**11. DHS-type systems applied for N2O elimination**

The emission of GHP-N2O is commonly observed in various mixed systems involved in nitrogen transformation, particularly during NH2OH oxidization [30], nitrifier denitrification [31], comammox [46] and NO2 − -N reduction [37]. Traditionally, the primary approach for N2O removal is carried out through its reduction in the denitrification, which requires an adequate carbon source to activate the microbes possessing the N2OR enzyme. However, this study explores an alternative possibility of N2O oxidation catalyzed by inhered ammonia oxidizers, utilizing the abundant atmospheric O2 as electron acceptor. The theoretically thermodynamic equations for these reactions are presented in Eqs. (1) and (2). These equations demonstrate the thermodynamic feasibility of N2O oxidation to NO3 − -N by ammonia oxidizers using O2 as the electron acceptor.

$$\mathrm{N\_2O} + \mathrm{O\_2} + \mathrm{H\_2O} \rightarrow 2\mathrm{NO\_2^-} + 2\mathrm{H^+}\\\mathrm{\\\Lambda G^0} = -5.33\,\mathrm{K} \text{J/molee}^- \tag{1}$$

$$\mathrm{N\_2O} + 2\mathrm{O\_2} + \mathrm{H\_2O} \to 2\mathrm{NO\_3^-} + 2\mathrm{H^+}\\\Delta\mathrm{G^0} = -2\mathrm{1.2 KJ/mole}\,\mathrm{e^-} \tag{2}$$

In order to assess the potential of N2O oxidation, a series of experiments were conducted, including batch assay and continuous systems such as DHS and USS. The batch assay exhibited an average N2O-removal rate of 107.1 mg N/g VSS (volatile suspended soilds) over a 60-day incubation period. During this time, NO3 − -N and N2-N production accounted for 6.66 and 1.86% of the total nitrogen, respectively. The microbial community in the batch culture consisted of 93% domain *Bacteria* and 7% domain *Archaea*. Notably, the population of ammonia-oxidizing *Nitrososphaera gargensis*-like group experienced a 3.45-fold increase, representing 68.4% of the total archaeal population. Moving on to the continuous-flow DHS system, a range of N2O-N loads, equivalent to HRT from 4 days to 12 hours, was applied under fully saturated O2 conditions. The DHS demonstrated an impressive N2O removal efficiency of 95% with a rate of 6.99 kg N/kg VSS-day. Concurrently, NO3 − -N production reached a maximum of 22.9 μmoles/day, while NH4 + -N and NO2 − -N were not detected throughout the entire operation. These findings indicate the effective capability of the system in removing N2O while promoting NO3 − -N production.

In the operation of the DHS, the concentration of NO3 − -N was accumulated in the effluent up to 7.79 mg N/L at the loadings below 1 mmole N2O-N/day, whereas gaseous N2 became the predominant end-product at the loadings over 2 mmoles N2O-N/day [103]. The mass balance analysis, as illustrated in **Figure 9**, revealed that the produced N2 was derived from two main pathways: the reduction of NO3 − formed from the N2O oxidation and the direct reduction of N2O itself. The conversion yield of mole N2-N per mole N2O-N (0.109 as average of phases II-V) was approximately twice as high as the conversion yield of mole NO3 − -N per mole N2O-N (0.052 in phase I). Furthermore, the anomalous increase in pH was observed in the final phase of the DHS operation, suggesting the potential accumulation of alkaline intermediates, such as NH2OH. This observation lends support to the hypothesis that N2O oxidation to NO3 − -N via NH2OH as the intermediate may occur in the system.

**Figure 9.** *The type of production from the transformed N2O in the downflow hanging sponge (DHS).*

In addition, a diverse community of the nitrogen-functional microbes coexisted in the DHS, including the dominant nitrite-oxidizing bacteria of the genus *Nitrospina* and ammonia-oxidizing archaea of the genus *Nitrosophaera* [104]. Furthermore, the family *Acidobacteriaceae* exhibited both denitrifying and DNRA activities [105]. A smaller population of ammonia-oxidizing bacteria was also detected (3.97 × 103 copies/μg DNA based on the *amoA* gene). The presence of the genera *Nitrosophaera* and *Nitrospira*, which are typically associated with marine environments, dominated in the aerobic DHS during N2O transformation, potentially serving as regulators of marine N2O production. Moreover, the contribution of nitrogen conversion through other redox reactions involving metals was estimated to be less than 10%. Therefore, we hypothesize that the major nitrogen loss observed in the DHS system could be attributed to the accumulation of unidentified intermediates resulting from N2O transformation such as NH2OH, nitrogen oxide (NOx), dinitrogen trioxide (N2O3) and dinitrogen tetroxide (N2O4) and possibly other compounds that were not analyzed in this study.

Another system utilized for cultivating the N2O-functional community is the submerged USS system, which received an average N2O concentration of 10.96 ppm with N2O load of 188 μmoles/day. Throughout the 240-day operation, the removal efficiency ranged from 13.6 to 37.5%, which resulted in effluent concentrations of NH4 + -N and NO2 − -N ranging from 0.08 mgN/L to 0.60 mgN/L and 0.12 mgN/L to 0.44 mgN/L, respectively. After a 30-day enrichment, the production of NO3 − -N commenced, reaching a maximum concentration of 2.74 mgN/L, equivalent to a production rate of 47.0 μmoles/day, with 70.98% originating from the oxidation of N2O on day 50, surpassing that of the aerobic DHS.

However, N2 production began while the transformed NO3 − -N concentration was below 10%, and the maximum N2 accumulation reached 53.6 μmoles/day, representing 5.4% of N2O conversion. In contrast to the DHS system, where N2 served as the end-product instead of NO3 − -N, the USS reactor exhibited N2 production in conjunction with the presence of NO3 − -N. Additionally, the average of O2 consumption during the 240-day operation ranged from 50.2 μmoles/day to 140.2 μmoles/day. Conversely,

### *The Contribution of Autotrophic Nitrogen Oxidizers to Global Nitrogen Conversion DOI: http://dx.doi.org/10.5772/intechopen.112709*

the concentrations of chloride and sulfate present in the cultivated medium experienced a reduction of over 78.2%, indicating their potential utilization by the nitrogenfunctional microbes. Additionally, the activity of the N2O oxidation, as determined by quantitative polymerase chain reaction (qPCR) analysis targeting functional genes, revealed a significant increase in the population of both ammonia oxidizers and nitrite oxidizers, reaching levels of approximately 105 cells/kg VSS. Notably, the growth rate of ammonia-oxidizing archaea outpaced that of ammonia-oxidizing bacteria, while the genus *Nitrospira* exhibited a higher growth rate compared to the genus *Nitrobacter*.

On the one hand, the four effect factors tested by the batch assay were discussed as follows. First, the influence of oxygen concentration was evaluated, and it was found that increasing the air input by 3-fold resulted in a 1.36-fold increase in NO3 − -N production compared to the aerobic DHS operated with 7% of air supply. This led to a NO3 − -N production rate of 10.16 mg/gVSS. Second, the addition of 34.5% methane (CH4) led to the highest NO3 − -N production, reaching a value of 15.19 mg NO3 − -N/ gVSS. Third, the enrichment of ammonia oxidizers using 7.5 mM NH4 + -N in conjunction with N2O-degrading microbes resulted in a conversion of 9.62 mg NO3 − -N/gVSS. Fourth, the addition of 1.4 mM manganese (Mn2+) aimed to convert N2O to NO3 − -N, but it achieved a lower conversion rate of 6.58 mg NO3 − -N/gVSS compared to the aerobic DHS. The microbial populations of both domains *Bacteria* and *Archaea* displayed an increase in the growth rate in response to O2, CH4 and NH4 + -N, except in the case of the assay with Mn2+ addition. Notably, the increase in the population of nitrite oxidizing genera *Nitrobacter* and *Nitrospira* was superior than that of ammonia oxidizers.

To further confirm N2O oxidation pathways involving NH4 + -N oxidation and NO2 − -N oxidation, pure cultures of the genera *Nitrosomonas* and *Nitrobacter* were employed for N2O elimination using sodium bicarbonate (NaHCO3) and carbon dioxide (CO2) as inorganic carbon sources. Ammonia-oxidizing *Nitrosomonas* demonstrated efficient catalysis of N2O transformation, resulting in the production of 0.20 to 0.64 mole NOx/mole N2O and 0.30 to 0.54 mole NH4 + -N/mole N2O. The addition of a two fold carbon source facilitated an increase in N2O oxidation. Notably, CO3 2− derived from NaHCO3 was more readily utilized for cell synthesis compared to gaseous CO2. In contrast, the nitrite-oxidizing *Nitrobacter* exhibited lower N2O transformation rates ranging from 25.94 to 53.84%. The addition of either inorganic carbon source resulted in high NOx production from the oxidized N2O, ranging from 0.90 to 0.91%. Based on these findings, it is suggested that the aerobic degradation of N2O follows a possible route involving the conversion of N2O to NO3 − -N via NH2OH and NO2 − -N as the intermediate. Additionally, aerobic nitrogen-functional microbes were the key contributors for N2O elimination.

## **12. Summary**

Both NH4 + -N and GHP-N2O are of great concern due to their impact on the globally ecological environment. This chapter introduces the application of the polyurethane sponge as a useful medium, providing a three-dimensional space for microbial growth. Two types of sponge-based systems, namely DHS and USS reactors, were utilized for various reactions of autotrophic nitrogen transformation. In terms of NH4 + -N conversion, processes, such as nitritation, anammox and one-stage nitrogen removal, demonstrated satisfactory rates of total nitrogen removal. Regarding N2O elimination, three potential routes were identified for N2O transformation, involving the production of NO3 − -N through the conversion of NO2 − -N, NH4 + -N or direct conversion to N2 as end-product. Five different autotrophic nitrogen-functional microbes cooperated synergistically within the expanded system, contributing to the reduction of nitrogenous compounds.
