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Article

Impact of Naproxen on Wastewater Biological Treatment: Focus on Reactor Performance and Mechanisms

by
Lidan Wei
and
Wenbin Zhang
*
School of Resource Engineering, Heilongjiang University of Technology, Jixi 158100, China
*
Author to whom correspondence should be addressed.
Water 2023, 15(14), 2615; https://doi.org/10.3390/w15142615
Submission received: 17 May 2023 / Revised: 4 July 2023 / Accepted: 9 July 2023 / Published: 19 July 2023
(This article belongs to the Section Wastewater Treatment and Reuse)
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Abstract

:
Pharmaceutical contamination has emerged as a significant environmental concern; yet, the impact and underlying mechanisms of widely detected naproxen (NPX) on wastewater biological treatment remain poorly understood. To address this knowledge gap, this study investigates the influence of NPX on biological nutrient removal in wastewater treatment under mesophilic conditions, and elucidates the associated mechanisms. The results demonstrate that NPX concentrations below 0.05 mg/L have no significant effect on the removal of pollutants and nutrients in wastewater, whereas the concentrations exceeding 0.5 mg/L hinder the removal of both chemical oxygen demand (COD) and nutrients, with a more pronounced inhibitory effect observed at higher NPX doses. When the concentration of NPX was 2.0 mg/L, the removal efficiency of COD, NH4+-N and phosphate decreased to 81.2~83.6%, 82.5~84.2% and 80.2~82.6%, respectively, which was much lower than that of the blank. Furthermore, NPX leads to a decrease in activated sludge concentration and organic matter content within the sludge. Additionally, NPX promotes the secretion of extracellular polymeric substances (EPS). Periodic investigations reveal that NPX inhibits the biosynthesis of intracellular polymer polyhydroxyalkanoate (PHA), thereby reducing energy production during later stages of degradation. Enzyme activity analysis indicates that high NPX concentrations suppress the activity of key enzymes associated with biological nitrogen and phosphorus removal. These findings provide theoretical insights for the treatment of NPX-containing wastewater using activated sludge processes.

1. Introduction

The traditional activated sludge process has been widely adopted by wastewater treatment plants worldwide due to its advantages of low operational costs, ease of management, and mature technology [1,2]. The performance of wastewater biological treatment is closely related to wastewater quality, process flow, and operational parameters. Previous studies have focused on optimizing operational parameters and process flow to improve the effectiveness of wastewater biological treatment [3,4]. However, there are few studies on the effects of emerging pollutants in sewage on the biological nutrients removal and its potential mechanism.
Chiral pharmaceuticals are emerging contaminants frequently detected in wastewater [5]. After metabolism in the human or animal body, chiral pharmaceuticals are excreted in the form of urine or feces and enter municipal sewage systems. Since the existing wastewater treatment technologies have difficulty effectively removing them, these pharmaceuticals enter the aquatic environment through wastewater treatment plant effluents. Naproxen (NPX), an effective anti-inflammatory drug used in the treatment of arthritis and rheumatic diseases, has attracted significant attention [6,7]. However, its extensive use has resulted in significant drug residues and water pollution, with evident toxicity to aquatic organisms. The reported detection concentrations of NPX in the UK and Australia in the WWTP influent reached 136 ng/L and 177.5 ng/L, respectively [8,9]. Furthermore, a recent study confirmed that NPX detection concentration could be as high as 41.25 ng/L in Chinese water bodies [10]. Prolonged exposure to NPX in the environment may cause various adverse reactions in organisms, including oxidative stress responses, developmental expressions, and disruption of vertebrate endocrine systems [11]. Stancova et al. (2015) [12] found that even at a concentration of only 0.001 mg/L in water, adult zebrafish exhibited mild oxidative stress responses, and NPX had a certain impact on the expression of antioxidant genes in the zebrafish gut. In addition, Chen et al. (2022) [13] investigated the impact of NPX on hydrogen production during anaerobic sludge fermentation, and the results showed that high doses of NPX reduced hydrogen production and the activity of key enzymes involved in organic matter degradation (protease and amylase). The main removal methods of NPX in the environment are photoconversion and advanced oxidation process [14,15]. However, the above methods may lead to the production of more toxic intermediates [16,17,18]. The environmental behavior of the emerging pollutant NPX has received much attention. Previous studies have mostly focused on the occurrence characteristics, migration and transformation laws of NPX in the environment [12,13], while there have been few reports on the impact of NPX on wastewater biological treatment. This work explored the impact of NPX on wastewater biological treatment, filling the gap in this research. In addition, the mechanism of the impact of NPX exposure on wastewater biological treatment has not been elucidated yet.
Therefore, the main objective of this study is to investigate the effects of the emerging pollutant NPX on wastewater biological treatment and reveal the associated mechanisms. Firstly, the influence of different NPX concentrations on nutrient removal in the mesophilic activated sludge system was investigated. Subsequently, the effects of NPX exposure on the characteristics of activated sludge, changes in extracellular polymeric substances (EPS), and the biological transformation of nutrients and intracellular polymers during a typical cycle were analyzed. Finally, the key enzyme activities related to the biological nutrients’ removal in the activated sludge under NPX exposure were investigated. The research findings will provide data support and a theoretical basis for wastewater treatment processes involving NPX.

2. Materials and Methods

2.1. Experimental Materials

The inoculum sludge used in this work was obtained from the secondary sedimentation tank of a wastewater treatment plant in Harbin, China. The plant adopts the conventional A2/O process for urban wastewater treatment, with removal efficiencies of chemical oxygen demand (COD), total nitrogen (TN), and phosphate reaching 92.3%, 72.6%, and 92.3%, respectively. The collected inoculum sludge was filtered through a 2.0 mm sieve to remove impurities and then stored for later use. The main characteristics of the inoculum sludge were as follows: pH 7.1, total suspended solids (TSS) 4.2 ± 0.2 g/L, and volatile suspended solids (VSS)/TSS ratio ranging from 0.68 to 0.72. The determination of the above experiment was completed in the Chemistry Laboratory of Heilongjiang University of Technology.
Synthetic wastewater was used in the experiments. The carbon source in the synthetic wastewater consisted mainly of sodium acetate and sodium propionate, with a mass ratio of 2:1. Ammonium chloride and potassium dihydrogen phosphate were used as the nitrogen and phosphorus sources, respectively. The concentrations of COD, NH4+-N, and soluble phosphate in the wastewater were controlled at 210 ± 5.0 mg/L, 45 ± 2.0 mg/L, and 10.0 ± 0.9 mg/L, respectively. In addition, in order to improve the metabolic activity of microorganisms in activated sludge, 0.5 mL/L of trace element reserve solution was also introduced into the synthetic wastewater. The composition of trace element reserves mainly refers to previous literature [19,20]. The main components of the trace element reserve solution are as follows (g/L): 1.5 FeCl3·6H2O, 0.03 CuSO4·5H2O, 0.12 MnCl2·4H2O, 0.06 Na2MoO4·2H2O, 0.12 ZnSO4·7H2O, 0.15 CoCl2·6H2O, 0.18 KI, 0.15 H3BO3, and 10 EDTA.
The reactors used in the work were made of resin glass and had an effective working volume of 5.0 L. The sequencing batch reactors (SBR) were equipped with mechanical stirring devices and evenly distributed aeration discs at the bottom. The dissolved oxygen concentration during the aerobic phase was controlled at 3.5~4.5 mg/L using a rotor flowmeter (LZB-15 glass). The SBRs operated three cycles per day, with each cycle consisting of 8 h. The operational mode for each cycle was slightly modified based on the previous literature [21]. The operational mode for each cycle was as follows: anaerobic phase 120 min, aerobic phase 150 min, anoxic phase 90 min, settling and effluent discharge 30 min, and idle period 90 min. The volume of effluent discharge during each cycle was controlled at 3.4 L to achieve a hydraulic retention time of 12 h, and a mixed sludge discharge at the end of the aerobic phase to achieve a sludge retention time of 12 days. The chemicals used in the experiment were purchased from Aladdin Reagent Co., Ltd., Shanghai, China. NPX was first dissolved in ethanol and prepared as a solution of 0.5 mg/L. Subsequently, the solution of NPX was added to the SBR.

2.2. Influence of NPX on Nutrient Removal in Wastewater

The experiment was conducted in five sets of SBRs, with each set consisting of three identical reactors. Initially, each reactor was filled with 1.5 L of inoculum sludge and 3.5 L of synthetic wastewater. Subsequently, different doses of NPX were added to each reactor to achieve concentrations of 0.05 mg/L, 0.5 mg/L, 1.0 mg/L, and 2.0 mg/L, respectively. The above reactors were defined as R1~R4. One reactor (R0) served as the blank without NPX. There are two reasons for selecting the concentration of NPX in this study. On the one hand, considering the large production and consumption of NPX, the concentration of NPX in wastewater will show an upward trend [17]. On the other hand, this study provides a reference for the future environmental ecological assessment of NPX. The initial pH of the influent was controlled at 7.0 ± 0.2 by manually adding 2.0 M NaOH or HCl, with adjustments made every 30 min. Once the effluent from each group stabilized, regular sampling was conducted for analysis during typical cycles to assess the impact of NPX on wastewater biological treatment. In addition, sludge samples were taken at 100 days for enzyme activity analysis.

2.3. Analytical Methods

The determination of COD, NH4+-N, VSS (volatile suspended solids), TSS (total suspended solids), and phosphate followed international standard methods [22]. Polyhydroxyalkanoates (PHA) was determined using gas chromatography (Agilent-7890B). The extraction of EPS (Extracellular Polymeric Substances) was performed using the thermal extraction method reported in this document [23]. The analysis of key enzymes (exopolyphosphatase kinase (PPX) and polyphosphate kinase(PPK), ammonia monooxygenase (AMO), nitriteoxido reductase (NOR), nitrate reductase (NAR), nitrite reductase(NIR)) related to biological nitrogen and phosphorus removal in the literature [20] was carried out. The extracted EPS was stored at −20 °C in a freezer. Polysaccharides (PS) were measured using the anthrone method with glucose as the standard, while protein (PN) was measured using a modified Lowry method protein concentration assay kit from Shanghai Sangon Biotech, and the sum of PS and PN content was considered as the total EPS content. The pre-treatment conditions for PHA were as follows: an appropriate amount of sludge-water mixture was centrifuged at 8000 g for 5 min, and the supernatant was discarded. The sediment was then placed in a freezer (−20 °C) for 24 h and subsequently treated in a freeze-dryer for 24 h. After the process, 10 mg of dried sludge was taken out, and its mass was recorded (accurate to 0.0001 g). Then, 1 mL of chloroform and 1 mL of esterification solution were added sequentially and thoroughly mixed. The esterification solution was prepared with 3 mL of H2SO4 (98%), 0.29 g of benzoic acid, and 97 mL of methanol. The detection conditions were as follows: the chromatographic column model (HP-5/19091J-413) was used with N2 as the carrier gas, with flow rates and split ratios of 40 mL/min and 10/1, respectively. The temperatures of the injection port and detector were both set at 250 °C, and the initial and final oven temperatures were set at 70 °C and 150 °C, respectively. The linear heating rate was 25 °C/min, and the furnace temperature was maintained for 2 min.

3. Results and Discussion

3.1. Effects of NPX on COD and Nutrient Removal

Figure 1 illustrates the impact of NPX on effluent COD concentration and removal efficiency. It can be observed that in the blank, the effluent COD concentration initially decreased and then stabilized, maintaining a range of 9.2 mg/L to 11.5 mg/L during the steady period, corresponding to COD removal efficiency of 94.6% to 96.2%. The presence of NPX at a concentration below 0.05 mg/L had little effect on effluent COD, and the COD removal efficiency ranged from 93.5% to 96.4%. However, when the NPX concentration exceeded 0.5 mg/L, the effluent COD concentration was lower than that of the blank, and the higher the NPX exposure concentration, the greater the effluent COD concentration and the lower the corresponding COD removal efficiency. When the NPX concentration reached 2.0 mg/L, the effluent COD concentration increased to 37.1~42.5 mg/L, and the COD removal efficiency decreased to 81.283.6%, indicating that a high concentration of NPX reduced the pollutant removal capacity of the activated sludge. Chen et al. (2022) [13] confirmed that NPX had an inhibitory effect on the metabolism of microorganisms in anaerobic sludge systems, and this work further discovered that a high dosage of NPX had an inhibitory effect on the pollutant removal capacity of the activated sludge microorganisms.
Figure 2a shows the effect of NPX on NH4+-N removal. Unlike the effect of NPX on COD removal, NPX concentrations below 0.5 mg/L had little impact on NH4+-N removal. In the blank and the groups with 0.05 mg/L and 0.5 mg/L NPX, the NH4+-N removal efficiencies remained high at 93.696.4%. However, when the NPX concentration exceeded 1.0 mg/L, it resulted in a decrease in NH4+-N removal. Moreover, when the NPX concentration reached 2.0 mg/L, the NH4+-N removal efficiency decreased to 82.584.2%, which was significantly lower than the other groups’. A certain amount of NPX can inhibit the nitrification process, thus reducing the removal of ammonia nitrogen [13]. Figure 2b shows the influence of NPX on phosphate removal, which is also closely related to the NPX concentration. As the NPX concentration increased from 0.5 mg/L to 2.0 mg/L, the phosphate removal efficiency decreased from 86.787.9% to 80.282.6%, indicating that a high dosage of NPX reduced the phosphate removal in this activated sludge system. Studies have shown that NPX exhibits certain ecological toxicity to anaerobic microorganisms [24,25], and the metabolism of polyphosphate-accumulating organisms (PAO) during the anaerobic phase may be affected by NPX, thus reducing their ability to metabolize phosphate.

3.2. Effect of NPX on Activated Sludge Concentration

The sludge concentration and the proportion of organic matter in the sludge directly reflect the metabolic activity of the sludge [26]. Figure 3 further illustrates the long-term effects of NPX exposure on sludge concentration and the proportion of organic matter. It can be observed that the influence of NPX concentrations below 0.05 mg/L on MLTSS and MLVSS/MLTSS can be ignored. In the blank and the group with 0.05 mg/L NPX, the MLTSS concentration and MLVSS/MLTSS ratio were 4.26~4.35 g/L and 0.74~0.78, respectively, with no significant difference between the two groups. However, a high concentration of NPX had inhibitory effects on sludge concentration and the proportion of organic matter. When the NPX exposure concentration increased from 0.5 mg/L to 2.0 mg/L, the sludge concentration decreased from 4.31~4.35 g/L to 3.85~3.92 g/L, and MLVSS/MLTSS decreased from 0.69~0.71 to 0.62~0.64 during the stable period. The metabolic inhibition of microorganisms by high concentrations of NPX reduced microbial proliferation. Furthermore, NPX also reduced the settleability of the sludge, leading to the loss of some sludge during the drainage process, thereby reducing the sludge concentration [17].

3.3. Effect of NPX on EPS Content in Activated Sludge

EPS is a complex high-molecular-weight polymer primarily composed of PN and PS, and its content and composition significantly affect the physicochemical properties of activated sludge [27,28]. This study also investigated the effects of NPX on EPS and the content of different layers of EPS (Figure 4). When the NPX concentration was below 0.05 mg/L, there were no significant changes in the total EPS content or the content of each layer, indicating that low-dose NPX had a negligible effect on EPS secretion. However, when the NPX concentration exceeded 0.5 mg/L, NPX stimulated EPS secretion. In the 2.0 mg/L NPX group, the EPS content increased to 109.8~112.3 mg/g, much higher than the blank. In addition, the presence of NPX also increased the content of PS in EPS, especially in the 2.0 mg/L NPX group, the content of PS increased to 34.5 mg/g, which was much higher than that in the blank. These experimental results confirm that high-dose NPX promotes the secretion of extracellular EPS by the sludge. NPX, as an exogenous toxic substance, can stimulate microbial metabolism to produce EPS for self-defense, thereby mitigating the toxic effects of NPX [26].
SB-EPS, which is the outer layer of EPS, plays an important role in the configuration of activated sludge. NPX concentration also affects the content of SB-EPS. During the stable period, the SB-EPS content remained at 24.1~26.5 mg/g in the blank and the 0.05 mg/L NPX group, indicating that low-dose NPX had no significant effect on SB-EPS. However, when the NPX concentration exceeded 0.5 mg/L, high-dose NPX stimulated the secretion of SB-EPS. In the 2.0 mg/L NPX group, the SB-EPS content increased to 32.6–35.3 mg/g, approximately 1.29–1.33 times higher than that in the blank.
Similar experimental results were also observed for the content of LB-EPS and TB-EPS. In the 2.0 mg/L NPX group, the LB-EPS and TB-EPS concentrations increased to 19.6~21.3 mg/g and 38.4~39.5 mg/g, respectively, which were significantly higher than the control group’s values of 18.6~16.9 mg/g and 30.3~31.8 mg/g. These experimental results confirm that high-dose NPX not only increases the content of SB-EPS but also promotes the content of LB-EPS and TB-EPS. The existence of NPX stimulates the secretion of EPS in each layer to protect itself.

3.4. Effect of NPX on the Nutrient and Intracellular Polymer Content during a Typical Cycle

Figure 5 illustrates the influence of NPX exposure on the variation in COD concentration during a typical cycle. It can be observed that the COD concentration decreases over time in all groups, and NPX can affect the variation in COD concentration during a typical cycle. In the blank and the 0.05 mg/L NPX group, the effluent COD concentrations remained at 11.3 mg/L and 11.9 mg/L, respectively, indicating that low concentrations of NPX had no significant impact on COD removal during a typical cycle. However, when the NPX concentration exceeded 0.5 mg/L, the effluent COD concentration gradually increased, especially in the 2.0 mg/L NPX group, where the effluent COD concentration reached 38.6 mg/L, indicating that high doses of NPX reduced COD consumption during a typical cycle. The reduction in COD removal during a typical cycle by high NPX concentrations would decrease the biosynthesis of intracellular polymer PHA in the later stages, thereby reducing nutrient removal.
The impact of NPX on the variations of NH4+-N and phosphate during a typical cycle is shown in Figure 6. During the anaerobic phase, there was minimal change in NH4+-N concentration within each group. The decrease in NH4+-N concentration during this phase was primarily attributed to its utilization for normal cellular metabolism. Towards the end of the anaerobic phase, the ammonia nitrogen concentrations across all groups were approximately 40.9~41.8 mg/L. During the aerobic phase, NH4+-N was oxidized through nitrification, leading to a rapid decline in ammonia nitrogen concentration. However, when the exposure concentration of NPX was below 0.5 mg/L, its impact on NH4+-N nitrification was not significant, and the NH4+-N concentration remained at 11.5~13.2 mg/L during the aerobic phase. However, at concentrations exceeding 0.5 mg/L, NPX hindered the nitrification process of NH4+-N, impeding its biological conversion. Subsequently, during the anoxic phase, the NH4+-N concentration further decreased. Figure 6b illustrates the influence of NPX on phosphate concentration during a typical cycle. During the anaerobic phase, the phosphate concentration increased within each group, but NPX was found to affect the release of phosphates. In the blank group and the 0.05 mg/L NPX group, the net release of phosphate during anaerobic conditions was 15.6 mg/L and 16.1 mg/L, respectively. However, high NPX concentrations resulted in a reduction in phosphate release. In the 1.0 mg/L NPX and 2.0 mg/L NPX groups, the net release of phosphates was 12.5 mg/L and 11.8 mg/L, which was significantly lower than that of the blank. The study confirmed that the efficiency of biological phosphorus removal is closely related to phosphate release [29,30,31], and NPX was found to reduce phosphate release, indicating a decrease in the metabolic activity of PAO with high doses of NPX. During the aerobic phase, there was a sharp decline in the phosphate concentration within each group, and high NPX concentrations similarly reduced the excessive absorption of phosphates during the aerobic phase. High-dose NPX synchronously reduces anaerobic phosphate release and aerobic phosphate absorption, thereby reducing the efficiency of biological phosphorus removal [31].
Figure 7 shows the effects of NPX on the content of intracellular polymers PHA and glycogen during a typical cycle. During the anaerobic phase, the content of PHA gradually increased in all groups, which was primarily related to the consumption of COD and its conversion to PHA through microbial processes. However, NPX could affect the biosynthesis of PHA. In the late anaerobic phase, the maximum synthesis of PHA in the 0.05 mg/L NPX group and the blank was 6.24 mmol/g and 6.27 mmol/g, respectively, with no significant difference between the two. When the NPX concentration was increased from 0.5 mg/L to 2.0 mg/L, the maximum synthesis of PHA decreased from 6.15 mmol/g to 5.94 mmol/g, indicating that high doses of NPX reduced the biosynthesis of PHA. Inhibited PHA synthesis during the anaerobic phase would reduce the capacity for subsequent aerobic phosphate uptake and denitrification in the anoxic phase [29]. During the subsequent aerobic and anoxic phases, PHA was biodegraded, leading to a gradual decrease in its content. In the aerobic phase, PHA degradation capacity was utilized for aerobic phosphate uptake, while in the anoxic phase, PHA served as an electron donor for denitrification. High doses of NPX reduced the degradation of PHA during the aerobic and anoxic phases. When the NPX concentration was increased from 0.5 mg/L to 2.0 mg/L, the degraded PHA content decreased from 1.09 mmol/g to 0.52 mmol/g, indicating that high doses of NPX not only reduced the biosynthesis of PHA but also inhibited its biodegradation.
Changes in intracellular polymer content can reflect the metabolic activity of microorganisms. The effects of NPX on the typical intracellular polymers PHA and glycogen content in an activated sludge system are shown in Figure 7. During the anaerobic phase, when the external carbon source is abundant, microorganisms, especially PAO, can uptake available carbon sources from the water and synthesize PHA intracellularly. NPX can influence the biosynthesis of PHA. In the 0.05 mg/L NPX group and the blank group, the content of PHA at the end of the anaerobic phase was 6.24 mmol/g and 6.27 mmol/g, respectively, with no significant difference between them, indicating that low doses of NPX had minimal impact on microorganisms’ uptake of carbon sources and biosynthesis of PHA. However, when the NPX concentration exceeded 0.5 mg/L, the presence of NPX inhibited the biosynthesis of PHA, and the higher the NPX exposure concentration, the lower the content of PHA at the end of the anaerobic phase. When the NPX concentration was 2.0 mg/L, the maximum synthesis of PHA was only 5.94 mmol/g, which was significantly lower than that in the blank group. NPX reduced the biosynthesis of PHA during the anaerobic phase, resulting in less energy being generated during subsequent aerobic and anoxic phases from PHA decomposition [32,33].
Figure 7b illustrates the impact of NPX exposure on glycogen content. It can be observed that glycogen content exhibits a decreasing-then-increasing trend throughout the entire cycle. In the blank, the initial glycogen content decreased from 6.51 mmol/g to 6.05 mmol/g at 120 min, and during the subsequent aerobic and anaerobic periods, glycogen content increased again to 6.34 mmol/g. When the NPX concentration was below 0.05 mg/L, the periodic variation in glycogen content was not significantly different from that of the blank group. However, when the NPX concentration exceeded 0.5 mg/L, the degradation of glycogen in the anaerobic phase was as high as 0.85 mmol/g, which was significantly higher than that of the blank group (0.46 mmol/g). In the subsequent aerobic and anaerobic periods, glycogen content was replenished, reaching 6.34 mmol/g in the anaerobic end phase, with a replenishment amount of 0.33 mmol/g. Part of the PHA decomposition was used to replenish glycogen, and the NPX-present group showed significantly higher glycogen replenishment compared to other groups, while the NPX-present group exhibited lower PHA biosynthesis. Therefore, in the group with high NPX concentration, there was less energy available from PHA decomposition for biological denitrification and aerobic phosphorus uptake, resulting in suboptimal nutrient removal. NPX can capture DNA polymerases on DNA, thereby blocking DNA replication [34,35]. On the other hand, the large amount of glycogen decomposition and synthesis metabolism during the cycle indicates a higher abundance of microorganisms that primarily utilize glycogen as their main intracellular polymer. These glycogen-accumulating organisms (GAOs) are a type of microorganism that mainly relies on glycogen as their main intracellular metabolic substrate [36]. These microorganisms do not contribute to phosphate removal but can compete with PAOs for limited carbon sources in wastewater. The experimental results demonstrate that high-dose NPX enhances the metabolism of glycogen in the activated sludge and increases the metabolic activity of microorganisms that primarily utilize glycogen as their main intracellular polymer.

3.5. Impact of NPX on the Activity of Nutrient Removal-Related Enzymes

Denitrification involves the conversion of ammonium to nitrate or nitrite, followed by the reduction in nitrate or nitrite to nitrogen gas. These biological processes are associated with the activity of several key enzymes. It has been reported that biological denitrification is mediated by various key enzymes, including AMO, NAR, and NIR, while phosphorus removal is related to the activity of PPX and PPK [37]. This study also investigated the effect of NPX on the activity of these key enzymes. As shown in Figure 8, high concentrations of NPX reduced the activity of all the key enzymes associated with biological denitrification and phosphorus removal. For example, in the 2.0 mg/L NPX group, the relative activities of PPX, PPK, AMO, NAR, and NIR were 86.5%, 82.1%, 90.2%, 82.3% and 86.1%, respectively. High NPX concentrations decreased the activity of these key enzymes, resulting in a reduction in the biological transformation of nitrogen and phosphorus elements, thereby affecting nutrient removal. It is noteworthy that in the low-dose NPX group, the differences in the activity of the aforementioned key enzymes compared to that of the blank group were not significant, which is consistent with the insignificant impact of low-dose NPX on nutrient removal. Previous studies have demonstrated that NPX can inhibit key enzymes involved in the anaerobic digestion system’s organic matter degradation, such as proteases and amylases [13]. The inhibition of key enzymes by NPX may be related to the interaction between NPX and the protein activity within the enzymes.

3.6. Implementation Significance and Research Limitations

NPX is a new pollutant detected at high frequency in wastewater. However, the effect of NPX on biological treatment of wastewater has not been explored yet. This work fills this gap, explores the effect of NPX dosage on wastewater biological treatment in SBR, and reveals the related mechanism from the point of view of nutrient transformation law, sludge characteristics and key enzyme activity in typical cycle. As far as we know, this is the first time to explore the effect of new pollutant NPX on biological treatment of wastewater. The results showed that low dose NPX had no obvious effect on biological treatment of wastewater, while more than 0.5 mg/L NPX reduced the biological removal of COD and nutrients. Therefore, in the future treatment containing high dose of NPX, we can consider increasing the pretreatment process to reduce the inhibitory effect of NPX on the subsequent biological treatment, and ensure the removal of higher COD and nutrients. Some low-cost advanced oxidation technologies can be considered in NPX pretreatment technology. This work provides some theoretical guidance for the treatment of wastewater containing NPX using traditional activated sludge.
In this work, the wastewater used to explore the effect of NPX on nutrient removal using the activated sludge process was synthetic wastewater rather than actual wastewater. There are a large number of other emerging pollutants in the actual wastewater, and the removal of nutrients via the combined stress of a variety of pollutants is still not clear. In addition, clarifying the changes in microbial community characteristics and key functional genes in activated sludge exposed to NPX is an important way to reveal the related mechanisms. In future work, the effects of NPX on microorganisms and functional genes will be revealed with the help of macrogenomics technology.

4. Conclusions

This work assessed the impact of the emerging pollutant NPX on the removal of pollutants and nutrients in activated sludge and revealed the underlying mechanisms by investigating the variations in pollutants and key enzyme activities during a typical cycle under NPX exposure. The results indicate that NPX concentrations below 0.05 mg/L have insignificant effects on wastewater treatment using activated sludge. However, NPX concentrations exceeding 0.5 mg/L reduce the removal of pollutants and nutrients in wastewater treatment, and the inhibitory effect becomes more pronounced with higher NPX exposure concentrations. When the concentration of NPX was 2.0 mg/L, the removal efficiency of COD, NH4+-N and phosphate decreased to 81.283.6%, 82.584.2% and 80.282.6%, respectively, which was much lower than that of the blank. High-dose NPX promotes the secretion of EPS and glycogen metabolism but decreases the biosynthesis of PHA. Enzyme activity analysis reveals that NPX reduces the activity of key enzymes involved in biological denitrification and phosphorus removal. These conclusions provide an important theoretical basis for understanding and controlling the environmental behavior and effects of NPX in the wastewater treatment process.

Author Contributions

L.W.: Writing—original draft preparation. W.Z.: Data analysis, financial support. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Effect of NPX on the effluent COD concentration (a) and removal efficiency (b) in the activated sludge system. The error bar represents the standard deviation of three measurements.
Figure 1. Effect of NPX on the effluent COD concentration (a) and removal efficiency (b) in the activated sludge system. The error bar represents the standard deviation of three measurements.
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Figure 2. Effect of long-term exposure to NPX on NH4+-N (a) and phosphate (b) removal efficiency in activated sludge system. The error bar represents the standard deviation of three measurements.
Figure 2. Effect of long-term exposure to NPX on NH4+-N (a) and phosphate (b) removal efficiency in activated sludge system. The error bar represents the standard deviation of three measurements.
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Figure 3. Effect of NPX on MLTSS concentration (a) and MLVSS/MLTSS (b) in activated sludge system. The error bar represents the standard deviation of the three measurements.
Figure 3. Effect of NPX on MLTSS concentration (a) and MLVSS/MLTSS (b) in activated sludge system. The error bar represents the standard deviation of the three measurements.
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Figure 4. Effect of NPX on EPS content (a) and delamination content (b–d) in activated sludge system. The error bar represents the standard deviation of the three measurements.
Figure 4. Effect of NPX on EPS content (a) and delamination content (b–d) in activated sludge system. The error bar represents the standard deviation of the three measurements.
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Figure 5. Variation in COD with time in typical cycle of activated sludge system under NPX exposure. The error bar represents the standard deviation of the three measurements.
Figure 5. Variation in COD with time in typical cycle of activated sludge system under NPX exposure. The error bar represents the standard deviation of the three measurements.
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Figure 6. Effect of NPX on the changes in ammonia nitrogen (a) and phosphate (b) concentration in typical cycles. The error bar represents the standard deviation of the three measurements.
Figure 6. Effect of NPX on the changes in ammonia nitrogen (a) and phosphate (b) concentration in typical cycles. The error bar represents the standard deviation of the three measurements.
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Figure 7. Variation in intracellular polymer PHA (a) and glycogen content (b) with time in typical cycle of activated sludge system under NPX exposure. The error bar represents the standard deviation of the three measurements.
Figure 7. Variation in intracellular polymer PHA (a) and glycogen content (b) with time in typical cycle of activated sludge system under NPX exposure. The error bar represents the standard deviation of the three measurements.
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Figure 8. Effect of NPX on relative activities of key enzymes in activated sludge. The error bar represents the standard deviation of the three measurements.
Figure 8. Effect of NPX on relative activities of key enzymes in activated sludge. The error bar represents the standard deviation of the three measurements.
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Wei, L.; Zhang, W. Impact of Naproxen on Wastewater Biological Treatment: Focus on Reactor Performance and Mechanisms. Water 2023, 15, 2615. https://doi.org/10.3390/w15142615

AMA Style

Wei L, Zhang W. Impact of Naproxen on Wastewater Biological Treatment: Focus on Reactor Performance and Mechanisms. Water. 2023; 15(14):2615. https://doi.org/10.3390/w15142615

Chicago/Turabian Style

Wei, Lidan, and Wenbin Zhang. 2023. "Impact of Naproxen on Wastewater Biological Treatment: Focus on Reactor Performance and Mechanisms" Water 15, no. 14: 2615. https://doi.org/10.3390/w15142615

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