Introduction

Nitrous oxide (N2O), a powerful greenhouse and ozone-depleting gas, has a lifetime of approximately 118 to 131 years and is 300–fold more potent than carbon dioxide (CO2)1,2. N2O contributes 6 to 8% of the anthropogenic greenhouse effect worldwide3. Moreover, the atmospheric concentration of N2O has increased at an annual rate of 0.2 to 0.3% over the past decade4. N2O can be produced in biological wastewater treatments, especially treatments involving biological nitrogen (N) removal5,6. Recently, wastewater treatment plants (WWTPs) were found to exhibit gradually rising N2O emissions due to increases in population density and industrial activity7. Therefore, studying the N2O emissions of biological N removal systems is beneficial for controlling the greenhouse effect and protecting the ozone layer.

The one-stage autotrophic N removal process is especially well suited for treating wastewater containing high ammonia but low organics, such as landfill leachate, livestock wastewater and agricultural effluent8, because it has several advantages: a low demand for aeration, no consumption of organic carbon and low sludge production9,10. In a spatial model of biofilm from a one-stage completely autotrophic N removal process, ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing bacteria (AnAOB) grew in different regions according to the concentration of dissolved oxygen (DO)11. In this case, ammonia was initially oxidized to nitrite by AOB located in an area of higher DO, i.e., the surface of the biofilm. Then, the nitrite and remaining ammonia are converted to N2 by AnAOB in anaerobic zones8. Kartal et al.12 presented Eq. 1 to describe the Anammox process.

Ammonium is the main N source during one-stage autotrophic N removal. Meanwhile, nitrite is produced by the oxidation of ammonia, and N2 forms through the pairing of one N atom from ammonium and another N atom from nitrite13. Although the Anammox process is not fully understood, it is generally thought to produce no N2O gas14,15. Thus, improving the Anammox activity would be beneficial for reducing N2O emissions. However, the Anammox activity and its contribution to the removal of total N (TN) have not been measured in one-stage autotrophic N removal, making reducing the N2O generated in this process difficult.

In addition, heterotrophic denitrifying bacteria were also found in the systems used to treat wastewater containing high levels of ammonia-N without organics16, which suggests that heterotrophic denitrification (HD) is likely an additional pathway for N removal in the one-stage autotrophic N removal process. Traditionally, AOB denitrification and HD have been considered the two main pathways responsible for N2O emissions from biological N removal processes when DO is limited17,18. The presence of AOB and HD bacteria in the system indicates that the one-stage autotrophic N removal process might be a potential source of N2O emissions. In HD, N2O is believed to be an intermediate produced during denitrification that can be converted into N2 by nitrous oxide reductase (N2OR)19. In contrast, AOB denitrification is thought to contribute the same level of N2O emissions as HD, or perhaps more, in terrestrial and marine ecosystems because of the lack of genes encoding traditional N2OR20,21. Typically, AOB denitrification can be influenced by the concentration of DO or elevated nitrite22,23, where as HD is closely related to nitrite accumulation, oxygen inhibition and the presence of biodegradable organic compounds24,25,26.

However, the contributions of AOB denitrification and HD to N2O emissions when the one-stage autotrophic N removal processis used to treat high-ammonia-N, organic-free wastewater remains unclear, especially under anaerobic conditions, such as non-aeration during the application of intermittent aeration or the inner space of the micro-biofilm environment when limited oxygen is supplied to the bulk liquid. Clearly, the emission of N2O under anaerobic conditions is an important contribution of the total N2O emissions of this system. Therefore, better understanding these mechanisms is essential for formulating operating strategies to minimize N2O.

This study was conducted to investigate the pathways of N removal and N2O emission from a one-stage autotrophic Nitrogen removal process under anaerobic conditions. Biofilm from a sequencing batch biofilm reactor (SBBR) was used for two groups of batch experiments, and the microbial composition was analysed. First, an 15N isotope tracer technique was applied to investigate the contributions of Anammox and denitrification to TN removal via a one-stage autotrophic N removal process (batch test 1). Then, the N2O emissions corresponding to AOB denitrification and HD were quantified using specific inhibitors in this system (batch test 2). Finally, the microbial diversity and functional microorganisms associated with N2O emissions were analysed via high-throughput sequencing technology.

Results and Discussion

Performance of N transformation in the SBBR

The SBBR operated for more than one year with a stable effluent nutrient level and TN removal efficiency exceeding 80%. Figure 1 presents the N transformation performance of the SBBR in the final month of operation. The effluent TN remained in the range of 37.9–40.4 mg N L−1, and the TN removal efficiency was 80.6 ± 0.6% (Fig. 1(A)). The N compounds involved in the cycle are also shown in Fig. 1(B). The NH4+-N concentration gradually decreased from 89.3 mg N L−1 to 0 mg N L−1 as NO3−-N production increased from 11.2 mg N L−1 to 31.2 mg N L−1, whereas the NO2−-N concentration did not exceed 5 mg N L−1 during this whole phase. In particular, NH4+-N exhibited a higher disappearance rate during aeration phases than during non-aeration followed by the increase of NO2−-N. This behaviour suggests that nitrosation occurred during the aeration phase, whereas during the non-aeration phase, NH4+-N and NO2−-N simultaneously disappeared via the Anammox process. These results indicate that nitrosation-Anammox is the main pathway for N removal in this system. However, during 22 to 24 h of NO2−-N degradation, the NH4+-N phase was completely removed, suggesting that denitrification occurred.

Figure 1: The N removal performance in the SBBR.
figure 1

(A) The TN removal efficiency during a recent month; (B) The concentrations of NH4+-N, NO2−-N, NO2−-N and TN in a cycle; and (C) the N2O emission in a cycle.

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The N2O emissions corresponding to a single cycle of the SBBR are shown in Fig. 1(C). According to Eq. 6, the N2O-N emission factor throughout the process (EF(total)) was 3.3% in the SBBR, which is similar to the result reported by Liu et al.27, and 2.7% of the TN input was converted to N2O-N in the simultaneous nitrification-denitrification (SND) process with intermittent aeration (aeration DO:1.5–2.0 mg/L). Jia et al.28, who used a lower DO (0.35–0.80 mg/L) during the aerobic phase, found that EF(total) was 7.7%. These results indicated that at the one-stage, completely autotrophic N removal and SND processes likely had similar sources of N2O emission, mainly during phases of low DO. However, the rates of N2O emission during the aeration intervals were much higher than those during the non-aerated intervals, probably because the later are associated with lower gas/liquid transfer coefficients. As a result, N2O emission occurs in both production processes, and stripping from the liquid arises during aerated intervals. Furthermore, the dissolved N2O increased during the non-aeration phase, suggesting that this phase is an important stage in N2O generation and may generate more N2O than the aeration phase. Specifically, the maximum rate of N2O emission was observed between 4 and 6 h, when the increase in nitrite was maximized. This finding indicates that N2O emission was affected by nitrite accumulation.

Pathways of N removal

Table 1 shows the substrate addition strategies and N removal performances of batch test 1. The rates of TN removal in group A and group B were −0.08 mg (L h)−1 and 0.07 mg (L h)−1, respectively, and were far below that of group C (5.82 mg (L h)−1), reflecting both the anaerobic conditions of the experiments and the negligible effect of endogenous metabolism on TN removal. The concentrations of nitrogenous compounds and rates of N transformation (i.e., the appearance or disappearance rates of TN, NH4+-N, NO2−-N, NO3−-N, and N2O-N) were measured in group C. The results (Fig. 2(B)) demonstrated that the rate of disappearance of NH4+-N(rNH4+-N) decreased gradually from 0.4 to 0.2 mg (g·MLSS·h)−1. Meanwhile, the rate of disappearance of NO2−-N (rNO2−-N) decreased gradually from 0.5 to 0.4 mg (g·MLSS·h)−1. rNH4+-N and rNO2−-N displayed similar, gradually reducing trends, but rNO2−-N was always higher than rNH4+-N. The average rNH4+-N and rNO2−-N were 0.3 and 0.4 mg (g·MLSS·h)−1, respectively, and the related ratio of rNO2−-N to rNH4+-N was 1.34, which is similar to the Anammox stoichiometry (1.32) for this ratio according to Strous et al.29 and van der Heijden et al.30. This finding indicates that Anammox plays the main role in the N removal process. During the test, the ratio of rNH4+-N to rNO2−-N decreased gradually from 91% to 60%, indicating a gradual increase in the relative contribution of denitrification to N removal.

Table 1 Strategies for substrate addition in isotope batch experiments and the N removal performance.
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Figure 2: Nitrogen transformation and isotopic analysis of the batch experiments using the 15N-NO2− isotopic tracer.
figure 2

(A) The concentrations of nitrogen compounds; (B) the rates of nitrogen transformation (the positive axis represents the apparent rate, and the negative axis represents the disappearance rate); (C) R30/29 (Square) and R46/45 (Triangle); and (D) Relative contributions of denitrification and Anammox to N2 production.

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The value of R30/29 (Fig. 2) was determined by IRMS, and the relative contributions of Anammox and denitrification were calculated (Fig. 2(B)) via Eqs 2 and 3. The results showed that R30/29 gradually increased from 0.09 to 0.19; thus, 83–91% of all N2 was produced by Anammox, and 9–17% was generated via denitrification. These results suggested that Anammox plays the primary role in N removal, consistent with the conclusion drawn above. In addition, the relative contribution of denitrification was found to gradually increase during the operation. Previous studies have shown that autotrophs supply heterotrophs with soluble microbial products (SMPs) for use as electron donors and carbon sources31,32; subsequently, in turn, autotrophs receive inorganic carbon from heterotrophs metabolizing SMPs33. Therefore, the increased denitrification was probably attributable to the synthesis of SMPs, which can act as a potential electron donor for denitrification, by AOB.

N2O emission under anaerobic conditions

The N2O-N emission from batch test 1 gradually decreased from 17.0 to 8.2 μg (g·MLSS·h)−1 (Fig. 2(B)), and the EF(total) was 1.6%, as calculated using Eq. 6. The EF(total) of batch test 1 was significantly lower than that of the SBBR (3.3%) because of the absence of nitrification, which is another source of N2O emission under aerobic conditions31,32. Furthermore, DO exerts an important influence on N2O emission from denitrification via HD bacteria and AOB33,34, and the DO concentration of batch test 1 differed substantially from that of the SBBR, which may also affect N2O emission. The isotopic composition of N2O from batch test 1was determined by IRMS (Fig. 2(C)). The results showed that R46/45 was much larger than R30/29, indicating that the pathways of N2O emission are quite different from those of N2 production. Additionally, the values of R46/45 gradually declined from 14.2 to 10.7, whereas according to Eq. 6, the ratio of D30 to D29 was equal to 26. This finding suggested that denitrification is not the only pathway to generate N2O. However, Anammox does not generate N2O. Thus, a pathway for N2O emission other than denitrification may exist and could potentially be an intermediate step in the denitrification process.

N2O emission from AOB denitrification and HD

To estimate the pathways of N2O emission during the process of denitrification, an approach using specific inhibitors was applied to determine the proportions of the total N2O emission corresponding to AOB denitrification and HD. No significant N2O emission was observed in group I without the addition of NO2−-N and inhibitors (Fig. 3). As NO2−-N was added to system (group II), AOB denitrification and HD occurred simultaneously, and the average N2O-N release rate was 11.6 μg (g·MLSS·h)−1. Meanwhile, with the addition of inhibitors (group III), AOB denitrification was inhibited, and the average release rate of N2O-N was 7.5 μg (g·MLSS·h)−1. Thus, the release rate reduction of 4.1 μg (g·MLSS·h)−1 reflects the activity of AOB denitrification. Calculations based on the N2O emissions results showed that 36% and 64% of N2O emissions were from AOB denitrification and HD, respectively, during the denitrification process, implying that HD is the main pathway of N2O emission under anaerobic conditions.

Figure 3: The rates of nitrogen transformation in batch experiments with inhibitors.
figure 3

(I) With no addition of nitrite or inhibitor; (II) with the addition of nitrite; and (III) with the addition of nitrite and inhibitors.

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Microbial distributions

Figure 4 presents the microbial composition of the biofilm based on the 16S rDNA amplicon pyrosequencing. These results suggest that the dominant microorganisms in the biofilm were Candidatus brocadia, Anaerolineaceae, Gemmatimonadaceae, Ardenticatenia, Nitrospira, Xanthomonadales, Nitrosomonas and Denitratisoma, with relative abundances of 11.2%, 10.4%, 10.1%, 8.7%, 7.0%, 4.2%, 4.1%, and 3.3%, respectively (Fig. 4(A)). C. brocadia, Nitrosomonas and Denitratisoma have been reported to be Anammox, AOB denitrification and HD bacteria, respectively35. In addition, Nitrospira has been shown to be distributed in the outer layers of biofilms and to possess the ability to convert nitrite into nitrate36, whereas Xanthomonadales was classified as a member of Gamma proteobacteria, which are regarded as a type of HD bacteria. However, the roles of some species in N removal remain unknown. Thus, each phylum was classified based on 16S rDNA to investigate the biological bases for N removal and N2O emissions (Fig. 4(B)). Chloroflexi, Proteobacteria, Acidobacteria, Planctomycetes, Gemmatimonadetes, Nitrospirae and Bacteroidetes were the main phyla. Most of the Anammox bacteria, HD bacteria and AOB for wastewater treatment could be classified as Proteobacteria, Planctomycetes and Nitrospirae, respectively37,38,39, which corresponded to 21%, 13%, and 7% of the total bacteria in the biofilm of this system. Thus, these bacteria might be the main sources of N2O emissions under anaerobic conditions.

Figure 4: Microbial composition of the biofilm.
figure 4

(A) Sequence assignment results at the genus level; and (B) sequence assignment results at the phylum level. All effective sequences in the biofilm sample were assigned, and only those with high relative abundances (>0.5%) are shown in this figure.

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Conclusions

The relative contributions of denitrification and Anammoxto N2 production were calculated to investigate the N removal pathways in a one-stage autotrophic N removal system under anaerobic conditions. Anammox played the most important role in N removal, and denitrification emitted the most N2O, despite contributing little to N removal. Furthermore, HD created more N2O emissions than AOB denitrification under anaerobic conditions, although AOB denitrification was expected to be the more worrisome source of these emissions. Therefore, improving Anammox and decreasing denitrification contributed to reducing the N2O emissions of the system.

Materials and Methods

SBBR operation and synthetic wastewater

The SBBR consisted of a rigid Plexiglas® cylinder with an effective volume of 30 L, including approximately 9 L (30%, V/V) of flexible medium for biofilm growth. The bioreactor was operated at 30 ± 2 °C with intermittent aeration (aeration:non-aeration = 2 h:2 h) and a cycle time of 24 h (i.e., 4 min of feeding, 23 h of reaction, 30 min of settling and 26 min of decanting). The DO concentration in the aeration phase was controlled at 1.5 to 2.0 mg L−1. In each cycle, approximately 10.5 L of wastewater was fed into the bioreactor, and the same amount of supernatant was with drawn after settling, resulting in a hydraulic retention time (HRT) of 48 h. The synthetic wastewater fed into the parent SBBR contained 1.13-g L−1NH4HCO3 (200-mg L−1NH4+-N), 583.61-mg L−1NaHCO3 and 20-mg L−1KH2PO4. NH4HCO3 and KH2PO4 were added as N and phosphorus sources, and NaHCO3 was used to regulate the pH between 7.8 and 8.2. In addition, an appropriate amount of trace elementswere added to support microorganism growth, as described by Jia et al.40.

Isotopic tracer experiment

To distinguish the contributions of Anammox and denitrification to N removal in the one-stage autotrophic N removal process, a 15N-NaNO2 isotopic tracer was added to a sealed bottle with an active volume of 100 ml that contained 10 g wet weight of biofilm from the SBBR and 90 ml of synthetic wastewater (Table 1). The biofilm had been previously incubated for 5 h to remove nitrate from the biofilm. Next, helium gas was introduced to eliminate DO from the sealed Erlenmeyer flask containing the biofilm and pure water, and the temperature was controlled at 30 ± 2 °C. Then, the pure water was replaced with synthetic wastewater that was continuously sparged with helium gas; all other conditions remained constant. The wastewater contained 100-mgN L−1 NH4HCO3 and 100-mgN L−115/14N-NaNO2. 15/14N-NO2− was provided by Sangon Biotech (Shanghai, China), and the 15N atomic percentage (100% × 15N/(15N + 14N), F) was 99%. The pH was controlled between 7.8 and 8.2 by the addition of NaHCO3. To evaluate the background N2O emissions from the biofilm and check the anaerobic conditions, two control groups were performed: (A) pure water and (B) synthetic wastewater with NH4HCO3 only. The off-gas was collected every 6 h for 24 h to simultaneously analyse the isotopic compositions of N2 and N2O, and 2-ml liquid samples were collected to determine the concentrations of NH4+-N, NO2−-N and NO3−-N. Finally, 100 μl of 50% ZnCl2 was added to the liquid samples to inhibit microbial activity.

The isotope composition of N2 was analysed to quantify the contributions of Anammox and denitrification to N2 production. In incubations with 15NO2− and NH4+, N2 production via Anammox consisted of one N atom from NO2− and another from NH4+, leading to the production of 29N2, whereas the denitrification of two N atoms from NO2− was assumed to produce 30N2. However, because the F of 15NO2− was not 100%, 28N2 and 29N2 were produced via Anammox, and 28N2, 29N2, and 30N2 were generated via denitrification. Therefore, the N2 production mass of Anammox and denitrification could be respectively calculated according to Thamdrup and Dalsgaard41, The calculations were described as Eqs 2 and 3:

where and represented the mass of N2 produced by denitrification and Anammox, respectively; P29 and P30 represent the production amount of 29N2 and 30N2, respectively, and F represents the fraction of 15N in the NO2− pool. In this system, Anammox and denitrification were the only two pathways of N removal, the relative contributions of denitrification (Cd) and Anammox (Ca) to N2 production can be described as the ratio of to plus and that of to plus , respectively. Therefore, Cd and Ca can be described by Eqs. 4 and 5, respectively:

In which R30/29 represents the ratio of 30N2 production to 29N2 production

The isotopic composition of N2O was also investigated. N2O was generated as an intermediatein both nitrification and denitrification during the process of biological N removal42. Therefore, denitrification should be the only pathway of N2O emission under anaerobic conditions, and N2O should possess an isotopic composition similar to that of the N2 produced by denitrification; that is, the ratio of 46N2O to 45N2O (R46/45) should be equal to the ratio of 30N2 to 29N2 of denitrification. The ratio of 30N2 to 29N2 corresponding to denitrification can be expressed using Eq. 6 according to Thamdrup and Dalsgaard41

where D30 and D29 represent the production of 30N2 and 29N2 via denitrification, respectively. Thus, if R46/45 was not equal to the ratio of D30 to D29, denitrification was not the only pathway for N2O emission.

Experiments involving specific inhibitors

The use of inhibitors can facilitate investigating the magnitudes of the various processes at the source of N2O production under anaerobic conditions. Allylthiourea (ATU) was used as the inhibitor of the nitrification of ammonia to nitrite, whereas NaClO3was used to inhibit the conversion of nitrite to nitrate catalysed by nitrite oxido-reductase28. The co-use of ATU and NaClO3 can effectively inhibit the production of N2O via AOB denitrification37, whereas N2O emissions by heterotrophic bacteria are not significantly affected by the presence of ATU and NaClO337. Therefore, the emission of N2O produced by HD alone and by both AOB denitrification and HD can be quantified by batch experiments with or without the inhibitors.

Thus, three batch experiments were conducted: (I) no addition of nitrite or inhibitor, (II) the addition of nitrite, and (III) the addition of both nitrite and nitrification inhibitors (ATU and NaClO3). Three devices were assembled for these the batch experiments using an isotopic tracer; then, a 1-L mixture containing 100 mg wet weight of biofilm and 900 ml of wastewater (NH4+-N: 9.7 mg L−1; NO2−-N: 1.8 mg L−1; and NO3−-N: 23.6 mg L−1) from the SBBR were introduced into a sealed Erlenmeyer flask, and then, NaNO2, ATU, and NaClO3 were added to the effluent at concentrations of 100.0 mgN L−1, 10.0 mg L−1, and 1.0 g L−1, respectively. Helium gas was introduced into the wastewater to ensure anaerobic conditions. The solution and off-gas in the devices were sampled every 6 h for 24 h, and the concentrations of NH4+-N, NO2−-N, NO3−-N and TN in the wastewater were measured to investigate the characteristics of N transformation. The N2O emissions were also detected to identify the contributions of AOB denitrification and HD. The amount of N2O emissions can be described as follows: II–I, the sum of AOB denitrification and HD; III–I, HD; and (II–I)–(III–I), AOB denitrification (Fig. 5).

Figure 5: Diagrams of expected nitrogen transformation under anaerobic conditions.
figure 5

(I) No NO2−-N or inhibitors, (II) with NO2−-N (100 mg L−1) addition only, and (c) with NO2−-N (100 mg L−1) and inhibitors (ATU and NaClO3). HD, heterotrophic denitrification; AD, AOB denitrification.

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Physicochemical analysis

The concentrations of TN, NH4+-N, NO2−-N, and NO3−-N were measured using a flow injection analyser (HachQuickchem 8500S2, Hach Inc., Loveland, CO, USA). Alkalinityand biomass dry weight (mixed liquid suspended solids, MLSS) were measured according to standard methods for water and wastewater43. The concentration of N2O was determined with an Agilent 7820A gas chromatograph (Agilent Technology Inc., Santa Clara, CA, USA) according to Jia et al.40. The dissolved N2O in wastewater was determined using the head space gas method described by Tsuneda et al.44. The values of R30/29 for N2 and R46/45 for N2O were measured by isotope-ratio mass spectrometry (IRMS;MAT253, Thermo Finnigan LLC, San Jose, CA, USA) according to the method described by Cao et al.45. The N2O-N emission factors per TN converted during the interval i–i + 1 (h) and the whole process were calculated using Eqs 7 and 8, respectively:

where r(i)N2O-N and r(i)TN represent the average rates of N2O emissions and TN removal, respectively during the interval i–i + 1 (h); and t(i) is the duration of interval i–i + 1 (h).

Microbial composition

To analyse the microbial composition in the one-stage autotrophic N removal process, biofilm from the SBBR was collected and centrifuged for to extract the DNA. The total genomic DNA was extracted using an E.Z.N.A.® Soil DNA Kit (OMEGA Bio-Tek, Inc., Norcross, GA, USA), and the bacterial 16S rDNA genes of the biofilm were sequenced using Illumina MiSeq technology at the Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Ultra-fast sequence analysis (USEARCH) was used to cluster the operational taxonomic units (OTUs) of a 16S DNA gene based on 97% similarity, and the statistical abundances of different OTUs in the samples reflect those of different microbial species. Then, the microbial composition was analysed according to sequencing information and data from the National Center of Biotechnology Information (NCBI) reference genome. Finally, microorganisms were classified as Anammox bacteria, AOB and HD bacteria based on the pathway of N metabolism. Simultaneously, the relative proportions of these microorganisms were calculated based on the OTU abundances.

Additional Information

How to cite this article: Li, K. et al. Pathways of N removal and N2O emission from a one-stage autotrophic N removal process under anaerobic conditions. Sci. Rep. 7, 42072; doi: 10.1038/srep42072 (2017).

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