The Confounding Effect of Nitrite on N₂O Production by an Enriched Ammonia-Oxidizing Culture

The effect of nitrite (NO₂^–) on the nitrous oxide (N₂O) production rate of an enriched ammonia-oxidizing bacteria (AOB) culture was characterized over a concentration range of 0–1000 mg N/L. The AOB culture was enriched in a nitritation system fed with synthetic anaerobic digester liquor. The N₂O production rate was highest at NO₂^– concentrations of less than 50 mg N/L. At dissolved oxygen (DO) concentration of 0.55 mg O₂/L, further increases in NO₂^– concentration from 50 to 500 mg N/L resulted in a gradual decrease in N₂O production rate, which maintained at its lowest level of 0.20 mg N₂O–N/h/g VSS in the NO₂^– concentration range of 500–1000 mg N/L. The observed NO₂^–-induced decrease in N₂O production was even more apparent at increased DO concentration. At DO concentrations of 1.30 and 2.30 mg O₂/L, the lowest N₂O production rate (0.25 mg N₂O–N/h/g VSS) was attained at a lower NO₂^– concentration of 200–250 mg N/L. These observations suggest that N₂O production by the culture is diminished by both high NO₂^– and high DO concentrations. Collectively, the findings show that exceedingly high NO₂^– concentrations in nitritation systems could lead to decreased N₂O production. Further studies are required to determine the extent to which the same response to NO₂^– is observed across different AOB cultures

Effect of H₂S on N₂O Reduction and Accumulation during Denitrification by Methanol Utilizing Denitrifiers

Sulfide is produced in sewer networks, and previous studies suggest that sulfide in sewage could alter the activity of heterotrophic denitrification and lead to N₂O accumulation during biological wastewater treatment. However, the details of this phenomenon are poorly understood. In this 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 exposure and adaptation to sulfide. Hydrogen sulfide was found to be strongly inhibitory to N₂O reduction, with 50% inhibition observed at H₂S concentrations of 0.04 mg H₂S–S/L and 0.1 mg H₂S–S/L for the unadapted and adapted cultures, respectively. In comparison, both nitrate and nitrite reduction was more tolerant to H₂S. A 50% inhibition of nitrite reduction was observed at approximately 2.0 mg H₂S–S/L for both unadapted and adapted cultures, while no inhibition of nitrate reduction occurred at the highest H₂S concentrations applied (2.0 mg H₂S–S/L) to either culture. N₂O accumulation was observed during nitrate and nitrite reduction by the adapted culture when H₂S concentrations were above 0.5 and 0.2 mg H₂S–S/L, respectively. Additionally, we reveal that hydrogen sulfide (H₂S), rather than sulfide, was likely the true inhibitor of N₂O reduction, and the inhibitory effect was reversible. These findings suggest that sulfide management in sewers could potentially have a significant impact on N₂O emission from wastewater treatment plants

Reducing N₂O Emission from a Domestic-Strength Nitrifying Culture by Free Nitrous Acid-Based Sludge Treatment

An increase of nitrite in the domestic-strength range is generally recognized to stimulate nitrous oxide (N₂O) production by ammonia-oxidizing bacteria (AOB). It was found in this study, however, that N₂O emission from a mainstream nitritation system (cyclic nitrite = 25–45 mg of N/L) that was established by free nitrous acid (FNA)-based sludge treatment was not higher but much lower than that from the initial nitrifying system with full conversion of NH₄⁺-N to NO₃^–-N. Under dissolved oxygen (DO) levels of 2.5–3.0 mg/L, N₂O emission from the nitritation stage was 76% lower than that from the initial stage. Even when the DO level was reduced to 0.3–0.8 mg/L, N₂O emission from the nitritation stage was still 40% lower. An investigation of the mechanism showed that FNA treatment caused a shift of the stimulation threshold of nitrite on N₂O emission. At the nitritation stage, the maximal N₂O emission factor occurred at ∼16 mg of N/(L of nitrite). However, it increased with increasing nitrite in the range of 0–56 mg of N/L at the initial stage. FNA treatment decreased the biomass-specific N₂O production rate, suggesting that the enzymes relevant to nitrifier denitrification were inhibited. Microbial analysis revealed that FNA treatment decreased the microbial community diversity but increased the abundances of AOB and denitrifiers

Molecular Dynamics Unlocks Atomic Level Self-Assembly of the Exopolysaccharide Matrix of Water-Treatment Granular Biofilms

Biofilm formation, in which bacteria are embedded within an extracellular matrix, is the default form of microbial life in most natural and engineered habitats. In this work, atomistic molecular dynamics simulations were employed to examine the self-assembly of the polysaccharide Granulan to provide insight into the molecular interactions that lead to biofilm formation. Granulan is a major gel forming matrix component of granular microbial biofilms found in used-water treatment systems. Molecular dynamics simulations showed that Granulan forms an antiparallel double helix stabilized by complementary hydrogen bonds between the β-glucosamine of one strand and the <i>N</i>-acetyl-β-galactosamine–2-acetoamido-2-deoxy-α-galactopyranuronic pair of the other in both the presence and absence of Ca²⁺. It is shown that Ca²⁺ binds primarily to the carboxyl group of the terminal hexuronic acid of the sugar branch and that interactions between branches mediated by Ca²⁺ suggest a possible mechanism for strengthening gels by facilitating interhelical bridging

Mathematical Modeling of Nitrous Oxide (N₂O) Emissions from Full-Scale Wastewater Treatment Plants

Mathematical modeling of N₂O emissions is of great importance toward understanding the whole environmental impact of wastewater treatment systems. However, information on modeling of N₂O emissions from full-scale wastewater treatment plants (WWTP) is still sparse. In this work, a mathematical model based on currently known or hypothesized metabolic pathways for N₂O productions by heterotrophic denitrifiers and ammonia-oxidizing bacteria (AOB) is developed and calibrated to describe the N₂O emissions from full-scale WWTPs. The model described well the dynamic ammonium, nitrite, nitrate, dissolved oxygen (DO) and N₂O data collected from both an open oxidation ditch (OD) system with surface aerators and a sequencing batch reactor (SBR) system with bubbling aeration. The obtained kinetic parameters for N₂O production are found to be reasonable as the 95% confidence regions of the estimates are all small with mean values approximately at the center. The model is further validated with independent data sets collected from the same two WWTPs. This is the first time that mathematical modeling of N₂O emissions is conducted successfully for full-scale WWTPs. While clearly showing that the NH₂OH related pathways could well explain N₂O production and emission in the two full-scale plants studied, the modeling results do not prove the dominance of the NH₂OH pathways in these plants, nor rule out the possibility of AOB denitrification being a potentially dominating pathway in other WWTPs that are designed or operated differently

Modeling of Nitrous Oxide Production by Autotrophic Ammonia-Oxidizing Bacteria with Multiple Production Pathways

Autotrophic ammonia oxidizing bacteria (AOB) have been recognized as a major contributor to N₂O production in wastewater treatment systems. However, so far N₂O models have been proposed based on a single N₂O production pathway by AOB, and there is still a lack of effective approach for the integration of these models. In this work, an integrated mathematical model that considers multiple production pathways is developed to describe N₂O production by AOB. The pathways considered include the nitrifier denitrification pathway (N₂O as the final product of AOB denitrification with NO₂^– as the terminal electron acceptor) and the hydroxylamine (NH₂OH) pathway (N₂O as a byproduct of incomplete oxidation of NH₂OH to NO₂^–). In this model, the oxidation and reduction processes are modeled separately, with intracellular electron carriers introduced to link the two types of processes. The model is calibrated and validated using experimental data obtained with two independent nitrifying cultures. The model satisfactorily describes the N₂O data from both systems. The model also predicts shifts of the dominating pathway at various dissolved oxygen (DO) and nitrite levels, consistent with previous hypotheses. This unified model is expected to enhance our ability to predict N₂O production by AOB in wastewater treatment systems under varying operational conditions

Free Nitrous Acid (FNA)-Based Pretreatment Enhances Methane Production from Waste Activated Sludge

Anaerobic digestion of waste activated sludge (WAS) is currently enjoying renewed interest due to the potential for methane production. However, methane production is often limited by the slow hydrolysis rate and/or poor methane potential of WAS. This study presents a novel pretreatment strategy based on free nitrous acid (FNA or HNO₂) to enhance methane production from WAS. Pretreatment of WAS for 24 h at FNA concentrations up to 2.13 mg N/L substantially enhanced WAS solubilization, with the highest solubilization (0.16 mg chemical oxygen demand (COD)/mg volatile solids (VS), at 2.13 mg HNO₂–N/L) being six times that without FNA pretreatment (0.025 mg COD/mg VS, at 0 mg HNO₂–N/L). Biochemical methane potential tests demonstrated methane production increased with increased FNA concentration used in the pretreatment step. Model-based analysis indicated FNA pretreatment improved both hydrolysis rate and methane potential, with the highest improvement being approximately 50% (from 0.16 to 0.25 d^–1) and 27% (from 201 to 255 L CH₄/kg VS added), respectively, achieved at 1.78–2.13 mg HNO₂–N/L. Further analysis indicated that increased hydrolysis rate and methane potential were related to an increase in rapidly biodegradable substrates, which increased with increased FNA dose, while the slowly biodegradable substrates remained relatively static

Modeling of Simultaneous Anaerobic Methane and Ammonium Oxidation in a Membrane Biofilm Reactor

Nitrogen removal by using the synergy of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) microorganisms in a membrane biofilm reactor (MBfR) has previously been demonstrated experimentally. In this work, a mathematical model is developed to describe the simultaneous anaerobic methane and ammonium oxidation by DAMO and Anammox microorganisms in an MBfR for the first time. In this model, DAMO archaea convert nitrate, both externally fed and/or produced by Anammox, to nitrite, with methane as the electron donor. Anammox and DAMO bacteria jointly remove the nitrite fed/produced, with ammonium and methane as the electron donor, respectively. The model is successfully calibrated and validated using the long-term (over 400 days) dynamic experimental data from the MBfR, as well as two independent batch tests at different operational stages of the MBfR. The model satisfactorily describes the methane oxidation and nitrogen conversion data from the system. Modeling results show the concentration gradients of methane and nitrogen would cause stratification of the biofilm, where Anammox bacteria mainly grow in the biofilm layer close to the bulk liquid and DAMO organisms attach close to the membrane surface. The low surface methane loadings result in a low fraction of DAMO microorganisms, but the high surface methane loadings would lead to overgrowth of DAMO bacteria, which would compete with Anammox for nitrite and decrease the fraction of Anammox bacteria. The results suggest an optimal methane supply under the given condition should be applied not only to benefit the nitrogen removal but also to avoid potential methane emissions

Determining Multiple Responses of Pseudomonas aeruginosa PAO1 to an Antimicrobial Agent, Free Nitrous Acid

Free nitrous acid (FNA) has recently been demonstrated as an antimicrobial agent on a range of micro-organisms, especially in wastewater-treatment systems. However, the antimicrobial mechanism of FNA is largely unknown. Here, we report that the antimicrobial effects of FNA are multitargeted. The response of a model denitrifier, Pseudomnas aeruginosa PAO1 (PAO1), common in wastewater treatment, was investigated in the absence and presence of inhibitory level of FNA (0.1 mg N/L) under anaerobic denitrifying conditions. This was achieved through coupling gene expression analysis, by RNA sequencing, and with a suite of physiological analyses. Various transcripts exhibited significant changes in abundance in the presence of FNA. Respiration was likely inhibited because denitrification activity was severely depleted, and decreased transcript levels of most denitrification genes occurred. As a consequence, the tricarboxylic acid (TCA) cycle was inhibited due to the lowered cellular redox state in the FNA-exposed cultures. Meanwhile, during FNA exposure, PAO1 rerouted its carbon metabolic pathway from the TCA cycle to pyruvate fermentation with acetate as the end product as a possible survival mechanism. Additionally, protein synthesis was significantly decreased, and ribosome preservation was evident. These findings improve our understanding of PAO1 in response to FNA and contribute toward the potential application for use of FNA as an antimicrobial agent