Stable-isotopic and metagenomic analyses reveal metabolic and microbial link of aerobic methane oxidation coupled to denitrification at different O2 levels.

Aerobic methane (CH4) oxidation coupled to denitrification (AME-D) can not only mitigate CH4 emission into the atmosphere, but also potentially alleviate nitrogen pollution in surface waters and engineered ecosystems, and it has attracted substantial research interest. O2 concentration plays a key role in AME-D, yet little is understood about how it impacts microbial interactions. Here, we applied isotopically labeled K15NO3 and 13CH4 and metagenomic analyses to investigate the metabolic and microbial link of AME-D at different O2 levels. Among the four experimental O2 levels of 21%,10%, 5% and 2.5% and a CH4 concentration of 8% (i.e., the O2/CH4 ratios of 2.62, 1.26, 0.63 and 0.31), the highest NO3--N removal occurred in the AME-D system incubated at the O2 concentration of 10%. Methanol and acetate may serve as the trophic linkage between aerobic methanotrophs and denitrifers in the AME-D systems. Methylotrophs including Methylophilus, Methylovorus, Methyloversatilis and Methylotenera were abundant under the O2-sufficient condition with the O2 concentration of 21%, while denitrifiers such as Azoarcus, Thauera and Thiobacillus dominated in the O2-limited environment with the O2 concentration of 10%. The competition of denitrifiers and methylotrophs in the AME-D system for CH4-derived carbon, such as methanol and acetate, might be influenced by chemotactic responses. More methane-derived carbon flowed into methylotrophs under the O2-sufficient condition, while more methane-derived carbon was used for denitrification in the O2-limited environment. These findings can aid in evaluating the distribution and contribution of AME-D and in developing strategies for mitigating CH4 emission and nitrogen pollution in natural and engineered ecosystems.