
==== Front
Water Res X
Water Res X
Water Research X
2589-9147
Elsevier

S2589-9147(24)00047-1
10.1016/j.wroa.2024.100257
100257
Full Paper
Isotope analysis of nitrogen removal pathways and N2O production potential in the SDAD-anammox system under different N/S ratios
Zhan Mengjia
Zeng Wei zengwei@bjut.edu.cn
⁎
Hao Xiaojing
Miao Haohao
Lu Yao
Jiang Wenzhuo
Meng Qingan
Gong Qingteng
National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Beijing University of Technology, Beijing 100124, China
⁎ Corresponding author at: Department of Environmental Engineering, Beijing University of Technology, Pingleyuan No.100, Chaoyang District, Beijing 100124, China. zengwei@bjut.edu.cn
11 9 2024
01 9 2024
11 9 2024
24 10025721 7 2024
10 9 2024
11 9 2024
© 2024 The Authors. Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• Optimal nitrogen removal was achieved at N/S ratios of 1.5–2.0 in the SDAD-anammox system.

• The lowest N2O emissions were recorded at an N/S ratio of 2.5.

• Isotope tracing technology revealed an increased anammox contribution to nitrogen removal as the N/S ratio rose.

• Higher hzsB gene expression at an N/S ratio of 2.5 indicated enhanced anammox activity.

• For balanced nitrogen removal efficiency and minimal N2O emissions, an N/S ratio of 2.5 is recommended.

This study explored the impact of varying nitrate to sulfide (N/S) ratios on nitrogen removal efficiency (NRE) in the sulfide-driven autotrophic denitrification and anammox (SDAD-anammox) system. Optimal nitrogen removal was observed at N/S ratios between 1.5 and 2.0. Isotope tracing results showed that the contribution of anammox to nitrogen removal was enhanced with increasing N/S ratios, reaching up to 37 % at the N/S ratio of 2.5. Additionally, complex nitrogen pathways were identified, including dissimilatory nitrate reduction to ammonium (DNRA). Furthermore, isotope tracing was innovatively applied to investigate N2O emissions, demonstrating that higher N/S ratios significantly reduced N2O emissions, with the lowest emissions at N/S ratio of 2.5. Gene expression analysis indicated that nitrogen and sulfide transformation genes decreased with increasing N/S ratios, while anammox-related genes first increased and then decreased, reflecting the system's microbial dynamics. These findings offer insights into nitrogen transformation pathways and N2O production mechanisms in the SDAD-anammox process.

Graphical abstract

Image, graphical abstract

Keywords

Denitrification desulfurization system
Anammox
Isotope effect
Nitrous oxide
Functional gene
==== Body
pmcIntroduction

Anaerobic ammonium oxidation (anammox) has attracted global interest as an efficient and innovative technology for wastewater treatment. Due to the limit of nitrite (NO2−-N) in wastewater, it is typically combined with partial nitrification (PN) and partial denitrification (PD) for nitrogen removal (Wu et al., 2022a). However, the instability of NO2−-N generation in PN restricts the widespread application of partial nitrification and anammox (PN/A) (Joss et al., 2009; Bunse et al., 2020). In contrast, coupling of PD with anammox (PD/A) is more feasible because PD can produce stable NO2−-N and enhance NRE by reducing the nitrate (NO3−-N) generated by anammox (Du et al., 2022, 2023). The development of PD/A has primarily focused on generating NO2−-N through heterotrophic partial denitrification, where organic carbon serves as the electron donor. However, the higher growth rates and biomass yield coefficients of heterotrophic denitrifying bacteria (HDB) compared to ammonia-oxidizing bacteria (AnAOB) may lead to excessive growth of heterotrophs, potentially inhibiting anammox activity (Kumar and Lin, 2010; Cao et al., 2013). Among the studies on autotrophic partial denitrification coupled with anammox, SDAD-anammox has garnered attention for its unique advantages. Firstly, AnAOB and sulfur-oxidizing bacteria (SOB) share similar ecological niches and exhibit intersecting growth conditions regarding temperature, pH, etc. (Kuypers et al., 2018; Strous et al., 1999). Studies have confirmed that coexistence of AnAOB and SOB facilitates simultaneous removal of nitrogen and sulfide (S2−) (Kalyuzhnyi et al., 2006; Wu et al., 2020). Secondly, the relatively low biomass of SOB helps alleviate conflicts between AnAOB and SOB (Lu et al., 2018). All evidence suggests that the SDAD-anammox is a promising approach for nitrogen removal.

The ratio of NO3−-N to S2− (N/S) is crucial to the efficiency of the SDAD-anammox system. Reyes-Avila et al. (2004) reported that at an N/S ratio of 1:1, NO3−-N was reduced to NO2−-N and S2− was oxidized to S0 (Eq. (1)). Theoretically, the N/S ratio should be controlled between 1.0 and 4.0 to achieve higher NO2−-N conversion ((1), (2)). Qin et al. (2019) found that in the SDAD-anammox system, total nitrogen (TN) removal by anammox could be maintained above 90 % under N/S ratios ranging from 0.96 to 4.56, supporting the theoretical values. However, the most favorable N/S ratio for the anammox reaction remains controversial. Deng et al. (2021) observed that the highest NRE via the anammox (82.8 %) occurred at an N/S ratio of 3.0, while Wu et al. (2020) achieved the highest ammonium conversion rate at N/S ratios of 1.8–2.0. Considering the competition between AnAOB and SOB for NO2−-N, thermodynamically, the NO2−-N reduction process driven by S2−/S0 is more favorable than anammox (Huo et al., 2022). However, AnAOB can induce higher NO2−-N affinity by virtue of its unique NO2−-N transporter protein and anammoxosome membrane, making them more competitive than SOB for NO2−-N under limited supply conditions (van Niftrik and Jetten, 2012). Consequently, the interaction details between anammox and SDAD under varying N/S conditions remain unclear.(1) S2− + NO3− + H2O → S0 + NO2− + 2OH−

(2) S2− + 4NO3− → 4NO2− + SO42−

Greenhouse gases produced during the urban wastewater treatment contribute to the carbon footprint of water utilities, preventing the achievement of carbon-neutral (Zhang et al., 2022). Among these gases, N2O has attracted wide attention due to its high global warming potential (273 times that of CO2) (Chen et al., 2020). Previous studies have mostly focused on exploring N2O emissions under different conditions in the SDAD-anammox system (Polizzi et al., 2022; Qian et al., 2018). In fact, N2O emissions result from the activities of denitrifying bacteria as N2O sources and bacteria possessing N2O reductase (NOS) as N2O sinks. Therefore, quantifying N2O production and consumption rates and their response to environmental variables can help develop strategies to reduce N2O emissions.

This study investigated the performance and mechanism of the SDAD-anammox system under different N/S ratios using activated sludge from a system stably operated for 120 days. The isotope tracing technique was employed to assess the nitrogen removal pathways and N2O production and consumption activities. By combining the transcriptional activity of relevant functional genes involved in N and S cycling, this study aimed to elucidate nitrogen removal capacity and N2O production potential in the SDAD-anammox system at different N/S ratios.

Results and discussion

Analysis of nutrient removal characteristics at the different N/S ratios

At a consistent influent TN concentration, the total nitrogen removal rate (TNRR) of the SDAD-anammox process (0.06–0.1 mg/(L min)) was significantly lower than that of the SDAD process (0.18 mg/(L min)) (Figs. 1 and S1). This was attributed to the thermodynamic advantage of SDAD over anammox. In the SDAD-anammox system (Fig. 1), the NRE decreased from 89.09 % to 52.42 % as the N/S ratio increased from 1.0 to 4.0, which was attributed to the insufficient sulfur-related electron donors (S2−/S0). This was consistent with previous studies (Dolejs et al., 2015; Chen et al., 2018), where the complete SDAD process occurred at N/S less than 1.5. NO3−-N and NO2−-N were undetectable in the effluent at N/S ratios of 1.0 and 2.0 (Fig. 1a and c). Notably, the ammonia removal rate (ARR) at N/S ratio of 2.0 (0.029 mg/(L min)) was 1.7 times higher than that at 1.0 (0.017 mg/(L min)), indicating that SDAD predominated at 1.0, while SDAD-anammox dominated at 2.0. As the N/S ratio increased to 2.5 (Fig. 1e), the NRE decreased to 83.4 %, while the ammonium removal efficiency (ARE) further increased to 83 %. A peak in NO2−-N concentration (6.2 mg/L) was observed at 15 min, confirming the conversion of NO3−-N to NO2−-N due to electron acceptor limitation. This is consistent with previous findings (Qin et al., 2019; Wu et al., 2020), which indicated that an N/S ratio of 2.0–2.5 was favorable for the SDAD-anammox process. As the N/S ratio further increased to 4.0 (Fig. 1g), both NRE and ARE decreased to 46 % and 62 %, respectively. Only part of NO3−-N was converted to NO2−-N due to the severe lack of S2− as electron donor, resulting in decreased NRE.Fig. 1 S2−, NO3−-N and NH4+-N removal and pH variation in typical cycles at the different N/S ratios (a, b: N/S ratio = 1.0; c, d: N/S ratio = 2.0; e, f: N/S ratio = 2.5; g, h: N/S ratio = 4.0).

Fig 1

Sulfide removal efficiency (SRE) reached 100 % in all conditions (Fig. 1b, d, f and h). Trace amount of S0 (5 mg/L) was detected in the effluent at the N/S ratio of 1.0 (Fig. 1b), whereas S2− was oxidized to SO42− at N/S ratios of 2.0–4.0 (Fig. 1d, f and h), indicating that N/S ratios determined the sulfide reduction products in SDAD-anammox. Additionally, sulfide was rapidly exhausted within 15–30 min. Compared to soluble S2−, the low solubility of S0 greatly limited its electron-donating capacity (Xu et al., 2014), consequently reducing the NO3−-N reduction rate. After sulfide exhausted, the accumulation of S0 peaked. It was hypothesized that sulfide was utilized in two steps: initially, S2− was oxidized to S0, and then S0 was further oxidized to SO42−. This has been widely confirmed in previous studies (Lee and Wong, 2014; Deng et al., 2021). Additionally, some studies (Fu et al., 2023; Liu et al., 2017) suggested that during the conversion of S2− to S0, NO3−-N was preferentially converted to NO2−-N. This study showed a different viewpoint, i.e., whether NO3−-N was first converted to NO2−-N depended on the N/S ratio, since the accumulation of NO2−-N was found only at N/S ratios of 2.5 and 4.0 (Fig. 1e and g). Since the S2− concentrations at N/S ratios of 2.5 and 4.0 were lower than those at 1.0 and 2.0 (Table 1), it was believed that the NO2−-N accumulation was unrelated to the inhibitory effect of S2− on AnAOB.Table 1 Experimental conditions for the batch tests.

Table 1Run	Concentration (mg/L)	N/S (moles/moles)	
NO3−-N	NH4+-N	S2−	
Ⅰ	80		120	1.5	
Ⅱ	60	20	125	1.0	
Ⅲ	50	30	58	2.0	
Ⅳ	50	30	45	2.5	
Ⅴ	50	30	30	4.0	

The pH value initially rose and then declined, reaching its peak when sulfide was completely depleted (Fig. 1b, d, f and h). This phenomenon was due to the consumption of protons during the oxidation of S2− to S0, followed by proton generation as S0 oxidized further to SO42−. Furthermore, as the N/S ratio increased from 1.0 to 4.0, the effluent pH value gradually decreased from 7.91 to 7.31. In the SDAD process, the consumption of 1.0 mol of sulfide required an additional 1.6 mol of protons (Xu et al., 2016), leading to an increase in effluent pH. As the N/S ratio increased, the sulfide concentration decreased, which reduced the proton consumption and consequently lowered the effluent pH.

Nitrogen transformation and contribution of anammox and SDAD to nitrogen removal

The isotope tracing technology revealed the contribution of SDAD and anammox to nitrogen removal (Fig. 2). The N2 was not detected in the blank control group, which implied that O2 and NOx− were completely consumed. The maximum N2 production was detected within 480 min at N/S ratio of 1.0 (Fig. 2c) and within 720 min at N/S ratio of 2.5 (Fig. 2g), suggesting that sufficient sulfide accelerates NRE. With calculation of 28N2, 29N2 and 30N2, the amount of N2 produced by denitrification and anammox at different time points was analyzed. The maximum denitrification rate at an N/S ratio of 1.0 (1.62 umol N2/min) was higher than that at an N/S of 2.5 (0.76 umol N2/min). Lower N/S ratios provided a richer supply of electron donors, leading to higher denitrification rates. Denitrification rates peaked earlier at an N/S ratio of 1.0 compared to 2.5. The increase in substrate concentration was attributed to the increase in reaction affinity, which accelerated the denitrification process. This finding has been confirmed by Polizzi et al. (2022). Within 240 min, 42–45 umol of N2 was produced via the anammox at N/S of 1.0 and 2.5, but the anammox rate at an N/S ratio of 2.5 (0.09 umol N2/min) was significantly higher than that at N/S of 1.0 (0.03 umol N2/min) in the subsequent reaction (Fig. 2b and f). This was mainly due to that insufficient S2− reduced the SDAD rate and enhanced anammox pathway at N/S ratio of 2.5.Fig. 2 Isotope analysis of the accumulation of N2 and the contribution of anammox and SDAD to nitrogen removal (a, b, c, d: N/S ratio = 1.0; e, f, g, h: N/S ratio = 2.5).

Fig 2

When the N/S ratio was 1.0, the SDAD process was the main nitrogen removal pathway with a contribution of more than 85 % (Fig. 2d). It is noteworthy that the contribution of anammox to nitrogen removal averaged 11.7 % over 120 min, which was slightly lower than the average contribution of 14.6 % from 120 to 720 min. It may be caused by the slight inhibition of AnAOB by S2− at the beginning of the reaction. The average contribution of anammox to nitrogen removal reached 36.1 % at an N/S ratio of 2.5 (Fig. 2h). In particular, it was as high as 52 % within the first 30 min, showing its remarkable activity. However, no NH4+-N degradation was observed within 30 min in the batch experimental (Fig. 1e). It was hypothesized that sulfide-induced reduction of NO3−-N to NH4+-N (DNRA) occurred at the beginning of the reaction leading to an increase in NH4+-N concentration, which made up for the decrease in NH4+-N concentration caused by anammox. The ability of sulfide to contribute to the DNRA has been widely found in lakes, oceans, and wetlands (Huang et al., 2024; Brunet and GarciaGil, 1996).

N2O emissions at the different N/S ratios

The isotopes of N2O (45N2O and 46N2O) changed noticeably throughout the reaction (Fig. 3). 46N2O (15N15N16O) consistently emerged as the dominant product, while 45N2O (14N15N16O) was virtually absent, indicating that N2O was primarily produced through denitrification in the SDAD-anammox system (Fig. 3f). Notably, trace amounts of 45N2O were detected at the end of the reaction, suggesting that 14NH4+ had participated in the synthesis of 45N2O. Given the substantial production of sulfate from the SDAD observed in this study, it is inferred that a sulfate-induced ammonium oxidation (SRAO) process might have oxidized a small amount of 14NH4+ to 14NOX−, which then combined with the existing 15NO2− in the system to produce 45N2O (Derwis et al., 2024).Fig. 3 Variations in N2O values (a: N/S ratio = 1.5 (without NH4+-N); b: N/S ratio = 1.0; c: N/S ratio = 2.0; d: N/S ratio = 2.5), percentage of N2O emissions (e) and N2O production pathways (f) in the isotope experiment.

Fig 3

As the reaction progressed, 46N2O initially increased and then decreased (Fig. 3a–d). The N2O emissions in the SDAD-anammox and SDAD accounted for 0.22–1.13 % and 3.78 % of nitrogen loss, respectively (Fig. 3e), demonstrating that SDAD-anammox is more effective in reducing N2O emissions compared to SDAD. In the SDAD-anammox system, N2O emissions decreased progressively with increasing N/S ratios (Fig. 3e). The increased N/S ratio led to higher anammox efficiency (Fig 2), which in turn resulted in reduced N2O emissions. Besides, lower N/S ratios corresponded to higher sulfide concentrations, which inhibited the expression of the nosZ gene. The inhibitory effect of sulfide on nosZ gene expression has been widely documented (Fortune et al., 2024; Martínez-Santos et al., 2018).

15N tracer test to detect N2O production activity

As an intermediate product of the denitrification pathway, N2O emissions (DN2Ototal) were determined by the N2O production (rN2Oprod) and N2O consumption (rN2Ocons) (Fig. 4a). The ratio of N2O consumption to N2O production (rN2Ocons/rN2Oprod) was 0.99, 0.99, 0.98, and 0.95 at N/S ratios of 2.5, 2.0, 1.0, and 1.5 (without NH4+-N addition), respectively. As the N/S ratio increased, the rN2Ocons/rN2Oprod ratio gradually approached 1.0, indicating a balance between N2O production and consumption, which in turn led to reduced N2O emissions.Fig. 4 N2O production and consumption pathways (a) and the kinetic characteristic of N2O production (b: N/S ratio = 1.5 (without NH4+-N); c: N/S ratio = 1.0; d: N/S ratio = 2.0; e: N/S ratio = 2.5) in the isotope experiment.

Fig 4

The production of N2O comprised both the existing N2O and the N2O that had already been converted to N2 (Fig. 4a). 15N tracer technology helped us reduce errors in the traditional N2O production calculation method (Suenaga et al., 2021; Ali et al., 2016) and calculate the real N2O production (rN2O, prod) in the SDAD-anammox system (Text. S3). The significant relationship between N2O yield and reaction time could be well described by a Logit model (Fig. 4b–e). N2Omax represented the maximum N2O production capacity, which was 284.28, 258.59, 247.51, and 240.63 µmol at N/S ratios of 1.5 (without NH4+-N addition), 1.0, 2.0 and 2.5, respectively. Lower N2Omax values indicated a reduced capacity for N2O production, which was beneficial for minimizing N2O emissions. The λ value represented the N2O production ratio. As the N/S ratio increased from 1.0 to 2.5, λ decreased from 0.024 to 0.006, suggesting that sufficient sulfide accelerated the gene expression related to the conversion of NO2−-N to N2O. Additionally, when the N/S ratio was 1.5 (without NH4+-N addition), the λ value for N2O (0.012) was lower than at an N/S ratio of 1.0 (0.024). Since at N/S ratio of 1.0, the generated NO2−-N could be used in time through the anammox process, thus avoiding the accumulation of NO2−-N to inhibit the conversion of NO2−-N to N2O.

Quantitative expression of functional genes at the different N/S ratios

The functional gene activities of nitrogen and sulfide metabolic pathways in the SDAD-anammox systems at different N/S ratios were investigated (Fig. 5). In the SDAD-anammox system, the abundance of napA (NO3−→NO2−) and nirS (NO2−→NO) genes decreased as the N/S ratio increased (Fig. 5a and b). This decline can be attributed to insufficient electron acceptors at higher N/S ratios, leading to reduced denitrification efficiency. The ratio of abundance of nitrate reductase genes and nitrite reductase genes was often used to evaluate the NO2−-N accumulation capacity of the system (Wu et al., 2022a,b; Liu et al., 2023). At N/S ratios of 2.5 and 4.0, the napA/nirS values were 21.1 and 14.4, respectively, significantly higher than those at N/S ratios of 1.0 (3.78) and 2.0 (7.56). This explained the NO2−-N accumulation observed at N/S ratios of 2.5 and 4.0 (Fig. 1). NosZ, which reduces N2O to N2, had expression levels that were closely related to N2O emissions (Jones et al., 2008). The highest expression level of NosZ was observed at an N/S ratio of 2.5 (7.49 × 106 copies/g sludge), indicating the fastest N2O reduction rate under this condition (Fig. 5c). This was consistent with the observed N2O peak being lower at an N/S ratio of 2.5 than that at 1.0 and 2.0 (Fig. 3), confirming that sulfide inhibited the expression of nosZ.Fig. 5 Abundance of the functional genes related to nitrogen reduction and sulfide oxidation under the conditions of N/S ratios of 1.5 (without NH4+-N), 1.0, 2.0, 2.5 and 4.0.

Fig 5

As a representative functional gene of anammox, hzsB expression levels increased from 1.5 × 105 copies/g sludge to 5.2 × 105 copies/g sludge with increasing N/S ratios (Fig. 5d). This was due to the enhancement of partial denitrification, leading to NO2−-N accumulation, which is a substrate for anammox. However, at N/S ratio of 4.0, hzsB expression decreased to 2.87 × 105 copies/g sludge. Although partial denitrification had an advantage under this condition, the overall denitrification efficiency was significantly reduced, leading to decreased anammox activity.

Sqr and soxb are functional genes associated with sulfide transformation, where sqr converts S2− into S0, and soxb further converts S0 into SO42− (Luo et al., 2011). In the SDAD-anammox system, the expression levels of both sqr and soxb decreased with increasing N/S ratios (Fig. 4e and f). This was due to the reduced sulfide concentration, leading to a subsequent decrease in their expression levels. Whether the final sulfur product existed in the form of S0 or SO42− depended on the sqr/soxb ratio, with a higher sqr/soxb ratio favoring the accumulation of S0 (Shi et al., 2024). At the N/S ratio of 1.0, the sqr/soxb ratio was 1.02, higher than other conditions, which explained why trace amounts of S0 were detected in the effluent only under this condition.

Additionally, in the SDAD system (without NH4+-N addition), the expression levels of nirS, napA, and nosZ were slightly lower than those in the SDAD-anammox system at an N/S ratio of 1.0, even though the N/S 1.5 system had higher NRE. This suggested that the anammox reaction could enhance the activity of denitrifying bacteria to some extent, possibly by reducing the accumulation of NO2−-N, which otherwise would inhibit the activity of denitrifying bacteria.

Conclusions

This study demonstrated that the N/S ratio significantly influenced the nitrogen and sulfide removal efficiency in the SDAD-anammox system. Optimal NRE was achieved at the N/S ratio between 1.5 and 2.0. Sulfide limitations affected overall removal efficiency at N/S ratios greater than 2.5. The lowest N2O emissions occurred at N/S ratio of 2.5 due to enhanced anammox activity. Gene expression analysis of the hzsB gene supported these findings, showing increased anammox activity at optimal N/S ratios. To balance efficiency and N2O emissions reduction, the N/S ratio of 2.5 was recommended, as it maintained 83 % NRE while minimizing N2O emissions.

Materials and methods

Characteristic of the parent reactor

Typical SDAD-anammox sludge was chosen for batch experiments in this study. The SDAD-anammox sludge was collected from a parent sequencing batch reactor (SBR). The parent SBR was fed with synthetic wastewater containing 70 mg/L NH4+-N, 110 mg/L NO3−-N and 100 mg/L S2−, where NH4+-N from ammonium chloride (NH4Cl), NO3−-N from sodium nitrate (NaNO3), and S2− from sodium sulfide nonahydrate (Na2S·9H2O). It was operated at 12 h hydraulic retention time (HRT) for more than 120 days steadily, with NRE as high as 89.7 % and specific anammox activity (SAA) of 2.42 mg NH4+-N/ (g VSS h). The other components of synthetic wastewater were: NaHCO3, 1.0 g/L; KH2PO4, 0.027 g/L; CaCl2·2H2O, 0.2 g/L; MgSO4·7H2O, 0.3 g/L. Trace element I and trace element II were each 1.0 ml/L (Qin et al., 2019). NaOH/HCl (1 mol/L) was used to maintain an influent pH of 8.0 ± 0.3.

Batch tests

To assess the effect of N/S ratios on the SDAD-anammox process, five serum bottles with an effective volume of 0.5 L were operated in a shaking incubator at 30 ± 1 °C and 200 rpm for batch assays. The inoculums were sampled from the parent reactor and washed twice with distilled water to remove residual substrate. The initial biomass concentration in the serum bottles was approximately 2.12 g VSS L−1. The serum bottles were purged with He gas for 15 min and then injected with sterile anaerobic stock solution. The initial pH was controlled at 8.0 ± 0.3 by NaOH/HCl (1 mol/L).

Five sets of batch assays were conducted under different N/S conditions (1.0, 1.5, 2.0, 2.5, and 4.0), with each condition tested in triplicate to ensure accuracy. The detailed assay design is shown in Table 1. For all conditions, the initial total nitrogen concentration was set at 80 mg/L. Notably, no NH4+-N was added under the N/S ratio of 1.5 to compare SDAD and SDAD-Anammox nitrogen removal pathways. Mixed liquor samples were collected periodically until the sulfur was consumed, then these samples were filtered for chemical analyses.

Analytical methods

The collected liquid samples were filtered through a 0.45 μm Millipore filter. The concentrations of NO3− - N, NO2− - N and NH4+ - N were measured according to the standard methods (APHA, 2012). SO42− and S2O32− were measured by ion chromatography (Metrosep A Supp 5–250/4.0). The concentrations of H2S, HS−1 and S2− were determined using the methylene blue method (APHA, 2005). The 3420 Multi Parameter Meter (WTW Company, Germany) was to monitor DO and pH on-line. N2O and N2 were quantitatively analyzed by isotope mass spectrometer (253Plus, Thermo, United States). S0 was analyzed according to the method descried by Seth et al. (1995).

Identification of nitrogen conversion pathways by isotopic tracer incubations

15N isotope labeling experiments were conducted to investigate the individual contributions and activity changes of anammox and SDAD in the nitrogen removal process. Na15NO3 (>99.1 %15N, CIL, USA) and 14NH4Cl were used as reaction substrates to evaluate the progress of anammox and SDAD reactions and their contribution to nitrogen removal. Different concentrations of S2− were added to achieve N/S ratios of 1.0 and 2.5 (Table S1). Additionally, a blank control group was set up to determine whether native oxygen (O2) and NOx− (i.e., NO2− and NO3−) were completely removed during the pre-incubation process. Three parallel tests were set up for each group to ensure the reliability of the results. The specific experimental procedures were as follows: Prior to cultivation, the activated sludge was washed three times with distilled water and pre-incubated anoxically at 30 °C in the dark for 24 h to remove O2 and NOx−. The pretreated sludge was divided equally into 2 mL each and placed into 20 mL headspace bottles. Deionized water containing nutrients was then added, and the bottles were purged with high purity He for more than 15 min. The headspace bottles were sealed with butyl rubber stoppers and crimped with aluminum caps. Cultivation was conducted at 30 ± 1 °C for 12 h, followed by the addition of ZnCl2 (0.1 mL, 7 M) to stop microbial activity. The sludge-water mixture of 4 mL was transferred from each sample into a 12 mL sealed vial that has been thoroughly purged with high purity He. Then, the produced 29N2 and 30N2 were quantified using isotope ratio mass spectrometers (253Plus, Thermo, United States). Calculations of 29N2 and 30N2 produced by anammox and SDAD were provided in the supplementary material (Text S1 and Text S2).

Determination of N2O production pathways and rates by 15N tracer tests

15N tracer tests using Na15NO3 (>99.1 %15N, CIL, USA) and 14NH4Cl were performed to determine the pathways of N2O production, as well as its production and consumption rates at different N/S ratios (Table S2). The specific experimental procedure for the labeling test was consistent with the nitrogen conversion pathway determination experiment. At the end of the incubation, 8 mL of the sludge-water mixture was withdrawn from each sample and equally transferred to two He-purged 12 mL headspace vials. One headspace vial was used directly to determine the N2 isotope (29N2 and 30N2), while the other was placed in a shaker for 30 min to transfer N2O from the solution into the headspace. Then, 1 mL of headspace gas was withdrawn into 20 mL He-purged headspace vials for the determination of the N2O isotope (44N2O, 45N2O, and 46N2O). The concentrations of N2O isotope (44N2O, 45N2O, and 46N2O) and N2 isotope (29N2 and 30N2) were determined using isotope ratio mass spectrometers (253Plus, Thermo, United States).

The total accumulated N2O (Dtotal, N2O) was the sum of all N2O isotope, which included 14N14NO (44N2O), 15N14NO + 14N15NO (45N2O), and 15N15NO (46N2O) (Eq (3)). The true N2O production (rN2O, prod) was calculated by (Eq (4)). The 30N2 production resulted from 46N2O consumption; therefore, the sum of the N2O production (DN2O, total) and 30N2 production (DN2, total) represented the true N2O production. The calculations for 30N2 production (DN2, total) were provided in the supplementary material (Text S1 and Text S2).(3) Dtotal, N2O = D44N2O + D45N2O + D46N2O

(4) rN2O, prod = DN2, total + DN2O, total

Kinetic analysis of N2O production

The kinetic analysis of the rN2O, prod of the system was carried out to understand the N2O production pattern at different N/S ratios. The Logistic model was used to analyze the generation of new organisms in typical cycles (Zhan et al., 2024). The kinetic models and parameters are as follows:

The Logistic model:N2Oprod=N2Omax1+exp⁡(−λ*(x−xa))

where N2Oprod represents N2O production value (µmol). N2Omax represents maximum N2O production value (µmol), λ represents N2O production rate (µmol/min), and xa is the time corresponding to 50 % of maximum N2O production value (min).

Quantitative reverse transcription PCR (qRT-PCR) of functional genes

To investigate the transcriptional responses of functional genes related to nitrogen (napA, nirS, nosZ, hzsB) and sulfide (sqr and soxb) transformation under different N/S conditions, 5 mL of mixed liquor was taken from the batch reactors at the end of the batch experiment, and then centrifuged to remove the supernatant. The sludge pellets were immediately stored at −80 °C until use. Total RNA was extracted using the RNA PowerSoil™ Total RNA Isolation Kit (MoBio Laboratories Inc, USA). RNA concentration and purity were measured with a Nanophotometer (P-class, Implen, Germany). A total RNA of 2 μg was reverse transcribed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). The concentration and purity of the cDNA were measured with a Nanophotometer (P-class, Implen, Germany). The qRT-PCR was performed on an ABI 7500TM Real-Time PCR System (Applied Biosystems, CA, USA). Each qPCR reaction was carried out in triplicate and consisted of 5 μL 2 × TB Green Premix Ex Taq II, 0.4 μL of each primer, 0.2 μL 50 × ROX Reference Dye, 1 μL of cDNA template, and 3 μL of ddH2O. The PCR procedures for the amplification of napA, nirS, nosZ, hzsB, sqr and soxb genes were described in detail in the Supplementary material (Table S4). The efficiencies of the real-time PCR assays were over 95 % and the r2 values were 0.99.

CRediT authorship contribution statement

Mengjia Zhan: Writing – original draft, Data curation, Conceptualization. Wei Zeng: Writing – review & editing, Funding acquisition. Xiaojing Hao: Methodology. Haohao Miao: Investigation. Yao Lu: Validation. Wenzhuo Jiang: Project administration. Qingan Meng: Visualization. Qingteng Gong: Formal analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

Data availability

No data was used for the research described in the article

Acknowledgments

This research was supported by the 10.13039/501100012166 National Key Research and Development Programme of China (2021YFC3200601 ) and the 10.13039/501100001809 National Natural Science Foundation of China (52070004 ).

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.wroa.2024.100257.
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