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标题: Nitrous oxide emissions in novel wastewater treatment processes: A comprehensive review
DOI: 10.1016/j.biortech.2023.129950
摘要:
The proliferation of novel wastewater treatment processes has marked recent years, becoming particularly pertinent in light of the strive for carbon neutrality. One area of growing attention within this context is nitrous oxide (N2O) production and emission. This review provides a comprehensive overview of recent research progress on N2O emissions associated with novel wastewater treatment processes, including Anammox, Partial Nitrification, Partial Denitrification, Comammox, Denitrifying Phosphorus Removal, Sulfur-driven Autotrophic Denitrification and n-DAMO. The advantages and challenges of these processes are thoroughly examined, and various mitigation strategies are proposed. An interesting angle that delve into is the potential of endogenous denitrification to act as an N2O sink. Furthermore, the review discusses the potential applications and rationale for novel Anammox-based processes to reduce N2O emissions. The aim is to inform future technology research in this area. Overall, this review aims to shed light on these emerging technologies while encouraging further research and development.
全文内容:
## Introduction
Nitrous oxide (N2O) emissions are currently a significant hurdle in meeting greenhouse effect control targets. N2O, a greenhouse gas with a 100-year global warming potential 273 times higher than CO2 and 28 times that of CH4, is the principal greenhouse gas emitted from wastewater treatment (de Haas and Andrews, 2022). Currently, the growth rate of global N2O emissions caused by human activities surpasses the estimates predicted by the Intergovernmental Panel on Climate Change (IPCC) and the Paris Agreement (Domingo-Félez et al., 2014). A 1% N2O emission factor (EF) would increase the carbon footprint of wastewater treatment plants (WWTPs) by approximately 30% (Ni and Yuan, 2015). As a result, it is imperative to develop effective strategies to mitigate N2O emissions.
N2O from WWTPs mainly originates from the biological nitrogen removal (BNR) process. In the conventional BNR process, N2O is primarily produced via nitrifier denitrification (ND), NH2OH oxidation, heterotrophic denitrification (HD) and abiotic pathways. However, the abiotic contribution is negligible (<3%) (Chen et al., 2020; Guo et al., 2021) (Fig. 1 ). The produced N2O is in a dynamic change between liquid and gas phase, and part of the N2O escapes to the atmosphere in gaseous form and part of it is discharged with the effluent in dissolved form (Foley et al., 2010; Massara et al., 2017), thus increasing the carbon footprint of the WWTPs (Su et al., 2019; Yang et al., 2009).
The management measures for N2O are mainly divided into emission source control and tail gas purification or recovery technologies. Adsorption or recovery of N2O after discharge requires a large investment, and although N2O has a certain recovery benefits, there is no widely applicable and economically feasible extraction method to extract N2O from wastewater, so controlling N2O emissions from the source would be the most cost-effective method. The control of N2O generation is based on the optimization of operating parameters on the one hand, and on the application of novel process technologies on the other hand. (Solis et al., 2022; Yu et al., 2019; Zhang et al., 2019b). As such, the focus of research remains on N2O emissions mitigation from the process perspective. In recent years, processes such as anaerobic ammonia oxidation (Anammox), partial denitrification (PD), complete ammonia oxidizer (Comammox), and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) have sprung up (Al-Hazmi et al., 2022; Valenzuela et al., 2021b; Zhu et al., 2022). These processes demonstrate substantial merits in energy-saving and consumption-reduction and are believed to generate negligible N2O emissions. Conversely, processes like partial nitrification (PN), denitrifying phosphorus removal (DPR), and sulfur-driven autotrophic denitrification (SDAD) perform well in nitrogen removal, but less so in mitigating N2O emissions, which potentially inhibits the advancement of these processes (Du et al., 2016; Kong et al., 2013). Therefore, it is necessary to summarise the N2O emission potential of these novel processes (Fig. 2 ).
Although numerous reviews on N2O emissions from traditional wastewater treatment systems have been published (Vasilaki et al., 2019), comprehensive research examining the potential application of novel wastewater treatment processes for N2O mitigation remains scarce. This review seeks to fill that gap by focusing on the Anammox, PN, PD, DPR, Comammox, SDAD and n-DAMO processes. It delves into the N2O emission characteristics, elucidates N2O production mechanisms, and explores the pivotal roles exerted by pertinent functional microorganisms in these processes. Then, practical strategies to reduce N2O emissions during nitrogen removal are explored. Finally, the novel Anammox-based processes, the potential of exogenous and endogenous denitrification (ED) as an N2O sink, and bioaugmentation strategies to enrich N2O-reducing bacteria for N2O mitigation are proposed.
## Sustainable innovations in biological nitrogen removal: N2O emissions and mitigation strategies
The Anammox process is known for its ability to convert ammonium (NH4 +) and nitrite (NO2 –) into N2 and a small amount of nitrate (NO3 –) under anoxic conditions (Duan et al., 2021; Hu et al., 2019). Anammox has the absolute advantage not only in terms of energy-saving and consumption-reduction, but current research has deemed that Anammox does not generate N2O (Kuenen, 2008; Suenaga et al., 2021). Nonetheless, the N2O production has been frequently monitored in Anammox-based reactors and therefore it cannot be ignored and deserves to be discussed (Table 1 ).
The most important metabolic feature of Anammox is its ability to utilize NO2 –, thereby preventing its accumulation and reducing N2O production from both ND and HD pathways (Yang et al., 2009). Studies have demonstrated that NO2 – accumulation is limited due to the dominance of Anammox in the aerobic phase (Zhang et al., 2021; Zhang et al., 2019a). NO2 – accumulation can result in the downregulation of the NosZ gene, which is responsible for the reduction of N2O to N2, and Anammox could alleviate the issues associated with NO2 – accumulation to some extent (Highton et al., 2022).
Studies have also found that some Anammox bacteria (AnAOB) are able to utilize volatile fatty acids as electron donors for dissimilatory nitrate reduction to ammonium (DNRA) metabolism (Wang et al., 2022). This metabolic pathway could be beneficial to enhance the tolerance of AnAOB to organic compounds and also compete with heterotrophic bacteria to mitigate N2O produced resulting from HD. (Castro-Barros et al., 2017; Li et al., 2020b).
Another significant feature of Anammox is its capacity to utilize NO (Wan et al., 2021). NO plays a crucial role in the microbial nitrogen-cycling network as an ozone-depleting agent and a precursor of the greenhouse gas N2O (Hu et al., 2019). The findings indicated that the total release of NO ranged from only 0.014% to 0.028% under the laboratory study conditions. The main releases occurred during high aeration periods and the dissolved NO concentration in the wastewater was<0.05% of the gaseous release due to the very low solubility of NO (Fuerhacker et al., 2001). AnAOB can grow through the oxidation of NH4 + and NO reduction, even in the absence of NO2 – (4NH4 ++6NO → 5 N2 + 6H2O + 4H+) (Hu et al., 2019). This unique capability, supported by lab-scale evidence, can be harnessed to manage NO and N2O discharges (Zhuang et al., 2020). Kartal et al. (2010) investigated the impact of extremely high NO flux on AnAOB and observed that AnAOB were not inhibited by NO. Instead, they observed simultaneous degradation of NH4 + and NO without N2O production, providing further support for NO as an intermediate in Anammox. Nevertheless, the mechanisms and metabolic kinetics of NO metabolism by AnAOB are still unknown, and further experimental characterization in this area is necessary for future research.
The microorganisms in wastewater treatment reactors often exist in three distinct aggregate forms: floc sludge, biofilm and granular sludge. Given the slow propagation rate, long doubling time, and sensitivity to ambient conditions, it is difficult for AnAOB to grow and enrich in flocs, and biomass retention strategies are often adopted in studies. Thus, current studies on N2O emissions focus predominantly on Anammox granules and biofilms, and have rarely been reported in flocs (Wang et al., 2018; Zhang et al., 2023).
Granules are compact, dense aggregates with a nearly spherical shape, and settle significantly faster than flocs (Lemaire et al., 2008). In Anammox granular systems, due to the lack of external carbon sources, the carbon source for denitrification mainly come from the hydrolysis and acidification of proteins and polysaccharides on the surface of the granular sludge. However, both are long-chain molecules, and the time required for hydrolysis and acidification is long. When the rate of NO3 – production from Anammox exceeds the rate of hydrolysis and acidification of proteins and polysaccharides, it leads to N2O accumulation (Zhu et al., 2018). Although microbial diversity increased with increasing sludge particle diameter (<2.5 mm), N2O also increased. (Luo et al., 2017). Besides, when the particle radius was larger than the maximum mass transfer distance, the Anammox activity in the particles was inhibited, thus impairing the nitrogen removal (Zhu et al., 2018). Therefore, the particle size should be controlled to improve the process efficiency and mitigate greenhouse gas production.
Biofilms are defined as densely aggregated biomass, which is generally attached to gravel or plastic media surfaces (Todt and Dörsch, 2016). Biofilms normally harbor aerobic and anoxic zones that mediate close interactions between different nitrogen-transforming organisms. Nitrifiers predominantly inhabit in the aerobic zone, whereas heterotrophic denitrifiers and AnAOB tend to enrich the anoxic zone (Jin et al., 2012). Peng et al. (2019) concluded that N2O emissions from Anammox biofilms were mainly caused by denitrifiers that rely on soluble microbial products for growth. Modelling results indicated that thicker biofilm thickness (1500 μm) minimized N2O emissions. This was attributed to the fact that the detachment rate decreased significantly with increasing biofilm thickness. At the same time the kinetics of NO3 – reduction to NO is slower than the reduction step from NO to N2, making the residence time of N2O in the biofilm shorter in thinner biofilms thus leading to higher emissions (Eldyasti et al., 2014). Moreover, enhancing Anammox activity not only facilitated high levels of nitrogen removal but also reduced NO and N2O generation in the membrane-aerated biofilm reactor (Ni et al., 2013). In the membrane bioreactor with 97.6% enrichment purity of AnAOB, NO and N2O were detected with nitrogen conversion below 0.01% (van der Star et al., 2008). In conclusion, controlling biofilm thickness and improving Anammox enrichment purity are essential for N2O mitigation in Anammox systems.
PN process, as an integral part of the nitrogen removal pathway in wastewater treatment, involves the oxidation of NH4 + to NO2 – without proceeding to NO3 –. The primary advantage of this approach lies in the reduced O2 and organic carbon demand, thereby saving energy and lowering operational costs (Duan et al., 2019). PN usually acts as the preliminary reaction to Anammox, which together form the partial nitrification-Anammox (PN/A) process, an innovative autotrophic denitrification technology (Wang et al., 2021). However, significant N2O generation during the PN process poses a serious environmental challenge.
N2O production during the PN process primarily occurs via two mechanisms: ND and NH2OH oxidation pathways. In ND, Ammonia-Oxidizing Bacteria (AOB) oxidize NH4 + to NO2 –, which in turn is reduced to N2O under oxygen-limited conditions (Zhao et al., 2022). In the NH2OH oxidation pathway, N2O is produced directly from NH2OH, an intermediate product of ammonia oxidation. Efforts have been made to mitigate N2O emissions during PN process, primarily through process control strategies that aim to minimize the conditions conducive to N2O production. One critical factor is DO, where low DO levels negatively impact N2O emissions, and N2O EF decreases from 2.35% to 0.57% with an increasing in DO from 0.35 mg/L to 0.85 mg/L (Lv et al., 2016). Another key factor is NO2 – concentration, where both NO2 – accumulation and low DO promote ND pathway, resulting in higher N2O production (Massara et al., 2017). Therefore, the key to control N2O emissions from PN process is to limit ND pathway.
N2O emission characteristics of one-stage and two-stage PN/A systems have been investigated. Generally, N2O emissions from one-stage systems are lower than those from two-stage systems. In one-stage systems, the N2O emissions are relatively lower because these systems minimize the exposure of NO2 – to conditions that promote N2O production. The integrated nature of one-stage systems ensures that the conditions remain less conducive to N2O formation. Conversely, two-stage systems involve separate reactors where NO2 – accumulation is possible. In the nitritation reactor of the two-stage system, conditions that favor N2O production can lead to comparatively higher N2O emissions (Kampschreur et al., 2009). N2O emissions from one-stage and two-stage PN/A systems are shown in Table 1. In a one-stage system of sequencing biofilm batch reactor (SBBR), HD was found to be the primary pathway for N2O production, with Anammox contributing to 83–91% of nitrogen removal (Li et al., 2017). On a related note, Ali et al. (2016) identified that 70% of N2O production on the aerobic surface was dominated by nitrifiers, with the remainder being produced in anoxic zones. In contrast, in two-stage PN/A reactors, N2O emissions were mainly observed in nitritation reactor, often related to NO2 – accumulation. Okabe et al. (2011) identified average N2O emissions in a two-stage PN/A reactor at 4.0 ± 1.5% and 0.1 ± 0.07% of the influent nitrogen load, respectively, and that N2O in the Anammox reactor was mainly originated from HD in the inner part of the granules. Furthermore, in a year-long pilot-scale mainstream two-stage PN/A system, Hausherr et al. (2022) noted that N2O emissions primarily occurred in the PN stage (1.2% of total influent nitrogen), while the Anammox stage accounted for only 4% of the N2O production in the PN stage. Interestingly, it was found that the addition of external carbon sources in the latter reactor can further reduce N2O emissions and remove the NO3 – produced by Anammox (Juan-Diaz et al., 2022). In summary, while PN/A process presents a compelling approach for nitrogen removal, understanding and mitigating N2O emissions remain critical challenges.
PD process is an intermediate stage in denitrification, during which NO3 – is reduced to NO2 – instead of being fully reduced to N2. PD is increasingly being recognized as an essential component of novel and energy-efficient nitrogen removal strategies. It is increasingly acknowledged for its potential role in providing substrate for Anammox, thereby leading to the development of partial denitrification-Anammox (PD/A) process (Al-Hazmi et al., 2023b; Du et al., 2022).
N2O production during PD process is mainly due to the HD pathway, where N2O may be produced as intermediate or final products of denitrification. Previous studies have shown that N2O production could almost be ignored when NO2 – accumulation occurs before complete depletion of NO3 –, which proved the potential of PD process in achieving N2O emission reduction (Du et al., 2016; Ma et al., 2017a). Furthermore, the types and enzyme metabolism mechanisms of denitrifiers can significantly affect N2O production (Zhang et al., 2020b). Despite the potential for PD to contribute to N2O emissions, the understanding of the mechanisms underlying these emissions and the conditions promoting them is still far from complete. Further research into the N2O emissions characteristics of PD process is needed in order to better predict and manage these emissions in the context of wastewater treatment.
Compared to PN, the most remarkable advantages of PD are its ability to provide a steady substrate for Anammox and its tolerance of environmental factors (He et al., 2023). NO2 – and NO produced in PD process can be utilized by Anammox, avoiding N2O precursors accumulation (Al-Hazmi et al., 2023a). The N2O emission characteristics across different PD/A reactor configurations and operational conditions are summarized in Table 1. Du et al. (2020) treated the real municipal wastewater by a one-stage PD/A process with a maximum N2O accumulation of only 0.7% of the influent nitrogen load, which was considerably lower than the BNR process (Wunderlin et al., 2012). Furthermore, He et al. (2023) demonstrated that in an integrated PD/A system with low C/N, PD/A and denitrification was the dominant nitrogen removal pathway, accounting for 91.3% and 8.7%, respectively, with N2O emissions peaking between 0.18 and 0.25 mg N/L. In an INPDA-SBBR system, the low N2O emissions were attributed to the high-level expression of functional genes associated with Anammox (Zhou et al., 2020). Overall, the collaboration between denitrifiers and AnAOB in one-stage PD/A process determined the reactor performance and N2O production. PD/A process offers a promising strategy for managing N2O emissions in the anoxic stage of wastewater treatment, but the delicate balance of conditions required for a stable and efficient PD/A process requires careful monitoring and control of process parameters (Al-Hazmi et al., 2023a).
DPR process is recognized as the most promising strategy for enhanced biological phosphorus removal (Qiu et al., 2019). Unlike traditional biological phosphorus removal, DPR utilizes denitrifying phosphorus accumulating organisms (DPAOs) that employ NO3 – or NO2 – as electron acceptors instead of O2 and use intracellular carbon as electron donor under anoxic conditions to simultaneously accomplish nitrogen removal and phosphorus absorption (Guo et al., 2018; Zhang et al., 2020a). A significant challenge in DPR systems was that N2O, rather than N2, tended to be the dominant denitrification product when poly-hydroxyalkanoates (PHA) serve as the carbon source. This phenomenon considerably increased the carbon footprint of WWTPs (Jia et al., 2012; Lemaire et al., 2006).
Studies have shown that weak competition of NOS for electrons and NO2 – accumulation are the primary reasons contributing to N2O production in DPR process (Li et al., 2013b). NO2 – accumulation could also severely inhibit NOR activity, leading to N2O accumulation (Kampschreur et al., 2009). PHA degradation kinetics directly reflected DPR efficiency and N2O production. Slower PHA degradation rates could lead to electron competition among denitrifying enzymes, potentially affecting enzymes expression. The N2O reduction is the final step in denitrification, which makes NOS more difficult to compete for electrons than other enzymes, ultimately leading to an increase in N2O production (Liu et al., 2015; Wang et al., 2011b). However, contradicting these findings, Wei et al. (2014) observed that even lower PHA degradation rates did not stimulate higher N2O generation, and the electron distribution for NOS was not affected by PHA oxidation rate. Moreover, PHA degradation rates are correlated with the amount of PHA synthesised, this issue can be mitigated by optimizing the anaerobic during to increase PHA synthesis (Wang et al., 2011b). The above results indicate that the effects of PHA on NOS in DPR process need to be further explored.
N2O emissions in DPR systems are significantly affected by organic shock loads, temperature and microbial communities. Variations in carbon source shocks significantly affect denitrifying enzyme activity, the amount and composition of anaerobically synthesized PHA, leading to oxidative stress and directly stimulating N2O production (Wang et al., 2011a). Li et al. (2013a) investigated the impact of organic shock loads on N2O production in a DPR system and found that N2O-N production ranged from 1.2% to 7.12% of TN removal. A continuous NO3 – dosing strategy has proven effective in mitigating this adverse effect on N2O production. Temperature has also been found to influence N2O production, with a 43.3% higher rate at 10 °C compared to 25 °C (Jia et al., 2016). DPAOs and denitrifying glycogen accumulating organisms (DGAOs) are the two main N2O-contributing microorganisms present in DPR systems, and both compete for carbon sources, which can affect the outcomes of denitrification (Ren et al., 2023). Interestingly, some studies suggested that DPAOs appeared to have higher denitrification activity, DGAOs were the culprits of N2O accumulation in DPR systems (Lemaire et al., 2006). This aligned with the findings of Roy et al. (2021), where Candidatus Accumulibacter organisms showed complete denitrification, while Candidatus Competibacter organisms were limited to reducing NO3 – to NO2 –. This observation highlights the low N2O emissions potential of DPR systems dominated by Accumulibacter, which could be a promising focus for designing carbon–neutral treatment processes.
Comammox are organisms that harbor all the necessary enzymes to execute the entire nitrification process, facilitating the oxidation from NH4 + to NO3 –. So far, Comammox are confined to phylogenetic lineage II of the genus Nitrospira. Importantly, due to the absence of NOR in its genome, Comammox do not produce N2O. Instead, any N2O generated was a result of abiotic NH2OH conversion, with levels released being similar to those of ammonia-oxidizing archaea (AOA), but considerably lower than AOB (Han et al., 2021; Kits et al., 2019). Previous studies reported N2O yields of 0.51% for AOB, a range of 0.068–0.158% for AOA, and 0.07–0.2% for Comammox (Han et al., 2021; Kits et al., 2019). In a pilot-scale oxidation ditch, the enrichment of Comammox on the biocarrier decreased the N2O emissions of the system by 36.77% (Tian et al., 2021). Further genomic and physiological investigations demonstrated that Comammox Nitrospira had extremely high ammonia affinity, growth yield and metabolic potential in comparison to AOB and AOA (Sakoula et al., 2021). Comammox have been documented to dominate the nitrification process (Cui et al., 2023). Given these findings, the potential replacement of AOB and Nitrite-Oxidizing Bacteria (NOB) with Comammox in aerobic process offers an intriguing strategy for reducing N2O production. This transition towards Comammox technology represents an innovative step forward in the attempts to curb greenhouse gas emissions from WWTPs.
SDAD is an autotrophic denitrification process that utilizes reductive sulfur as electron donors and NO3 – or NO2 – as electron acceptors, converting these to N2 under anoxic conditions (Cao et al., 2021). Sulfur (S0), sulfide (S2-) and thiosulfate (S2O3 2-) have been demonstrated to serve as effective electron donors for SDAD (Di Capua et al., 2015).
Modelling results showed that SDAD process could accumulate quantities of N2O due to low autotrophic N2O reduction rates, but N2O emissions could be dramatically mitigated by increasing SRT or S/N (Liu et al., 2016b). The N2O release levels in a SDAD granular sludge reactor increased from 0.002% to 0.405% of the influent N load as the sulfide/nitrogen mass ratio decreased from 2.1 to 1.4 (Yang et al., 2016). Sulfides, being prevalent in SDAD systems, critically impact N2O emissions. Previous studies proposed that sulfide inhibition of N2O reduction was attributed to the formation of metal sulfide precipitates by copper and sulfur ions in the periplasm, thereby affecting NOS activity. (Richardson et al., 2009). However, some studies suggested that dissolved sulfide present during SDAD neither exerted toxicity nor inhibited soluble copper-containing NOS, which instead could significantly mitigate N2O emissions (Pan et al., 2013b; Yang et al., 2016). Pan et al. (2013b) verified that H2S, rather than sulfide, was the actual inhibitor of N2O reduction and that its inhibitory effect was reversible. Therefore, in sulfide-containing systems, the combined effect of pH and sulfide inhibited the N2O reduction. Yang et al. (2016) derived the same results and demonstrated that dissolved sulfide stimulated N2O reduction (rather than inhibiting it) regardless of SDAD sludge granule size. This stimulation effectively alleviated electron competition among various denitrifying enzymes.
In recent years, coupling SDAD with Anammox to enhance nitrogen removal has been reported (Qian et al., 2018; Qin et al., 2019). The specific process involves the reduction of NO3 – to NO2 – by autotrophic sulfur-oxidizing nitrate-reducing bacteria, providing the substrate for Anammox while further reducing the NO3 – produced by Anammox (Zhang et al., 2022). Within SDAD-Anammox systems, autotrophic denitrification was probably the dominant N2O production pathway, as corroborated by various studies (Qian et al., 2018; Yang et al., 2016). Cao et al. (2021) discovered that with decreasing pH, the N2O EF was only 0.06% to 0.71% in an innovative Anammox coupled with sulfite-driven autotrophic denitrification system. Qian et al. (2018) achieved a 29.6% reduction in N2O emissions by coupling Anammox with a thiosulfate-driven denitrification system. Theoretically, the SDAD-Anammox process holds significant potential to mitigate N2O emissions. However, the cooperative interactions of the relevant bacterial genera involved in this process remain under-explored and warrant further investigation.
Recently, the discovery of the n-DAMO process has opened up a new avenue to utilize CH4 as an alternative carbon source for denitrification (Ettwig et al., 2010; Liu et al., 2021a). This process can simultaneously mitigate CH4 and N2O emissions from aquatic ecosystems by oxidizing CH4 to provide electrons for denitrification, thereby bridging the carbon–nitrogen cycles in the biogeochemical sphere (Wang et al., 2017).
Two distinct microbial groups were involved in the n-DAMO process: n-DAMO archaea and n-DAMO bacteria (Haroon et al., 2013; Nie et al., 2021). N-DAMO archaea were able to utilize CH4 to provide electrons to reduce NO3 – to NO2 –, meanwhile the methane-driven DNRA process is also performed by n-DAMO archaea. N-DAMO bacteria can reduce NO2 – to N2 and possess part of the genes required for denitrification. Although these bacteria lack the gene NosZ, encoding NOS, they have NO dismutase, which dismutates NO into N2 and O2 (Ettwig et al., 2010). Consequently, N2O is rarely produced during the metabolism of n-DAMO microorganisms. To date, no pure n-DAMO strains have been cultured, so there is no absolute evidence that N2O is not produced as an intermediate product of the n-DAMO reaction, but the N2O yields in n-DAMO reactors studied so far have been extremely low (Ettwig et al., 2010; Ma et al., 2017b). In addition, macrogenomics analysis revealed that N2O stimulated the activity and abundance of methanotrophic bacteria, favoring electron transport and ATP production, and thus spurring the n-DAMO process (Cheng et al., 2019). This suggests that the n-DAMO process may have the potential to utilize N2O as an electron acceptor.
Research into the N2O emissions characteristics of n-DAMO process has yielded promising results. Valenzuela et al. (2021a) identified that no N2O production was detected at NO3 – loading rates of 22–66 mgN/(m3·h) in a continuous n-DAMO system, presumably mainly attributed to the N2O reducing ability of the system flora and the strict control of DO and pH. Furthermore, minimal N2O production was detected in an n-DAMO-dominated anaerobic downflow hanging sponge reactor, possibly originating from denitrifiers or specific methanotrophic bacteria (Tran et al., 2020). Ma et al. (2017b) also reported N2O conversion rates from 0.03% to 0.11% in an n-DAMO system, considerably lower than the conventional processes. However, it is important to note that while the available findings on N2O emissions in n-DAMO reactors are promising, there is a relative paucity of studies on this topic.
## Anammox process: N2O production, mitigation mechanisms, and aggregate forms
The Anammox process is known for its ability to convert ammonium (NH4 +) and nitrite (NO2 –) into N2 and a small amount of nitrate (NO3 –) under anoxic conditions (Duan et al., 2021; Hu et al., 2019). Anammox has the absolute advantage not only in terms of energy-saving and consumption-reduction, but current research has deemed that Anammox does not generate N2O (Kuenen, 2008; Suenaga et al., 2021). Nonetheless, the N2O production has been frequently monitored in Anammox-based reactors and therefore it cannot be ignored and deserves to be discussed (Table 1 ).
The most important metabolic feature of Anammox is its ability to utilize NO2 –, thereby preventing its accumulation and reducing N2O production from both ND and HD pathways (Yang et al., 2009). Studies have demonstrated that NO2 – accumulation is limited due to the dominance of Anammox in the aerobic phase (Zhang et al., 2021; Zhang et al., 2019a). NO2 – accumulation can result in the downregulation of the NosZ gene, which is responsible for the reduction of N2O to N2, and Anammox could alleviate the issues associated with NO2 – accumulation to some extent (Highton et al., 2022).
Studies have also found that some Anammox bacteria (AnAOB) are able to utilize volatile fatty acids as electron donors for dissimilatory nitrate reduction to ammonium (DNRA) metabolism (Wang et al., 2022). This metabolic pathway could be beneficial to enhance the tolerance of AnAOB to organic compounds and also compete with heterotrophic bacteria to mitigate N2O produced resulting from HD. (Castro-Barros et al., 2017; Li et al., 2020b).
Another significant feature of Anammox is its capacity to utilize NO (Wan et al., 2021). NO plays a crucial role in the microbial nitrogen-cycling network as an ozone-depleting agent and a precursor of the greenhouse gas N2O (Hu et al., 2019). The findings indicated that the total release of NO ranged from only 0.014% to 0.028% under the laboratory study conditions. The main releases occurred during high aeration periods and the dissolved NO concentration in the wastewater was<0.05% of the gaseous release due to the very low solubility of NO (Fuerhacker et al., 2001). AnAOB can grow through the oxidation of NH4 + and NO reduction, even in the absence of NO2 – (4NH4 ++6NO → 5 N2 + 6H2O + 4H+) (Hu et al., 2019). This unique capability, supported by lab-scale evidence, can be harnessed to manage NO and N2O discharges (Zhuang et al., 2020). Kartal et al. (2010) investigated the impact of extremely high NO flux on AnAOB and observed that AnAOB were not inhibited by NO. Instead, they observed simultaneous degradation of NH4 + and NO without N2O production, providing further support for NO as an intermediate in Anammox. Nevertheless, the mechanisms and metabolic kinetics of NO metabolism by AnAOB are still unknown, and further experimental characterization in this area is necessary for future research.
The microorganisms in wastewater treatment reactors often exist in three distinct aggregate forms: floc sludge, biofilm and granular sludge. Given the slow propagation rate, long doubling time, and sensitivity to ambient conditions, it is difficult for AnAOB to grow and enrich in flocs, and biomass retention strategies are often adopted in studies. Thus, current studies on N2O emissions focus predominantly on Anammox granules and biofilms, and have rarely been reported in flocs (Wang et al., 2018; Zhang et al., 2023).
Granules are compact, dense aggregates with a nearly spherical shape, and settle significantly faster than flocs (Lemaire et al., 2008). In Anammox granular systems, due to the lack of external carbon sources, the carbon source for denitrification mainly come from the hydrolysis and acidification of proteins and polysaccharides on the surface of the granular sludge. However, both are long-chain molecules, and the time required for hydrolysis and acidification is long. When the rate of NO3 – production from Anammox exceeds the rate of hydrolysis and acidification of proteins and polysaccharides, it leads to N2O accumulation (Zhu et al., 2018). Although microbial diversity increased with increasing sludge particle diameter (<2.5 mm), N2O also increased. (Luo et al., 2017). Besides, when the particle radius was larger than the maximum mass transfer distance, the Anammox activity in the particles was inhibited, thus impairing the nitrogen removal (Zhu et al., 2018). Therefore, the particle size should be controlled to improve the process efficiency and mitigate greenhouse gas production.
Biofilms are defined as densely aggregated biomass, which is generally attached to gravel or plastic media surfaces (Todt and Dörsch, 2016). Biofilms normally harbor aerobic and anoxic zones that mediate close interactions between different nitrogen-transforming organisms. Nitrifiers predominantly inhabit in the aerobic zone, whereas heterotrophic denitrifiers and AnAOB tend to enrich the anoxic zone (Jin et al., 2012). Peng et al. (2019) concluded that N2O emissions from Anammox biofilms were mainly caused by denitrifiers that rely on soluble microbial products for growth. Modelling results indicated that thicker biofilm thickness (1500 μm) minimized N2O emissions. This was attributed to the fact that the detachment rate decreased significantly with increasing biofilm thickness. At the same time the kinetics of NO3 – reduction to NO is slower than the reduction step from NO to N2, making the residence time of N2O in the biofilm shorter in thinner biofilms thus leading to higher emissions (Eldyasti et al., 2014). Moreover, enhancing Anammox activity not only facilitated high levels of nitrogen removal but also reduced NO and N2O generation in the membrane-aerated biofilm reactor (Ni et al., 2013). In the membrane bioreactor with 97.6% enrichment purity of AnAOB, NO and N2O were detected with nitrogen conversion below 0.01% (van der Star et al., 2008). In conclusion, controlling biofilm thickness and improving Anammox enrichment purity are essential for N2O mitigation in Anammox systems.
## Potential mechanisms for N2O mitigation in the Anammox process
The most important metabolic feature of Anammox is its ability to utilize NO2 –, thereby preventing its accumulation and reducing N2O production from both ND and HD pathways (Yang et al., 2009). Studies have demonstrated that NO2 – accumulation is limited due to the dominance of Anammox in the aerobic phase (Zhang et al., 2021; Zhang et al., 2019a). NO2 – accumulation can result in the downregulation of the NosZ gene, which is responsible for the reduction of N2O to N2, and Anammox could alleviate the issues associated with NO2 – accumulation to some extent (Highton et al., 2022).
Studies have also found that some Anammox bacteria (AnAOB) are able to utilize volatile fatty acids as electron donors for dissimilatory nitrate reduction to ammonium (DNRA) metabolism (Wang et al., 2022). This metabolic pathway could be beneficial to enhance the tolerance of AnAOB to organic compounds and also compete with heterotrophic bacteria to mitigate N2O produced resulting from HD. (Castro-Barros et al., 2017; Li et al., 2020b).
Another significant feature of Anammox is its capacity to utilize NO (Wan et al., 2021). NO plays a crucial role in the microbial nitrogen-cycling network as an ozone-depleting agent and a precursor of the greenhouse gas N2O (Hu et al., 2019). The findings indicated that the total release of NO ranged from only 0.014% to 0.028% under the laboratory study conditions. The main releases occurred during high aeration periods and the dissolved NO concentration in the wastewater was<0.05% of the gaseous release due to the very low solubility of NO (Fuerhacker et al., 2001). AnAOB can grow through the oxidation of NH4 + and NO reduction, even in the absence of NO2 – (4NH4 ++6NO → 5 N2 + 6H2O + 4H+) (Hu et al., 2019). This unique capability, supported by lab-scale evidence, can be harnessed to manage NO and N2O discharges (Zhuang et al., 2020). Kartal et al. (2010) investigated the impact of extremely high NO flux on AnAOB and observed that AnAOB were not inhibited by NO. Instead, they observed simultaneous degradation of NH4 + and NO without N2O production, providing further support for NO as an intermediate in Anammox. Nevertheless, the mechanisms and metabolic kinetics of NO metabolism by AnAOB are still unknown, and further experimental characterization in this area is necessary for future research.
## Effects of Anammox aggregate forms on N2O production
The microorganisms in wastewater treatment reactors often exist in three distinct aggregate forms: floc sludge, biofilm and granular sludge. Given the slow propagation rate, long doubling time, and sensitivity to ambient conditions, it is difficult for AnAOB to grow and enrich in flocs, and biomass retention strategies are often adopted in studies. Thus, current studies on N2O emissions focus predominantly on Anammox granules and biofilms, and have rarely been reported in flocs (Wang et al., 2018; Zhang et al., 2023).
Granules are compact, dense aggregates with a nearly spherical shape, and settle significantly faster than flocs (Lemaire et al., 2008). In Anammox granular systems, due to the lack of external carbon sources, the carbon source for denitrification mainly come from the hydrolysis and acidification of proteins and polysaccharides on the surface of the granular sludge. However, both are long-chain molecules, and the time required for hydrolysis and acidification is long. When the rate of NO3 – production from Anammox exceeds the rate of hydrolysis and acidification of proteins and polysaccharides, it leads to N2O accumulation (Zhu et al., 2018). Although microbial diversity increased with increasing sludge particle diameter (<2.5 mm), N2O also increased. (Luo et al., 2017). Besides, when the particle radius was larger than the maximum mass transfer distance, the Anammox activity in the particles was inhibited, thus impairing the nitrogen removal (Zhu et al., 2018). Therefore, the particle size should be controlled to improve the process efficiency and mitigate greenhouse gas production.
Biofilms are defined as densely aggregated biomass, which is generally attached to gravel or plastic media surfaces (Todt and Dörsch, 2016). Biofilms normally harbor aerobic and anoxic zones that mediate close interactions between different nitrogen-transforming organisms. Nitrifiers predominantly inhabit in the aerobic zone, whereas heterotrophic denitrifiers and AnAOB tend to enrich the anoxic zone (Jin et al., 2012). Peng et al. (2019) concluded that N2O emissions from Anammox biofilms were mainly caused by denitrifiers that rely on soluble microbial products for growth. Modelling results indicated that thicker biofilm thickness (1500 μm) minimized N2O emissions. This was attributed to the fact that the detachment rate decreased significantly with increasing biofilm thickness. At the same time the kinetics of NO3 – reduction to NO is slower than the reduction step from NO to N2, making the residence time of N2O in the biofilm shorter in thinner biofilms thus leading to higher emissions (Eldyasti et al., 2014). Moreover, enhancing Anammox activity not only facilitated high levels of nitrogen removal but also reduced NO and N2O generation in the membrane-aerated biofilm reactor (Ni et al., 2013). In the membrane bioreactor with 97.6% enrichment purity of AnAOB, NO and N2O were detected with nitrogen conversion below 0.01% (van der Star et al., 2008). In conclusion, controlling biofilm thickness and improving Anammox enrichment purity are essential for N2O mitigation in Anammox systems.
## Partial nitrification process: An energy-efficient step in nitrification with implications for N2O emissions
PN process, as an integral part of the nitrogen removal pathway in wastewater treatment, involves the oxidation of NH4 + to NO2 – without proceeding to NO3 –. The primary advantage of this approach lies in the reduced O2 and organic carbon demand, thereby saving energy and lowering operational costs (Duan et al., 2019). PN usually acts as the preliminary reaction to Anammox, which together form the partial nitrification-Anammox (PN/A) process, an innovative autotrophic denitrification technology (Wang et al., 2021). However, significant N2O generation during the PN process poses a serious environmental challenge.
N2O production during the PN process primarily occurs via two mechanisms: ND and NH2OH oxidation pathways. In ND, Ammonia-Oxidizing Bacteria (AOB) oxidize NH4 + to NO2 –, which in turn is reduced to N2O under oxygen-limited conditions (Zhao et al., 2022). In the NH2OH oxidation pathway, N2O is produced directly from NH2OH, an intermediate product of ammonia oxidation. Efforts have been made to mitigate N2O emissions during PN process, primarily through process control strategies that aim to minimize the conditions conducive to N2O production. One critical factor is DO, where low DO levels negatively impact N2O emissions, and N2O EF decreases from 2.35% to 0.57% with an increasing in DO from 0.35 mg/L to 0.85 mg/L (Lv et al., 2016). Another key factor is NO2 – concentration, where both NO2 – accumulation and low DO promote ND pathway, resulting in higher N2O production (Massara et al., 2017). Therefore, the key to control N2O emissions from PN process is to limit ND pathway.
N2O emission characteristics of one-stage and two-stage PN/A systems have been investigated. Generally, N2O emissions from one-stage systems are lower than those from two-stage systems. In one-stage systems, the N2O emissions are relatively lower because these systems minimize the exposure of NO2 – to conditions that promote N2O production. The integrated nature of one-stage systems ensures that the conditions remain less conducive to N2O formation. Conversely, two-stage systems involve separate reactors where NO2 – accumulation is possible. In the nitritation reactor of the two-stage system, conditions that favor N2O production can lead to comparatively higher N2O emissions (Kampschreur et al., 2009). N2O emissions from one-stage and two-stage PN/A systems are shown in Table 1. In a one-stage system of sequencing biofilm batch reactor (SBBR), HD was found to be the primary pathway for N2O production, with Anammox contributing to 83–91% of nitrogen removal (Li et al., 2017). On a related note, Ali et al. (2016) identified that 70% of N2O production on the aerobic surface was dominated by nitrifiers, with the remainder being produced in anoxic zones. In contrast, in two-stage PN/A reactors, N2O emissions were mainly observed in nitritation reactor, often related to NO2 – accumulation. Okabe et al. (2011) identified average N2O emissions in a two-stage PN/A reactor at 4.0 ± 1.5% and 0.1 ± 0.07% of the influent nitrogen load, respectively, and that N2O in the Anammox reactor was mainly originated from HD in the inner part of the granules. Furthermore, in a year-long pilot-scale mainstream two-stage PN/A system, Hausherr et al. (2022) noted that N2O emissions primarily occurred in the PN stage (1.2% of total influent nitrogen), while the Anammox stage accounted for only 4% of the N2O production in the PN stage. Interestingly, it was found that the addition of external carbon sources in the latter reactor can further reduce N2O emissions and remove the NO3 – produced by Anammox (Juan-Diaz et al., 2022). In summary, while PN/A process presents a compelling approach for nitrogen removal, understanding and mitigating N2O emissions remain critical challenges.
## Unraveling the mechanism of N2O emissions in partial nitrification
N2O production during the PN process primarily occurs via two mechanisms: ND and NH2OH oxidation pathways. In ND, Ammonia-Oxidizing Bacteria (AOB) oxidize NH4 + to NO2 –, which in turn is reduced to N2O under oxygen-limited conditions (Zhao et al., 2022). In the NH2OH oxidation pathway, N2O is produced directly from NH2OH, an intermediate product of ammonia oxidation. Efforts have been made to mitigate N2O emissions during PN process, primarily through process control strategies that aim to minimize the conditions conducive to N2O production. One critical factor is DO, where low DO levels negatively impact N2O emissions, and N2O EF decreases from 2.35% to 0.57% with an increasing in DO from 0.35 mg/L to 0.85 mg/L (Lv et al., 2016). Another key factor is NO2 – concentration, where both NO2 – accumulation and low DO promote ND pathway, resulting in higher N2O production (Massara et al., 2017). Therefore, the key to control N2O emissions from PN process is to limit ND pathway.
## N2O emissions in PN/A systems: Insights from reactor configurations and performance
N2O emission characteristics of one-stage and two-stage PN/A systems have been investigated. Generally, N2O emissions from one-stage systems are lower than those from two-stage systems. In one-stage systems, the N2O emissions are relatively lower because these systems minimize the exposure of NO2 – to conditions that promote N2O production. The integrated nature of one-stage systems ensures that the conditions remain less conducive to N2O formation. Conversely, two-stage systems involve separate reactors where NO2 – accumulation is possible. In the nitritation reactor of the two-stage system, conditions that favor N2O production can lead to comparatively higher N2O emissions (Kampschreur et al., 2009). N2O emissions from one-stage and two-stage PN/A systems are shown in Table 1. In a one-stage system of sequencing biofilm batch reactor (SBBR), HD was found to be the primary pathway for N2O production, with Anammox contributing to 83–91% of nitrogen removal (Li et al., 2017). On a related note, Ali et al. (2016) identified that 70% of N2O production on the aerobic surface was dominated by nitrifiers, with the remainder being produced in anoxic zones. In contrast, in two-stage PN/A reactors, N2O emissions were mainly observed in nitritation reactor, often related to NO2 – accumulation. Okabe et al. (2011) identified average N2O emissions in a two-stage PN/A reactor at 4.0 ± 1.5% and 0.1 ± 0.07% of the influent nitrogen load, respectively, and that N2O in the Anammox reactor was mainly originated from HD in the inner part of the granules. Furthermore, in a year-long pilot-scale mainstream two-stage PN/A system, Hausherr et al. (2022) noted that N2O emissions primarily occurred in the PN stage (1.2% of total influent nitrogen), while the Anammox stage accounted for only 4% of the N2O production in the PN stage. Interestingly, it was found that the addition of external carbon sources in the latter reactor can further reduce N2O emissions and remove the NO3 – produced by Anammox (Juan-Diaz et al., 2022). In summary, while PN/A process presents a compelling approach for nitrogen removal, understanding and mitigating N2O emissions remain critical challenges.
## Partial denitrification process: An energy-efficient step in denitrification with implications for N2O emissions
PD process is an intermediate stage in denitrification, during which NO3 – is reduced to NO2 – instead of being fully reduced to N2. PD is increasingly being recognized as an essential component of novel and energy-efficient nitrogen removal strategies. It is increasingly acknowledged for its potential role in providing substrate for Anammox, thereby leading to the development of partial denitrification-Anammox (PD/A) process (Al-Hazmi et al., 2023b; Du et al., 2022).
N2O production during PD process is mainly due to the HD pathway, where N2O may be produced as intermediate or final products of denitrification. Previous studies have shown that N2O production could almost be ignored when NO2 – accumulation occurs before complete depletion of NO3 –, which proved the potential of PD process in achieving N2O emission reduction (Du et al., 2016; Ma et al., 2017a). Furthermore, the types and enzyme metabolism mechanisms of denitrifiers can significantly affect N2O production (Zhang et al., 2020b). Despite the potential for PD to contribute to N2O emissions, the understanding of the mechanisms underlying these emissions and the conditions promoting them is still far from complete. Further research into the N2O emissions characteristics of PD process is needed in order to better predict and manage these emissions in the context of wastewater treatment.
Compared to PN, the most remarkable advantages of PD are its ability to provide a steady substrate for Anammox and its tolerance of environmental factors (He et al., 2023). NO2 – and NO produced in PD process can be utilized by Anammox, avoiding N2O precursors accumulation (Al-Hazmi et al., 2023a). The N2O emission characteristics across different PD/A reactor configurations and operational conditions are summarized in Table 1. Du et al. (2020) treated the real municipal wastewater by a one-stage PD/A process with a maximum N2O accumulation of only 0.7% of the influent nitrogen load, which was considerably lower than the BNR process (Wunderlin et al., 2012). Furthermore, He et al. (2023) demonstrated that in an integrated PD/A system with low C/N, PD/A and denitrification was the dominant nitrogen removal pathway, accounting for 91.3% and 8.7%, respectively, with N2O emissions peaking between 0.18 and 0.25 mg N/L. In an INPDA-SBBR system, the low N2O emissions were attributed to the high-level expression of functional genes associated with Anammox (Zhou et al., 2020). Overall, the collaboration between denitrifiers and AnAOB in one-stage PD/A process determined the reactor performance and N2O production. PD/A process offers a promising strategy for managing N2O emissions in the anoxic stage of wastewater treatment, but the delicate balance of conditions required for a stable and efficient PD/A process requires careful monitoring and control of process parameters (Al-Hazmi et al., 2023a).
## Unraveling the mechanism of N2O emissions in partial denitrification
N2O production during PD process is mainly due to the HD pathway, where N2O may be produced as intermediate or final products of denitrification. Previous studies have shown that N2O production could almost be ignored when NO2 – accumulation occurs before complete depletion of NO3 –, which proved the potential of PD process in achieving N2O emission reduction (Du et al., 2016; Ma et al., 2017a). Furthermore, the types and enzyme metabolism mechanisms of denitrifiers can significantly affect N2O production (Zhang et al., 2020b). Despite the potential for PD to contribute to N2O emissions, the understanding of the mechanisms underlying these emissions and the conditions promoting them is still far from complete. Further research into the N2O emissions characteristics of PD process is needed in order to better predict and manage these emissions in the context of wastewater treatment.
## N2O emissions in PD/A systems: Insights from reactor configurations and performance
Compared to PN, the most remarkable advantages of PD are its ability to provide a steady substrate for Anammox and its tolerance of environmental factors (He et al., 2023). NO2 – and NO produced in PD process can be utilized by Anammox, avoiding N2O precursors accumulation (Al-Hazmi et al., 2023a). The N2O emission characteristics across different PD/A reactor configurations and operational conditions are summarized in Table 1. Du et al. (2020) treated the real municipal wastewater by a one-stage PD/A process with a maximum N2O accumulation of only 0.7% of the influent nitrogen load, which was considerably lower than the BNR process (Wunderlin et al., 2012). Furthermore, He et al. (2023) demonstrated that in an integrated PD/A system with low C/N, PD/A and denitrification was the dominant nitrogen removal pathway, accounting for 91.3% and 8.7%, respectively, with N2O emissions peaking between 0.18 and 0.25 mg N/L. In an INPDA-SBBR system, the low N2O emissions were attributed to the high-level expression of functional genes associated with Anammox (Zhou et al., 2020). Overall, the collaboration between denitrifiers and AnAOB in one-stage PD/A process determined the reactor performance and N2O production. PD/A process offers a promising strategy for managing N2O emissions in the anoxic stage of wastewater treatment, but the delicate balance of conditions required for a stable and efficient PD/A process requires careful monitoring and control of process parameters (Al-Hazmi et al., 2023a).
## Denitrifying phosphorus removal process: A promising approach for sustainable biological nitrogen and phosphorus removal
DPR process is recognized as the most promising strategy for enhanced biological phosphorus removal (Qiu et al., 2019). Unlike traditional biological phosphorus removal, DPR utilizes denitrifying phosphorus accumulating organisms (DPAOs) that employ NO3 – or NO2 – as electron acceptors instead of O2 and use intracellular carbon as electron donor under anoxic conditions to simultaneously accomplish nitrogen removal and phosphorus absorption (Guo et al., 2018; Zhang et al., 2020a). A significant challenge in DPR systems was that N2O, rather than N2, tended to be the dominant denitrification product when poly-hydroxyalkanoates (PHA) serve as the carbon source. This phenomenon considerably increased the carbon footprint of WWTPs (Jia et al., 2012; Lemaire et al., 2006).
Studies have shown that weak competition of NOS for electrons and NO2 – accumulation are the primary reasons contributing to N2O production in DPR process (Li et al., 2013b). NO2 – accumulation could also severely inhibit NOR activity, leading to N2O accumulation (Kampschreur et al., 2009). PHA degradation kinetics directly reflected DPR efficiency and N2O production. Slower PHA degradation rates could lead to electron competition among denitrifying enzymes, potentially affecting enzymes expression. The N2O reduction is the final step in denitrification, which makes NOS more difficult to compete for electrons than other enzymes, ultimately leading to an increase in N2O production (Liu et al., 2015; Wang et al., 2011b). However, contradicting these findings, Wei et al. (2014) observed that even lower PHA degradation rates did not stimulate higher N2O generation, and the electron distribution for NOS was not affected by PHA oxidation rate. Moreover, PHA degradation rates are correlated with the amount of PHA synthesised, this issue can be mitigated by optimizing the anaerobic during to increase PHA synthesis (Wang et al., 2011b). The above results indicate that the effects of PHA on NOS in DPR process need to be further explored.
N2O emissions in DPR systems are significantly affected by organic shock loads, temperature and microbial communities. Variations in carbon source shocks significantly affect denitrifying enzyme activity, the amount and composition of anaerobically synthesized PHA, leading to oxidative stress and directly stimulating N2O production (Wang et al., 2011a). Li et al. (2013a) investigated the impact of organic shock loads on N2O production in a DPR system and found that N2O-N production ranged from 1.2% to 7.12% of TN removal. A continuous NO3 – dosing strategy has proven effective in mitigating this adverse effect on N2O production. Temperature has also been found to influence N2O production, with a 43.3% higher rate at 10 °C compared to 25 °C (Jia et al., 2016). DPAOs and denitrifying glycogen accumulating organisms (DGAOs) are the two main N2O-contributing microorganisms present in DPR systems, and both compete for carbon sources, which can affect the outcomes of denitrification (Ren et al., 2023). Interestingly, some studies suggested that DPAOs appeared to have higher denitrification activity, DGAOs were the culprits of N2O accumulation in DPR systems (Lemaire et al., 2006). This aligned with the findings of Roy et al. (2021), where Candidatus Accumulibacter organisms showed complete denitrification, while Candidatus Competibacter organisms were limited to reducing NO3 – to NO2 –. This observation highlights the low N2O emissions potential of DPR systems dominated by Accumulibacter, which could be a promising focus for designing carbon–neutral treatment processes.
## Unraveling the mechanism of N2O emissions in denitrifying phosphorus removal
Studies have shown that weak competition of NOS for electrons and NO2 – accumulation are the primary reasons contributing to N2O production in DPR process (Li et al., 2013b). NO2 – accumulation could also severely inhibit NOR activity, leading to N2O accumulation (Kampschreur et al., 2009). PHA degradation kinetics directly reflected DPR efficiency and N2O production. Slower PHA degradation rates could lead to electron competition among denitrifying enzymes, potentially affecting enzymes expression. The N2O reduction is the final step in denitrification, which makes NOS more difficult to compete for electrons than other enzymes, ultimately leading to an increase in N2O production (Liu et al., 2015; Wang et al., 2011b). However, contradicting these findings, Wei et al. (2014) observed that even lower PHA degradation rates did not stimulate higher N2O generation, and the electron distribution for NOS was not affected by PHA oxidation rate. Moreover, PHA degradation rates are correlated with the amount of PHA synthesised, this issue can be mitigated by optimizing the anaerobic during to increase PHA synthesis (Wang et al., 2011b). The above results indicate that the effects of PHA on NOS in DPR process need to be further explored.
## Factors influencing N2O emissions in denitrifying phosphorus removal systems
N2O emissions in DPR systems are significantly affected by organic shock loads, temperature and microbial communities. Variations in carbon source shocks significantly affect denitrifying enzyme activity, the amount and composition of anaerobically synthesized PHA, leading to oxidative stress and directly stimulating N2O production (Wang et al., 2011a). Li et al. (2013a) investigated the impact of organic shock loads on N2O production in a DPR system and found that N2O-N production ranged from 1.2% to 7.12% of TN removal. A continuous NO3 – dosing strategy has proven effective in mitigating this adverse effect on N2O production. Temperature has also been found to influence N2O production, with a 43.3% higher rate at 10 °C compared to 25 °C (Jia et al., 2016). DPAOs and denitrifying glycogen accumulating organisms (DGAOs) are the two main N2O-contributing microorganisms present in DPR systems, and both compete for carbon sources, which can affect the outcomes of denitrification (Ren et al., 2023). Interestingly, some studies suggested that DPAOs appeared to have higher denitrification activity, DGAOs were the culprits of N2O accumulation in DPR systems (Lemaire et al., 2006). This aligned with the findings of Roy et al. (2021), where Candidatus Accumulibacter organisms showed complete denitrification, while Candidatus Competibacter organisms were limited to reducing NO3 – to NO2 –. This observation highlights the low N2O emissions potential of DPR systems dominated by Accumulibacter, which could be a promising focus for designing carbon–neutral treatment processes.
## Comammox process: The green solution for nitrification in wastewater treatment
Comammox are organisms that harbor all the necessary enzymes to execute the entire nitrification process, facilitating the oxidation from NH4 + to NO3 –. So far, Comammox are confined to phylogenetic lineage II of the genus Nitrospira. Importantly, due to the absence of NOR in its genome, Comammox do not produce N2O. Instead, any N2O generated was a result of abiotic NH2OH conversion, with levels released being similar to those of ammonia-oxidizing archaea (AOA), but considerably lower than AOB (Han et al., 2021; Kits et al., 2019). Previous studies reported N2O yields of 0.51% for AOB, a range of 0.068–0.158% for AOA, and 0.07–0.2% for Comammox (Han et al., 2021; Kits et al., 2019). In a pilot-scale oxidation ditch, the enrichment of Comammox on the biocarrier decreased the N2O emissions of the system by 36.77% (Tian et al., 2021). Further genomic and physiological investigations demonstrated that Comammox Nitrospira had extremely high ammonia affinity, growth yield and metabolic potential in comparison to AOB and AOA (Sakoula et al., 2021). Comammox have been documented to dominate the nitrification process (Cui et al., 2023). Given these findings, the potential replacement of AOB and Nitrite-Oxidizing Bacteria (NOB) with Comammox in aerobic process offers an intriguing strategy for reducing N2O production. This transition towards Comammox technology represents an innovative step forward in the attempts to curb greenhouse gas emissions from WWTPs.
## Sulfur-driven autotrophic denitrification process: Unlocking novel N2O mitigation strategies
SDAD is an autotrophic denitrification process that utilizes reductive sulfur as electron donors and NO3 – or NO2 – as electron acceptors, converting these to N2 under anoxic conditions (Cao et al., 2021). Sulfur (S0), sulfide (S2-) and thiosulfate (S2O3 2-) have been demonstrated to serve as effective electron donors for SDAD (Di Capua et al., 2015).
Modelling results showed that SDAD process could accumulate quantities of N2O due to low autotrophic N2O reduction rates, but N2O emissions could be dramatically mitigated by increasing SRT or S/N (Liu et al., 2016b). The N2O release levels in a SDAD granular sludge reactor increased from 0.002% to 0.405% of the influent N load as the sulfide/nitrogen mass ratio decreased from 2.1 to 1.4 (Yang et al., 2016). Sulfides, being prevalent in SDAD systems, critically impact N2O emissions. Previous studies proposed that sulfide inhibition of N2O reduction was attributed to the formation of metal sulfide precipitates by copper and sulfur ions in the periplasm, thereby affecting NOS activity. (Richardson et al., 2009). However, some studies suggested that dissolved sulfide present during SDAD neither exerted toxicity nor inhibited soluble copper-containing NOS, which instead could significantly mitigate N2O emissions (Pan et al., 2013b; Yang et al., 2016). Pan et al. (2013b) verified that H2S, rather than sulfide, was the actual inhibitor of N2O reduction and that its inhibitory effect was reversible. Therefore, in sulfide-containing systems, the combined effect of pH and sulfide inhibited the N2O reduction. Yang et al. (2016) derived the same results and demonstrated that dissolved sulfide stimulated N2O reduction (rather than inhibiting it) regardless of SDAD sludge granule size. This stimulation effectively alleviated electron competition among various denitrifying enzymes.
In recent years, coupling SDAD with Anammox to enhance nitrogen removal has been reported (Qian et al., 2018; Qin et al., 2019). The specific process involves the reduction of NO3 – to NO2 – by autotrophic sulfur-oxidizing nitrate-reducing bacteria, providing the substrate for Anammox while further reducing the NO3 – produced by Anammox (Zhang et al., 2022). Within SDAD-Anammox systems, autotrophic denitrification was probably the dominant N2O production pathway, as corroborated by various studies (Qian et al., 2018; Yang et al., 2016). Cao et al. (2021) discovered that with decreasing pH, the N2O EF was only 0.06% to 0.71% in an innovative Anammox coupled with sulfite-driven autotrophic denitrification system. Qian et al. (2018) achieved a 29.6% reduction in N2O emissions by coupling Anammox with a thiosulfate-driven denitrification system. Theoretically, the SDAD-Anammox process holds significant potential to mitigate N2O emissions. However, the cooperative interactions of the relevant bacterial genera involved in this process remain under-explored and warrant further investigation.
## Factors influencing N2O emissions in sulfur-driven autotrophic denitrification systems
Modelling results showed that SDAD process could accumulate quantities of N2O due to low autotrophic N2O reduction rates, but N2O emissions could be dramatically mitigated by increasing SRT or S/N (Liu et al., 2016b). The N2O release levels in a SDAD granular sludge reactor increased from 0.002% to 0.405% of the influent N load as the sulfide/nitrogen mass ratio decreased from 2.1 to 1.4 (Yang et al., 2016). Sulfides, being prevalent in SDAD systems, critically impact N2O emissions. Previous studies proposed that sulfide inhibition of N2O reduction was attributed to the formation of metal sulfide precipitates by copper and sulfur ions in the periplasm, thereby affecting NOS activity. (Richardson et al., 2009). However, some studies suggested that dissolved sulfide present during SDAD neither exerted toxicity nor inhibited soluble copper-containing NOS, which instead could significantly mitigate N2O emissions (Pan et al., 2013b; Yang et al., 2016). Pan et al. (2013b) verified that H2S, rather than sulfide, was the actual inhibitor of N2O reduction and that its inhibitory effect was reversible. Therefore, in sulfide-containing systems, the combined effect of pH and sulfide inhibited the N2O reduction. Yang et al. (2016) derived the same results and demonstrated that dissolved sulfide stimulated N2O reduction (rather than inhibiting it) regardless of SDAD sludge granule size. This stimulation effectively alleviated electron competition among various denitrifying enzymes.
## N2O emission characteristics in coupling sulfur-driven autotrophic denitrification and Anammox
In recent years, coupling SDAD with Anammox to enhance nitrogen removal has been reported (Qian et al., 2018; Qin et al., 2019). The specific process involves the reduction of NO3 – to NO2 – by autotrophic sulfur-oxidizing nitrate-reducing bacteria, providing the substrate for Anammox while further reducing the NO3 – produced by Anammox (Zhang et al., 2022). Within SDAD-Anammox systems, autotrophic denitrification was probably the dominant N2O production pathway, as corroborated by various studies (Qian et al., 2018; Yang et al., 2016). Cao et al. (2021) discovered that with decreasing pH, the N2O EF was only 0.06% to 0.71% in an innovative Anammox coupled with sulfite-driven autotrophic denitrification system. Qian et al. (2018) achieved a 29.6% reduction in N2O emissions by coupling Anammox with a thiosulfate-driven denitrification system. Theoretically, the SDAD-Anammox process holds significant potential to mitigate N2O emissions. However, the cooperative interactions of the relevant bacterial genera involved in this process remain under-explored and warrant further investigation.
## Nitrate/nitrite-dependent anaerobic methane oxidation process: Revolutionizing nitrogen removal and greenhouse gas reduction
Recently, the discovery of the n-DAMO process has opened up a new avenue to utilize CH4 as an alternative carbon source for denitrification (Ettwig et al., 2010; Liu et al., 2021a). This process can simultaneously mitigate CH4 and N2O emissions from aquatic ecosystems by oxidizing CH4 to provide electrons for denitrification, thereby bridging the carbon–nitrogen cycles in the biogeochemical sphere (Wang et al., 2017).
Two distinct microbial groups were involved in the n-DAMO process: n-DAMO archaea and n-DAMO bacteria (Haroon et al., 2013; Nie et al., 2021). N-DAMO archaea were able to utilize CH4 to provide electrons to reduce NO3 – to NO2 –, meanwhile the methane-driven DNRA process is also performed by n-DAMO archaea. N-DAMO bacteria can reduce NO2 – to N2 and possess part of the genes required for denitrification. Although these bacteria lack the gene NosZ, encoding NOS, they have NO dismutase, which dismutates NO into N2 and O2 (Ettwig et al., 2010). Consequently, N2O is rarely produced during the metabolism of n-DAMO microorganisms. To date, no pure n-DAMO strains have been cultured, so there is no absolute evidence that N2O is not produced as an intermediate product of the n-DAMO reaction, but the N2O yields in n-DAMO reactors studied so far have been extremely low (Ettwig et al., 2010; Ma et al., 2017b). In addition, macrogenomics analysis revealed that N2O stimulated the activity and abundance of methanotrophic bacteria, favoring electron transport and ATP production, and thus spurring the n-DAMO process (Cheng et al., 2019). This suggests that the n-DAMO process may have the potential to utilize N2O as an electron acceptor.
Research into the N2O emissions characteristics of n-DAMO process has yielded promising results. Valenzuela et al. (2021a) identified that no N2O production was detected at NO3 – loading rates of 22–66 mgN/(m3·h) in a continuous n-DAMO system, presumably mainly attributed to the N2O reducing ability of the system flora and the strict control of DO and pH. Furthermore, minimal N2O production was detected in an n-DAMO-dominated anaerobic downflow hanging sponge reactor, possibly originating from denitrifiers or specific methanotrophic bacteria (Tran et al., 2020). Ma et al. (2017b) also reported N2O conversion rates from 0.03% to 0.11% in an n-DAMO system, considerably lower than the conventional processes. However, it is important to note that while the available findings on N2O emissions in n-DAMO reactors are promising, there is a relative paucity of studies on this topic.
## Unraveling the metabolic mechanisms of n-DAMO microorganisms
Two distinct microbial groups were involved in the n-DAMO process: n-DAMO archaea and n-DAMO bacteria (Haroon et al., 2013; Nie et al., 2021). N-DAMO archaea were able to utilize CH4 to provide electrons to reduce NO3 – to NO2 –, meanwhile the methane-driven DNRA process is also performed by n-DAMO archaea. N-DAMO bacteria can reduce NO2 – to N2 and possess part of the genes required for denitrification. Although these bacteria lack the gene NosZ, encoding NOS, they have NO dismutase, which dismutates NO into N2 and O2 (Ettwig et al., 2010). Consequently, N2O is rarely produced during the metabolism of n-DAMO microorganisms. To date, no pure n-DAMO strains have been cultured, so there is no absolute evidence that N2O is not produced as an intermediate product of the n-DAMO reaction, but the N2O yields in n-DAMO reactors studied so far have been extremely low (Ettwig et al., 2010; Ma et al., 2017b). In addition, macrogenomics analysis revealed that N2O stimulated the activity and abundance of methanotrophic bacteria, favoring electron transport and ATP production, and thus spurring the n-DAMO process (Cheng et al., 2019). This suggests that the n-DAMO process may have the potential to utilize N2O as an electron acceptor.
## N2O emission characteristics of n-DAMO systems: A new path to sustainability
Research into the N2O emissions characteristics of n-DAMO process has yielded promising results. Valenzuela et al. (2021a) identified that no N2O production was detected at NO3 – loading rates of 22–66 mgN/(m3·h) in a continuous n-DAMO system, presumably mainly attributed to the N2O reducing ability of the system flora and the strict control of DO and pH. Furthermore, minimal N2O production was detected in an n-DAMO-dominated anaerobic downflow hanging sponge reactor, possibly originating from denitrifiers or specific methanotrophic bacteria (Tran et al., 2020). Ma et al. (2017b) also reported N2O conversion rates from 0.03% to 0.11% in an n-DAMO system, considerably lower than the conventional processes. However, it is important to note that while the available findings on N2O emissions in n-DAMO reactors are promising, there is a relative paucity of studies on this topic.
## Mitigation strategies for N2O emissions during nitrogen removal
DO is a critical parameter affecting N2O production and emission during nitrification. In the conventional nitrification process, N2O EF was significantly higher at low aeration rates (10.1%) than at high aeration rates (2.3%) (Wang et al., 2016a). In contrast, Wen et al. (2020) managed to enhance TN removal efficiency and reduce N2O emissions from 0.65% to 0.15% of TN removal in a Comammox-enriched bioreactor by adjusting DO downward from 3.5 mg/L to 0.5 mg/L. The N2O emissions reduction under low DO conditions was mainly attributed to the predominant role of Comammox in the system over canonical AOB (Li et al., 2021a). Furthermore, in a Comammox system, N2O EF was lower under the intermittent aeration strategy compared to continuous aeration (Liu et al., 2021c). Therefore, applications of the Comammox process are valuable in optimising the aeration strategy to reduce energy consumption and N2O emissions.
Carbon sources provide electron acceptors for HD, and insufficient carbon supply could exacerbate the competition among denitrifying electron acceptors, leading to ID (Pan et al., 2013a). In the case of an adequate supply of external carbon sources, N2O accumulation was extremely low and not significantly correlated with the type of carbon source. (Zhou et al., 2012). However, the addition of carbon sources not only increases the cost of wastewater treatment, but also leads to excessive sludge production. Autotrophic nitrogen removal processes (e.g., Anammox, SDAD, etc.) do not utilize organic carbon sources, but only inorganic substances to provide electron donors for nitrogen removal, effectively solving the problems of N2O accumulation caused by insufficient carbon sources.
The composition of the microbial community structure within activated sludge in wastewater treatment processes significantly affects both nitrogen removal capacity and N2O emission capacity (Lin et al., 2022). Mitigation of N2O emissions may require strategic adaptation of the microbial community, in particular by eliminating N2O producers and increasing N2O consumers. In Anammox-based systems, AOB and denitrifiers were the main contributors to N2O production, and AnAOB were able to mitigate N2O emissions indirectly (Lotti et al., 2014). AOB protect AnAOB from DO toxicity (Yan et al., 2010), while denitrifiers contribute to NO3 – reduction and protect AnAOB from organic carbon toxicity (Langone et al., 2014). N2O production could be significantly reduced by AnAOB through the consumption of NO and NO2 – produced by denitrifiers (Wan et al., 2021). Furthermore, the potential substitution of AOB by Comammox, which have a lower N2O production rate, could provide the substrate NO2 – to Anammox-dominated systems (Kits et al., 2019; Lin et al., 2022). DNRA bacteria can reduce NO3 – to provide the substrate NO2 – and NH4 + to AnAOB, thereby enhancing the nitrogen removal capacity of AnAOB (Li et al., 2020b; Zhou et al., 2023). In the calcium nitrate-added systems, a mutualistic symbiosis existed between denitrifiers, DNRA bacteria and AnAOB, which promoted the rapid start-up of Anammox (Sheng et al., 2021). AnAOB and endogenous denitrifiers also exhibit well synergistic relationships (Gao et al., 2023; Wang et al., 2023a). Clearly, AnAOB play an active role in complex microbial systems. The inoculation of mature Anammox granular sludge into wastewater treatment systems may be a potential microbial control of N2O emissions. Finally, drastic changes in microbial community processes are associated with impaired nitrification and elevated N2O emissions (Gruber et al., 2021). Therefore, maintaining a stable microbial community is an essential factor in avoiding abrupt elevations in N2O emissions.
Dissolved N2O in wastewater treatment plays a significant role in the overall N2O emissions from WWTPs. It's important to understand the role of dissolved N2O and its impact on emissions. N2O is produced within wastewater treatment systems primarily during nitrification and denitrification processes. It can accumulate in the liquid phase of the treatment system, leading to dissolved N2O. This dissolved N2O is in equilibrium with the gaseous N2O phase, but its concentration depends on factors like temperature, pressure, and the presence of other gases (Wunderlin et al., 2012). Dissolved N2O is transported along with the treated wastewater, including the effluent and any discharge. It can be found in various compartments of the treatment process, such as aeration zones, anoxic zones, and settling tanks, where denitrification occurs. The distribution of dissolved N2O can vary depending on the system's design and operational conditions. The presence of dissolved N2O can potentially contribute to emissions during effluent discharge and air transfer in aeration zones (Kampschreur et al., 2009; Wunderlin et al., 2012). Factors affecting emissions include dissolved N2O concentration, temperature, and treatment configuration. Higher dissolved N2O concentrations in wastewater increase the potential for emissions. Higher temperatures tend to reduce the solubility of N2O in water, potentially increasing emissions (Kampschreur et al., 2009). The design and configuration of the treatment system, including the presence of specific N2O reduction mechanisms, can affect emissions. To reduce emissions, strategies like effluent degassing, optimized aeration, and biological N2O reduction can be employed. In summary, dissolved N2O in wastewater treatment is a crucial contributor to overall N2O emissions, and efficient management and mitigation strategies are essential for minimizing its environmental impact.
## Optimize operating parameters
DO is a critical parameter affecting N2O production and emission during nitrification. In the conventional nitrification process, N2O EF was significantly higher at low aeration rates (10.1%) than at high aeration rates (2.3%) (Wang et al., 2016a). In contrast, Wen et al. (2020) managed to enhance TN removal efficiency and reduce N2O emissions from 0.65% to 0.15% of TN removal in a Comammox-enriched bioreactor by adjusting DO downward from 3.5 mg/L to 0.5 mg/L. The N2O emissions reduction under low DO conditions was mainly attributed to the predominant role of Comammox in the system over canonical AOB (Li et al., 2021a). Furthermore, in a Comammox system, N2O EF was lower under the intermittent aeration strategy compared to continuous aeration (Liu et al., 2021c). Therefore, applications of the Comammox process are valuable in optimising the aeration strategy to reduce energy consumption and N2O emissions.
Carbon sources provide electron acceptors for HD, and insufficient carbon supply could exacerbate the competition among denitrifying electron acceptors, leading to ID (Pan et al., 2013a). In the case of an adequate supply of external carbon sources, N2O accumulation was extremely low and not significantly correlated with the type of carbon source. (Zhou et al., 2012). However, the addition of carbon sources not only increases the cost of wastewater treatment, but also leads to excessive sludge production. Autotrophic nitrogen removal processes (e.g., Anammox, SDAD, etc.) do not utilize organic carbon sources, but only inorganic substances to provide electron donors for nitrogen removal, effectively solving the problems of N2O accumulation caused by insufficient carbon sources.
## Regulation of microbial community structure
The composition of the microbial community structure within activated sludge in wastewater treatment processes significantly affects both nitrogen removal capacity and N2O emission capacity (Lin et al., 2022). Mitigation of N2O emissions may require strategic adaptation of the microbial community, in particular by eliminating N2O producers and increasing N2O consumers. In Anammox-based systems, AOB and denitrifiers were the main contributors to N2O production, and AnAOB were able to mitigate N2O emissions indirectly (Lotti et al., 2014). AOB protect AnAOB from DO toxicity (Yan et al., 2010), while denitrifiers contribute to NO3 – reduction and protect AnAOB from organic carbon toxicity (Langone et al., 2014). N2O production could be significantly reduced by AnAOB through the consumption of NO and NO2 – produced by denitrifiers (Wan et al., 2021). Furthermore, the potential substitution of AOB by Comammox, which have a lower N2O production rate, could provide the substrate NO2 – to Anammox-dominated systems (Kits et al., 2019; Lin et al., 2022). DNRA bacteria can reduce NO3 – to provide the substrate NO2 – and NH4 + to AnAOB, thereby enhancing the nitrogen removal capacity of AnAOB (Li et al., 2020b; Zhou et al., 2023). In the calcium nitrate-added systems, a mutualistic symbiosis existed between denitrifiers, DNRA bacteria and AnAOB, which promoted the rapid start-up of Anammox (Sheng et al., 2021). AnAOB and endogenous denitrifiers also exhibit well synergistic relationships (Gao et al., 2023; Wang et al., 2023a). Clearly, AnAOB play an active role in complex microbial systems. The inoculation of mature Anammox granular sludge into wastewater treatment systems may be a potential microbial control of N2O emissions. Finally, drastic changes in microbial community processes are associated with impaired nitrification and elevated N2O emissions (Gruber et al., 2021). Therefore, maintaining a stable microbial community is an essential factor in avoiding abrupt elevations in N2O emissions.
## Dissolved N2O in wastewater: The silent contributor to N2O emissions
Dissolved N2O in wastewater treatment plays a significant role in the overall N2O emissions from WWTPs. It's important to understand the role of dissolved N2O and its impact on emissions. N2O is produced within wastewater treatment systems primarily during nitrification and denitrification processes. It can accumulate in the liquid phase of the treatment system, leading to dissolved N2O. This dissolved N2O is in equilibrium with the gaseous N2O phase, but its concentration depends on factors like temperature, pressure, and the presence of other gases (Wunderlin et al., 2012). Dissolved N2O is transported along with the treated wastewater, including the effluent and any discharge. It can be found in various compartments of the treatment process, such as aeration zones, anoxic zones, and settling tanks, where denitrification occurs. The distribution of dissolved N2O can vary depending on the system's design and operational conditions. The presence of dissolved N2O can potentially contribute to emissions during effluent discharge and air transfer in aeration zones (Kampschreur et al., 2009; Wunderlin et al., 2012). Factors affecting emissions include dissolved N2O concentration, temperature, and treatment configuration. Higher dissolved N2O concentrations in wastewater increase the potential for emissions. Higher temperatures tend to reduce the solubility of N2O in water, potentially increasing emissions (Kampschreur et al., 2009). The design and configuration of the treatment system, including the presence of specific N2O reduction mechanisms, can affect emissions. To reduce emissions, strategies like effluent degassing, optimized aeration, and biological N2O reduction can be employed. In summary, dissolved N2O in wastewater treatment is a crucial contributor to overall N2O emissions, and efficient management and mitigation strategies are essential for minimizing its environmental impact.
## Research prospects
There were numerous advanced next-generation processes based on Anammox that showed great potential for reducing N2O emissions (Ren et al., 2022), and the following three coupled processes are discussed in this section: integration of Comammox and Anammox process, integration of n-DAMO and Anammox process and dual-coupled PN/A-PD/A process (Fig. 3 ).
Comammox and AnAOB have been reported to exhibit a well-synergistic relationship, holding promising potential for N2O mitigation (Annavajhala et al., 2018; Shao and Wu, 2021). Gottshall et al. (2021) successfully developed a unique hydrogel format, which fostered the coupling of Comammox and Anammox in a granular structure. Within this structure, Comammox acted to deplete O2, creating an anoxic environment, while also providing NO2 – to support the Anammox process, instigating a cooperative and symbiotic relationship. Moreover, biocarriers provided suitable ecological niches for both Comammox and AnAOB due to the O2 gradient between the inner and outer layers formed by DO. Comammox thrived in micro-oxygen environments, predominantly growing on the outer side of the biofilm, while AnAOB resided on the inner side, their synergistic effect played a crucial role in reducing N2O emissions. The integration of Comammox and Anammox process potentially paves the way for achieving nitrogen removal with lower energy consumption and diminished carbon footprints. However, despite these advantages, this process remains under-studied, and the mechanisms driving N2O emissions remain unclear. Comprehensive further research is necessary to unravel a more profound understanding of this process. Although co-occurrence and cooperation between Comammox and AnAOB have been successfully implemented in a full-scale WWTP, direct evidence supporting N2O production in these systems remains elusive (Vilardi et al., 2023).
The integration of n-DAMO and Anammox process holds great promise in treating wastewater, specifically in removing nitrogen and dissolved CH4. This potential stems from the unique characteristics of the n-DAMO microorganisms and AnAOB.
Studies have shown that n-DAMO microorganisms have extremely low growth rates and long doubling times (Raghoebarsing et al., 2006). However, the introduction of AnAOB could help speed up the enrichment time of these microorganisms, subsequently increasing the activity of n-DAMO archaea (Ding et al., 2014). Several researchers have successfully integrated the n-DAMO and Anammox processes. Liu et al. (2021a) utilized mature Anammox granules as biotic carriers to rapidly cultivate combined Anammox and n-DAMO granules. This strategy not only realized high-level nitrogen removal performance, but also curbed the carbon footprint caused by CH4 and N2O emissions. Similarly, Liu et al. (2019) successfully coupled PN, Anammox and n-DAMO processes in a single membrane biofilm reactor with N2O EF of only 0.34 ± 0.01%, which was mainly attributed to the AOB metabolism. In an attempt to limit greenhouse gas emissions, Valenzuela et al. (2021a) constructed a strictly anaerobic process that coupled n-DAMO with NO3 – reduction, resulting in negligible N2O emissions. In the face of increasing environmental concerns, the focus should shift towards the use of such anaerobic processes in biological treatments. In conclusion, the integration of n-DAMO and Anammox process offers a promising approach to wastewater treatment (Wang et al., 2017).
The integration of PN/A and PD/A process, known as dual-coupled PN/A-PD/A process, has recently gained the attention of researchers. This combination addresses the drawbacks of each individual process while enriching the AnAOB more effectively with dual NO2 – supply.
Li et al. (2020a) achieved a high nitrogen removal efficiency (94.6%) in a one-stage bioreactor with dual-coupled PN/A-PD/A process. Building upon this, Cao et al. (2022) proposed an innovative Sidestream Enhanced Mainstream Anammox process that could integrate sidestream treatment into mainstream Anammox process. This process adeptly combines PN and PD, effectively mitigating N2O emissions caused by nitrous acid formation. This strategy provides a promising approach for transitioning Anammox processing from sidestream to mainstream operations. The main advantage of the dual-coupled PN/A-PD/A process lies in its potential to curb N2O emissions. This is achieved by increasing the proportion of nitrogen removal contributed by the Anammox process, while reducing NO2 – accumulation in both aerobic and anoxic zones, thereby lessening N2O production by AOB and HB. The effectiveness of the dual-coupled PN/A-PD/A process in nitrogen removal has been validated in various studies (Kao et al., 2022; Li et al., 2021b). However, the specific pathways of N2O production within this system need further investigation to gain a deeper understanding.
The dynamics of N2O in wastewater treatment systems can be characterized by its production and utilization in both liquid and gas phases. The N2O that escapes into the atmosphere is largely uncontrolled due to the affinity constant of the culture and gas–liquid mass transfer (Conthe et al., 2019). However, the high solubility of N2O in water provides an opportunity for further removal by denitrification in the anoxic zone. The microbial communities involved in denitrification possess the capability to reduce N2O beyond their production levels, thereby having the potential to eliminate additional N2O produced via other pathways aside from their denitrification. Consequently, this establishes denitrification as a potential N2O sink. Notably, the location of the anoxic zone affects the different sources of electron donors utilized by the denitrification metabolism (Fig. 4 ).
Exogenous denitrification relies mainly on external carbon sources. Overcapacity for N2O reduction is widespread among denitrifying communities (Conthe et al., 2019). Bollon et al. (2016) found that in denitrifying biofilters, denitrification diminished the dissolved N2O flux from the upstream nitrification stage by an average of 86%. In alternating anoxic-aerobic reactors, part of the N2O produced in the aerobic phase could be removed by denitrification in the anoxic zone. The addition of external carbon sources or step feeding could further provide electron donors for N2O reduction, reducing emissions (Hu et al., 2011). However, limited organics tend to intensify the competition for electrons between NOR and NOS, leading to higher N2O accumulation (Pan et al., 2013a). Consequently, the amount of carbon sources determines the potential of exogenous denitrification as an N2O sink.
ED utilizes intracellular carbon as an electron donor for nitrogen removal without the addition of external carbon sources (Gong et al., 2021). This process conserves energy by eliminating the need for mixed liquid return flow and enhances nitrogen removal by utilizing intracellular carbon, such as PHA or glycogen (Wang et al., 2016b). However, it's important to note that N2O, not N2, was the major product of denitrification in a DGAOs-dominated ED system, which might be attributed to the inhibition of NOS by elevated levels of NO2 – accumulated during ED (Zeng et al., 2003). Although it has been reported that more N2O was produced by ED with intracellular PHA as carbon sources (Schalk-Otte et al., 2000; Zhou et al., 2012), models have indicated that N2O was mainly produced by nitrification, and the fraction of N2O potentially produced by ED was much lower than that produced by nitrification (Ding et al., 2016). Furthermore, N2O production did not necessarily increase under famine conditions when relying on intracellular polymers unless intracellular carbon was insufficient (Ding et al., 2016). In such cases, N2O accumulation in the anoxic zone is inevitable, whereas an adequate amount of intracellular storage products would produce N2 rather than N2O. Furthermore, it was found that higher intracellular polymers storage in the anaerobic zone resulted in higher ED efficiency and lower N2O production in the anoxic zone (Ding et al., 2022; Wang et al., 2011b). In addition. PHA with a higher PHV proportion could serve as superior electron donors for ED to mitigate N2O emissions (Zhu and Chen, 2011). However, the current research on ED, particularly concerning N2O emissions, is far from comprehensive, calling for further exploration in the future.
N2O-reducing microorganisms are the only known microorganisms in the ecosystem that reduce N2O production, and their abundance and activity can strongly influence the net N2O emissions from WWTPs (Jones et al., 2013; Wang et al., 2023b). The isolation and enrichment of N2O-reducing bacteria can be applied in bioaugmentation technology. This strategy promoted a symbiotic relationship between the newly formed NosZ gene-enriched microbial community in the biocarriers and the original microbial community in the activated sludge, which significantly boosted the N2O emissions reduction in the system (Hong et al., 2019; Liu et al., 2016a). Moreover, this technology could also offset the adverse effects of ammonia overload and aeration failure shock, improving the robustness of the system in mitigating N2O emissions (Tian et al., 2021). However, the composition of real wastewater is complex, and the performance of bioaugmented strains is hardly to fully exploit their unique advantages. Therefore, future research should prioritize improving the adaptability of these strains to more effectively reduce N2O emissions in diverse wastewater contexts.
## Novel Anammox-based processes
There were numerous advanced next-generation processes based on Anammox that showed great potential for reducing N2O emissions (Ren et al., 2022), and the following three coupled processes are discussed in this section: integration of Comammox and Anammox process, integration of n-DAMO and Anammox process and dual-coupled PN/A-PD/A process (Fig. 3 ).
Comammox and AnAOB have been reported to exhibit a well-synergistic relationship, holding promising potential for N2O mitigation (Annavajhala et al., 2018; Shao and Wu, 2021). Gottshall et al. (2021) successfully developed a unique hydrogel format, which fostered the coupling of Comammox and Anammox in a granular structure. Within this structure, Comammox acted to deplete O2, creating an anoxic environment, while also providing NO2 – to support the Anammox process, instigating a cooperative and symbiotic relationship. Moreover, biocarriers provided suitable ecological niches for both Comammox and AnAOB due to the O2 gradient between the inner and outer layers formed by DO. Comammox thrived in micro-oxygen environments, predominantly growing on the outer side of the biofilm, while AnAOB resided on the inner side, their synergistic effect played a crucial role in reducing N2O emissions. The integration of Comammox and Anammox process potentially paves the way for achieving nitrogen removal with lower energy consumption and diminished carbon footprints. However, despite these advantages, this process remains under-studied, and the mechanisms driving N2O emissions remain unclear. Comprehensive further research is necessary to unravel a more profound understanding of this process. Although co-occurrence and cooperation between Comammox and AnAOB have been successfully implemented in a full-scale WWTP, direct evidence supporting N2O production in these systems remains elusive (Vilardi et al., 2023).
The integration of n-DAMO and Anammox process holds great promise in treating wastewater, specifically in removing nitrogen and dissolved CH4. This potential stems from the unique characteristics of the n-DAMO microorganisms and AnAOB.
Studies have shown that n-DAMO microorganisms have extremely low growth rates and long doubling times (Raghoebarsing et al., 2006). However, the introduction of AnAOB could help speed up the enrichment time of these microorganisms, subsequently increasing the activity of n-DAMO archaea (Ding et al., 2014). Several researchers have successfully integrated the n-DAMO and Anammox processes. Liu et al. (2021a) utilized mature Anammox granules as biotic carriers to rapidly cultivate combined Anammox and n-DAMO granules. This strategy not only realized high-level nitrogen removal performance, but also curbed the carbon footprint caused by CH4 and N2O emissions. Similarly, Liu et al. (2019) successfully coupled PN, Anammox and n-DAMO processes in a single membrane biofilm reactor with N2O EF of only 0.34 ± 0.01%, which was mainly attributed to the AOB metabolism. In an attempt to limit greenhouse gas emissions, Valenzuela et al. (2021a) constructed a strictly anaerobic process that coupled n-DAMO with NO3 – reduction, resulting in negligible N2O emissions. In the face of increasing environmental concerns, the focus should shift towards the use of such anaerobic processes in biological treatments. In conclusion, the integration of n-DAMO and Anammox process offers a promising approach to wastewater treatment (Wang et al., 2017).
The integration of PN/A and PD/A process, known as dual-coupled PN/A-PD/A process, has recently gained the attention of researchers. This combination addresses the drawbacks of each individual process while enriching the AnAOB more effectively with dual NO2 – supply.
Li et al. (2020a) achieved a high nitrogen removal efficiency (94.6%) in a one-stage bioreactor with dual-coupled PN/A-PD/A process. Building upon this, Cao et al. (2022) proposed an innovative Sidestream Enhanced Mainstream Anammox process that could integrate sidestream treatment into mainstream Anammox process. This process adeptly combines PN and PD, effectively mitigating N2O emissions caused by nitrous acid formation. This strategy provides a promising approach for transitioning Anammox processing from sidestream to mainstream operations. The main advantage of the dual-coupled PN/A-PD/A process lies in its potential to curb N2O emissions. This is achieved by increasing the proportion of nitrogen removal contributed by the Anammox process, while reducing NO2 – accumulation in both aerobic and anoxic zones, thereby lessening N2O production by AOB and HB. The effectiveness of the dual-coupled PN/A-PD/A process in nitrogen removal has been validated in various studies (Kao et al., 2022; Li et al., 2021b). However, the specific pathways of N2O production within this system need further investigation to gain a deeper understanding.
## Integration of Comammox and Anammox process
Comammox and AnAOB have been reported to exhibit a well-synergistic relationship, holding promising potential for N2O mitigation (Annavajhala et al., 2018; Shao and Wu, 2021). Gottshall et al. (2021) successfully developed a unique hydrogel format, which fostered the coupling of Comammox and Anammox in a granular structure. Within this structure, Comammox acted to deplete O2, creating an anoxic environment, while also providing NO2 – to support the Anammox process, instigating a cooperative and symbiotic relationship. Moreover, biocarriers provided suitable ecological niches for both Comammox and AnAOB due to the O2 gradient between the inner and outer layers formed by DO. Comammox thrived in micro-oxygen environments, predominantly growing on the outer side of the biofilm, while AnAOB resided on the inner side, their synergistic effect played a crucial role in reducing N2O emissions. The integration of Comammox and Anammox process potentially paves the way for achieving nitrogen removal with lower energy consumption and diminished carbon footprints. However, despite these advantages, this process remains under-studied, and the mechanisms driving N2O emissions remain unclear. Comprehensive further research is necessary to unravel a more profound understanding of this process. Although co-occurrence and cooperation between Comammox and AnAOB have been successfully implemented in a full-scale WWTP, direct evidence supporting N2O production in these systems remains elusive (Vilardi et al., 2023).
## Integration of n-DAMO and Anammox process
The integration of n-DAMO and Anammox process holds great promise in treating wastewater, specifically in removing nitrogen and dissolved CH4. This potential stems from the unique characteristics of the n-DAMO microorganisms and AnAOB.
Studies have shown that n-DAMO microorganisms have extremely low growth rates and long doubling times (Raghoebarsing et al., 2006). However, the introduction of AnAOB could help speed up the enrichment time of these microorganisms, subsequently increasing the activity of n-DAMO archaea (Ding et al., 2014). Several researchers have successfully integrated the n-DAMO and Anammox processes. Liu et al. (2021a) utilized mature Anammox granules as biotic carriers to rapidly cultivate combined Anammox and n-DAMO granules. This strategy not only realized high-level nitrogen removal performance, but also curbed the carbon footprint caused by CH4 and N2O emissions. Similarly, Liu et al. (2019) successfully coupled PN, Anammox and n-DAMO processes in a single membrane biofilm reactor with N2O EF of only 0.34 ± 0.01%, which was mainly attributed to the AOB metabolism. In an attempt to limit greenhouse gas emissions, Valenzuela et al. (2021a) constructed a strictly anaerobic process that coupled n-DAMO with NO3 – reduction, resulting in negligible N2O emissions. In the face of increasing environmental concerns, the focus should shift towards the use of such anaerobic processes in biological treatments. In conclusion, the integration of n-DAMO and Anammox process offers a promising approach to wastewater treatment (Wang et al., 2017).
## Dual-coupled PN/A-PD/A process
The integration of PN/A and PD/A process, known as dual-coupled PN/A-PD/A process, has recently gained the attention of researchers. This combination addresses the drawbacks of each individual process while enriching the AnAOB more effectively with dual NO2 – supply.
Li et al. (2020a) achieved a high nitrogen removal efficiency (94.6%) in a one-stage bioreactor with dual-coupled PN/A-PD/A process. Building upon this, Cao et al. (2022) proposed an innovative Sidestream Enhanced Mainstream Anammox process that could integrate sidestream treatment into mainstream Anammox process. This process adeptly combines PN and PD, effectively mitigating N2O emissions caused by nitrous acid formation. This strategy provides a promising approach for transitioning Anammox processing from sidestream to mainstream operations. The main advantage of the dual-coupled PN/A-PD/A process lies in its potential to curb N2O emissions. This is achieved by increasing the proportion of nitrogen removal contributed by the Anammox process, while reducing NO2 – accumulation in both aerobic and anoxic zones, thereby lessening N2O production by AOB and HB. The effectiveness of the dual-coupled PN/A-PD/A process in nitrogen removal has been validated in various studies (Kao et al., 2022; Li et al., 2021b). However, the specific pathways of N2O production within this system need further investigation to gain a deeper understanding.
## Denitrification as an effective N2O sink
The dynamics of N2O in wastewater treatment systems can be characterized by its production and utilization in both liquid and gas phases. The N2O that escapes into the atmosphere is largely uncontrolled due to the affinity constant of the culture and gas–liquid mass transfer (Conthe et al., 2019). However, the high solubility of N2O in water provides an opportunity for further removal by denitrification in the anoxic zone. The microbial communities involved in denitrification possess the capability to reduce N2O beyond their production levels, thereby having the potential to eliminate additional N2O produced via other pathways aside from their denitrification. Consequently, this establishes denitrification as a potential N2O sink. Notably, the location of the anoxic zone affects the different sources of electron donors utilized by the denitrification metabolism (Fig. 4 ).
Exogenous denitrification relies mainly on external carbon sources. Overcapacity for N2O reduction is widespread among denitrifying communities (Conthe et al., 2019). Bollon et al. (2016) found that in denitrifying biofilters, denitrification diminished the dissolved N2O flux from the upstream nitrification stage by an average of 86%. In alternating anoxic-aerobic reactors, part of the N2O produced in the aerobic phase could be removed by denitrification in the anoxic zone. The addition of external carbon sources or step feeding could further provide electron donors for N2O reduction, reducing emissions (Hu et al., 2011). However, limited organics tend to intensify the competition for electrons between NOR and NOS, leading to higher N2O accumulation (Pan et al., 2013a). Consequently, the amount of carbon sources determines the potential of exogenous denitrification as an N2O sink.
ED utilizes intracellular carbon as an electron donor for nitrogen removal without the addition of external carbon sources (Gong et al., 2021). This process conserves energy by eliminating the need for mixed liquid return flow and enhances nitrogen removal by utilizing intracellular carbon, such as PHA or glycogen (Wang et al., 2016b). However, it's important to note that N2O, not N2, was the major product of denitrification in a DGAOs-dominated ED system, which might be attributed to the inhibition of NOS by elevated levels of NO2 – accumulated during ED (Zeng et al., 2003). Although it has been reported that more N2O was produced by ED with intracellular PHA as carbon sources (Schalk-Otte et al., 2000; Zhou et al., 2012), models have indicated that N2O was mainly produced by nitrification, and the fraction of N2O potentially produced by ED was much lower than that produced by nitrification (Ding et al., 2016). Furthermore, N2O production did not necessarily increase under famine conditions when relying on intracellular polymers unless intracellular carbon was insufficient (Ding et al., 2016). In such cases, N2O accumulation in the anoxic zone is inevitable, whereas an adequate amount of intracellular storage products would produce N2 rather than N2O. Furthermore, it was found that higher intracellular polymers storage in the anaerobic zone resulted in higher ED efficiency and lower N2O production in the anoxic zone (Ding et al., 2022; Wang et al., 2011b). In addition. PHA with a higher PHV proportion could serve as superior electron donors for ED to mitigate N2O emissions (Zhu and Chen, 2011). However, the current research on ED, particularly concerning N2O emissions, is far from comprehensive, calling for further exploration in the future.
## Bioaugmentation technologies for enhanced N2O reduction and system resilience
N2O-reducing microorganisms are the only known microorganisms in the ecosystem that reduce N2O production, and their abundance and activity can strongly influence the net N2O emissions from WWTPs (Jones et al., 2013; Wang et al., 2023b). The isolation and enrichment of N2O-reducing bacteria can be applied in bioaugmentation technology. This strategy promoted a symbiotic relationship between the newly formed NosZ gene-enriched microbial community in the biocarriers and the original microbial community in the activated sludge, which significantly boosted the N2O emissions reduction in the system (Hong et al., 2019; Liu et al., 2016a). Moreover, this technology could also offset the adverse effects of ammonia overload and aeration failure shock, improving the robustness of the system in mitigating N2O emissions (Tian et al., 2021). However, the composition of real wastewater is complex, and the performance of bioaugmented strains is hardly to fully exploit their unique advantages. Therefore, future research should prioritize improving the adaptability of these strains to more effectively reduce N2O emissions in diverse wastewater contexts.
## Conclusions
This review delves into novel wastewater treatment processes, centering on the intricate issue of N2O emissions. These novel processes have their individual strengths and challenges in addressing N2O emissions. N2O emissions can be effectively mitigated by optimizing operating parameters and regulating microbial community structure in these processes. Notably, Anammox-based processes such as Comammox-Anammox, n-DAMO-Anammox, and PN/A-PD/A show promising potential for simultaneous nitrogen removal and N2O mitigation. Moreover, denitrification, whether exogenous or endogenous, has potential as an N2O sink, propelled by N2O-reducing bacteria, and the ability of the denitrification functional zone for N2O redution can be further enhanced by bioaugmentation technology.
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