Nitrous oxide (N 2 O) production pathway in a signal-stage nitritation-anammox sequencing batch reactor (SBR) was investigated based on a multilateral approach including real-time N 2 O monitoring, N 2 O isotopic composition analysis, and in-situ analyses of spatial distribution of N 2 O production rate and microbial populations in granular biomass. N 2 O emission rate was high in the initial phase of the operation cycle and gradually decreased with decreasing NH 4 + concentration. The average emission of N 2 O was 0.98 ± 0.42% and 1.35 ± 0.72% of the incoming nitrogen load and removed nitrogen, respectively. The N 2 O isotopic composition analysis revealed that N 2 O was produced via NH 2 OH oxidation and NO 2 - reduction pathways equally, although there is an unknown influence from N 2 O reduction and/or anammox N 2 O production. However, the N 2 O isotopomer analysis could not discriminate the relative contribution of nitrifier denitrification and heterotrophic denitrification in the NO 2 - reduction pathway. Various in-situ techniques (e.g. microsensor measurements and FISH (fluorescent in-situ hybridization) analysis) were therefore applied to further identify N 2 O producers. Microsensor measurements revealed that approximately 70% of N 2 O was produced in the oxic surface zone, where nitrifiers were predominantly localized. Thus, NH 2 OH oxidation and NO 2 reduction by nitrifiers (nitrifier-denitrification) could be responsible for the N 2 O production in the oxic zone. The rest of N 2 O (ca. 30%) was produced in the anammox bacteria-dominated anoxic zone, probably suggesting that NO 2 - reduction by coexisting putative heterotrophic denitrifiers and some other unknown pathway(s) including the possibility of anammox process account for the anaerobic N 2 O production. Further study is required to identify the anaerobic N 2 O production pathways. Our multilateral approach can be useful to quantitatively examine the relative contributions of N 2 O production pathways. Good understanding of the key N 2 O production pathways is essential to establish a strategy to mitigate N 2 O emission from biological nitrogen removal processes.
Bibliographical noteFunding Information:
This research was financially supported by Japan Science and Technology Agency (JST) CREST, Nagase Science and Technology Foundation , and Institute for Fermentation, Osaka (IFO), which were granted to S. Okabe. Authors express gratitude to the Gene Science Division, Natural Science Center for Basic Research and Development , Hiroshima University for their technical support for FISH analysis. Authors are thankful for Yoshitaka Uchida (Assistant Professor, Hokkaido University ) for useful discussion and providing technical support for dissolved N 2 O measurements.
© 2016 Elsevier Ltd.
- N O isotopomer analysis
- N O production pathway
- Nitritation-anammox reactor