Production of Nitrous Oxide from Nitrite in Stable Type II Methanotrophic Enrichments

The coupled aerobic–anoxic nitrous decomposition operation is a new process for wastewater treatment that removes nitrogen from wastewater and recovers energy from the nitrogen in three steps: (1) NH₄⁺ oxidation to NO₂^–, (2) NO₂^– reduction to N₂O, and (3) N₂O conversion to N₂ with energy production. Here, we demonstrate that type II methanotrophic enrichments can mediate step two by coupling oxidation of poly­(3-hydroxybutyrate) (P3HB) to NO₂^– reduction. Enrichments grown with NH₄⁺ and NO₂^– were subject to alternating 48-h aerobic and anoxic periods, in which CH₄ and NO₂^– were added together in a “coupled” mode of operation or separately in a “decoupled mode”. Community structure was stable in both modes and dominated by Methylocystis. In the coupled mode, production of P3HB and N₂O was low. In the decoupled mode, significant P3HB was produced, and oxidation of P3HB drove reduction of NO₂^– to N₂O with ∼70% conversion for >30 cycles (120 d). In batch tests of wasted cells from the decoupled mode, N₂O production rates increased at low O₂ or high NO₂^– levels. The results are significant for the development of engineered processes that remove nitrogen from wastewater and for understanding of conditions that favor environmental production of N₂O.