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

Aerobic methane (CH) oxidation coupled to denitrification (AME-D) can not only mitigate CH emission into the atmosphere, but also potentially alleviate nitrogen pollution in surface waters and engineered ecosystems, and it has attracted substantial research interest. O concentration plays a key role in AME-D, yet little is understood about how it impacts microbial interactions. Here, we applied isotopically labeled KNO and CH and metagenomic analyses to investigate the metabolic and microbial link of AME-D at different O levels. Among the four experimental O levels of 21%,10%, 5% and 2.5% and a CH concentration of 8% (i.e., the O/CH ratios of 2.62, 1.26, 0.63 and 0.31), the highest NO-N removal occurred in the AME-D system incubated at the O 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 O-sufficient condition with the O concentration of 21%, while denitrifiers such as Azoarcus, Thauera and Thiobacillus dominated in the O-limited environment with the O concentration of 10%. The competition of denitrifiers and methylotrophs in the AME-D system for CH-derived carbon, such as methanol and acetate, might be influenced by chemotactic responses. More methane-derived carbon flowed into methylotrophs under the O-sufficient condition, while more methane-derived carbon was used for denitrification in the O-limited environment. These findings can aid in evaluating the distribution and contribution of AME-D and in developing strategies for mitigating CH emission and nitrogen pollution in natural and engineered ecosystems.