Enzymatic regulation of N 2 O production by denitrifying bacteria in the sludge of biological nitrogen removal process

This study analyzed the activities of all denitrifying enzymes involved in the denitrification process under different organic loads in a continuously operating sequencing batch reactor (SBR), to reveal how the denitrifying enzymes performed while the denitrifying bacteria facing changes in organic load, and leading to nitrous oxide (N 2 O) production by fine-tuning enzyme activities. Results show that the activities of nitrate reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Nor) and nitrous oxide reductase (N 2 OR) increased with the increase of organic loads, and the increase of the activity of different enzymes promoted by the organic load increase were as Nar > Nir > Nor > N 2 OR. Compared with the Nar and Nir, the catalytic processes of the Nor and N 2 OR were more susceptible to the influence of the substrate concentration and the content of internal and external carbon sources. The Nor usually maintained “excess” catalytic activity to ensure the smooth reduction of nitric oxide when the electron donor and substrate were sufficient. Otherwise, it reduced to a relatively lower catalytic activity and remained stable. The activities of the N 2 OR were generally weaker than that of other denitrifying enzymes. More N 2 O was produced in the period feeding with low organic loads (COD/NO 3 − -N ≤ 4.9). The mechanism of the enzyme activities (Nor and N 2 OR) regulating the total concentrations of N 2 O was clarified. When the organic load was relatively low (COD/NO 3 − -N ≤ 2.5), the N 2 OR activity was inhibited due to its inability to acquire enough electrons, resulting the production of N 2 O. When the organic load was moderate (2.5 < COD/NO 3 − -N ≤ 4.9), the N 2 OR activity was lower than the Nor activity due to the different activation rates of Nor and N 2 OR by the substrate in bacteria, resulting the production of N 2 O.