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. 2024 Dec 27;27:100299. doi: 10.1016/j.wroa.2024.100299

Smaller sizes of polyethylene terephthalate microplastics mainly stimulate heterotrophic N2O production in aerobic granular sludge systems

Yingrui Liu a, Yanying He a, Qian Lu a, Tingting Zhu a, Yufen Wang a, Yindong Tong a, Yingxin Zhao a, Bing-Jie Ni b, Yiwen Liu a,⁎
PMCID: PMC11758821  PMID: 39867741

Highlights

  • •

    Effects of PET MPs with different particle sizes on N2O turnovers are firstly uncovered.

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    PET MPs (especially 0.1 mm) stimulate total N2O production by denitrification in the AGS system.

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    0.1 mm PET MPs induce N2O accumulation by decreasing electron transport during denitification.

  • •

    0.1 mm PET MPs further restrain N2O-sink capability by reducing key genes related to N2O reduction.

Keywords: Nitrous oxide (N2O), Microplastics (MPs), Particle sizes, Aerobic granular sludge (AGS), Polyethylene terephthalate (PET)

Abstract

Widespread polyethylene terephthalate microplastics (PET MPs) have played unintended role in nitrous oxide (N2O) turnovers (i.e., production and consumption) at wastewater treatment plants (WWTPs). Mainstream aerobic granular sludge (AGS) systems possess potentially strong N2O-sink capability, which may be reduced by PET MPs stress through altering N2O-contributing pathways, electron transfer, and microbial community structures. In this study, the effects of PET MPs with two common particle sizes of effluent from WWTPs (0.1 and 0.5 mm) on N2O turnovers, production pathways and N2O-sink capability were systematically disclosed in AGS systems by a series of biochemical tests and molecular biological means to achieve the goal of carbon neutrality. The results indicated that 0.1 mm PET MPs could more significantly stimulate N2O production in AGS systems by inhibiting denitrifying metabolism, compared with control and 0.5 mm PET MPs systems. Specifically, 0.1 mm PET MPs slightly increased the relative abundance of Nitrosomonas, reducing N2O yields via promoting the hydroxylamine (NH2OH) oxidation pathway during nitrification. Also, 0.1 mm PET MPs inhibited the electron transport system activities and the relative abundance of N2O reductase, hindering N2O reduction during denitrification. Most importantly, 0.1 mm PET MPs more apparently reduced the N2O-sink capability based on the ratio of N2O reductase gene and nitrite reductase gene.

Graphical abstract

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1. Introduction

Nitrous oxide (N2O), as a potent greenhouse gas with global warming potential of approximately 300 times greater than that of carbon dioxide (CO2) (Tong et al., 2024), potentially contributes to carbon footprints at wastewater treatment plants (WWTPs) (Yu et al. 2022). During the biological nitrogen removal (BNR) process, nitrification and denitrification play a significant role in regulating N2O emissions at WWTPs (Dai et al. 2021; He et al. 2023a; Su et al. 2023; Zheng et al. 2024). Nitrification contributes to N2O production by ammonia-oxidizing bacteria (AOB) via two primary pathways including the hydroxylamine (NH2OH) oxidation pathway (He et al. 2023b), i.e., NH2OH→nitrosyl radical (NOH)/nitric oxide (NO)→N2O, and AOB denitrification pathway, i.e., nitrite (NO2-)→NO→N2O. Denitrification, as a N2O sink (Conthe et al. 2019), involves four respective specific reductases of heterotrophic denitrifying bacteria (HB), i.e., nitrate (NO3-), NO2-, NO, and N2O reductase (Nar, Nir, Nor and Nos) for four sequential reductive reactions, i.e., NO3-→NO2-→NO→N2O→N2, respectively, through a complete electron transfer chain. Recently, several reports have suggested that aerobic granular sludge (AGS) systems demonstrated lower N2O emissions compared to conventional activated sludge systems (Liu et al. 2024). However, extreme environmental stress could intensify N2O emissions in aquatic environments by altering the abundance and activity of related bacteria, enzymes and key genes of AGS in terms of N2O turnovers (i.e., production and consumption) (Li et al. 2023).

Microplastics (MPs), as an emerging pollutant, have been commonly detected in the influent of the mainstream BNR system in WWTPs (Yang et al. 2019), (Mahon et al. 2017). Thus, the dominant position of mainstream AGS system for lower N2O production could be threatened because of MPs stress. Specifically, MPs can alter the biomass (Zheng et al. 2022), extracellular polymeric substance (EPS) contents (Jachimowicz et al. 2022), granule structure (Qin et al. 2020) and microbial abundance (Dai et al. 2020) of AGS as well as the biochemical nitrogen conversion process of the system, all of which are closely related to N2O turnovers. Recently, He et al. explored the effects of polyethylene terephthalate (PET) MPs (He et al. 2022) and polyvinyl chloride (PVC) MPs (He et al. 2024) with various doses on N2O emissions during complete nitrogen removal and partial nitrification in activated sludge systems. However, they ignored another important influence characteristic of MPs, particle size (Li et al. 2022; Shi et al. 2024; Zhang et al. 2022). Reportedly, over 90 % of MPs in the effluent of WWTPs were smaller than 0.5 mm and around 60 % of MPs were smaller than 0.1 mm (Mintenig et al. 2017). He et al. (He et al. 2021) studied the inhibition effect of polystyrene MPs (0–0.3 mm) in activated sludge systems on nitrogen conversion, whereas did not further investigate the influence of particle sizes on N2O emissions. In addition, the interaction between MPs with different particle sizes and granular sludge could alter the structure of AGS and the adhesion by EPS on AGS surfaces (Jachimowicz et al. 2022), which may indirectly affect N2O emissions. Therefore, systematically exploring the impacts of MPs with different particle sizes on N2O turnovers in AGS systems could fill the knowledge gaps.

Therefore, this study aimed to reveal the N2O emissions principle and N2O turnovers mechanism during nitrification and denitrification in long-term PET MPs-exposed AGS systems by integrating real-time N2O detecting, related N2O calculations, isotopic measurements and molecular biological means. This study offered a thorough insight into the influence of PET MPs stress on N2O turnovers within AGS systems, potentially aiding in assessing the environmental risk of MPs on N2O footprint at WWTPs in the future.

2. Results and discussion

2.1. Effects of PET MPs with different particle sizes on N2O production during a typical cycle and sole nitrification

The nitrogen removal and N2O production within three AGS systems were illustrated over a typical 6-h cycle (Fig. 1A-D). The results showed that PET MPs stress deteriorated the TN removal efficiency (ca. 0.6 %−15.3 %) and exacerbated N2O production (ca. 0.13–0.2 %) in AGS systems, especially smaller PET MPs (0.1 mm) exposure (Text S1). This was likely because PET MPs could potentially influence N2O -linked microbial activity through leaching toxic chemicals (i.e., additives) (Seeley et al. 2020) and increasing the generation of reactive oxygen species (2022, Wei et al. 2020).

Fig. 1.

Fig 1

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(A) removal efficiency of total nitrogen (TN) and NH4+-N, (B) gas-phase N2O and (C) N2O emission factors in three AGS systems including the control and exposure to two PET MPs particle sizes (i.e., AGS, AGS_0.5mmPET and AGS_0.1mmPET) during a typical 6-h cycle.

Fig. 2A and S3 depicted the nitrogen compounds, DO and pH profiles for the three AGS systems during nitrification batch test 1. The N2O concentration was gradually produced during 45-min experimental phase. In Fig. 2B, N2O production appeared to decrease in the following order: AGS>AGS_0.5mmPET>AGS_0.1mmPET, i.e., the N2O production rate (Fig. 2C) decreased by 0.01–0.015 mg-N/h/g VSS as the PET MPs exposure (p < 0.01), and N2O emission factors in PET MPs-exposed AGS systems decreased by 0.45–0.56 % (Fig. 2D). From Fig. 2E, PET MPs stress increased the SP value by ca.1.9–2.3 ‰, confirming that PET MPs (especially smaller PET MPs) altered N2O production pathways driven by AOB (Fig. 2H). Specifically, the AOB denitrification pathway accounted for 54.9 % in the control system (Liu et al. 2024), while the contribution of NH2OH oxidation pathway gradually increased from 51.6 % to 52.8 % with the decreasing particle sizes of PET MPs. This was likely because MPs might increase the media oxygen content within the nitrifying AGS system (Guo et al. 2024), where oxygen was used for further NO2- oxidation to NO3- instead of reduction to N2O through the AOB denitrification pathway, thus leading to the decreasing contribution of AOB denitrification pathway (Liu et al. 2024; Peng et al. 2015).

Fig. 2.

Fig 2

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Batch test 1 during nitrification: (A) NH4+, NO2-, NO3- DO and N2O profiles in AGS_0.1mmPET; (B) gas-phase N2O profiles, (C) N2O production rate, (D) N2O emission factors, (E) site preference of N2O and (F) relative contributions of different N2O production pathways in AGS, AGS_0.5mmPET and AGS_0.1mmPET.

2.2. Impacts of PET MPs with different particle sizes on N2O turnovers and N2O-sink capability during denitrification

2.2.1. N2O turnovers, reduction rates and electron consumption rates

From Fig. 3A-C, compared to the control system, PET MPs (especially 0.1 mm PET MPs) stress inhibited N2O reduction (He et al. 2022) in schemes c and g (i.e., ca.3.82–6.61 mg-N/L) and promoted N2O accumulation in scheme d (i.e., 0.04–0.2 mg-N/L) during experiment 1 (Text S2). Moreover, PET MPs significantly decreased N2O reduction rates by 2.3–4.8 mg/g VSS/h and electron consumption rates of Nos 0.17–0.34 m mole/g VSS/h, and its inhibitory effects was intensified with the decreasing particle sizes from 0.5 to 0.1 mm (Fig. 3D-I, Text S3).

Fig. 3.

Fig 3

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Batch test 2 during denitrification in AGS (R1), AGS_0.5mmPET (R2) and AGS_0.1mmPET (R3): liquid-phase N2O profiles from experiment 1 of three AGS systems in scheme (A) c, (B) d and (C) g. NOx reduction rate and electron consumption rate in schemes a-g under non-carbon-limiting conditions during (D and G) experiment 1, and under carbon-limiting conditions during (E and H) experiment 2 and (F and I) experiment 3.

2.2.2. Electron competition, distribution, transfer and N2O-sink capability

In Fig. 4A, the susceptibility of rN2O under non-carbon-limiting condition was significantly weakened with PET MPs adding (Pan et al. 2013b), especially in AGS_0.1mmPET (i.e. 6.1 % (e1), 6.2 % (f1) and 13.2 % (g1)). The electron competition intensity under carbon-limiting condition gradually increased (ca. 0.4–0.6) in PET MPs-exposed systems (Fig. 4A). In Fig. 4B, the average γ (Conthe et al. 2019) decreased from 3.6 in the control system to 3.3 in AGS_0.5mmPET and 3.2 in AGS_0.1mmPET, suggesting an apparent inhibition of PET MPs to N2O-sink capability.

Fig. 4.

Fig 4

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Batch test 2 during denitrification in AGS, AGS_0.5mmPET and AGS_0.1mmPET: (A) degree of decrease in N2O reduction rates (rN2O) from experiment 1 and the intensity of electron competition during experiment 1–3; (B) N2O-sink capability represented by the ratio γ; percentages of electrons distribution in scheme d (C), e (D), f (E), g (F) for experiments 1, 2 and 3, respectively. (G) ETSA, (H) Cyt-c and (I) NADH concentrations.

For electron distribution, decreasing electrons distributed to Nos with PET MPs exposure were observed in scheme d (Fig. 4C), inducing N2O accumulation described above (Fig. 3B) (Pan et al. 2013a). The highest decreasing rates of electron distributed to Nos in schemes e (Fig. 4D), f (Fig. 4E) and g (Fig. 4F) were observed in AGS_0.1mmPET. Most typically, in scheme g, electrons allocated to Nos in AGS_0.1mmPET reduced by 8.5–10.5 % compared to that of the control system (Fig. 4F). Such observations were possibly due to the inhibitory influence of PET MPs on bioenergy conversion (Text S4) (Almeida et al. 1995; Oberoi et al. 2021), electron transfer and the activity of several key enzymes.

Reduced nicotinamide adenine dinucleotide (NADH) was mainly involved in the front-end electron transfer process (Chen and Strous 2013; Sazanov and Hinchliffe 2006). In Fig. 4G, the NADH concentration of 187.3 ng/L in the control system obviously decreased to 175.6 ng/L in AGS_0.5mmPET, and further declined to 134.2 ng/L in AGS_0.1mmPET, indicating an inhibitory effect of PET MPs on the front-end electron transfer process. Periplasmic cytochrome c (Cyt-c), as an electron carrier, directly provided electrons to Nir and Nos in the downstream electron pool (Qi et al. 2012). In Fig. 4H, after exposure to 0.5 and 0.1 mm PET MPs, the Cyt-c concentration decreased by 2.77 and 6.44 nmol/L, respectively, compared to the control. The results indicated that PET MPs inhibited the activity of electron carriers (i.e., Cyt-c) in the downstream electron pool, further reducing the number of electrons distributed to Nos. From Fig. 4I that electron transport system activity (ETSA) of 1.39 mg TF/g VSS/h in the control decreased to 1.24 mg TF/g VSS/h in AGS_0.5mmPET and 1.19 mg TF/g VSS/h in AGS_0.1mmPET. This delineated that PET MPs significantly hindered electron transfer efficiency in the AGS system.

2.3. Effects of PET MPs with different particle sizes on microbial community and functional gene abundance

The rarefaction curves and alpha diversity for three samples (i.e., in two PET-exposed groups were higher than the control group) indicated that PET MPs could enhance microbial richness and diversity in AGS systems ((Fig. S4–5). At the phylum level (Fig. 5A), an increasing relative abundance of Bacteroidota (ca. 3.3–9.5 %) was observed in PET MPs-exposed systems (Dai et al. 2020; He et al. 2022; Wang et al. 2023; Zhang et al. 2017). In addition, the highest relative abundance of Bacteroidota (27.5 %) was observed in AGS_0.1mmPET, indicating that smaller PET MPs were more easily adhered to AGS surfaces (Wang et al. 2021). At the genus level (Fig. 5B), Thauera was plausibly associated with N2O reduction activity during the complete denitrification (Chourey et al. 2013) and its abundance was decreased by 4.5 % in AGS_0.1mmPET. Also, Rhodobacter was documented to be associated with N2O emission and the expression of the nirK gene (Vieira et al. 2019). The decreasing relative abundance of Rhodobacter (i.e., decreased by 0.16 % in AGS_0.1mmPET) could induce NO2- accumulation, and then lead to N2O accumulation. Moreover, the relative abundance of Nitrosomonas (i.e., AOB) increased from 0.8 % in the control system to 1.1 % in AGS_0.1mmPET (He et al. 2022).

Fig. 5.

Fig 5

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Relative abundance of bacterial populations in AGS, AGS_0.5mmPET and AGS_0.1mmPET: (A) at phylum level, (B) at genus level, (C) the predicted relative abundances of key enzymes, (D) the nitrogen metabolism KEGG pathway related to N2O turnovers and (E) ratio of nosZ and nirK+nirS from qPCR.

Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database were utilized to explore the relative abundance of crucial genes and enzymes associated with N2O production and reduction (He et al. 2022). From Fig. 5C and Table S1, ammonia mono-oxygenase (AMO), represented by EC 1.14.99.39 (K10944, K10945 and K10946), was slightly elevated by 0.2 % in AGS_0.1mmPET, consistent with the variation of Nitrosomonas. The relative abundance of hydroxylamine oxidoreductase (HAO) represented by EC 1.7.2.6 (K10535) also increased in AGS_0.1mmPET, further explaining the increasing contribution of NH2OH oxidation pathway (Fig. 2F). The relative abundance of all four denitrification reductases (i.e., Nap/Nar, Nir, Nor and Nos) showed a decreasing trend in AGS_0.1mmPET (Fig. 5C-D). For example, the abundance of Nos (K00376) decreased from 2.2 % in the control system to 2.1 % in AGS_0.5mmPET and 1.8 % in AGS_0.1mmPET, further confirming the inhibitory effect of PET MPs (especially 0.1 mm) on N2O reduction. For quantitative polymerase chain reaction (qPCR), the abundance of NosZ in AGS_0.5mmPET and AGS_0.1mmPET was reduced by 13.6 % and 27.3 %, respectively (Table S2). The reduced abundance of Nir (ca. 11.2 %−13.8 % under PET MPs stress) was regarded as mainly ascribed to HB. Additionally, decreased NosZ/(NirS+NirK) (Fig. 5E) was observed in PET MPs-exposed systems (especially 0.1 mm), suggesting that PET MPs exposure weakened the N2O-sink capacity in AGS systems (Chen et al. 2020).

2.4. Mechanism of PET MPs with different particle sizes influencing N2O emissions in AGS systems

During a typical cycle, PET MPs (especially smaller particle sizes) inhibited total nitrogen removal (Fig. 1A) and stimulated N2O production (Fig. 1C) in AGS systems. For nitrification, PET MPs exposure faintly increased the relative abundance of Nitrosomonas and AMO/HAO (Fig. 5B-D and Table S1), thus slightly reducing N2O yields produced by AOB (Fig. 2D) in the AGS system via promoting the contribution of the NH2OH oxidation pathway (Fig. 2F). Therefore, the upward trend and the downward trend of N2O emission factors in cyclic and nitrification batch tests, respectively (Fig. 1C and 2D), indicated that PET MPs stimulated the total N2O production in AGS systems mainly by inhibiting the denitrification process. For denitrification, PET MPs (especially smaller particle sizes) inhibited the activity of NADH (Fig. 4I) and Cyt-c (Fig. 4H) to reduce the electron transport activity (Fig. 4G), reduced electron affinity of Nos and intensified the electron competition intensity, thus inducing N2O accumulation. In addition, PET MPs reduced the distribution of electrons to Nos, thereby hindering N2O reduction (Fig. 3D). Subsequently, PET MPs reduced the relative abundance of denitrifiers and enzymes, especially key genes associated with N2O turnovers (NirK, NirS, NosZ), restraining the N2O-sink capability during denitrification in AGS systems (Fig. 4B, 5E).

Smaller PET MPs (0.1 mm) would more stimulate N2O emission and inhibited N2O reduction. Specifically, PET MPs first increased the relative abundance of Bacteroidota conducive to their adhesion to AGS surfaces (Fig. 5A), laying the foundation for the interaction between PET MPs and AGS. The surface of AGS observed by SEM collected from the control system was dense and complete (Fig. S6A), while that exposed to PET MPs was damaged and had many cracks (Fig. S6B-C). Previous studies have reported that microplastics could attach to the surface of AGS (Wang et al. 2021), which prevented the transfer of nutrient matrix, resulting in a decrease of EPS content secreted by AGS (Zhang et al. 2020). The result of EPS further indicated that smaller PET MPs would be easier to accumulate in large quantities on AGS (Fig. S7). Therefore, smaller PET MPs (0.1 mm) should be given attention in N2O emissions during nitrogen removal in AGS systems, because after all, 60 % of MPs smaller than 0.1 mm in the effluent of WWTPs were still detected.

2.5. Implications

This work exhibited that smaller PET MPs significantly stimulated N2O production via inhibiting denitrification in the AGS system. The effect of different concentrations of MPs on N2O production in wastewater treatment has been preliminatively understood (He et al. 2022; Li et al. 2020), while the influence of MPs structural morphology (e.g., particle sizes) is still unknown. This work explored whether N2O turnovers were promoted or inhibited by PET MPs with different particle sizes during nitrification and denitrification in AGS systems, ultimately identifying particle sizes and underlying mechanisms that stimulated higher N2O emissions. Specifically, the relative abundance of denitrifiers and reductase activity in AGS are inhibited by PET MPs (especially 0.1 mm). Flocs could promote N2O reduction during denitrification (Liu et al. 2023c), thus the lack of denitrifiers in AGS can be compensated by the addition of returned floc sludge to establish the suspend/attach-based system. In addition, PET MPs (especially smaller sizes) also inhibit the electron transport chain. As the main source of electron donors during denitrification in the AGS system, extra organic carbon may help to alleviate the negative effect of PET MPs on N2O turnovers. Additionally, over 90 % of MPs in the effluent were smaller than 0.5 mm (around 60 % of MPs were smaller than 0.1 mm) at WWTPs (Mintenig et al. 2017; Zhang et al. 2024a; Ziajahromi et al. 2017). Therefore, a finer screen or diverter can be set before entering the AGS system to avoid AGS treating wastewater containing smaller MPs and further alleviate N2O footprint in AGS systems.

3. Conclusions

This work aimed to uncover effects and mechanisms of PET MPs with different particle sizes on N2O turnovers in mainstream AGS systems. 0.1 mm PET MPs further exacerbated N2O emissions by 8.2 % compared to 0.5 mm PET MPs during cyclic studies. PET MPs slightly reduced N2O production by weakening the contribution of the AOB denitrification pathway (by 11.8 %−14 %). PET MPs inhibited the electron transfer activity, thus hindering N2O reduction and inducing N2O accumulation during denitrification in AGS systems. Also, PET MPs (especially 0.1 mm) reduced the relative abundance of nirK, nirS, nosZ, restraining the N2O-sink capability during denitrification in AGS systems. Therefore, this study provided a theoretical basis for the effect of MPs with different particle sizes on N2O emissions at WWTPs, and various particle sizes of MPs are worth studying in the future.

4. Materials and methods

4.1. Experimental granules, PET MPs, setup and cyclic study

The inoculated AGS with granule size of ca. 1.0 mm was obtained from a lab-scale mother AGS sequencing batch reactor (AGS-SBR) fed with synthetic wastewater (Table S3). The PET MPs with two particle sizes (i.e., 0.5 and 0.1 mm) were obtained from HuaChuang Co., Ltd. (Dongguan, China). Two experimental AGS-SBRs fed with 0.5 and 0.1 mm PET MPs (5 mg/L) named AGS_0.5mmPET and AGS_0.1mmPET, and a control AGS-SBR (0 mg/L of PET MPs) were operated for over 120 days in this study. (Text S5). A typical 6 h cyclic test was complemented on day 120 to evaluate N2O emissions in each AGS-SBR (Text S6).

4.2. Batch tests design

4.2.1. N2O heterotrophic consumption tests

To verify no N2O turnovers by heterotrophs during nitrification batch tests, three essential N2O heterotrophic consumption tests by heterotrophic bacteria under aerobic conditions without organic carbon were carried out. In order to eliminate the interference of endogenous denitrification, the 1-h experiment was selected to uncover N2O production mechanism by AOB. These consumption tests utilized the same liquor as described above and were performed at DO levels of 3.0 mg-O2/L. The tests were conducted in a full-sealed reactor at 25 ± 1 °C, eliminating the headspace. Each test contained evenly mixed aerobic granules. Once the appointed DO concentration was reached, the aeration was stopped immediately. Afterward, the freshly prepared saturated N2O solution was transferred to the reactor to obtain an N2O concentration of 1.1 mg-N/L. Fig. S1 showed that the concentration of DO was almost stable within 1 h, since no additional organic carbon, NH4+, or NaHCO3 was added.

4.2.2. Batch tests during nitrification

For nitrification (batch test 1), batch tests were conducted to investigate N2O production and its pathways by AOB under different particle sizes of PET MPs stress at 25 mg-N/L NH4+, 3.0 mg-O2/L of dissolved oxygen (DO) and pH=7.5 (Table S4). All batch tests were conducted after the N2O heterotrophic consumption test (Section 4.2.1), to exclude the interference of heterotrophic bacteria (Fig. S1). The batch reactor operation, granules and samples extraction, DO and pH control, as well as the selection of experiment duration and phase were all consistent with our previous study[]. Aerobic granules were individually extracted from the three reactors during the decanting period and washed six times. Each batch test was conducted in a reactor with a working volume of 800 mL and a sealable lid. DO and pH (adjusted by 1 M HCl or NaHCO3) was dynamically maintained through a programmed logic controller (PLC), and monitored online using a DO probe (MIK-DO-7012–7 M) and a pH probe (MIK-pH-5018-BBL5), respectively. Mixed liquor volatile suspended solids (MLVSS) from the end of each batch test were measured in triplicate. NH4+ and DO levels were controlled to be relatively stable during the entire experiment, which were separately achieved through the ammonia oxidation rate (AOR) calculation based on a KLa test (Text S7), and two air/N2 mass flow controllers (AST10-DLCMX, Asert, Text S8). The total gas flow rate, regulated by these controllers, was consistently maintained at 0.5 L/min. Each batch test had a duration of 60 min. The first 15 min (i.e., the control phase) were conducted to ensure that any observed N2O production originated exclusively from heterotrophic denitrification bacteria. During the subsequent 45 min (i.e., the experimental phase), a solution containing 39.43 g/L of NH4Cl and 83.3 g/L of NaHCO3 was added to achieve an initial NH4+ concentration of approximately 25 ± 4 mg-N/L, along with adequate HCO3-. Sample collection for the analysis of NH4+, NO2-, and NO3- concentrations occurred every 15 min. The gas-phase N2O levels were monitored online by a N2O analyzer (AO2020 series, ABB, Text S9).

4.2.3. Batch tests during denitrification

Batch tests during denitrification with the addition schemes of nitrogen compounds including seven combinations of NO3-, NO2- and N2O (schemes a, b, c, d, e, f, g) under conditions of sufficient and insufficient carbon sources were designed in Table S5. All batch tests were performed using glucose/sodium acetate as the electron donor. The initial NO3-, NO2- and N2O concentrations of ca. 20 mg-N/L were achieved by injecting NaNO3, NaNO2 or N2O stock solution, respectively. For the non-carbon-limiting condition (experiment 1), the COD stock solution (glucose/sodium acetate=1/3) was added to obtain an initial COD/N ratio of 10. Separately, schemes a, b and c in experiment 1 were compared to evaluate the maximum specific reduction rates of NO3-, NO2- and N2O for the three PET MPs-exposed AGS systems. Individually, schemes d and g in experiment 1 were compared to assess varying N2O accumulation and reduction in the presence of multiple nitrogen oxide (NOx, i.e., suitable for the actual wastewater treatment) for the three AGS systems. For the carbon-limiting condition, the COD stock solution was added to the batch reactor through a peristaltic pump with a slow feeding mode to achieve a carbon loading rate of 60 mg-COD/g VSS/h in experiment 2 and 20 mg-COD/g VSS/h in experiment 3. Such design was aimed to explore the NOx reduction rates, N2O reduction capability, electron competition and electron distribution for the three AGS systems. The batch reactor operation, granules and samples extraction, pH control and N2O stock solution acquisition were demonstrated in detailed in Text S10. The liquid-phase N2O concentrations were measured continuously by an Unisense microsensor (N2O-R-100, Unisense A/S, Text S9).

4.3. Calculations

During nitrification batch tests, the specific N2O production rate (mg-N/g VSS/h) was computed by dividing the gaseous N2O concentration by the MLVSS concentration of AGS, and then multiplying by the gas flow rate. The average N2O production rate was determined by calculating the average during the period with stable values of N2O production rate (relatively constant in all cases). The N2O emission factor was calculated as the ratio of emitted N2O—N to converted NH4+-N. The converted NH4+-N concentration was determined based on the added and measured NH4+-N profiles according to our previous study (Liu et al. 2024). The calculations of the SP-based quantification of two N2O production pathways during nitrification are presented in Text S11.

During denitrification batch tests, the maximum NO3-, NO2- and N2O consumption rates were calculated via linear regression of NO3-, NO2- and N2O profiles, respectively. Such rates were divided by the MLVSS concentration to obtain the specific reduction rates. The calculation of NOx reduction rate, electron consumption rate and electron distribution were demonstrated in Text S12. Susceptibility of rN2O and the intensity of electron competition were calculated according to previous studies (He et al. 2024).

NO3- reduction rates in the presence of sole NO3- scheme (rNO3) and N2O reduction rates under the absence of other nitrogen compounds (rN2O) were regarded as estimates of the maximum N2O production rates (VmaxNO3-→N2O=V1) and the maximum N2O reduction rates (VmaxN2O→N2=V2), respectively, assuming no obvious intermediates accumulation. The ratio γ = V2/V1 was calculated to express the N2O-sink capacity of AGS (Conthe et al. 2019). Additionally, the N2O-sink capacity was also represented by the ratio of nosZ and Nir (i.e., nirK+nirS) from quantitative PCR (He et al. 2024).

4.4. Analytical methods

NH4+, NO2-, NO3-, MLVSS, and MLSS measurements were carried out using standard methods (Association and Washington 1995). Surface morphologies of MPs and sludge were examined utilizing scanning electron microscopy (SEM, Hitachi S4800, Japan) (Wang et al. 2024b). The EPS extraction and component determination were according to a previous study (Liu et al. 2023b; Wang et al. 2024a; Zhang et al. 2024b). The electron transport system activity (ETSA) at the end of the typical cycle in each AGS-SBR was determined by using 2, 3, 5-triphenyltetrazolium chloride (Text S13) (Guo et al. 2023; Guo et al. 2022). Enzyme-linked biological nicotinamide adenine dinucleotide (NADH) kit was used to determine the NADH content in AGS-based samples (Xi et al. 2013). The periplasmic Cyt-c was collected by centrifugating (8000 rpm, 5 min) the effluent from the end of typical cycle in each AGS system, subsequently measured using an UV–VIS spectrophotometer at 520 nm (Liu et al. 2023a). High-throughput sequencing was employed to discern variations in the microbial community following PET MPs exposure. The real-time PCR amplification was conducted using universal primers 515F and 806R targeting the V4 region of bacterial 16S DNA genes (Xiang et al. 2023). Functional gene abundances were assessed by quantitative PCR with the primers detailed in Table S6 (Han et al. 2022; Zheng et al. 2023). Data analysis was performed using SPSS 26.0 (Wang et al., 2022), and statistical significance was considered for p < 0.05 (Tian et al. 2022).

CRediT authorship contribution statement

Yingrui Liu: Writing – original draft. Yanying He: Conceptualization. Qian Lu: Data curation. Tingting Zhu: Formal analysis. Yufen Wang: Investigation. Yindong Tong: Methodology. Yingxin Zhao: Project administration. Bing-Jie Ni: Validation. Yiwen Liu: Writing – review & editing.

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.

Acknowledgements

This study was funded by National Natural Science Foundation of China through projects (No. 52470108). The authors are grateful to the research collaboration.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.wroa.2024.100299.

Appendix. Supplementary materials

mmc1.docx (1.1MB, docx)

Data availability

Data will be made available on request

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (1.1MB, docx)

Data Availability Statement

Data will be made available on request

Articles from Water Research X are provided here courtesy of Elsevier

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