Nitrate input simplified community composition
PCoA well substantiated the community dissimilarities between two groups (Figure 1A), where the nitrate group fell into distinct subgroups per sampling time but control samples showed no significant difference over time. For nitrate group, a longer incubation led to a larger dissimilarity between subgroups. This suggested that the notable community succession was in response to elevated nitrate but not to the one-month incubation. We performed one-way ANOSIM to further compare the pairwise dissimilarities between subgroups with respect to dissimilarity significance (P ) and distribution (R2). For convenience, we used “C” to represent control sediments, and “N” to represent nitrate-amended sediments. For example, “4-C” refered to the control sediment on Day 4. Most subgroups were statistically different from each other except 4-C vs. 16-C and 8-C vs. 16-C (Figure 1B). Any pairwise dissimilarity between C-N and N-N subgroups was significant (P < 0.001), as PCoA suggested. In contrast, most control subgroups had comparable within- and between-subgroup dissimilarities (Figure 1C).
The relative abundance of categorized OTUs at the phylum or class level suggested a progressive dominance of beta-Proteobacteria in nitrate-amemded sediments, while classes Anaerolineae, delta-Proteobacteria and Euryarchaeota dominated the control (Figure 1D). The enrichment of beta-proteobacteria (from 1.79% to 61.81%, ~35 folds) accounted for the dissimilarities among nitrate subgroups. Only ~1/3 of total 4990 OTUs could be assigned to a specific genus (Figure 1E). Among those, syntrophic bacteria (e.g. Smithella , Syntrophphorhabdus ,Syntrophphus and Syntrophobater ) and methanogenic archaea (e.g. Methanothrix , Methanoregula , Methanolinea ,Methanobacterium and Methanomassiliicoccus ) dominated control and early nitrate communities. Compositions of control communities were rather time-steady so we only screened genera significantly changing over time within the nitrate group by paired T-test. We found that most genera were gradually outcompeted alongside the rise of genus Thiobacillus and Luteimonas (Figure 1F), and the core microbiomes of late nitrate subgroups (i.e., 16N and 32N) greatly differed in taxonomy and abundance from control and the early nitrate subgroups.