Hydroxylamine Diffusion Can Enhance N₂O Emissions in Nitrifying Biofilms: A Modeling Study

Abstract

Wastewater treatment plants can be significant sources of nitrous oxide (N2O), a potent greenhouse gas. However, little is known about N2O emissions from biofilm processes. We adapted an existing suspended-growth mathematical model to explore N2O emissions from nitrifying biofilms. The model included N2O formation by ammonia-oxidizing bacteria (AOB) via the hydroxylamine and the nitrifier denitrification pathways. Our model suggested that N2O emissions from nitrifying biofilms could be significantly greater than from suspended growth systems under similar conditions. The main cause was the formation and diffusion of hydroxylamine, an AOB nitrification intermediate, from the aerobic to the anoxic regions of the biofilm. In the anoxic regions, hydroxylamine oxidation by AOB provided reducing equivalents used solely for nitrite reduction to N2O, since there was no competition with oxygen. For a continuous system, very high and very low dissolved oxygen (DO) concentrations resulted in lower emissions, while intermediate values led to higher emissions. Higher bulk ammonia concentrations and greater biofilm thicknesses increased emissions. The model effectively predicted N2O emissions from an actual pilot-scale granular sludge reactor for sidestream nitritation, but significantly underestimated the emissions when the NH2OH diffusion coefficient was assumed to be minimal. This numerical study suggests an unexpected and important role of hydroxylamine in N2O emission in biofilms.

Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission

“…S10). This lack of reactivity is inconsistent with the hypothesis of Bari and coworkers (34) that the hydrolysis of an {Fe-NO} 6 generates an Fe II -ONOH adduct that is poised for proton-coupled, oneelectron oxidation to NO − 2 because cyt P460 does not produce NO − 2 under anaerobic conditions. Taken together, these data support the assignment of the 455-nm intermediate as an {FeNO} 6 .…”

“…The reduction of cyt c was monitored by the increase in absorption at 550 nm (e 550 = 19,600 M −1 ·cm −1 (39). These experiments show that 3 electrons are required to oxidize Fe III -NH 2 OH to the 455-nm intermediate, suggesting an {FeNO} 6 or Fe II -ONOH species (33). Consistent with this observation, treatment of Fe III -OH 2 with NO supplied either as the gas or via the NO donor PROLI-NONOate [1-(hydroxy-NNOazoxy)-L-proline] produces an EPR-silent species with an absorption spectrum identical to the absorption spectrum of the 455-nm intermediate (Fig.…”

“…The stability of the {FeNO} 6 intermediate in the absence of NH 2 OH and the NH 2 OH dependence of its decay suggests that {FeNO} 6 reacts with NH 2 OH to form N 2 O. A so-called shunted {FeNO} 6 species was prepared by adding 2 eq of NO supplied via PROLI-NONOate to cyt P460.…”

“…By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH 2 OH to N 2 O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an Fe III -NH 2 OH adduct of cyt P460 is oxidized to an {FeNO} 6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH 2 OH, forming the N-N bond of N 2 O during a bimolecular, rate-determining step.…”

“…This species subsequently undergoes nucleophilic attack by a second equivalent of NH 2 OH, forming the N-N bond of N 2 O during a bimolecular, rate-determining step. We propose that NO − 2 results when nitric oxide (NO) dissociates from the {FeNO} 6 intermediate and reacts with dioxygen. Thus, NO − 2 is not a direct product of cyt P460 activity.…”