Metagenomic Analysis of the Microbial Community in the Underground Coal Fire Area (Kemerovo Region, Russia) Revealed Predominance of Thermophilic Members of the Phyla Deinococcus-Thermus, Aquificae, and Firmicutes

Underground burning of coal seams accompanied by release of gases leads to development of local thermal ecosystems. We investigated the microbial community of the ground heated to 72°C in the release area of hot gases resulting from underground combustion of coal mining waste at the Bungurskiy-Severny coal deposit in the Kemerovo region of Russia. Analysis of the composition of the microbial community by 16S rRNA gene profiling revealed predominance of thermophilic bacteria of the phyla Deinococcus-Thermus, Aquificae, and Firmicutes. As a result of metagenomic analysis, 18 genomes of the main members of the microbial community were assembled, including the complete genomes of Hydrogenobacter thermophiles, a member of the candidate genus UBA11096 of the phylum Aquificae (RBS10-58), Thermoflexus hugenholtzii, and Thermus antranikianii. Analysis of the RBS10-58 genome indicates that this bacterium can autotrophically fix carbon in the reductive tricarboxylic acid cycle and obtain energy via oxidation of hydrogen and sulfur compounds with oxygen or nitrate as electron acceptors. Genome analysis of the two dominant Firmicutes species, Hydrogenibacillus schlegelii and an uncultured member of the class Thermaerobacteria, showed that these bacteria could grow aerobically by oxidizing hydrogen and carbon monoxide. Overall, the community was dominated by aerobic bacteria capable of growing autotrophically and obtaining energy via oxidation of the main components of coal gases, hydrogen and carbon monoxide. Thermus antranikianii, which makes up about a half of the microbial community, probably uses organic matter produced by autotrophic members of Firmicutes and Aquificae.

Studies of thermophilic microorganisms have expanded our knowledge of microbial diversity, evolution, and mechanisms of adaptation to extreme environmental conditions (Urbieta et al., 2015;Counts et al., 2017). Most research on thermophilic microorganisms has focused on thermal ecosystems associated with volcanic activity, such as terrestrial hot springs and deep-sea hydrothermal vents, or on technogenic biotopes (e.g., high-temperature bioreactors). Similarly to volcanic activity, natural combustion of fossil hydrocarbons and coal can also underlie formation of local thermal ecosystems. Characterization of microbial communities in such ecosystems contributes to our understanding of the diversity of thermophilic microorganisms and processes mediated by them.
Underground burning of coal seams is a widespread natural phenomenon and has been observed in Australia, Germany, the United States, China, Russia, India, and other countries (Stracher and Taylor, 2004). Such underground fires can last for centuries; for example, the coal seam in Dudweiler (Saarland, Germany) has been burning since 1668. An example of long-term natural underground coal combustion is the Burning Mountain in Australia, which is estimated to have burned for about 6000 years (Rattigan, 1967).
Coal burning under conditions of oxygen deficit and in the presence of water leads to the formation of coal gases according to the following reactions (Shafirovich and Varma, 2009): Underground coal combustion is a natural analog of the synthesis gas production by coal gasification. In addition to CO 2 , coal gases contain mainly hydrogen, CO, and gaseous hydrocarbons (Stracher and

EXPERIMENTAL ARTICLES
2004; Engle et al., 2012). These gases can also contain hydrogen sulfide, sulfur oxides, and other toxic compounds, such as benzene, xylene, or aliphatic and halogenated compounds (Engle et al., 2012;Zhang et al., 2013). Sulfur and other elements present in coal seams can be transferred to the surface with the gas flow (Pone et al., 2007). Local extreme ecosystems developing in areas where hot coal gases come to the surface are characterized by high temperatures (>50°C) and the presence of toxic substances (Tammy et al., 2005). The highenergy compounds contained in coal gases, such as hydrogen and CO, can be used by microorganisms as substrates, while oxygen is used as an electron acceptor, which enables formation of specific communities of thermophilic microorganisms. However, relatively little is known about the composition of microbial communities of such ecosystems and the genetic potential of their microorganisms.
The phenomenon of underground coal combustion is also found in coal deposits in Russia. The Kuznetsk coal basin (Kuzbass) located in the south of Western Siberia is one of the largest coal mining areas in the world. Currently, coal in this region is mainly mined by open pit mining, which produces large quantities of waste, including overburden. Overburden containing residual coal is stored as dumps directly at the mining sites, where ignition and long-term burning can occur due to natural and anthropogenic causes. A well-known example of an environmental disaster is the burning of coal dumps near the mining town of Kiselevsk in Kuzbass (Russia) (Kadnikov et al., 2021).
Start new paragraph here we studied the soil microbial community associated with the zone of underground coal combustion and the release of hot coal gases at the dumps of the Bungurskiy-Severny coal deposit in the Novokuznetsk district of the Kemerovo region (Russia). The goal of the present work was to investigate the composition and the genetic potential of this microbial community. We present data on the composition of the community obtained by highthroughput sequencing of 16S rRNA gene amplicons and the soil metagenome in the area of hot coal gas release to the surface. As a result of metagenomic analysis, high-quality genomes (metagenome-assembled genomes, MAGs) of most members of the community were obtained, which made it possible to characterize the metabolic potential of the corresponding microorganisms.

MATERIALS AND METHODS
Sampling and DNA extraction. Samples were taken at the storage site of coal mining waste at the Bungurskiy-Severny coal deposit near the Apanas village of the Novokuznetsk district, Kemerovo region (53.542314 N, 86.862370 E). A ground sample col-lected at a depth of 5-10 cm and designated RBS10 represented wet ground on the slope of a mining waste dump near the site of hot steam and gas release. The sample collected was a finely dispersed rock containing coal. Metagenomic DNA was isolated using a MO BIO Power Soil DNA Kit (MO BIO Laboratories, Qiagen, United States).
Sequencing and analysis of 16S rRNA gene fragments. PCR amplification of 16S rRNA gene fragments including the hypervariable regions V3-V6 was performed using the universal primers 341F (5'-CC-TAYGGGDBGCWSCAG-3') and 806R (5'-GGAC-TACNVGGGTHTCTAAT-3') (Frey et al., 2016). The obtained PCR fragments were barcoded using a Nextera XT Index Kit v.2 (Illumina, United States) and purified using Agencourt AMPure beads (Beckman Coulter, United States); the amount of DNA was determined using a Qubit dsDNA HS Assay Kit (Invitrogen, United States). Next, the amplicons were sequenced using Illumina MiSeq (paired-end reads, 2 × 300 bp). Overlapping reads were merged using FLASH v.1.2.11 (Magoč and Salzberg, 2011). Filtering by quality and clustering of sequences into operational taxonomic units (OTUs) at the level of 97% sequence identity was performed using the Usearch software (Edgar, 2010). Chimeric sequences and singletons were removed during clustering by the Usearch algorithm. To calculate the relative abundance of OTUs, all reads (including singletons and low-quality ones) were mapped using Usearch on OTU sequences with an identity threshold of 97%.
Taxonomic identification of OTUs was carried out by searching the SILVA v.132 rRNA sequence database using the VSEARCH algorithm (Rognes et al., 2016).
Metagenomic DNA was additionally sequenced on a MinION (Oxford Nanopore, United Kingdom) using a 1D Genomic DNA by Ligation kit (SQK-LSK108). Sequencing this library using a MinION device with an R9.4 flowcell (FLO-MIN106) yielded 8280228 reads with a total length of 16.12 billion bp.
All Illumina (about 25 billion bp in total) and Nanopore reads obtained were de novo assembled into contigs using the metaSPAdes hybrid assembler v.3.13.0 software (Nurk et al., 2017). Contigs longer than 1500 bp were binned into clusters representing MAGs using MetaBAT v.2.12.1 (Kang et al., 2015). To improve MAG assembly, MinION reads were mapped to the contigs included in MAG using the BWA v.0.7.15 software (Li and Durbin, 2010). Next, Npscarf v.1.0 (Cao et al., 2017) was used to form chains of contigs (scaffolds) and to fill the gaps between contigs using Illumina consensus sequences from the metaSPAdes assembly graph.
Gene search and MAG annotation were performed using the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al., 2016) or the RAST server 2.0 (Brettin et al., 2015) with subsequent correction of the annotation by comparing the predicted protein sequences to the databases of the National Center for Biotechnology Information (NCBI). N-terminal signal peptides were predicted using Signal P v.5.0, and the presence of transmembrane domains was predicted using the TMHMM v.2.0 (http://www. cbs.dtu.dk/services/TMHMM/).

Evaluation of genome similarity and phylogenetic
analysis based on complete genome data. The levels of average nucleotide identity (ANI) and average amino acid identity (AAI) between selected genomes were calculated using scripts from the Enveomics Collection toolbox (Rodriguez-R and Konstantinidis, 2016).
GTDB-Tk v.0.3.2 software was used to search for single-copy marker genes in a given MAG and to construct multiple alignments of concatenated singlecopy marker gene sequences from this MAG and all species from GTDB. A part of the multiple alignment created using the GTDB-Tk software was used to build a phylogenetic tree using PhyML v.3.3 (Guindon et al., 2010) with default parameters. Internal branching support was assessed using a Bayesian test in the PhyML.
Deposition of nucleotide sequences. The primary data obtained by sequencing 16S rRNA gene fragments and by metagenome sequencing were deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRX10881305, SRX10881306 and SRX10881307. The annotated MAG sequences were deposited in the GenBank database and are available via BioProject PRJNA728906.

Composition of the microbial community based on analysis of 16S rRNA gene amplicons.
A ground sample was collected from a burning coal mining dump with numerous vents releasing combustion products to the surface. The ground temperature at the sampling site was 72°C.
The composition of the microbial community was characterized based on 57213 sequences of 16S rRNA gene fragments. As a result of clustering these sequences, 29 OTUs were identified with an identity threshold of 97%. All identified OTUs belonged to bacteria; no archaea were detected. The results of the taxonomic classification of the OTUs are shown in Fig. 1.
The community was dominated by members of three phyla: Deinococcus-Thermus (47.7% of the total number of 16S rRNA gene sequences), Firmicutes (34.9%), and Aquificae (16.5%). Members of the phyla Bacteroidetes (0.48%), Proteobacteria (0.27%), Chlo-  100 % roflexi (0.02%), and Actinobacteria (0.01%) were found in minor amounts. The phylum Deinococcus-Thermus was represented by two OTUs, one of which dominated the community, accounting for 47.4% of the total number of 16S rRNA gene sequences. This OTU belonging to the genus Thermus had 98.7% sequence identity to the thermophilic heterotrophic bacterium Thermus antranikianii isolated from hot springs in Iceland (Chung et al., 2000).
The phylum Firmicutes was represented by 12 OTUs. The most abundant of them (14.1%) belonged to the genus Brevibacillus and had 98.73% 16S rRNA gene sequence identity with Brevibacillus borstelensis, a moderately thermophilic heterotrophic spore-forming bacterium found in soils and hydrothermal habitats (Shida et al., 1995;Khalil et al., 2018). Approximately 11% of the sequences represented one OTU belonging to the genus Hydrogenibacillus (order Thermicanales, family Thermicanaceae according to the Genome Taxonomy Database). A cultured representative of this genus, Hydrogenibacillus schlegelii, is a facultative chemolithotrophic aerobic thermophilic bacterium capable of oxidizing hydrogen and, presumably, CO (Schenk and Aragno, 1979;Kämpfer et al., 2013). About 6.3% of the community belonged to an OTU phylogenetically distant from the cultured members of the phylum Firmicutes (<87% 16S rRNA gene sequence identity).
The phylum Aquificae was represented by one OTU phylogenetically close to the genus Hydrogenobacter of the family Aquificaceae. Members of Hydrogenobacter, typical inhabitants of high-temperature thermal springs, are chemolithoautotrophic thermophiles capable of aerobic hydrogen oxidation (Reysenbach et al., 2000;Takacs-Verbach et al., 2013).
Metagenome sequencing and MAGs assembly. To obtain the genomes of the microbial community members, we sequenced the metagenome of RBS10 sample using a combination of Illumina and Oxford Nanopore technologies. The assembled contigs were binned into 18 MAGs with completeness of more than 80% and contamination (redundancy) of less than 10% as assessed by CheckM based on the analysis of a set of conserved single-copy marker genes (Table 1). Altogether, these MAGs represented approximately 80% of the total metagenome of the community. The taxonomic affiliation of the obtained MAGs was determined based on phylogenetic analysis of concatenated sequences of conserved marker genes according to the Genome Taxonomy Database (Parks et al., 2018).
The taxonomic classification of the MAGs identified the same main bacterial phyla that were found using 16S rRNA profiling. The relative abundances of some groups differed between the pool of 16S rRNA gene sequences and the complete metagenome ( Fig. 1), probably due to the different number of 16S rRNA gene copies in the genomes and different sizes of the genomes. Nearly a half of the total metagenome was represented by a single MAG, RBS10-92, assigned to the phylum Deinococcus-Thermus. About 26% of the metagenome were represented by ten MAGs of the phylum Firmicutes; three MAGs were attributed to Aquificae (2.4% of the metagenome); three belonged to Chloroflexi (3.3% of the metagenome); two were assigned to Proteobacteria (0.5% of the metagenome), and 0.02% of the metagenome was the MAG of the candidate phylum WOR-3 (Table 1).
To characterize the metabolic capabilities of the dominant members of the microbial community, we performed a detailed analysis of several MAGs.
Genomes of members of the phylum Firmicutes. Two bacteria of the phylum Firmicutes, represented by MAG RBS10-35 and MAG RBS10-49, were abundant members of the microbial community. The RBS10-35 genome represented 11.0% of the total metagenome and was identified only at the class level as belonging to Thermaerobacteria according to the genomic taxonomy system. Probably, this genome corresponded to the OTU that constituted 6.3% of the 16S rRNA sequences and was assigned to unclassified Firmicutes. A GenBank search for closest relatives of RBS10-35 revealed only one 16S rRNA gene sequence (FN687452) with 91% identity found in a thermal aerobic bioreactor for processing activated sludge waste (Hayes et al., 2011). Analysis of the RBS10-35 genome showed that this bacterium had a complete aerobic respiratory chain and CO dehydrogenase, which indicated the possibility of CO oxidation.
The organism represented by the genome RBS10-49 was the second most abundant among Firmicutes (10.7% of the metagenome). This genotype was identified as Hydrogenibacillus schlegelii based on 97.68% AAI with H. schlegelii strain MA48 (Maker et al., 2017). Analysis of the RBS10-49 genome revealed the presence of the genes for the aerobic respiratory chain, membrane-bound uptake [NiFe]hydrogenase of group 1d, and respiratory CO dehydrogenase, which indicates the ability of this bacterium to obtain energy by oxidation of coal gas components, hydrogen and CO. The genome also encoded a complete Calvin cycle, which can enable autotrophic carbon fixation.
It should be noted that among the assembled MAGs, there was no genome belonging to the genus Brevibacillus, although its members accounted for about 15% of the 16S rRNA gene sequences. Brevibacillus were represented by several closely related phylotypes, which made it difficult to assemble long contigs and, accordingly, to obtain their MAGs.
Complete genome of Thermus antranikiani RBS10-92. As a result of metagenome sequencing, the complete genome of the bacterium RBS10-92 was assembled. This genome had 97.79% AAI with Thermus antranikianii DSM 12462 (GCF_000423905), and therefore was assigned to this species. The genome of T. antranikianii RBS10-92 was sequenced with 5030-fold average coverage and was 2424424 bp long. It is the first known complete genome of T. antranikianii. The relative abundance of RBS10-92 in the metagenome was 48.8%, which was in good agreement with the 47.64% share of the corresponding OTU among the 16S rRNA gene reads. Annotation of the RBS10-92 genome identified 2636 potential proteincoding genes. The functions were predicted for only a half of the genes revealed. Two copies of the rRNA operon and 50 transfer RNA (tRNA) genes were identified in the genome. Analysis of the genome of T. antranikianii RBS10-92 showed that this bacterium is probably an aerobic heterotroph capable of hydrolyzing various carbohydrates. This microorganism also has the ability to grow under anaerobic conditions using nitrate as an electron acceptor. These predictions are consistent with the microbiological characteristics of T. antranikianii isolates (Chung et al., 2000).
Complete genome of Hydrogenobacter thermophilus RBS10-74. Three genomes of representatives of the phylum Aquificae were assembled with completeness of more than 97% and contamination of less than 2%. Complete circular genomic sequences were obtained for two of these organisms. RBS10-74, which accounts for 0.17% of the total metagenome, was classified as Hydrogenobacter thermophilus based on 95.7% ANI with H. thermophilus TK-6 (GCA_000010785) isolated from a hot spring in Japan (Arai et al., 2010).
Analysis of the genome of H. thermophiles RBS10-74 predicted that, like cultured strains of H. thermophiles, it is an obligate autotrophic organism capable of oxidizing hydrogen and fixing CO 2 through the reverse tricarboxylic acid cycle. When growing under anaerobic conditions, this bacterium can use nitrate as an electron acceptor. RBS10-74 can also oxidize elemental sulfur or thiosulfate.
Complete genome of MAG RBS10-58, a member of the candidate genus UBA11096. The second complete genome of a member of the phylum Aquificae was assigned to the genus UBA11096 of the family Aquificaceae according to the genomic taxonomy system. To date, no cultured members of this genus have been isolated; for this reason, its definition is based on several draft genomes assembled from metagenomes that have not been analyzed previously.
The RBS10-58 genome was sequenced with 309fold average coverage and assembled into a 1722082bp-long circular chromosome. The relative abundance of this genotype in the metagenome was 2.13%. Annotation of the RBS10-58 genome identified 1848 potential protein-coding genes, as well as one 16S-23S-5S rRNA operon and 42 tRNA genes; gene functions were predicted for a half of the protein-coding genes revealed.
To determine the phylogenetic position of the bacterium RBS10-58, a phylogenetic tree was constructed based on concatenated amino acid sequences of conserved marker genes belonging to members of all genera of the family Aquificaceae determined in the genomic taxonomy system. It was shown that RBS10-58, together with several other MAGs, represented a separate genus-level lineage, along with the genera Aquifex, Hydrogenivirga, Hydrogenobacter, and Thermocrinis, as well as Thermocrinis minervae, which represents a separate genus Thermocrinis_A in the genomic taxonomy (Fig. 2).
Analysis of the RBS10-58 genome revealed a complete set of genes for the reverse tricarboxylic acid cycle, which is used in Aquificae for autotrophic CO 2 fixation (Hügler et al., 2007), as well as genes of the gluconeogenesis pathway. The pentose phosphate pathway was absent in RBS10-58. The genome contained a complete set of genes for oxidative phosphorylation, including NADH dehydrogenase, succinate dehydrogenase, cytochrome bc1 complex, and several cytochrome c oxidases. The presence of membranebound uptake [NiFe]-hydrogenases of the groups 2a and 1d indicates the possibility of using molecular hydrogen as an electron donor during respiration, which is typical for members of the phylum Aquificae. Formate can also serve as energy source, as evidenced by the presence of membrane-bound formate dehydrogenase.
Under anaerobic conditions, nitrate can serve as an electron acceptor, as indicated by the presence of membrane-bound nitrate reductase, which reduces nitrate to nitrite. Further, nitrite can be reduced by cytochrome cd1 nitrite reductase to form nitric oxide(II). Subsequent denitrification steps can be carried out by nitric oxide reductase and nitrous oxide reductase to form molecular nitrogen.
The bacterium RBS10-58 can also use sulfur compounds as an electron donor. Oxidation of hydrogen sulfide can be carried out by sulfide:quinone oxidoreductase and flavocytochrome c:sulfide dehydrogenase. The genome also encoded a Sox-Hdr-Soe variant of the pathway for the oxidation of sulfur compounds to sulfate (Watanabe et al., 2019), including a cluster of sox genes soxYZAXB, sulfite dehydrogenase genes soeABC, and a gene cluster encoding subunits of heterosulfide reductase hdrCBAhyphdrCB.
Thus, the bacterium RBS10-58 is a chemolithoautotroph capable of obtaining energy by oxidation of molecular hydrogen and sulfur compounds through aerobic respiration, while under anaerobic conditions it can perform all stages of denitrification.

DISCUSSION
Despite the fact that one of the first described thermophiles, Thermoplasma acidophilum, was isolated from coal waste samples by Thomas Brock's group in the late 1960s (Darling et al., 1970), the composition of the microbial communities of these ecosystems remains poorly understood. The composition of soil microbial communities in areas of underground coal burning was studied in Pennsylvania (United States) (Tobin-Janzen et al., 2005;Lee et al., 2017). Analysis of microbial communities by 16S rRNA gene sequencing revealed the presence of archaea of the phylum Crenarchaeota, as well as bacteria of the phyla Chloro-  (Sorensen et al., 2019). Microbial communities of heated soil near hot coal gas release sites in Xinjiang (China) were investigated using T-RFLP analysis and 16S rRNA gene clone libraries (Zhang et al., 2013). Predominant groups of microorganisms included members of the phyla Firmicutes, Proteobacteria, Acidobacteria, Bacteroidetes, Planctomycetes, and Actinobacteria. The most abundant group was Firmicutes, mainly represented by the genera Bacillus and Paenibacillus.
In both these cases, the objects of study were "ordinary," organic-rich soils heated due to release of hot gases, which led to predominance of soil groups of microorganisms in the communities. In the present work, the object of study was not soils, but dumps of open-pit coal mining composed of coal-bearing rocks. It is assumed that coal gases can sustain development of specific communities of thermophiles in such ecosystems. The closest analog of the object studied in the present work was the microbial community of heated rocks in the area of underground coal burning in the Altai Mountains (Russia) (Kadnikov et al., 2018). It had a simple composition and included only three dominant phylotypes, all of which represented the phylum Firmicutes. It was the aerobic heterotroph Ca. Carbobacillus altaicus, the anaerobic chemolithoautotroph Brockia lithotrophica, and the aerobic bacterium Hydrogenibacillus schlegelii capable of both using organic compounds and growing autotrophically. All these microorganisms can obtain energy by oxidation of molecular hydrogen, and some of them can also oxidize CO (Kadnikov et al., 2018). This community was also found to contain uncultured lineages of Firmicutes related to Thermaerobacteria.
Another close analog was the burning dumps of a coal mine near the city of Kiselevsk, Kemerovo region (Russia). The microbial community of the surface ground layer heated to 58°C was dominated by members of Ktedonobacteria (phylum Chloroflexi) capable of oxidizing hydrogen and CO, while thermophilic hydrogenotrophic Firmicutes constituted a minor share of the community (Kadnikov et al., 2021). The differences between this community and the object studied in the present work were probably due to differences in temperature and humidity.
In the RBS10 community studied in the present work, Firmicutes accounted for about one third of the community and were represented by thermophilic groups. H. schlegelii and uncultured Thermaerobacteria were among the dominant groups, while Brockia lithotrophica was found in small numbers. However, along with Furmicutes, the dominant groups in the RBS10 community were members of the genus Thermus and the family Aquificaceae, which are character- istic of hydrothermal ecosystems (Counts et al., 2017;Bonch-Osmolovskaya, 2020). Previously, their presence in soils of underground coal burning areas has not been reported. Aquificaceae are capable of autotrophic carbon fixation and energy production through oxidation of hydrogen and sulfur compounds; along with Firmicutes, they represent the autotrophic component of the community sustained by coal gases. In turn, the organic substances produced by autotrophic Firmicutes and Aquificaceae are used as substrates by T. antranikianii, which constitutes about a half of the microbial community.
The thermal ecosystem associated with burning coal dumps that was studied in this work, as well as other similar objects in the Kemerovo region and Altai Mountains, formed no more than a few decades ago; they are young in comparison to geothermal objects. Previously, it was suggested that thermophilic Firmicutes with spores that can spread over long distances (Bonjour et al., 1988;Aullo et al., 2013) may be the first colonizers of such novel thermal ecological niches (Kadnikov et al., 2018). The discovery of members of Thermus and Aquificaceae indicates that the spread of non-spore-forming thermophiles, the source of which may be hot springs and other geothermal objects found in southern Siberia, can also be fast.

FUNDING
This work was partially supported by the Russian Science Foundation (project no. 19-74-00142).

COMPLIANCE WITH ETHICAL STANDARDS
The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

AUTHOR CONTRIBUTION
Sampling and DNA isolation were carried out by V.V. Kadnikov. Sequencing of 16S rRNA genes and metagenomic DNA was carried out by V.V. Kadnikov and A.V. Mardanov. Bioinformatic analysis of sequencing results was carried out by A.V. Beletsky. Data analysis and preparation of the article were carried out by V.V. Kadnikov, O.V. Karnachuk and N.V. Ravin. All authors participated in the discussion of the results.

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