Methane dependent denitrification- from ecosystem to laboratory-scale enrichment for engineering applications.

Managing nitrogen and carbon cycles in engineered and natural ecosystems is an environmental challenge. In this manuscript, we report a process which connects these two cycles with immense ecological and engineering significance. Sediments, collected from Jordan River in Salt Lake City, Utah were used as seed to start a laboratory-scale denitrification coupled to anaerobic methane oxidation (n-DAMO) reactor fed with methane (CH4) and nitrite (NO2(-)). Methane (CH4)-dependent denitrification in sediments of a nutrient-impaired river was found to be in the range of 40 nmol kg(-1) d(-1) to 70 nmol kg(-1) d(-1). Post 19 months of operation of the lab scale reactor, the n-DAMO reactor achieved nitrite removal rate of 2.88 mmol L(-1) d(-1). Enrichment of n-DAMO prokaryotes was evident from the increase in 16S rRNA gene copy number of bacteria belonging to the NC10 phylum in the reactor, corroborating with increase in the oxidation rates of CH4 coupled with NO2(-)-N removal from 21 μM to 190 μM of CH4 d(-1). Based on stable isotope experiments by other researchers, nitric oxide dismutase (nod) functional gene was hypothesized to be responsible for splitting nitric oxide to nitrogen and oxygen and this internally generated oxygen is utilized by n-DAMO prokaryotes to oxidize methane gas. Primers targeting the unique nitric oxide dismutase (nod) gene were developed and tested on the enrichment culture for the first time. This revealed that n-DAMO organisms are closely related yet distinct from, the M. oxyfera which had been enriched in earlier studies. The results emphasize tremendous future promise to use these novel organisms for wastewater treatment purposes, especially to take advantage of the dissolved methane present in anaerobic digester effluents.