Haili Zhang’s research while affiliated with Nanjing Agricultural University and other places

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.

Methanotrophs of the genus Methylocystis are frequently found in rice paddies. Although more than ten facultative methanotrophs have been reported since 2005, none of these strains was isolated from paddy soil. Here, a facultative methane-oxidizing bacterium, Methylocystis iwaonis SD4, was isolated and characterized from rhizosphere samples of rice plants in Nanjing, China. This strain grew well on methane or methanol but was able to grow slowly using acetate or ethanol. Moreover, strain SD4 showed sustained growth at low concentrations of methane (100 and 500 ppmv). M. iwaonis SD4 could utilize diverse nitrogen sources, including nitrate, urea, ammonium as well as dinitrogen. Strain SD4 possessed genes encoding both the particulate methane monooxygenase and the soluble methane monooxygenase. Simple and rapid genetic manipulation methods were established for this strain, enabling vector transformation and unmarked genetic manipulation. Fast growth rate and efficient genetic tools make M. iwaonis SD4 an ideal model to study facultative methanotrophs, and the ability to grow on low concentration of methane implies its potential in methane removal.