Characterization and application of methane cometabolism for methanol production in ammonia oxidizing bacteria

The requirement of external carbon for conventional biological nitrogen removal (BNR) process has stimulated research on more resource-efficient treatment technologies. Bioconversion of methane to methanol using ammonia oxidizing bacteria (AOB), a process relies on the activity of ammonia monooxygenase expressed by AOB, offers an alternative and sustainable carbon source as all the components (ammonia, AOB, and methane) are often available within the water resource recovery facilities. Though the concept of biomethanol production using AOB has been proposed decades ago, previous studies have been primarily focused on the kinetics of methane oxidation in axenic batch cultures. In this study, the overall objectives were to (1) develop a platform for biomethanol production using AOB in a continuous process and (2) investigate the mechanism of methane cometabolism in AOB under chemostat conditions. Pure culture AOB (Nitrosmonas europaea and Nitrosomonas eutropha) and mixed culture nitrifying consortia were cultivated in bioreactors and continuously exposed to methane to study the responses of nitrifying bacteria at reactor level (nitrification performance) and molecular level (genes and proteins expression). The mixed culture experiments successfully demonstrated continuous biomethanol production with two types of bioreactor configurations. Acclimation of the mixed culture nitrifying consortia to co-exposure and co-oxidation of ammonia and methane were shown at multiple levels (reactor performance, biomass concentrations, bacterial activities, and genes expression). Furthermore, the accumulation of nitrite and a more substantive impact on nitrite oxidizing bacteria growth (compared to AOB) indicated the possibility for partial nitrification (ammonia-N to nitrite-N) coupled with biomethanol production, thereby opening the prospect for even more resource-efficient concurrent carbon and nitrogen management and removal. On the other hand, experiments with axenic AOB culture showed that methane exposure negatively and reversibly affected ammonia oxidation and AOB growth. Proteomics and gene expression data from pure culture experiments suggested that AOB modulating their catabolic (energy synthesis) and anabolic (biomass synthesis) pathways in response to methane exposure. Moreover, N. eutropha experiments demonstrated the potential adaptation of AOB to methane supplementation. Comparative transcriptomic analysis of methane cometabolism in N. europaea and N. eutropha showed that AOB upregulated genes involved in ATP production while downregulated genes involved in NADH production, organic molecules synthesis and cell division. In conclusion, this work provides insights for the process optimization for integrated AOB-mediated biomethanol production and (full- or partial-) nitrification processes as well as structured process modeling to facilitate such optimization and process scale-up and adoption. The whole transcriptomic analysis delineates a comprehensive and detailed view regarding methane cometabolism in AOB