Understanding nitrous oxide production during denitrification by methanol-utilizing denitrifying activated sludge

Nitrous oxide (N2O) is a potent greenhouse gas, which could be produced during biological nitrogen removal in wastewater treatment. Both nitrification and denitrification processes can generate N2O. Although nitrification is lately revealed to be the primary source of N2O emitted from WWTPs due to on-going stripping when N2O is produced, N2O production by denitrification cannot be ignored. Even low levels of N2O accumulation during denitrification could lead to significant N2O emission if it is stripped in the subsequent aerobic. Thus, it is crucial to study the mechanism and identify the key parameters that lead to N2O accumulation during denitrification.Methanol is a commonly used external carbon source to enhance denitrification in WWTPs either as a supplementary carbon source in the main reactor or as the sole carbon source in a separate denitrification system. Therefore, a good understanding of N2O production by methanol utilizing denitrifiers will have significant practical implications to the operation of wastewater treatment plants receiving methanol.By using an enriched culture of methanol utilizing denitrifiers, this thesis firstly investigated the effect of pH on N2O reduction. The pH dependency of nitrate and nitrite reduction was also investigated. The maximum biomass-specific N2O reduction rate (ranging from 120 to 350 mg N2O-N/(L×hour)) was always higher than the corresponding maximum nitrate and nitrite reduction rates (ranging from 37 to 45 mg NO3-- N/(L×hour) and 35 to 85 NO2-- N/(L×hour)). However, the maximum biomass-specific N2O reduction rate was much more sensitive to pH variation outside of the optimal range (pH 7.5 – pH 8.0) than the maximum biomass-specific nitrate and nitrite reduction rates. The half-saturation coefficient of the N2O reductase increased from 0.10 mg N2ON/L to 0.92 mg N2O-N/L as the pH increased from pH 6.0 to 9.0. At pH 6.0, approximately 20% and 40% of the denitrified nitrate accumulated as N2O in the presence and absence of methanol (as an exogenous carbon source), respectively. However, at pH 6.5, these fractions decreased to 0% and 30%, respectively. No N2O accumulation occurred at pH 7.0 - 9.0, independent of the availability of methanol. The experimental results observed at pH 6.5 suggest that the competition for electrons among different nitrogen oxides reductases likely plays a role in N2O accumulation at low pH conditions.To further investigate the role of electron competition in N2O accumulation, the effect of carbon source availability on N2O production was investigated in the second study. The results showed that electron competition occurred under not only carbon limiting but also carbon abundant conditions. The electron distribution among the nitrogen oxide reductases was affected by the carbon loading -IIrate, with a lower fraction of electrons distributed to the N2O reductase with reduced carbon loading rate. Consequently, N2O accumulation occurred when the electron flux going to nitrite reduction is higher than that going to N2O reduction.Mathematical models are gaining more attention for the prediction of N2O accumulation and emission during nitrification and denitrification in wastewater treatment processes. However, the current widely used four-step denitrification model does not consider the possible electron competition. A new mathematical model is developed, calibrated and validated to describe the electron competition among nitrogen oxides reduction and N2O accumulation in denitrification, through decoupling the carbon oxidation and nitrogen reduction processes. Electron carriers are introduced in the model, with carbon oxidation donating electrons to these carriers and nitrogen oxides reduction consuming electrons from these carriers. Model calibration and validation results demonstrate that the developed model is able to reasonably describe the nitrate, nitrite and N2O data measured in experiments. The model proposed forms a conceptual basis for the development of denitrification models that are able to predict N2O accumulation in denitrification.Sulfide is often produced biologically in sewer pipes and could enter the denitrification tank in a biological wastewater treatment reactor. However, the details of this phenomenon still need to be fully revealed and clarified. In the fourth study, the potential inhibitory effects of sulfide on nitrate, nitrite and N2O reduction were assessed with a methanol utilizing denitrifying culture, both prior to and after its adaptation to sulfide. Hydrogen sulfide was found to be strongly inhibitory to N2O reduction, with 50% inhibition observed at H2S concentrations of 0.04 and 0.1 mg H2S-S/L for the unadapted and adapted cultures, respectively. In comparison, both nitrate and nitrite reduction were much more tolerant to H2S. About 50% inhibition on nitrite reduction was observed at approximately 2.0 mg H2S-S/L for both cultures, while nitrate reduction was not affected by H2S at up to 2.0 mg H2S-S/L (the highest concentration studied) for either culture. N2O accumulation was observed during nitrate and nitrite reduction by the adapted culture when the H2S concentration was above 0.5 and 0.2 mg H2S-S/L, respectively. The results also showed that hydrogen sulfide (H2S), rather than sulfide was likely the true inhibitor of N2O reduction, and the inhibitory effect was reversible.