4.3.1 C cycling
Changes in hydrodynamic conditions from runoff area to stagnant area significantly impacted a number of microbial functional groups important for C decomposition. 25 genes associated with decomposition of labile or recalcitrant C were detected. Among them, 9 genes exhibited higher relative abundance in stagnant area than runoff area samples (p < 0.05, Fig. 9a), including cellulose 1,4-beta-cellobiosidase (P = 2.64E-07), licheninase (P = 5.03E-06), alpha-mannosidase (P = 8.89E-05), 6-phospho-beta-glucosidase (P = 0.000490162), alpha-glucuronidase (P = 0.000582225), endo-1,4-beta-galactosidase (P = 0.000920978), endoglucanase (P = 0.002333825), chitinase (P = 0.002343304), beta-mannosidase (P = 0.018552947). Increases of the genes involved in recalcitrant C decomposition suggested the possible degradation of old recalcitrant C in stagnant area.
This study held the idea that these recalcitrant C came from the plant debris on the ground. With the atmospheric precipitation moving from the eastern outcrop to the western stagnant environment in the study area, the temperature and pressure in the stagnant area were increased from the runoff area, which is more favorable for the degradation of recalcitrant C. The relative abundance of recalcitrant C decomposition genes was more than that of methanogenesis genes. In the case that the high rank coal in the study area was difficult to be degraded, the degradation of recalcitrant C from the surface would be an important substrate for microorganisms, and the monosaccharide produced could further provide substrates for other carbohydrate metabolism. As genes associated with mannose metabolism, carbohydrate hydrolases, lactose and galactose uptake and utilization, L-fructose utilization, xylose utilization and chitin utilization were all increased in the stagnant area (Supplementary Fig. 1). Overall, as these functional genes directly participate in C degradation, their higher abundance could enhance C decomposition and enhance methanogenesis and other anaerobic heterotrophic microorganisms such as nitrate reducing bacteria and sulfate reducing bacteria.
4.3.2 Methane metabolism
For all the methanogenesis genes resulted in Picrust2 analysis, the relative abundance of methanogenesis genes were increased from runoff area to stagnant area (Fig.9d), including mcrA, mcrB, mcrG, the key enzyme in all types of methanogenesis (p < 0.00005). FwdA-FwdH (p < 0.00001), mtd (p < 0.00002), mer (p < 0.000002), mtrA-mtrH (p < 0.00002), MvhADG–HdrABC (p < 0.05) in hydrogenotrophic methanogenesis showed more relative abundance in stagnant area too. Codh–Acs (p < 0.00007) in aceticlastic methanogenesis and MtaA-MtaC (p < 0.00008) in methylotrophic methanogenesis were also more abundant in stagnant area. What’s more, fbiC (p < 0.03), cofH, cofG, cofC, cofD and cofE (p <0.0006) in F420 biosynthesis, mfnB, mfnD, mfnE, mfnF (p < 0.02) in methanofuran biosynthesis and comC, comE, comD (p <0.00002) in Coenzyme M biosynthesis were increased in the stagnant area. Suggesting an increase of all types of methanogenesis in the western stagnant area.
In both the runoff area and stagnant area samples there was a greater relative abundance of pmoB (a gene encoding particulate methane monooxygenase subunit B) than any methanogenesis genes, suggesting that of the biogenic methane could be oxidized aerobically (Treude et al., 2014). Indeed, the exploitation of CBM wells, including drilling and hydraulic fracturing, increased the opening degree and oxygen content of the coal seam, resulting in a high relative abundance of methane oxidizing bacteria in the samples. These aerobic bacteria grew in the wellbore or drainage outlet, consuming the dissolved methane of the produced water, which explained why there was methanogenesis microorganisms in the study area, but biogenic methane had not been effectively preserved. The aerobic and anaerobic oxidation of methane might have an important consumption mechanism of biogenic gas in the process of CBM generation history, although anaerobic methane oxidizing bacteria (ANME and M. oxyfera) were not detected in the 16S sequencing of our recently collected water samples.
Because the isotope characteristics of both the methane gas samples and the water-soluble methane samples showed complete thermogenic (Fig.7b), there were two possibilities that biogenic gas does not exist: (1) In most of the historical methane generation process, due to the unsuitable temperature and pressure conditions, methanogens did not exist or exist in the low abundance. The methanogens began to grow only when the outcrop of uplifted coal seams received meteoric water supply of organic matter and minerals. (2) In the gas generation stage of coalbed methane, the existence of methanogens was accompanied by aerobic and anaerobic oxidation methylotrophic bacteria, and biogenic methane was consumed by these microorganisms (Evans et al., 2019). In addition, because methanogens and sulfate reducing bacteria shared the same substrate H2, this competition mechanism further compressed the living space of methanogens, resulting in that biogenic methane is not rich in the study area.