Variations in methane (CH) and carbon dioxide (CO) emissions in municipal sewer driven by pollution sources are complex and multifaceted. It is important to investigate the role of dissolved organic matter (DOM) components and microbiota to better understand what and how those variations occurred. For this purpose, this study provides a systematic assessment based on short-term in-sewer conditioned cultivations, in conjunction with a field survey in four typical sewers in Shanghai Megacity. The results are as follows: (1) Sediment plays a main role in driving the sewer carbon emission behavior owing to its strong associations with the utilized substrates and predominant microbes that significantly promoted the gas fluxes (genera Bacteroidete_vadinHA17, Candidatus_competibacter, and Methanospirillum). (2) Aquatic DOM in overlying water is an indispensable factor in promoting total carbon emissions, yet the dominant microbes present there inversely correlated with gas fluxes (genera Methanothermobacter and Bacteroides). (3) The total fluxes of both CH and CO enhanced by pavement runoff were limited. Its high COD-CH/CO conversion efficiencies can be ascribed to its dominant anthropogenic humic-like components and the emerged aquatic tyrosine-like components. (4) Domestic sewage can significantly enhance the total fluxes because of its high concentration of bioavailable DOM. However, these substrates, which were more suitable for supporting microbial growth, as well as the substrate competition caused by sulfate reduction and the nitrogen cycle (revealed by the dominant functional microbes genera Acinetobacter, Pseudomonas, Dechloromona, and Candidatus_competibacter and their correlations with indicators), seemed to be responsible for the low COD-CH/CO conversion efficiencies of domestic sewage. (5) A field survey indicated the distinct features of carbon emissions of sewer sewage discharged from different catchments. An extreme hydraulic condition in a sewer in the absence of influent showed unexpectedly high levels of CO, while a small amount of CH emissions.

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