gene was amplified with the specific 515f/806 primer system [28]. Using the Cleanup Standard gel extraction kit (Evrogen, Russia), 16S rRNA gene fragments were amplified on the template of DNA isolated in three replicates, which then were combined and purified by electrophoresis in 2% agarose gel. The libraries were prepared as described previously [29]. High-throughput sequencing was conducted with a MiSeq system (Illumina, San Diego, CA, USA) and MiSeq Reagent Kit v2 (500 cycles) (Illumina, San Diego, CA, USA), according to the manufacturer's recommendations. The obtained 250-bp reads were then processed using workflows implemented the USEARCH v.10 scripts [30]. Reads were demultiplexed, trimmed to remove the primer sequences, and quality filtered. UNOISE3 [31,32]

Bacterial and Archaeal Strains
Bacterial strain HO-Ch2 T and methanogenic strain HO-Met1 were isolated from the HO-5600 anaerobic methanogenic enrichment grown on heavy oil. The aerobic strain HO-62b1 was isolated from the aerobic organotrophic enrichment. The strains HO-Ch2 T and HO-62b1 were isolated from the highest dilution of respective enrichments by successive plating on the R2A medium containing per liter distilled water: 0.5 g casein hydrolyzate, 0.5 g yeast extract, 0.5 g peptone, 0.5 g glucose, 0.5 g starch, 0.3 g K 2 HPO 4 , 0.02 g MgSO 4 , 0.3 g pyruvate, 5.0 g NaCl, and 18 g agar-agar; pH 7.0-7.2 [34]. Strains were incubated at 28-30 • C. The purity of the strains was checked by microscopy of colonies and by sequencing of the 16S rRNA genes. Strain HO-Ch2 T was deposited at the All-Russian collection of microorganisms (VKM; Pushchino, Moscow Region, Russia) under the number VKM Ac-2850, and at the Korean Collection for Type Cultures (KCTC; Jeongeup-si, Korea) under the number KCTC 49656. Strain Actinotalea ferrariae CF5-4 T (= KCTC 29134 T ) obtained from the KCTC (Jeongeup-si, Korea) was used as a reference strain. Methanogenic strain HO-Met1 was isolated by successive transfer from the highest dilution of the methanogenic enrichment in the liquid MS medium amended with ampicillin (10 mg·l-1) and with H 2 /CO 2 (4:1, v/v) mixture as a gas phase.
2.4. DNA Extraction from New Strains, 16S rRNA Gene Sequencing, and Phylogenetic Analysis DNA for the 16S rRNA gene or genome sequencing was extracted from biomass of the strains HO-Ch2 T and HO-62b1 grown aerobically on the R2A medium with 2.0% (w/v) NaCl at 28 • C. Cells were harvested after 7 days of cultivation. The cetyltrimethylammonium bromide (CTAB) method [35] was used to purify DNA from cell biomass. The 1492R and 27F primers were used to amplify the 16S rRNA genes of the strains HO-Ch2T and HO-62b2 [36]. The PCR products were sequenced using the Big Dye Terminator reagent kit, v. 3.1, at the ABI Prism 3730 DNA analyzer (Applied Biosystems, Waltham, MA, USA). Analysis of the 16S rRNA gene sequences was carried out using EzBioCloud [37]. The sequences were analyzed using the maximum-likelihood, neighbor-joining, and maximum-parsimony algorithms. First, the sequences were aligned by MUSCLE [38], and a maximum-likelihood tree was constructed using the model GTR+F+I+G4 recommended by ModelFinder [39] in IQ-Tree [40]. The MEGA7 software package was used to reconstruct the neighbor-joining and maximum-parsimony trees [41]. Bootstrap values were calculated from 1000 alternative trees. The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequence of strains HO-Ch2 T , HO-62b1, and HO-Met1 are MT225794, OK336454, and MT218393, respectively.

Genome Sequencing and Analyses
To construct DNA libraries for strain HO-Ch2 T , the NEBNext DNA library prep reagent kit for Illumina (New England Biolabs, Waltham, MA, USA) was used. Sequencing of genomic DNA was performed using the Illumina HiSeq 2500 platform (Illumina, Inc., San Diego, CA, USA). The quality of raw sequence reads was checked with FastQC v. 0.11.7 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 29 September 2021), and low-quality reads were trimmed with Trimmomatic v. 0.36 [42]. The quality-filtered reads were de novo assembled using SPAdes v. 3.13.0 [43]. The estimated completeness and contamination were evaluated using CheckM v1.0.18 [44]. Primary annotation and identification of protein-coding sequences were performed using the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAP) [45]. Additional gene prediction and functional annotation were performed in the Rapid Annotation using Subsystems Technology (RAST) server [46]. For the comparative metabolic study, an automatic assignment of KEGG Orthology (KO) identifiers to the proteins of Actinotalea type strains was completed using BlastKOALA [47]. The genome of strain HO-Ch2 T was deposited in GenBank/EMBL/DDBJ under the accession number VTTP00000000.1.

Morphological and Physiological Characterization
Strains HO-Ch2 T and HO-62b1 were characterized using a polyphasic taxonomic approach and compared with the reference strain Actinotalea ferrariae CF5-4 T . Cell morphology was studied by epifluorescence microscopy of 5-day cultures under an Axio Imager.D1 microscope (Carl Zeiss, Germany) and by scanning electron microscopy of metal-sprayed dry cells under a Camscan-S2 scanning electron microscope (Cambridge, UK) at 20 kV accelerating voltage. Cells negatively stained with 1% (w/v) phosphotungstic acid were studied also under a JEM-100C transmission electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV. Gram staining was performed by using a Gram staining kit (Biovitrum, Saint-Petersburg, Russia) according to the manufacturer's instructions. Growth at different temperatures (5,10,15,23,28,37, and 42 • C) was determined in the liquid R2A medium containing 2.0% (w/v) NaCl after incubation for 5-15 days. Salinity optimum and ranges for growth were determined in the liquid R2A medium containing 0, 0.1, 0.5, 1.0, 1.5, 2, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0% (w/v) NaCl for 7 days at 28 • C. The pH range for growth was determined in the Luria-Bertani (LB) medium at pH 5.2-9.0 with increments of~0.3-0.7 pH unit, using the appropriate citrate/phosphate (pH 5.2-7.5) and Tris/HCl (pH 8.0-9.0) buffers at optimal temperature (28 • C) and 2.0% (w/v) NaCl. Growth criteria were the change in the OD 660 of the medium, as well as microscopy. Increases of <10%, 10-50%, and >50% in the optical density (OD) at 660 nm of the liquid media after 5-15 days of growth were scored as no utilization (-), weak utilization (w), and good utilization (+), respectively. Biochemical and enzyme characteristics of strains HO-Ch2 T , HO-62b1 and CF5-4 T were determined by using API 50CH, API ® ZYM, and API 20E kits (Bio-Mérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. Catalase activity was determined by the standard method with H 2 O 2 . Oxidase activity was determined using the oxidase reagent (bioMérieux, Marcy l'Etoile, France). The aerobic and anaerobic utilization of carbon sources by the strains HO-Ch2 T and HO-62b1 was additionally tested in MS medium without Na 2 S·9H 2 O; yeast extract was replaced with 0.3 g·L −1 Casamino acids. Sugars, peptone, tryptone, and yeast extract were added at a concentration of 0.5% (w/v); alcohols, salts of organic acids, at 0.2% (w/v); amino acids, at 0.1-0.2% (w/v). Inoculated medium without the relevant substrate served as the control. Strains were tested for ability to use thiosulfate (3.2 g·L −1 ), nitrate (0.85 g·L −1 ), and Fe(3+) citrate (16.0 g·L −1 ) as electron acceptors for anaerobic growth with acetate (2.0 g·L −1 ). Anaerobic growth was tested by incubation in Hungate's tubes with Ar as a gas phase at 28 • C for 2 weeks. Sulfide was measured colorimetrically [55]; nitrite was determined using the Griess reagent. Products of glucose (5.0 g·L −1 ) fermentation in MS medium were analyzed by gas chromatography as described previously [56]. Antibiotic susceptibility was estimated in duplicate by spreading bacterial suspensions on Plate Count Agar (PCA, Merck, Darmstadt, Germany) medium with 2.0% (w/v) NaCl and applying filter paper disks (BD BBL sensi-disc antimicrobial susceptibility test discs, Becton, Dickinson and Company, USA) containing ampicillin (10 µg), chloramphenicol (30 µg), penicillin (10 µg), ciprofloxacin (5 µg), erythromycin (15 µg), gentamicin (10 µg), and kanamycin (30 µg). Susceptibility results were recorded as positive at zones with diameters higher than 10 mm after incubation at 28 • C for 2 days.

Chemotaxonomic Characterization
For chemotaxonomic characterization, strains HO-Ch2 T and Actinotalea ferrariae CF5-4 T were grown in TSB at 28 • C for 5 days. The cell biomass was dried with methanol and subjected to acidic methanolysis (1.2 M HCl/MeOH, 80 • C, 45 min). The fatty acid composition was analyzed using a Maestro gas chromatograph-mass spectrometer (Interlab, Russia) as described earlier [57]. The analysis of respiratory quinones of strains HO-Ch2 T and A. ferrariae CF5-4 T was performed at the All-Russian Collection of Microorganisms. Isoprenoid quinones were extracted from wet cells, purified according to Collins and Jones [58] and analyzed with a LCQ Advantage MAX mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Membrane lipids were analyzed as described in Supplementary Materials [59][60][61][62][63]. Peptidoglycan and sugars in the whole cell-wall of strains HO-Ch2 T and CF5-4 T were analyzed as described in Supplementary Materials [64,65].

Gas Chromatography
The n-alkanes and iso-alkanes content of oil were determined by gas-liquid chromatography on a Kristall 5000.1 chromatograph (Khromatek, Yoshkar-Ola, Russia) with a flame ionization detector and a ZB-FFAP 15 m capillary column as described earlier [66]. The oil samples were also analyzed by GC-MS on Agilent 5977A MSD fitted with HP-5MS (30 m × 0.25 mm i.d. × 0.25 µm film) capillary column (Agilent Technologies, Santa Clara, CA, USA). The carrier gas was helium (1 mL·min −1 ). For saturated oil components the initial column temperature was held for 3 min at 60 • C, then was increased at a rate 27 • C·min −1 to 180 • C and then was increased at 6 • C·min −1 to a final temperature of 300 • C, which was maintained for 25 min. To assess the extent of microbial oil degradation, the relative content (%) of n-alkanes was analyzed with relation to the control sample.

Nucleotide Sequence Accession Number
The library of 16S rRNA gene fragments of the HO-5600 methanogenic enrichment culture was deposited in NCBI SRA, project no. SRR17034681. The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequence of strains HO-Ch2 T , HO-62b1, and HO-Met1 are MT225794, OK336454, and MT218393, respectively. The whole-genome shotgun project of strain HO-Ch2 T has been deposited at DDBJ/EMBL/GenBank under the accession VTTP00000000.1. The raw FASTQ reads have been deposited in the NCBI SRA database under the accession no. SRR10092497.

Methane Production and Phylogenetic Diversity of Prokaryotes in the Anaerobic Oil-Degrading Enrichment
Dynamics of methane production by the HO-5600 oil-degrading enrichment obtained from production water of the heavy oil reservoir was monitored ( Figure 1). Production of H 2 was observed during the initial stage of incubation; it was subsequently consumed, and methane became the main gaseous product formed at the rate of 12.4 µmol·g of oil −1 ·day −1 . After 270 days of incubation, methane concentration in the gas phase was 17,460 ppm and 80 mg·l −1 acetate was found in the medium of the oil-degrading enrichment. HO-5600 methanogenic enrichment utilized the narrow range of C 16 -C 19 n-alkanes in crude oil ( Figure S1a-c).
The composition of the HO-5600 enrichment was determined by high-throughput sequencing of the V4 region of the 16S rRNA gene after 36 weeks of incubation with crude oil. A total of 72,601 16S rRNA gene fragments were obtained for the library. The sequences were grouped into 244 zOTUs. The oil-degrading enrichment was characterized by a low prokaryotic diversity. Archaea (1% of the sequences in the library) were represented by hydrogenothrophic methanogens of the genus Methanobacterium of the phylum Euryarchaeota. Bacteria (99%) predominated in the enrichment and belonged to the phyla Bacillota [67] (53%, including the genus Sedimentibacter, 49.1%), Actinomycetota (42%, including the genera Actinotalea, 26.8%, and Nocardioides, 8.3%), and Pseudomonadota (5%) ( Figure S2).
Members of the genus Sedimentibacter are known as rod-shaped, Gram-positive, amino acid-and pyruvate-utilizing, anaerobic bacteria, requiring yeast extract for growth and not utilizing carbohydrates [68]. Members of this genus have been detected in oilfields, in hydrocarbon-degrading enrichments [69,70], and in methanogenic enrichments from marine sediments [71]. It was suggested that bacteria of the genus Sedimentibacter act as syntrophs in such consortia. Other bacteria dominating in the enrichment belonged to the genus Actinotalea comprising aerobic or facultatively anaerobic organotrophic mesophilic bacteria [72][73][74][75][76]. Bacteria of the genera Pseudomonas, Tepidimonas, Paeniclostridium, Rhodoferax, and Pusillimonas, which often occur in subsurface environments, were among the minor components of the HO-5600 enrichment. The composition of the HO-5600 enrichment was determined by high-throughput sequencing of the V4 region of the 16S rRNA gene after 36 weeks of incubation with crude oil. A total of 72,601 16S rRNA gene fragments were obtained for the library. The sequences were grouped into 244 zOTUs. The oil-degrading enrichment was characterized by a low prokaryotic diversity. Archaea (1% of the sequences in the library) were represented by hydrogenothrophic methanogens of the genus Methanobacterium of the phylum Euryarchaeota. Bacteria (99%) predominated in the enrichment and belonged to the phyla Bacillota [67] (53%, including the genus Sedimentibacter, 49.1%), Actinomycetota (42%, including the genera Actinotalea, 26.8%, and Nocardioides, 8.3%), and Pseudomonadota (5%) ( Figure S2).
Members of the genus Sedimentibacter are known as rod-shaped, Gram-positive, amino acid-and pyruvate-utilizing, anaerobic bacteria, requiring yeast extract for growth and not utilizing carbohydrates [68]. Members of this genus have been detected in oilfields, in hydrocarbon-degrading enrichments [69,70], and in methanogenic enrichments from marine sediments [71]. It was suggested that bacteria of the genus Sedimentibacter act as syntrophs in such consortia. Other bacteria dominating in the enrichment belonged to the genus Actinotalea comprising aerobic or facultatively anaerobic organotrophic mesophilic bacteria [72][73][74][75][76]. Bacteria of the genera Pseudomonas, Tepidimonas, Paeniclostridium, Rhodoferax, and Pusillimonas, which often occur in subsurface environments, were among the minor components of the HO-5600 enrichment.

Isolation of Fermentative and Methanogenic Strains and Analysis of 16S rRNA Genes
Three strains were isolated in this study: the fermentative bacterial strain HO-Ch2 T and the methanogenic strain HO-Met1 isolated from the HO-5600 anaerobic methanogenic enrichment and aerobic strain HO-62b1 isolated from aerobic organotrophic enrichments. Strain HO-Met1 grew only on H2/CO2 with methane production. Based on the 99.9% sequence similarity of its 16S rRNA gene (GenBank accession number MT218393) with that of Methanobacterium aarhusense H2-LR T [77], the new strain HO-Met1 was assigned to this species. The 16S rRNA gene sequences of strains HO-Ch2 T and HO-62b1 had 100% similarity with the zOTU of Actinotalea sp., determined from the HO-5600 methanogenic enrichment and could be assigned to one Actinotalea species. On the phylogenetic tree (Figure 2), these sequences formed a separate lineage within the genus Actinotalea with a high level of bootstrap support. Strain HO-Ch2 T was chosen for further

Isolation of Fermentative and Methanogenic Strains and Analysis of 16S rRNA Genes
Three strains were isolated in this study: the fermentative bacterial strain HO-Ch2 T and the methanogenic strain HO-Met1 isolated from the HO-5600 anaerobic methanogenic enrichment and aerobic strain HO-62b1 isolated from aerobic organotrophic enrichments. Strain HO-Met1 grew only on H 2 /CO 2 with methane production. Based on the 99.9% sequence similarity of its 16S rRNA gene (GenBank accession number MT218393) with that of Methanobacterium aarhusense H2-LR T [77], the new strain HO-Met1 was assigned to this species. The 16S rRNA gene sequences of strains HO-Ch2 T and HO-62b1 had 100% similarity with the zOTU of Actinotalea sp., determined from the HO-5600 methanogenic enrichment and could be assigned to one Actinotalea species. On the phylogenetic tree ( Figure 2), these sequences formed a separate lineage within the genus Actinotalea with a high level of bootstrap support. Strain HO-Ch2 T was chosen for further in-depth study.
The 16S rRNA gene sequence similarity of 98.8% with A. ferrariae was slightly higher than 98.65%, the threshold accepted for species delineation [78]. Phylogenetic analysis of the 16S rRNA gene also revealed numerous discrepancies in the monophyletic nature of the genera within the family Actinotaleaceae. According to our results, type strains of the species Cellulomonas carbonis [79] and Cellulomonas bogoriensis [80] formed a cluster with members of the genus Actinotalea, which may indicate erroneous genus identification of these species. Moreover, the 16S rRNA gene sequences of Actinotalea caeni ERB-4-2 T clustered together with those of Pseudactinotalea, which indicated the probably required reclassification of this bacterium. It should be also noted that the branch uniting the Pseudactinotalea bacteria is anomalously long and may indicate incorrect position of this branching due to the Long Branch attraction [81] artifact. For elucidation of the taxonomic position of strain HO-Ch2 T , elucidation of phylogenetic inconsistencies within the family Actinotaleaceae, and determination of its possible functional role in the oil-degrading community, morphological, physiological, and chemotaxonomic properties of the strain were studied, and its genome was sequenced and analyzed. Gray circles indicate that the corresponding nodes were recovered in the tree reconstructed based on the maximum parsimony algorithm; black circles indicate that the corresponding nodes were also recovered based on the neighbor-joining and maximum-parsimony algorithms. Bootstrap values (>50%) are listed as percentages at the branching points. The tree was rooted using Nakamurella multipartita JCM 9543 T as the outgroup. GenBank accession numbers for the 16S rRNA gene sequences are indicated in parentheses. Bar, 0.05 substitutions per nucleotide position. Bacteria that have been reclassified are underlined.
The 16S rRNA gene sequence similarity of 98.8 % with A. ferrariae was slightly higher than 98.65%, the threshold accepted for species delineation [78]. Phylogenetic analysis of the 16S rRNA gene also revealed numerous discrepancies in the monophyletic nature of the genera within the family Actinotaleaceae. According to our results, type strains of the species Cellulomonas carbonis [79] and Cellulomonas bogoriensis [80] formed a cluster with members of the genus Actinotalea, which may indicate erroneous genus identification of these species. Moreover, the 16S rRNA gene sequences of Actinotalea caeni ERB-4-2 T

Phenotypic Characterization
Strains HO-Ch2 T and HO-62b1 and the closely related species, Actinotalea ferrariae CF5-4 T , were phenotypically characterized. At the time of writing, the genus Actinotalea comprised four species: A. fermentans, A. ferrariae, A. caeni, and A. solisilvae, isolated from coal seams, iron mining powder, sludge of a biofilm reactor, and forest soil, respectively [72][73][74][75]. Recently, the genera Actinotalea and Pseudactinotalea were placed within the new family Actinotaleaceae of the order Cellulomonadales of the class Actinomycetia [76].
The respiratory quinones of strains HO-Ch2 T and A. ferrariae CF5-4 T contained MK-9(H 4 ) as the major menaquinone, and MK-9(H 6 ) and MK-9(H 2 ) as the minor menaquinones at the ratios 10: 3: 1 and 10: 1.5: 0.5, respectively. The electron-impact mass spectrum of the isoprenoid quinone of strains HO-Ch2 T and CF5-4 T showed a base peak at m/z 211, and the peak of the molecular ion at m/z 789.4 ( Figure S7). The corresponding values for MK-9(H 4 ) were m/z 225 and 788, respectively. Traces of MK-8(H 4 ) were also detected in both strains. Menaquinone MK-10(H 4 ) has been found as a major menaquinone in the members of genus Actinotalea; however, A. fermentans DSM 3133 T contained MK-10(H 4 ), MK-9(H 4 ), and MK-8(H 4 ) in the ratio 56: 2: 1 [72]. The spectrum of major menaquinones may vary among Actinotalea species, so that menaquinone composition may be unsuitable for accurate differentiation of members of the genus Actinotalea from Cellulomonas containing MK-9(H 4 ). Amino acid analysis of the peptidoglycan preparation of HO-Ch2 T strain showed the presence of aspartic acid, serine, glutamic acid, alanine, and ornithine/lysin at an approximate molar ratio of 0.55: 2.0: 4.0: 6.1: 1.05/1.5, as well as glycin (5.8). The peptidoglycan of HO-Ch2 T strain corresponded to the A4β type containing L-Orn (Lys)-D-Ser-D-Glu. The molar ratio of ornithine, alanine, serine, D-glutamate, and aspartate in the peptidoglycan of A. ferrariae CF5-4 T was 0.8: 2.3: 0.9: 1.5: 1.1 (Table S5). Rhamnose was the major cell-wall sugar of strains HO-Ch2 T and CF5-4 T which is in accordance with features of the members of the genus Actinotalea. The composition of other polysaccharides varied and included galactose, mannose, and glucose in HO-Ch2 T ; and 3-O-methylgalactose (=madurosa), glucose, and traces of galactose and mannose were detected in A. ferrariae CF5-4 T .

Whole Genome Sequencing and Phylogenomic Analysis
The final assembled 4,027,363-bp-long genome of the strain HO-Ch2T comprised 28 scaffolds, with an N50 value of 335,339 bp, and coverage of 197×. The genomic DNA G + C content of strain HO-Ch2T was 73.4%, which was slightly below the expected range reported for members of the genus Actinotalea (73.8-75.2%) ( Table 2).
The genome of the strain HO-Ch2 T contained 3678 genes, of which 3589 were proteincoding sequences, 38 were pseudogenes, and 51 were RNA genes. Functional annotation of the genome performed via the RASTtk pipeline revealed that 415 of the genes were associated with carbohydrate metabolism, 288 genes, with metabolism of amino acids and their derivatives, 224 genes, with protein metabolism, and 198 genes, with metabolism of cofactors, vitamins, and pigments ( Figure S8). On the phylogenomic tree, strain HO-Ch2 T was placed within the genus Actinotalea (Figure 3). The ANI and dDDH values of 79.8-82.0% and 20.5-22.2%, respectively, to the Actinotalea genomes (Table 2) were below the species cutoff (95-96% for ANI and 70% for dDDH) [83], which indicated that the strain HO-Ch2 T belonged to a new species with proposed name Actinotalea subterranea sp. nov.
On the phylogenomic tree, as well as on the tree constructed using 16S rRNA gene alignment, type strains of Cellulomonas carbonis and Cellulomonas bogoriensis clustered together with members of the genus Actinotalea, which indicated the need for reclassification of these bacteria as members of the genus Actinotalea. Another confirmation of the need to reclassify these bacteria is the Average amino acid identity (AAI) values. Cellulomonas carbonis and Cellulomonas bogoriensis had AAI values with Actinotalea spp. in the range 69.7-76.3%, while only 65.4-67.7% with bacteria of the genus Cellulomonas. Based on phylogenetic analysis of the 16S rRNA gene, phylogenomic analysis and values of genomic indices, it was proposed to reclassify Cellulomonas carbonis and Cellulomonas bogoriensis as Actinotalea carbonis comb. nov. and Actinotalea bogoriensis comb. nov., respectively.  On the phylogenomic tree, as well as on the tree constructed using 16S rRNA gene alignment, type strains of Cellulomonas carbonis and Cellulomonas bogoriensis clustered together with members of the genus Actinotalea, which indicated the need for reclassification of these bacteria as members of the genus Actinotalea. Another confirmation of the need to reclassify these bacteria is the Average amino acid identity (AAI) values.  The phylogenomic tree confirmed the assumption that the taxonomic definition of Actinotalea caeni was incorrect. According to the results of phylogenetic studies and AAI values (Figure 3), this bacterium belongs to the genus Pseudactinotalea [84]. Moreover, despite their clustering on a 16S rRNA-based tree with bacteria of the genus Actinotalea, the phylogenomic tree indicates that the genus Pseudactinotalea belongs to the family Ruaniaceae [85]. The AAI values between Pseudactinotalea and Ruania are in the range of 63.1-64.2%, while between Pseudactinotalea and Actinotalea they are in the range 58.9-61.9%. Based on the above, it is proposed to reclassify Actinotalea caeni as Pseudactinotalea caeni comb. nov. and to transfer the genus Pseudactinotalea from the family Actinotaleaceae (Cellulomonadales) to the family Ruaniaceae of the order Ruaniales.

Pangenomic Analysis
A total of twenty-one genomes were used for the pangenomic analysis of Actinotaleaceae and Cellulomonadaceae species. The pangenome comprised 77,059 genes in 16,878 gene clusters (Figure 4). Of these 1137 gene clusters occurred in all bacterial genomes and were identified as the core ones for both families. Among these clusters were the genes responsible for complete pathways of carbohydrate metabolism: glycolysis (Embden-Meyerhof pathway), citrate cycle (TCA cycle), pentose phosphate cycle, UDP-N-acetyl-Dglucosamine biosynthesis. In addition, 32 gene clusters involved in energy metabolism (NADH:quinone oxidoreductase, succinate dehydrogenase, cytochrome bc1 complex, cytochrome c oxidase, F-type ATPase) were identified. All genomes of Actinotaleaceae and Cellulomonadaceae harbored also the nirBD nitrite reductase, which is responsible for the reduction of nitrite to ammonium. gene clusters (Figure 4). Of these 1137 gene clusters occurred in all bacterial genomes and were identified as the core ones for both families. Among these clusters were the genes responsible for complete pathways of carbohydrate metabolism: glycolysis (Embden-Meyerhof pathway), citrate cycle (TCA cycle), pentose phosphate cycle, UDP-N-acetyl-D-glucosamine biosynthesis. In addition, 32 gene clusters involved in energy metabolism (NADH:quinone oxidoreductase, succinate dehydrogenase, cytochrome bc1 complex, cytochrome c oxidase, F-type ATPase) were identified. All genomes of Actinotaleaceae and Cellulomonadaceae harbored also the nirBD nitrite reductase, which is responsible for the reduction of nitrite to ammonium. The core genome of Actinotaleaceae comprised 1454 gene clusters, and 52 of them were unique to this family and mainly with hypothetical functions. All Actinotaleaceae genomes harbored the genes involved in sulfonate utilization (ssuABCDE), also sulfide:quinone oxidoreductase (sqr), and assimilatory nitrate reductase (nasAB). Strain HO-Ch2 T had 543 gene clusters unique for Actinotaleaceae and Cellulomonadaceae, and 106 of them were functionally annotated. Unique genes were involved in signaling and cellular processes (24), in metabolism of carbohydrates (16), amino acids (5), vitamins/cofactors (4), and energy (4). Unlike other bacteria of the Actinotaleaceae and Cellu-T Figure 4. Pangenome analysis of Actinotaleaceae and Cellulomonadaceae calculated with Anvi'o versionv. 6.2. Dendrogram at the center represents the relationship between the 16,878 gene clusters (77,059 genes) found in analyzed genomes. Dark regions in colored circles represent genes found in that area for each genome. ANI heatmap in green squares vary between 70 and 100%. Phylogenomic tree reconstructed using the single copy genes.

Discussion
The biogeochemical processes of anaerobic transformation of oil with methane generation in reservoirs nor containing sulfate in waters or with hydrogen sulfide generation in reservoirs with sulfate-containing waters are well documented [1,7,10,86].
Degradation of oil n-alkanes has long been considered the process carried out mainly by members of the class Deltaproteobacteria, which use fumarate predominantly to activate alkane molecules [5,7,18]. Some members of this class, such as sulfate-reducing bacteria of the genera Desulfatibacillum, Desulfosarcina/Desulfococcus, Desulfoglaeba, and Desulfatiferula, grow with sulfide production in sulfate-containing environments on n-alkanes or n-alkenes as a sole carbon source [18]. Bacteria of the genera Smithella and Syntrophus degrade alkanes in consortia with methanogens [11]. Recently the archaeon 'Candidatus Methanoliparum' was shown to be able to combine the degradation of long-chain alkanes with methanogenesis [22].
In the present work, oil-degrading anaerobic methanogenic enrichment was obtained from a petroleum reservoir, its composition was determined by molecular 16S rRNA genebased survey; methanogenic and fermentative components of the enrichment were isolated in pure cultures and taxonomically characterized.
Growth of the studied HO-5600 enrichment resulted in accumulation of H 2 and methane in the gas phase and of acetate in the medium. The average rate of methane production by the studied culture for 275 days was 12.4 µmol·g of oil −1 ·day −1 . This value was closed to the methane yield rate (2.9-8.8 µmol·g of oil −1 ·day −1 ) during heavy oil degradation by methanogenic consortium from the Shengli oil field, which preferentially degraded long-chain alkyl substituted hydrocarbons [87]. In this consortium, bacteria of the genera Sedimentibacter, Soehngenia, Dehalococcoidetes, Actinobacteria, Anaerolineaceae, Clostridiales, and unclassified bacteria and methanogenic archaea of the genera Methanothrix, Methanosarcina and of class Methanomicrobia were stable components of consortium persisting for 4 successive transfers.
The HO-5600 methanogenic enrichment harbored obligate anaerobes of the genera Methanobacterium, Sedimentibacter, and Paeniclostridium and aerobic or facultatively anaerobic organothrophic bacteria of the genera Actinotalea, Pseudomonas, Tepidimonas, and Rhodoferax. Using traditional cultivation techniques, two strains were isolated in pure cultures from a methanogenic enrichment maintained for a long time: strain HO-Met1, a hydrogenotrophic methanogen phylogenetically closed to Methanobacterium aarhusense (100% 16S rRNA sequence similarity), and strain HO-Ch2 T , a facultatively anaerobic organothrophic bacterium belonging to the genus Actinotalea. While methanogenesis was the role Methanobacterium aarhusense strain HO-Met1 played in the enrichment [77], the roles of other components of the oil-degrading community remain unclear. Bacteria of the genera Sedimentibacter and Actinotalea predominated in the enrichment (49.1 and 26.8% of 16S rRNA gene sequences in the library, respectively). Some Sedimentibacter strains are known to utilize phenol, catechol, and benzoate, a central intermediate in several aromatic degradation pathways [68,71]. The major end products of amino acids utilization by Sedimentibacter sp. include acetate, propionate, and butyrate [71]. Anaerobic bacteria of the genus Sedimentibacter probably degraded the aromatic components of oil, releasing the products used by other members of the microbial community.
We should stress the predisposition of actinobacteria to oil-contaminated habitats. Bacteria of the genus Actinotalea were part of microbial biofilms (>1% of the community composition), degrading polycyclic aromatic hydrocarbons and naphthalene under microaerobic conditions [88]. Actinotalea ferrariae was the predominant component of the microbial community in the sample of oil-contaminated desert soil in some periods of the bioremediation process [89]. Actinotalea were numerous representatives of communities that degraded petroleum hydrocarbons in soil microbial fuel cells supplemented with biochar [90].
In our study, strain HO-62b1 identical to Actinotalea sp. strain HO-Ch2 T (100% similarity) was isolated from an aerobic organotrophic enrichment. On the phylogenetic tree (Figure 2), their sequences formed a separate lineage within the genus Actinotalea with the highest similarity (98.8%) to the sequences of the type strain of Actinotalea ferrariae CF5-4 T . This value was slightly below the value 98.65% accepted for species delineation [75]. Physiology and phylogenetic position of strains HO-Ch2 T and HO-62b1 were determined, and the genome of strain HO-Ch2 T was sequenced for elucidation of their taxonomy and functioning in the oil-degrading community.
The results of phenotypic analysis of the facultatively anaerobic Actinotalea strains HO-Ch2 T and HO-62b1 revealed that they utilized a broad range of protein-and sugarcontaining substrates under oxic and anoxic condition. Growth of both strains in the medium with crude oil was accompanied by slight changes in the C 16 -C 19 alkane fraction and in dimethylnaphthalenes content compared to those in the sterile control. The probable ecophysiological function of these bacteria is fermentation of carbohydrate-and proteincontaining substrates, including necromass (i.e., dead biomass). Fermentation of biomass by Actinotalea strains could results in generation of carbon sources (e.g., acetic, propionic, iso-butyric, and iso-valeric acids and CO 2 ), supporting a microbial carbon turnover and recycling of other nutrients (e.g., N and P). Low-molecular-mass metabolic products of organotrophic bacteria are used by methanogenic members of the community, which carry out the terminal stage of oil biodegradation with release of methane.
Our conclusion correlates with results of previous study of facultative anaerobic enrichment cultures derived from the coal basins of eastern Australia [91]. Vik and coworkers isolated Actinotalea strain SUR-A1 from enrichment and based on phenotypic and genomics studies suggested potential metabolic and ecological roles of these bacteria in coal seams. It was believed that Actinotalea cannot directly participate in biodegradation of coal compounds in situ, but may be involved in the degradation of accumulated biomass in coal seams, providing with fermentation products other members of microbial community degrading coal to methane.
Results of the 16S rRNA gene sequence and core-genome analysis, the average nucleotide identity (ANI), and in silico DNA-DNA hybridization (dDDH), as well as the phenotypic and chemotaxonomic characterization supported the classification of strains HO-Ch2 T and HO-62b1 as belonging to a novel species of the genus Actinotalea, for which the name Actinotalea subterranea sp. nov. is proposed. Using the 16S rRNA gene sequences and genome sequences from the reference type strains of the class Actinomycetia from the GenBank database, the phylogenomic and pangenomic comparison was performed. These data elucidated the phylogenetic relationships among the genera Actinotalea, Pseudactinotalea, and Cellulomonas and confirmed the reclassification of Cellulomonas carbonis and Cellulomonas bogoriensis as new combinations within the genus Actinotalea and the transfer of the genus Pseudactinotalea to the family Ruaniaceae of the order Ruaniales.

Conclusions
Our data show that the anaerobic methanogenic enrichment obtained from a petroleum reservoir comprises anaerobic prokaryotes of the genera Methanobacterium, Sedimentibacter, and Paeniclostridium and facultatively anaerobic organothrophic bacteria of the genera Actinotalea, Pseudomonas, Tepidimonas, and Rhodoferax. Anaerobic growth on crude oil resulted in accumulation of H 2 and CH 4 in the gas phase and of acetate in the medium.
Strain HO-Ch2 T , isolated from the methanogenic enrichment, was capable of fermenting carbohydrate-and protein-containing components of microbial biomass with production of volatile fatty acids and CO 2 , and participated in carbon turnover in the oil-degrading enrichment. The polyphasic taxonomic study and phylogenomic analysis demonstrated that strain HO-Ch2 T constituted a novel species within the genus Actinotalea, for which the name Actinotalea subterranea sp. nov. is proposed. Genome analysis of the novel strain and of the closely related strains of the genera Actinotalea, Pseudactinotalea, and Cellulomonas supports 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. The taxonomic descriptions of the new species and new combinations are enclosed below.
The type strain is HO-Ch2 T (= VKM Ac-2850 T = KCTC 49656 T ), isolated from the methanogenic enrichment obtained from a petroleum reservoir (Nurlat, Russia). The DNA G + C content of the genome of the type strain HO-Ch2 T is 73.4% and the genome size is 4.0 Mb. The EMBL/GenBank accession numbers for the 16S rRNA gene sequence and genome sequence of strain HO-Ch2 T are MT225794 and GCA_008364845.1, respectively.
The description is as given before (Salam et al., 2020) with the following modification. The genomic G+C content is around 70-74%. Genome sizes vary from 4.00 to 4.75 Mb. The type species is Pseudactinotalea terrae. The genus Pseudactinotalea is phylogenetically positioned within the family Ruaniaceae (Tang et al., 2010), order Ruaniales (Salam et al., 2020), class Actinomycetia of the phylum Actinobacteria.