Combined experimental and computational analyses of microbial ecology in engineered systems functioning as $N_2O$ source or sink

Abstract

Whether eco- or engineered systems act as sources or sinks of $N_2O$ is determined by the net of $N_2O$ production and reduction rate of the systems. The biological $N_2O$ reduction is mediated by the enzyme nitrous oxide reductase (NosZ) expressed by $N_2O$ reducing organisms, and $N_2O$ sink capacity of the systems is governed by the $N_2O$ reducing population and the behaviors of $N_2O$ reducing organisms under specific niches. Thus, attempts have been made to investigate the distribution of $nosZ$ to predict $N_2O$ sink potential of the systems. Moreover, physiological studies of $N_2O$ reducing organisms have been performed to identify $N_2O$ reducing organisms having high affinity for $N_2O$. The previous results suggested the activity of $N_2O$ reduction would be not just distinguished by clade types of $nosZ$, raising a necessity for subdivided quantification of $nosZ$. In the first research topic of the thesis, the primer and probe sets for qPCR assays were designed to target $nosZ$ group-specifically, and the performance was validated with the metagenome analysis performed in parallel. $nosZ$ genes and transcripts in activated sludge microbiome were investigated using the qPCR assays, and $nosZ$ belonged to NosZG3 (Flavobacterium-like) and NosZG5 (Dechloromonas-like) showed high abundance and expression.

In the second research topic, activated sludge was incubated in chemostat reactor with extremely low $N_2O$ flux, and the genomic and transcriptomic analysis were performed on the final enrichment to identify high affinity $N_2O$ reducers capable of reducing $N_2O$ at extremely low concentration. Unexpected oxygen contamination into the reactor was observed providing a serendipitous opportunity to simulate more a realistic anoxic environment, where trace oxygen continuously seeps in. The results suggested $N_2O$ reducing organisms with $nosZ$ of NosZG5 showed the most active $N_2O$ reduction activities under such circumstances while $N_2O$ reducing organisms with $nosZ$ of NosZG2 preferably utilized trace $O_2$ as an electron acceptor.