Coal bed methane (CBM) is oftentimes regarded as the fuel that bridges the gap between conventional fossil fuels and sustainable energy sources. Methane is known to have a lesser adverse effect on the environment and human health due to its cleaner combustion and higher calorific value than coal. Both thermogenic and biogenic pathways can produce methane in coalbeds (Stolper et al., 2014). High temperatures and pressures (157–221°C) during coalification lead to the formation of thermogenic methane, whereas biogenic methane is predominantly produced by methanogens via microbial degradation of coal at moderate temperatures (50°C) (Wei et al., 2014).

A sizeable fraction of the world’s natural gas reserves is made up of coalbed methane (CBM), and it has been speculated that up to 20% of all natural gas, which includes CBM, emanates from microbes (Rice and Claypool, 1981). Enhanced CBM, also known as microbially enhanced CBM (MECoM), is the technique of stimulating microbes to produce more methane from currently producing wells. It may be possible to facilitate the microbial communities involved in the dynamic process of methanogenesis that produces CBM to further increase the methane production from the biodegradation of coal over timescales that are relevant to the industry. The lifespans of deficient microbial CBM wells could be increased if microbial CBM production could be enhanced, and possibly new microbial methane could be produced in sites with no history of gas production. In addition to the commercial gain, enhancing microbial methane production could also lower the adverse environmental effects of CBM production as the existing infrastructure would be used instead of drilling new wells when the old wells are depleted. Similar techniques might be utilized to produce methane in gas deficient shales from coal waste or deep or thin, potentially unmineable coal seams (Ritter et al., 2015).

It is the current belief that the abundance and diversity of the microbial community present in the coal seams are crucial to the production of biogenic methane in the coal beds. Research in the past has repeatedly shown that the interaction of several microbe groups involved in bio-methanation, such as fermentative bacteria, syntrophs, and methanogens, is necessary for the microbial mineralization of organic materials to methane (Strąpoć et al., 2011; Wawrik et al., 2012; Park and Liang, 2016; Wang et al., 2016). Numerous studies have thus far investigated the microbial community of coalbeds from various geographical regions, including Japan (Shimizu et al., 2007), the United States (Barnhart et al., 2013), Vietnam (Hoang et al., 2021), Canada (Lawson et al., 2015), and China (Guo et al., 2012). In contrast, not much is known about the microbial population in coalbeds located in geologically differing regions of India.

Therefore, the principal objective of this study is to fill the knowledge gap by providing a thorough analysis of the microbial community and their potential functions in the formation water collected from the Raniganj CBM block located in Durgapur, West Bengal. Further, to understand the role of geochemical factors in shaping the microbial diversity within the reservoir, the relationship between the microbial communities and geochemical factors is evaluated. The study also illustrated significant enhancement in the production of biogenic methane at the lab scale. To the best knowledge of the authors, this study is a prime investigation elucidating the diversity of microbial communities present in this region by 16S rRNA-based metagenomic analyses. Understanding the role of the indigenous microbial population in methanogenesis would help highlight the potential of comparable high temperature CBM reservoirs for biogenic methane production via in situ stimulation in the future.

Numerous studies have examined the microbiological communities of coal/formation water for comprehending the methanogenic potential of coal beds in the USA, Canada, Japan, and Australia (Mochimaru et al., 2007; Shimizu et al., 2007; Green et al., 2008; Klein et al., 2008; Li et al., 2008; Strąpoć et al., 2008; Midgley et al., 2010; Penner et al., 2010). These microbial communities detected in coal beds have undergone culture-independent investigations that have revealed the identities of many bacterial species that are key players in the biodegradation of coal as well as archaea that produce methane via hydrogenotrophic or acetoclastic pathways. In this investigation, the 16S rRNA Amplicon sequencing revealed the relative abundance of the microbes present in the wells, showing that the most abundant phyla included Proteobacteria, Firmicutes, Bacteriodetes, and Actinobacteria. These phyla have been reported to be involved in the biotransformation of coal into methane in earlier studies (Strąpoć et al., 2011; Singh et al., 2012). The presence of Gammaproteobacteria and Alphaproteobacteria in the wells indicated different hydrocarbon-degrading pathways. It is widely recognized that bacteria from the phylum Alphaproteobacteria can break down hydrophobic compounds such as polycyclic aromatic hydrocarbons (Meslé et al., 2013). According to certain reports, Gammaproteobacteria can break down hydrocarbons when nitrate serves as the electron acceptor (Alain et al., 2012). In the past, Bacteroidetes have also been mentioned as pentachlorophenol degraders (Singh et al., 2012). The relative abundance shows the dominance of Euryarchaeota in Well-E. Euryarchaeota majorly consists of acetoclastic and hydrogenotrophic methanogens. Evidence suggesting that syntrophic acetate oxidation is coupled with hydrogenotrophic methanogenesis in the high temperature petroleum reservoirs, such as the Yabase oil field (Japan), has also been documented (Mayumi et al., 2011). Similar to the present study, a dominance of Spirochaetes has also been found by Singh et al. in the Jharia coal reservoir and in the coal bed of the Illinois basin (Strąpoć et al., 2008). As seen in the study by Green et al. (2008), the methanogenic consortium in the Powder River Basin included Spirochaetes in addition to Firmicutes, which predominated the bacterial community in coal bed methane wells there.

The relative abundance of class Gammaproteobacteria, Alphaproteobacteria, and Deltaproteobacteria in the taxonomic analysis of our samples at the class level is of significance, as members of Gammaproteobacteria and Alphaproteobacteria have been studied to be involved in the degradation of various organic compounds, including petroleum hydrocarbons (Zhou et al., 2020; Sudiana et al., 2022). Members of Deltaproteobacteria can anaerobically metabolize a wide variety of normal, iso-, and cyclic alkanes, as well as mono- and polycyclic aromatic hydrocarbons (Davidova et al., 2018). The hydrocarbonoclastic Deltaproteobacteria can take part in syntrophic consortia that ultimately transmit reducing equivalents to methanogenic Archaea without a respiratory electron acceptor. This shows their role in methane generation (Davidova et al., 2018). The presence of class Bacteroidia and Clostridia in all the samples is noteworthy, as previous studies have shown significant enhancement in methane production in consortia rich in Bacteroides and Clostridium (Tukanghan et al., 2021). Also, class Clostridium contains many hydrogen-producing bacteria, and since hydrogen is the limiting factor in the hydrogenotrophic mode of methanation, their presence is highly significant for enhanced CBM production (Palacios et al., 2021; Wang and Yin, 2021). The presence of Methanobacteria in the samples suggests hydrogenotrophic methanogenesis as a significant pathway of methane generation in our study.

There is a noteworthy variation in the genus level abundance within the reservoir. We speculate that this variation is due to the age of the coalbed, the depth of sampling, geological conditions, and the availability of substrates in the coalbed and formation water (Barnhart et al., 2016). Well-A is rich in Smithella and Syntrophobacter, which are syntrophic bacteria. These bacteria have been associated with coal degradation and with providing a substrate for methanogens to produce methane from mesophilic coal reservoirs in earlier research (Strąpoć et al., 2011; Singh et al., 2012). Well-B shows the presence of Hydrogenophaga, a typical hydrogen-utilizing bacteria which is also able to utilize biphenyl. In the study by Wei et al. (2014), the most dominant genus identified in abandoned coal mines was Hydrogenophaga. Another study highlighted that Hydrogenophaga, which was found in an oil-degrading consortium, is capable of metabolizing 4-aminobenzenesulfonate (Beckmann et al., 2011). It has been found in coal samples from the Liulin District (Guo et al., 2012) and in methanogenic enrichment modified with coal (Beckmann et al., 2011). The presence of Phenylobacterium was also abundant, and it also has been detected in the biogenic coal bed methane reserves of the Southern Qinshui Basin, China (Guo et al., 2014). The presence of sulfate-reducing bacteria such as Thermodesulfovibrio, Desulfotomaculum, and Desulfitibacter, which are known to be capable of degrading volatile organic matter (i.e., hydrocarbons and organic acids) in the presence of sulfate as the terminal electron acceptor, is attributed to the presence of sulfate ions and their concentration in the formation water. The prevalence of Methylobacillus, a methanotrophic bacterium, in the formation water of Well-C correlates with the findings of the study by Zhang et al. (2018) that methanogenic and methanotrophic pathways are present in the formation water of the coal reservoir, responsible for the carbon metabolism.

Thauera is amongst the most abundant in Well-D and is known to be a nitrate-reducing, hydrocarbon-degrading bacterium. It has been found in several other basins, including the Jharia basin (Singh et al., 2012), the Surat basin in Australia (Li et al., 2008), as well as the coal beds of Western Canada (Penner et al., 2010). The presence of Coprothermobacter is also notable in this study. Coprothermobacter sp. of the Thermodesulfobiaceae was previously identified as a thermophilic, chemo-organotrophic proteolytic acetogen in methanogenic enrichment culture from a thermophilic digester (55°C) (Ollivier et al., 1985). These proteolytic bacteria can convert yeast extract protein into acetate, hydroxyl, and carbon dioxide, as well as other acids in lower amounts, like isobutyric, isovaleric, and propionic acids. A recent study by Kobayashi et al. (2012) also claimed that Coprothermobacter works as a possible syntrophic acetate oxidizer in high-temperature methanogenic enrichment cultures. Moreover, Singh et al. noted that Coprothermobacter was dominant in their methanogenic enrichment culture.

The prevalence of Methanobacterium and Methanothermobacter in Well-E indicates that hydrogenotrophic pathways constitute the primary means of methane production. The presence of Methanothermobacter, which is also a thermophilic CO₂-reducing methanogen, was found in similar studies, including the coal reservoir of Ruhr Basin, Germany, a biogenic methane-producing source, and the Powder River Basin, USA (Flores et al., 2008). The predominance of the genera Pseudomonas has been reported for hydrocarbon degradation and has been found in the coal seams of Western Canada (Penner et al., 2010). Similarly, in the present study, the presence of Pseudomonas was observed. According to various reports, Pseudomonas is involved in the breakdown of complex organic substances such as alkanes, alkenes, and polycyclic aromatic hydrocarbons into fatty acids like formate, lactate, acetate, ethanol, and CO₂. Furthermore, the production of biosurfactants by the Pseudomonas genus can promote the breakdown of polycyclic aromatic hydrocarbons because these substances enable a decrease in surface and interfacial tensions between coal molecules to enhance solubility (Singh and Tripathi, 2013; Gründger et al., 2015; Colosimo et al., 2016; Mehboob et al., 2016; Davis and Gerlach, 2018). Desulfotomaculum, a methylotrophic sulfate-reducing bacteria found in our samples, has been previously reported in the formation water of the Jharia basin (Singh et al., 2012).

The results of the predictive functional analysis support the microbial diversity analysis, showing the presence of methanogenic functions in the reservoir. This indicates toward the potential for CBM enhancement in the studied wells. The highest methanogenic activity is observed in Well-E. The degradation of hydrocarbon compounds is an important step in the process of methanogenesis (Liu et al., 2022). A high abundance of aromatic degradation in Well-D, Well-E, and Well-A shows the potential of indigenous microbes to convert complex organic compounds into substrates used in the process of methane generation. The analysis reveals the existence of sulfate-reducing metabolism in the reservoir. Studies have also shown the coexistence of methanogens with sulfate-reducing bacteria in various environments (Sela-Adler et al., 2017). The presence of chitin and xylan degraders also represents the abundance of organic compounds degrading microbes, thus reflecting good potential of CBM enhancement in the wells.

The predictive functional analysis is also supported by the flowchart diagram showing the pathway of degradation of coal into methane (Figure 9). The diagram showed various microbes present in the formation water that are known to be involved in various steps of coal degradation. The analysis revealed the abundance of hydrolytic, fermentative, and syntrophic microbes in the wells. These are the major key players in the process of methanogenesis (Nazaries et al., 2013). The analysis also revealed the presence of Methanobacterium and Methanothermobacter as the main methanogens in the wells; thus, hydrogenotrophic is the main route in the process of methane generation. Acinetobacterium is a well-known bacterial genus involved in the degradation of complex organic compounds (Zhang et al., 2021). The high abundance of Acinetobacter in the wells shows the capability of coal degradation in the wells. Genus Fervidobacterium, Gelria, and Pseudomonas are the hydrolytic and fermenting bacteria involved in methane generation (Harirchi et al., 2022). Genus Rhodobacter and Rhodococcus are involved in the degradation of various complex organic and inorganic compounds (Do et al., 2003; Margesin et al., 2005). Species of Aquabacterium are known to be involved in the biodegradation of oils; thus, they have the capability of degrading complex hydrocarbons (Wang et al., 2019). Genus Thermoanaerobacter and Treponema present in the wells are known to be involved in acetogenesis (Harirchi et al., 2022). Various syntrophic bacteria were found in the wells essential for methane generation (Figure 6).

Several biotic and abiotic factors play huge roles in the shaping of microbial communities (Wang et al., 2020; Rajeev et al., 2021). In the case of CBM wells, the physicochemical parameters of the formation water significantly impact the distribution and diversity of microbial communities in the sample (Davis and Gerlach, 2018). Previous studies have shown the impact of geochemical factors like salinity, temperature, and pH significantly impacting the diversity of methanogens in their natural environments (Hoehler et al., 2010; Wen et al., 2017). In the present study, the results of the canonical corresponding analysis identify the effect of geochemical factors on the microbial diversity of the reservoir. The analysis shows a relation between various classes found in the samples. A correlation analysis further corroborates this relation, indicating a significant correlation between Iron, Electrical Conductivity, and TDS with classes Aminicenantia, Coprothermobacteria, Planctomycetacia, Thermodesulfovibriona, and Ignavibacteria. Members of these classes play an important role in the initial step of methanogenesis. They are involved in hydrocarbon degradation, fermentative, and hydrogen-producing metabolism (Liu et al., 2012; Kadnikov et al., 2019; Pavan et al., 2019; Storesund et al., 2020). A study has shown the relation between high bulk conductivities and TDS with enhanced mineral weathering, interlinked with the activities of hydrocarbon-degrading microbes in aquifers contaminated with hydrocarbons (Atekwana et al., 2004). Another study has reported that Fe(III) can be used as an electron acceptor in microbial hydrocarbon degradation, zero-valent iron can donate electrons for improved methanogenesis, and conductive iron oxides may help electron transfers in methanogenic processes. These pieces of evidence show how the geochemical parameters can impact the metabolisms and distribution of hydrocarbon degraders in the ecosystem (Castro et al., 2022). These findings validate the results of the correlation analysis done in the present study.

To further emphasize the role and functions of the indigenous archaea and bacteria present in the CBM reservoir of the Raniganj block, indigenous microbes from the formation water were enriched in methanogen-specific nutrient media, and methane gas produced by the consortium via coal conversion was quantified. The results clearly indicate the real potential of the indigenous microbes present in the formation water for the bioconversion of coal into methane. They also confirm the capability of the nutrient media to support the growth of a selective methanogenic consortium that can utilize bituminous coal present in thermogenic CBM reservoirs as a carbon source and lead to in situ methane enhancement.

As per the authors’ knowledge, this is the first study that provides the microbial analysis of the thermogenic gas-producing CBM reservoir in the Raniganj block and its biogenic methane potential. The microbial diversity found in the formation water samples in this investigation comprised abundant syntrophic bacteria (e.g., Coprothermobacter), methylotrophic bacteria (e.g., Methylobacterium, Methylobacillus), fermentative bacteria (e.g., Microbacterium, Pseudomonas, Acinetobacter) and methanogenic archaea. Proteobacteria, Bacteroidetes, and Firmicutes were the three bacterial phyla that were consistent in all formation water samples, with Proteobacteria being the most prevalent of them. The archaeal domain at the genus level showed the presence of methanogenic archaea, including Methanobacterium and Methanothermobacter, which are two well-known hydrogenotrophic methanogens, indicating that the primary pathway of biogenic methane production in the Raniganj CBM block is hydrogenotrophic. This analysis lays the groundwork for greater comprehension of the functional pathways that predominate and the underlying geomicrobiological diversity, enabling us to examine how it affects methane generation on a lab scale. A deeper understanding of the processes at play could be beneficial in the development of strategies for enhanced methane production.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

ML conceptualized the complete project and designed the experiments. NSi and ML provided the resources to set up and perform the experiments. MC conducted all the experiments, recorded and analyzed the data, and wrote the original draft. NSa conceptualized the idea of metagenomic analysis. MC, NSa, and ML reviewed and edited the manuscript. SS and KS participated in sampling. AM, MI, and SK provided the geological data. All authors contributed to the article and approved the submitted version.

AM, MI, and SK were employed by Essar Oil and Gas Exploration and Production Limited. SS and KS were employed by ONCG Energy Centre.

The authors declare that this study received financial support from Essar Oil and Gas Exploration and Production Limited (EOGEPL). The funder had the following involvement with the study: EOGEPL provided technical support in the manuscript, including well production history, and geographical and geological content of the studied CBM wells; Figures 1 and 2 in the manuscript were also provided by EOGEPL.

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