The mechanism by which O2:CH4 controls microbial community assembly in the process of aerobic methane oxidation coupled to denitrification (AMED) remains largely uncharacterized, which hinders the design of engineering microbiomes for the AMED. In this study, the changes in the bacterial community in fed-batch serum bottle reactors under different O2:CH4 ratios were systematically characterized. The ratios of CH4 consumption to the amount of nitrate removal in the treatment with O2:CH4 = 1.5:1, O2:CH4 = 0.5:1, and O2:CH4 = 0.25:1 were 13.1 ± 3.4, 4.7 ± 1.1, and 5.9 ± 3.0 mol-CH4 mol−1-NO3−, respectively. The α-diversity of the bacterial community increased as O2:CH4 decreased. Significantly different selection patterns were found for the high and low O2:CH4 ratios. The coherence process dominated the selection at high O2:CH4 ratios, while the diversification process played a role when O2:CH4 was low. Differences were also observed in the composition of CH4-derived carbon between treatments with O2:CH4 = 1.5:1 and O2:CH4 = 0.5:1. Compared with the treatments with O2:CH4 = 1.5:1, the concentrations of methanol, formaldehyde, acetate, and ethanol in the treatment with O2:CH4 = 0.5:1 were significantly higher, while the concentration of formate was significantly lower. The heterogeneity of CH4-derived carbon induced by O2:CH4 was likely to be responsible for the differences in the selection patterns. Our findings bridge the gaps between the observations of bacterial community perturbations and ecological community assembly theories, highlighting the potential of the bottom-up design approach to improve the nitrate removal rate of the AME-D.
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