Unraveling and overcoming the ammonia toxicity in methanotrophs for sustainable biomanufacturing and methane removal

Replacement of nitrate with ammonium at large scale cultivation of methanotrophs can improve the economic feasibility of these bacteria in methane-based biomanufacturing and methane removal. However, ammonia toxicity and N ₂ O emission impede this option. The mechanism of ammonia oxidation in methanotrophs remains elusive, limiting the effort to detoxify ammonia via genetic engineering. Using an industrially promising methanotroph as a model, we identified a porin PorA that facilitated ammonium uptake. Inactivation of PorA remarkably relieved ammonia toxicity and reduced N ₂ O production. Meanwhile, we demonstrated that haoA , cytL and hcp contributed to ammonia detoxification and cytL was involved in the conversion of NH ₂ OH to N ₂ O. A mutant strain with increased ammonium-utilizing ability and decreased N ₂ O emission was constructed. High growth rate and cell biomass were achieved in fed-batch fermentation with this strain using ammonium. These results deepen our understanding of ammonia oxidation in methanotrophs and promote their applications in biomanufacturing and methane removal.