Metabolic network modelling and metabolomics to analyse nitrous oxide production in biological nitrogen removal processes

Abstract

Biological nitrogen removal processes used to treat wastewater are known to emit nitrous oxide (N2O), a potent atmospheric greenhouse gas and ozone depletion substance. The specific triggering mechanisms behind the production, accumulation and emission of N2O have been difficult to elucidate due to different production pathways operating in parallel during microbial metabolism. The primary aim of this research was to establish a relationship between the activity of microbial N2O production metabolism and operational parameters of biological nitrogen removal processes in wastewater treatment. Metabolic network models and metabolomic analysis were developed and applied to quantitatively characterize the microbial respiration pathways of nitrifying and denitrifying cultures producing N2O in laboratory settings. Two simulation studies were performed to investigate N2O production in nitrifying systems, one for pure and another for mixed microbial cultures. The pure cultures (Nitrosomonas europaea) simulation study results showed that N2O is produced due to electron flow imbalances in nitrifying cells; and that electron carriers play a key role by distributing electron equivalents to N2O and NO formation reactions. The mixed culture simulation study results revealed two key aspects of N2O formation in nitrifying microbial communities: (i) microbes can dissipate NO (an N2O precursor molecule) and thereby lower N2O emissions; and (ii) the structure, i.e. species richness and abundance, of the microbial community influences the amount of N2O produced and emitted. N2O formation during denitrification was investigated in laboratory-scale experiments using denitrifying batch cultures and identifying the biomass metabolite profile, and then modelling the nitrogen respiration and carbon assimilation pathways of denitrifier cells. The results highlight that the carbon to nitrogen ratio in the growth medium significantly influences the metabolic state of the cells that determine the amount of N2O emitted during denitrification. This research concludes that operational conditions that promote an imbalance between a cell’s electron donor and electron acceptor potential lead to N2O accumulation. Specifically, in nitrification processes, an excess of electron donor potential leads to N2O accumulation. Conversely, for denitrification processes, insufficient electron donor potential results on N2O accumulation. This research demonstrates procedures to apply systems biology and metabolomics to bioprocess engineering of wastewater treatment and creates a platform for further investigation in these disciplines.