Flocs are the main source of nitrous oxide in a high-rate anammox granular sludge reactor: insights from metagenomics and fed-batch experiments.

Nitrous oxide (NO) emissions from anammox-based processes are well documented but insight into source of the NO emission in high-rate anammox granular sludge reactors (AGSR) is limited. In this study, metagenomics and fed-batch experiments were applied to investigate the relative contributions of anammox granules and flocs to NO production in a high-rate AGSR. Flocs, which constitute only ~10% of total biomass contributed about 60% of the total NO production. Granules, the main contributor of nitrogen removal (~95%), were responsible for the remaining ~40% of NO production. This result is inconsistent with reads-based analysis that found the gene encoding clade II type nitrous oxide reductase (nosZ) had similar abundances in both granules and flocs. Another notable trend observed was the relatively higher abundance of the gene for NO-producing nitrite reductase (nir) in comparison to the gene for the nitric oxide reductase gene (nor) in both granules and flocs, indicating nitric oxide (NO) may accumulate in the AGSR. This is significant since NO and NO pulse assays demonstrated that NO could lead to NO production from both granules and flocs. However, since anammox bacteria, which were shown to be in higher abundance in granules than in flocs, have the capacity to scavenge NO this provides a mechanism by which its inhibitory effects can be mitigated, limiting NO release from the granules, consistent with experimental observation. These results demonstrate flocs are the main source of NO emission in AGSR and provide lab-scale evidence that NO-dependent anammox can mitigate NO emission.