The enriched strains of the three groups of coal samples in the anaerobic incubation experiments were all from the sewage collected from the corresponding deep coal seams, which indicated that the differences in the in situ microorganisms of the coal seams resulted in different metabolic pathways expressed by the functional microorganisms in the experimental incubation process, which corresponded to the differences in the metabolites produced by the microorganisms during the whole incubation process. Methanogenic bacteria play an important role in the formation of biomethane from bituminous coals, and the dominant archaeon detected in this experiment was Methanosarcina, which is an acetic acid-nutrient methanogenic archaeon that cleaves acetic acid to produce methyl and carboxyl groups, and then oxidises the carboxyl groups to produce an electron donor, H2, which is used to reduce the methyl groups (FANG Xiaoyu 2015). This chemical process leads to the formation of formylmethane furans (CHO-MFR), which is facilitated in the presence of tungsten-containing formylmethane furan dehydrogenase (Fwd) (Wang et al. 2019). This could also explain the differences in methane production between groups in genome sequencing due to differences in the abundance of methanogenic bacteria contained in each group (Akimbekov et al. 2024).
At the same time, through the investigation of the original coal quality parameters of the three groups of coal seams used and the content of trapped gas, it can be seen that different coal seams in the same mine have different physicochemical properties, and the five parameters with greater relevance, Mad, Aad, pH, Vad, and Vi, have an impact on the key archaeal species of the entire reaction process. A study of pH was carried out and it was concluded that higher pH would inhibit the progress of gas production and that the ideal conditions for the reaction would be a neutral environment (Xian-bo et al. 2011; Li et al. 2022). And the optimal reaction condition is to be carried out in a neutral environment, and Desulfosporosinus (Desulfurising Bacillus spp.) has been detected in all three coal seams, and the proportion of this bacterial genus is the highest in Group 1#, reaching 14.50%, and the three groups of coal seams all contain the element sulfur, and during the reaction process, the culture environment will be acidic, and this bacterium mainly works by removing organic sulfur from the coal (Xiangzhao 2024). All three coal seams contain elemental sulfur, and the culture environment tends to be acidic during the reaction process, and this bacterium mainly removes organic sulfur from the coal. For the specular composition score, the three groups of coal seams evolved under the same geological conditions, and the coal quality is basically the same, but the specular group responds to the adsorption effect of different coal seams on CBM, and as the adsorption capacity of coal on CBM is enhanced with the higher specular composition score, the original CBM storage capacity increases, which corresponds to the difference in the original gas content of the coal seams in the original data (YONGyong, et al. 2022)
In addition, during anaerobic incubation, organic matter in the culture broth and coal is broken down into small molecules by extracellular enzymes, and the production of glycolysis / glycolysis metabolic pathway provides methanogenic bacteria with more available substrates, thus increasing the efficiency of gas production (Hua et al. 2019). For glycolysis, which is mainly divided into two phases, activation and energy release, the key enzymes in this process include glucokinase (C00845), 6-phosphofructokinase 1 (C00850), and pyruvate kinase (C00873), and the activity of these key intermediates directly affects the metabolic pathway's rate and direction (Guo et al. 2023). The expression of C00845 in group 1# was 6.74-fold and 6.85-fold higher than that of group 6# and group 9#, C00850 was 1.37-fold and 1.42-fold higher than that of group 6# and group 9#, and C00873 was 1.02-fold and 1.37-fold higher than that of group 6# and group 9# in group 1#. Thus group 1# had higher abundance about glycolytic enzyme genes than group 6# and group 9#. And during fatty acid degradation, β-oxidation may be an important pathway for organic matter decomposition in coal seams, which mediates the conversion of organic matter from long-chain fatty acids to short-chain fatty acids and oxidation to acetic acid (Devignes et al. 2022). The reconstructed metabolic pathway reveals that TCA-cycle acts as a bridge between fatty acid degradation and glycolysis/glycolysis, and with the key intermediates Malate (C00149) and pyruvate kinase (C00873), TCA-cycle converts the long-chain fatty acids generated from fatty acid degradation to acidification to sugars for glycolysis and metabolism (Lin et al. 2008). Metabolism, which provides energy for microbial activities and further decomposes and utilises the undegradable carbon long chains in the coal. Throughout microbial metabolism. By supplying molecular building blocks for protein synthesis, amino acid metabolism complements the bioenergetic pathways driven by glucose and fatty acids. This interplay ensures a comprehensive and efficient utilization of available resources for cellular processes. Protein synthesis heavily relies on amino acids, which are obtained through various metabolic pathways. In doing so, amino acid metabolism synergizes with glucose and fatty acid metabolism, enabling the synthesis of proteins necessary for cellular structure, function, and regulation. This integrated approach ensures the harmonious functioning of bioenergetic and biosynthetic processes within the cell. This is important because one of the primary roles of amino acid metabolism is to support protein synthesis. (Ruan et al. 2022). Amino acids are used not only in protein synthesis but also in other cellular processes. They can be directly incorporated into macromolecules such as nucleotides or used to fuel other metabolic pathways after enzymatic breakdown. Amino acids play a crucial role in biosynthesis by serving as key substances that provide metabolic intermediates. These intermediates serve as links between various important molecules, such as lipids, nucleotides, and nucleotide coenzymes like NAD and NADP. Through their involvement in metabolic pathways, amino acids contribute to the synthesis of these essential molecules, which are vital for cellular processes and energy metabolism. The metabolic intermediates derived from amino acids act as building blocks and precursors, enabling the efficient production of lipids, nucleotides, and nucleotide coenzymes. This interconnected network of metabolic pathways ensures the availability of these important molecules for cellular functions and maintains the overall metabolic balance. In addition to ribose-5-phosphate produced by the pentose phosphate pathway, nucleotide biosynthesis relies on feeder pathways that provide carbon or nitrogen precursors. These pathways include the amino acids glutamine, aspartic acid, serine, and glycine. (Morita and Ikeda 2022). In addition, tryptophan is used for the generation of quinolinic acid esters, which are the slave precursors of NAD and its phosphate derivative NADP pyridine ring. Glutamate, glycine, and cysteine are essential for maintaining cellular redox balance through the synthesis of glutathione (GSH), which is the most potent non-enzymatic cellular antioxidant. Additionally, single-carbon metabolism driven by serine via the folate cycle contributes to the production of NADPH. This conversion of glutathione to its reduced form is essential for redox regulation under hypoxic conditions (Mentch and Locasale 2016). In specific circumstances, amino acids can function as substrates independent of adrenal hormones, driving the activity of the tricarboxylic acid (TCA) cycle. This serves two primary purposes: maintaining mitochondrial ATP production and supporting the synthesis of lipids. The most studied is the bloodless metabolism of glutamine, which produces α-ketoglutarate (αKG) and subsequently fuels the TCA cycle. Oxidation of (branched-chain) amino acids in the TCA cycle is also important for the production of acetyl coenzyme A to support lipid biosynthesis (Mercatali et al. 2016). Indeed, DNA and histone methylation, which are essential epigenetic modifications, are regulated by metabolites associated with the methionine cycle/one-carbon metabolism. Methionine, serine, and glycine are key players in this regulatory process. The process of promoting gene transcription through histone acetylation requires acetyl coenzyme A, which can be derived from (iso)leucine, valine or glutamine. Absolutely, the catabolism of specific amino acids, including glutamate and arginine, generates metabolic precursors that are crucial for collagen synthesis (proline) and cell proliferation (polyamines). (Chen et al. 2020). The presence of these key products throughout the metabolic pathway in all three experiments suggests that in the amino acid metabolic pathway, the energy produced by glycolysis is utilised to feed back to the other modules, and that amino acid metabolism is involved in the synthesis of proteins in the cell in the process, which aids in the proliferation of methanogenic bacteria. For methane metabolism, it is mainly methanogenic bacteria that play a role in this part, and the methanogenic pathways can be subdivided into four categories, M00357 (decarboxylation of acetic acid), M00567 (reduction of CO2), M00563(methanogenesis of methylamine/dimethylamine/trimethylamine), and M00356 (methanol methanogenesis) (Wang et al. 2022). The methanogenic pathway can be divided into four categories. The methanogenic enzymes were acetate kinase (ack, EC: 2.7.2.1), phosphoacetyltransferase (pta, EC: 2.3.1.8), acetyl coenzyme a synthase (acs, EC: 6.2.1.1) and acetyl coenzyme a synthase (ACDS), respectively. Acetic acid can undergo two processes: the conversion of acetic acid to acetyl coenzyme a and the decarboxylation of acetyl coenzyme a to produce the methane precursor (5-methyl- thmpt) and finally to methane in the presence of ACDS. Among them, the abundance of genes of the ack-pta system, which has a lower affinity for acetic acid, was higher than that of the acs system, suggesting that the main pathway for the conversion of acetic acid to acetyl coenzyme a in the reaction is involved in the ack-pta system. In the CO2 reduction pathway, CO2 is reduced by different enzymes to a series of intermediates such as methyl, methylene, and formyl groups, and finally the methyl group is transferred to CoM to form methyl CoM, which is then reduced to CH4 (Wang et al. 2022). There was little difference in gene abundance among the three coal seams in the acetic acid decarboxylation pathway, but in the CO2 reduction pathway, the gene abundance of group 1# was 152.91% and 184.39% of that of group 6# and group 9#, respectively, suggesting that the enzyme activity of the CO2 reduction pathway was higher in group 1#, thus promoting the formation of biogenic methane, and that the dominant genus Methanosarcina played an important Role.