4.3 Microbial community functional structure
For the 23 water samples collected from CBM wells, 5 samples located in
the runoff areas and 18 samples in the stagnant areas (Fig.5a). We
generated a bacterial community profile for each sample via PCR
amplification of the 16S ribosomal RNA (rRNA) gene targeting regions
V3-V4 using primers 341F and 805R, followed by Illumina sequencing.
1966435 high-quality sequences from 23 samples were obtained (average,
85497; range, 53843-147971 reads per sample). High quality reads were
analyzed with USEARCH, removing chimeric and organelle sequences, to
produce 8264 operational taxonomic units (OTUs).
In all the samples, at the class level, Gammaproteobacteria,
Betaproteobacteria, Deltaproteobacteria and Alphaproteobacteria were the
most abundant bacteria, made up more than 85% of all microorganisms.
Clostridia, Bacteroidia, Flavobacteriia, Bacilli, Methanobacteria,
Actinobacteria were also present at higher relative abundance, but much
lower than the four most abundant classes (Fig.8a).
90 most highly abundant microorganisms related to methanogenesis,
denitrification, sulfate reduction and methane oxidation were selected.
Maximum likelihood phylogenetic was constructed based on 16s V3-V4
sequences, shown in Fig.8b. There were three orders of Methanogens in
study area, including Methanobacteriales, Methanomicrobiales and
Methanosarcinales. Methane oxidizing bacteria included Methylococcales
and Methylophilales. Denitrifying bacteria included Burkholderiales and
Rhodobacterales. Sulfate reduction bacteria included Desulfobacterales
and Desulfovibrionales. Aerobic ammonia oxidation bacteria (AOB)
included Nitrosomonadales. Nitrification bacteria included Nitrospirales
and Nitrosomonadales. The order of anammox bacteria was belonged to
Candidatus Brocadiales.
Consistent with this expectation, the microbial community functional
gene structure was different between runoff area and stagnant area. All
of the nonparametric multivariate statistical tests of dissimilarity
(MRPP, ANOSIM, and Adonis) showed that the overall functional structure
of 3# coal production water’s microbial communities was different
between runoff area and stagnant area (p < 0.005, Table 1).
Cluster analysis of C-N-S function genes showed that the samples from
runoff area and stagnant area were completely clustered into two groups,
which further indicated the functional differences of microorganisms
between the two groups (Fig.11a).
The relative abundance of genes
associated to C, N and S cycling was increased from the runoff area to
stagnant area (Fig. 9). In contrast, only few functional genes, which
mainly belong to functions related to assimilatory nitrate reduction,
were significantly (p < 0.005) decreased in relative abundance
in the stagnant area. Next, it will be discussed in detail.