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

Abstract

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 N2O 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 N2O production. Meanwhile, we demonstrated that haoA, cytL and hcp contributed to ammonia detoxification and cytL was involved in the conversion of NH2OH to N2O. A mutant strain with increased ammonium-utilizing ability and decreased N2O 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. IMPORTANCEM. buryatense 5GB1C is becoming a methanotrophic model for both fundamental research and practical applications. Here, the genetic determinants of ammonia toxicity were identified and a porin gene that facilitated ammonium uptake was described for the first time, providing new understanding regarding the mechanism of nitrification in methanotrophs. Moreover, this study dramatically reduced ammonia toxicity and N2O production in this strain and enabled the use of ammonium as nitrogen source in fermentation, improving the sustainability of this strain in practical applications.