Coal bed methane (CBM) refers to methane generated by either thermogenic or biogenic processes in coal beds (Moore, 2012). This gas trapped in the coal bed is recovered by using production wells that cut coal beds, allowing the migration of gas from the coal beds to the wells, as is illustrated by Figure 1. The stable carbon (δ¹³C) and deuterium (δ D) isotopic signatures and gas composition analyses in numerous basins worldwide have shown important microbial CBM occurrence (Strapoc et al., 2011), generating much interest in CBM technology. CBM generation through bio-stimulation and bio-augmentation have been documented as a potential technology for methane production (Jones et al., 2010). Currently, CBM is supplying 6% of the total natural gas consumed in the United States of America (U.S. Energy Information Agency, 2018).

Figure 1

Analysis of 16S rRNA gene sequences of metagenome samples from coal bed cores or aquifers has enlarged knowledge on the microbial diversity in coal reservoirs throughout world. Coal beds showed a high prokaryotic diversity represented by species of Firmicutes, Spirochetes, Bacteroidetes, and all subgroups of Proteobacteria; as well as methanogens, including Methano-sarcinales, Methanomicrobiales and Methanobacteriales species, which represent all the known methanogenic pathways (Strapoc et al., 2011; Meslé et al., 2013).

Coal methanogenesis is a process involving complex consortia that degrade fossil organic matter present in coal beds. Briefly, hydrolytic and fermentative bacteria hydrolyze complex organic compounds to more simple monomers and oligomers. Then the fermenters, syntrophs and/or acetogens ferment and/or convert these monomers and oligomers mainly to hydrogen (H₂), carbon dioxide (CO₂) and acetate (Wang et al., 2010). Finally, methanogens produce methane by hydrogen-otrophic ( CO₂ reduction), acetoclastic, or methylotrophic methanogenic pathways. Ex situ coal-enrichment cultures studies showed the Methanosarcina, Methanocorpusculum and Methanosaeta species as predominant methanogens and a wide diversity of hydrolytic and fermentative bacteria in the methanogenic consortia (Green et al., 2008; Kruger et al., 2008; Strqpoc et al., 2008; Orem et al., 2010; Penner et al., 2010; Barnhart et al., 2013). Methane production by microbial consortia appears to be influenced by coal micronutrient availability (Ünal et al., 2012), coal rank (Robbins et al., 2016) and coal oxidation state (Gallagher et al., 2013).

Because Colombia has the largest coal reserves in South America, CBM exploitation could contribute significantly to increase methane production in the country. The coal-bearing Guaduas formation of Maastichtian to Paleo-cene age is present in the Bogotá Basin, Eastern Cordillera of Colombia. Stable carbon (δ ¹³C) and deuterium (δ D) isotopic signatures indicate that methane gas in the Guaduas formation has a mixture of thermogenic and biogenic gases (Garcia-Gonzalez, 2010). Since knowledge on coal mine methanogens is essential for the establishment of CBM technologies, the present work aimed to identify the microbial consortia involved in coal biogenic methanogenesis in the "La Ciscuda" coal mine. Using coal-enrichment cultures, 16S rRNA gene metagenome and gas chromatography (GC) analyses, we identified the methanogenic microbial consortia from this coal mine involved in coal degradation and subsequent gas production.

Compared with standard gas profiles (Figure 2a), GC-TCD-FID analysis indicated that, after one-month, cultures at 37 ^oC (Figure 2b) produced a de novo gas mixture composed mainly of carbon dioxide (CO₂), methane (CH₄) and carbon monoxide (CO), while cultures at 60 ^oC (Figure 2c) only produced CO₂. As expected, control experiments (coal powder placed in sterile water) did not produce de novo biogenic gas (Figure 2d). These results indicated that a methanogenic consortium obtained from La Ciscuda coal sample was responsible for biogenic gas generation in the cultures.

Figure 2

A total of fourteen 16S rRNA gene sequences were obtained from bacteria isolates (7) and from bacteria libraries (7) developed from cultures (Table 2). BLAST analyses of the isolate sequences showed identity values between 94.05-99.77% with NCBI database Bacillus sequences; four of these (MH057206.1, MH057208.1, MH057210.1 and MH057211.1) matched Bacillus licheniformis sequences with identity values higher than 98.7%. One sequence (MH057077.1) from a bacteria clone library also matched B. licheniformis species sequences with an identity value of 99.56%. Further, BLAST analysis of other bacteria clone library sequences (MH057075.1, MH057073.1, MH057074.1 and MH057076.1) showed identity values (93.84-95.15%) with NCBI database Gracilibacteraceae sequences. One sequence (MH057075.1) matched Gracilibacter thermo-tolerans sequences, the type species of the genus Gracili-bacter (Lee et al., 2006), with identity values higher than 95.0%. Similarly, BLAST analysis of the sequences from the archaea clone libraries (MH057078.1 and MH197101.1) showed high identity values (>98.7%) with NCBI database Methanothermobacter thermautotrophicus (NR074260) and Methanothermobacter wolfeii (LT996592) sequences. In summary, the bacterial isolates and clone libraries obtained from coal-enriched cultures indicated that a minimal methanogenic consortium was formed by specie from two bacteria genera (Bacillus and Gracilibacter) and one archaea genus (Methanothermobacter) species. A UPGMA tree based on all 16S rRNA gene sequences (including type species sequences from the NCBI database) defined the same three main prokaryotic groups (Figure 3).

Figure 3

This work constitutes the first effort to identify the composition of microbial consortia involved in methane production in a coal mine from the Bogotá Basin in Colombia. Our results supported de novo biogenic nature of methane gas produced at the La Ciscuda coal mine as previously indicated using δ¹³C and δ D isotopic signatures (Garcia-Gonzalez 2010). Further, the study identified a minimal methanogenic consortium that inhabited this coal mine, formed by the bacteria species Bacillus licheniformis and Gracilibacter sp., possibly, G. thermo-tolerans (Lee et al., 2006), and the methanogens Methan-othermobacter thermautotrophicus and M. wolfeii (Wasserfallen et al., 2000). Excepting Gracilibacter, these microbial genera have been previously identified from coal-enrichment cultures experiments (Table 3).

Although methanogens from coal-enrichment cultures were not isolated, they did grow as a methanogenic consortium (Figure 2b). RCM is a very rich medium that provided multiple carbon and nitrogen sources and growth conditions required for methanogenesis such as osmotic balance and low redox potentials. Under these growth conditions, hydrolytic and fermentative bacteria (i.e., B. licheniformis) can enzymatically hydrolyze starch to saccharides such as dextrose (Komolprasert and Ofoli 1991), as well as, can ferment this dextrose through mixed-acid fermentation pathways to organic acids and alcohols (Shariati et al., 1995). Bacillus species, including B. licheniformis, can also solubilize or biodegrade coal lignite into aromatic and aliphatic compounds (Polman et al., 1994). Moreover, G. thermotolerans grows well in medium with similar carbon and nitrogen sources existing in RCM and their growth on media containing glucose produced acetate, lactate, and ethanol as main fermentation end products (Lee et al., 2006). It also has been reported (Sakai et al., 2010) that G. thermotolerans formed a methanogenic consortium with Methanocella arvoryzae, a hydrogenotrophic methanogen isolated from rice field soil. These authors also indicated that G. thermotolerans fermentation products (acetate, H₂ and CO₂) were required by Methanocella arvoryzae for methane production. We believe that in our study Bacillus and Gracilibacter species, especially the latter, provided substrates (H₂ and CO₂) to Methanothermobacter species (M. thermautotrophicus and M. wolfeii) for methane production. Gracilibacter thermotolerans cannot grow above 58 ^oC (Lee et al., 2006), explaining why our methanogenic consortium produced methane at 37 ^oC, but not at 60 ^oC.

Table 3

†, The genera were not denned.

Based on our results from coal-enrichment cultures we can speculate on how biogenic methane could be generated in the La Ciscuda coal mine. As indicated in Table 1, this coal mine is located at 200 m depth from surface, where anoxic and saline conditions prevail. Under these conditions the coal mine yields 636 cm³ of methane gas per kg of coal. Parkes et al. (2011) showed that prokaryotes stimulate mineral H₂ formation for the deep biosphere and for subsequent microbial activity, including CO₂ and CH₄ production. We believe that infiltration of meteoric waters into coal mines can stimulate microbial degradation of coal lignite to aromatic and other compounds (Chang et al., 2005), producing H₂ and CO₂ as final products that are, in this case, the substrates for methanogenesis by Methanothermobacter species. Meth-anothermobacter thermautotrophicus, formerly Methano-bacterium thermoautotrophicum (Smith et al., 1997), and M. wolfei, are representative subsurface methanogen species (Wasserfallen et al., 2000) that previously have been described to produce methane by reduction of CO₂ in coal mine methanogenic environments (Ward et al., 2004; Penner et al., 2010).

In this work, we identified bacteria (Bacillus and Gracilibacter) and archaea (Methanothermobacter) species forming a minimal methanogenic consortium from La Ciscuda coal mine as a first step for evaluation of CBM generation technologies. Based on this consortium we suggested that methane was produced by hydrogenotrophic or CO₂ reduction pathways.