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.