Mechanisms of Nitrous Oxide (N2O) Formation in Biofilm Processes

Nitrous oxide (N2O) is a powerful greenhouse gas that can be emitted during wastewater nitrification and denitrification. While much has been learned about N2O emissions from suspended growth processes, little is known about emissions from biofilms, which are of increasing interest for wastewater treatment. Since biofilms exhibit complex substrate gradients and microbial stratification, it very difficult to predict their behavior.

This research used mathematical modeling to address the mechanisms of emissions from nitrifying biofilms, denitrifying biofilms, and combined nitrifying and denitrifying (SND) biofilms. For the nitrifying biofilm, the model included N2O formation by ammonia-oxidizing bacteria (AOB) via the hydroxylamine (NH2OH) and the nitrifier denitrification pathways. N2O emissions from AOB biofilms of different thicknesses were explored as a function of bulk dissolved oxygen (D.O.). The results suggested that N2O emissions from thicker nitrifying biofilms were greater than those from suspended-growth systems. The main cause is the diffusion of NH2OH, an AOB nitrification intermediate, from the outer, aerobic regions of biofilm to the inner, anoxic regions. The presence of NOB also enhanced N2O emissions. This was due to an increased D.O. gradient in the biofilm.

The denitrifying biofilm model included N2O formation by heterotrophs via conventional denitrification pathway. The denitrifying model showed that, in presence of excess of electron donor, denitrifying biofilms have two distinct layers of activity: an outer layer with net production of NO2- and N2O, and an inner layer with a net consumption. The biofilm thickness affects N2O emissions, and rates are influenced by the electron availability within the biofilm. The presence of D.O. in the bulk led to a spike of N2O formation in the outer biofilm, due to diffusion of NO2- from the inner biofilm and its high affinity for reduced electron carriers relative to the other nitrogen oxides in the denitrification pathway. The model results were compared to those of an experimental system. Also, a model of N2O emissions in a denitrifying filter was developed and used to explore emissions in a biofilm reactor.

A SND model was created by combining the nitrifying and denitrifying biofilm models. The SND model showed that denitrifying bacteria can mitigate N2O formed by nitrifying bacteria, but also can increase total N2O emissions. Additionally, co-diffusional biofilms were compared with counter-diffusional biofilms. Co-diffusional biofilms had higher emissions N2O, due to the proximity of N2O production to the aerobic bulk. In counter-diffusional biofilms, the N2O formation was deeper within the biofilm, surrounded by a denitrifying zone where N2O could be reduced.

This research provided insights into the mechanisms of N2O production and consumption in biofilms under a variety of conditions relevant to wastewater treatment. The results suggest that N2O formation in biofilms is complex, and often distinct from suspended growth systems.