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.