4.3.3 N cycling
Nitrogen, including nitrate, nitrite and ammonium, is an important
element for all microorganisms and is required for the biosynthesis of
key cellular components such as amino acid and nucleotide (Wenk et al.,
2014; Kuypers et al., 2018; Tian et al., 2016; Shen et al., 2015; Hu et
al., 2014). 25 genes associated with N cycling were detected, including
nitrogen fixation, anaerobic oxidation of ammonia, nitrification,
anammox, dissimilatory nitrate reduction, assimilatory nitrate reduction
and denitrification. There were also different genes involved in N
cycling between runoff area and stagnant area (Fig. 9b), Most (56%) of
the Picrust2-detected functional genes involved in N cycling were
increased from runoff area to stagnant area (p < 0.05; Fig.
9b), consistent with the previous prediction that weakened hydrodynamic
conditions enhances nutrient cycling.
For example, the abundance of
N2-fixing genes
was higher in response to hydrodynamic conditions (p < 0.05;
Fig. 9b), the nifD (p = 0.00007),
nifH (p = 0.0001), nifK (p = 0.0007) had higher abundance in the
stagnant samples. In addition, weakened hydrodynamic strength from
runoff area to stagnant area seemed to decrease dissimilatory nitrate
reduction and increase nitrification, denitrification processes, as
indicated by decreased narG (p = 0.017), narH (p = 0.02), narI (p =
0.01) gene, and increased nirK (p = 0.008), nosZ (p = 0.00002), norC (p
= 0.001), napA (p = 0.0008), napB (p = 0.0005), nrfA (p = 0.000004), and
amoB (p = 0.04) gene from runoff area to stagnant area (Fig. 9b). The
increase in nitrification gene amoB and N2-fixing gene
nifDH would lead to higher nitrite and nitrate concentrations, which was
also supported by the greater abundance of genes for various reductive
processes which used nitrate as an electron acceptor, such as nirK,
nosZ, norC for denitrification, nrfA for dissimilatory nitrate reduction
to ammonium (narG, narH, narI, napA, napB shared by denitrification and
dissimilatory nitrate reduction) and nirA for assimilatory nitrate
reduction (Fig. 9b). Significant different N-cycle related genes between
the two groups were labeled on the N pathway map, as shown in Fig.11b.
Almost all the N cycling genes were more abundant in the stagnant area,
except dissimilatory nitrate reduction, were more abundant in the runoff
area. This was possible, as NO3-concentration decreased from runoff area to stagnant area, suggesting
the consumption of nitrate in runoff area by the first step of
dissimilatory nitrate reduction. Reduction products such as nitrite
would continue to converge into the stagnant area, and then be reduced
by denitrification microorganisms, resulting in the increase of
denitrification genes, such as nirK, norC and nosZ. The increased
relative abundance of N cycling genes (N fixation and nitrification) and
other nutrient-cycling genes could increase nutrient (especially N)
availability in stagnant coal reservoirs, which is important for
ecosystem C dynamics because N is a limiting factor for microorganism
growth in most groundwater ecosystems. The enhanced N uptake could in
turn affect C metabolism such as cellulose, mannose metabolism,
carbohydrate metabolism, which increased in response to hydrodynamic
conditions.
As the nitrogen and oxygen isotopes of nitrate shows that nitrate may
come from surface soil and fertilizer (Fig. 7c), which seep into
stagnant area with meteoric water, some nitrate may also come from the
nitrogen fixation and of in-situ microorganisms in coal seam itself.