Impacts of Polylactic Acid Microplastics on Performance and Microbial Dynamics in Activated Sludge System

: A large number of microplastics (MPs) have been found in various stages of wastewater treatment plants, which may affect the functional microbial activity in activated sludge and lead to unstable pollutant removal performance. In this study, the effects of different concentrations of polylactic acid microplastics (PLA MPs) on system performance, nitriﬁcation and phosphorus (P) removal activities, and extracellular polymeric substances (EPS) were evaluated. The results showed that under the same inﬂuent conditions, low concentrations (50 particles/(g TS)) of PLA MPs had no signiﬁcant effect on efﬂuent quality. The average removal efﬁciencies of chemical oxygen demand, phosphate, and ammonia were all above 80%, and the average removal efﬁciencies of total nitrogen remained above 70%. High concentrations (200 particles/(g TS)) of PLA MPs inhibited the activities of polyphosphate-accumulating organisms (PAOs) and nitrifying bacteria. The speciﬁc anaerobic P release rate decreased from 37.7 to 23.1 mg P/(g VSS · h), and the speciﬁc aerobic P uptake rate also signiﬁcantly decreased. The speciﬁc ammonia oxidation rate decreased from 0.67 to 0.34 mg N/(g VSS · h), while the change in the speciﬁc nitrite oxidation rate was not signiﬁcant. The dosing of PLA MPs decreased the total EPS and humic acid content. As the concentration of PLA MPs increased, microbial community diversity increased. The relative abundance of potential PAOs (i.e., Acinetobacter ) increased from 0.08 to 12.57%, while the relative abundance of glycogen-accumulating organisms (i.e., Competibacter and Deﬂuviicoccus ) showed no signiﬁcant changes, which would lead to improved P removal performance. The relative abundance of denitrifying bacteria (i.e., Pseudomonas ) decreased from 95.43 to 58.98%, potentially contributing to the decline in denitriﬁcation performance.


Introduction
Microplastics (MPs) are non-uniform plastic particles of less than 5 mm in diameter that enter the ecosystem through various pathways.Industrial production, personal care products, and the decomposition of large plastic debris are the main sources of MPs [1].
The widespread use of plastic products and poor waste management have led to the ubiquity of MPs in aquatic water bodies, including rivers, lakes, estuaries, shorelines, and marine ecosystems.Previous research has estimated that a large number of MPs from domestic producers enter wastewater treatment plants (WWTPs) through the drainage system [2].Therefore, WWTPs could represent the last barrier before the discharge of MPs into an aquatic ecosystem.A recent survey of 95 WTTPs in 12 countries found that the concentrations of microplastics in the influent and effluent were 0.28-18,285 particles/L and 0-447 particles/L, respectively [3].Due to the mostly high hydrophobicity of activated sludge in biological treatment plants [4], a large proportion of MPs in raw wastewater would potentially be captured by sludge.However, the accumulation of MPs in activated sludge has the potential to impact sludge characteristics, extracellular polymeric substance (EPS) fractions, microbial activities and compositions, and, eventually, system treatment performance.
The most common MPs in activated sludge are polystyrene (PS), polyvinyl chloride (PVC), polyethersulfone (PES), polyethylene (PE), polyethylene terephthalate (PET), and polypropylene (PP) [5].These MPs and smaller nanoplastics (NPs) may not only physically harm micro-organisms through ingestion and destroy proteins and lipids [6], but also release or adsorb toxic and harmful chemicals, inhibiting microbial activity in wastewater treatment systems.He et al. [7] showed that when the particle size of PS MPs was 150-300 µm and the concentration was 0.01-0.10g/L, the nitrifying activity in activated sludge was significantly inhibited and the ammonia oxidation rate decreased to 71-92% compared to the control.Wei et al. [8] investigated the effect of PS NPs on anaerobic granular sludge and found that higher concentrations of PS NPs caused changes in EPS composition of sludge, while micro-organisms produced more proteins to defend against potential harm [9].In another study, PS NP concentrations were found to be associated with an increase in intracellular reactive oxygen species (ROS) levels, which led to cell rupture and lactate dehydrogenase (LDH) leakage [10].Zhang et al. [11] found that PVC MPs (50 µm diameter) at concentrations of 20-100 mg/L significantly inhibited the denitrification capacity of aerobic granular sludge (AGS) and reduced total nitrogen (TN) removal by 3.8-17.8%.Meanwhile, PES MPs could adsorb the AGS surface or enter the interior and release benzene, phenols, or other additive chemicals, which affected the normal morphology, removal efficiency, and physical and chemical properties of AGS [12].In addition, the presence of PE MPs plays a screening role for micro-organisms, potentially reducing the diversity of microbial communities [13].
However, most studies of the effects of various MPs on activated sludge systems have focused on traditional non-degradable plastics, such as PVC, PE, and PP, while there are few and insufficient studies on biodegradable plastics.To alleviate plastic pollution, biodegradable plastics are widely used as environmentally friendly products [14] in medical, food packaging, agriculture, and other industries [15].The global production capacity of biodegradable bio-based plastics was 0.732 million tons in 2018 and is expected to increase to 1.3 million tons per year by 2030 [16].Biodegradable plastics are usually classified by product type as polyhydroxyalkanoate (PHA), polylactic acid (PLA), polybutylene succinate (PBS), and polycaprolactone (PCL) [16].PLA is one of the current focuses in the field of biodegradable plastics.The enzymatic polymerization or azeotropic dehydration of monomeric lactic acid is used to form PLA [17], and lactic acid can be produced by fermenting renewable resources, such as bagasse, corn, starch, and food waste [18].With its high mechanical strength, good biocompatibility and solvent-carrying properties, and low sale price, PLA is widely used in food packaging industry, automotive industry, textile industry, medical industry, and agriculture, and the global market has been steadily expanding with increasing demand [19,20].The huge future consumption of PLA means that a large number of PLA MPs will be produced and enter the WWTPs, and PLA plastics may produce more MPs and oligomer NPs than traditional plastics with potential risk [14], though there is still a lack of research on whether they and their degradation products have an impact on the activated sludge system.
In this study, the impacts of different concentrations of PLA MPs on a typical labscale activated sludge system were investigated by analyzing effluent water quality, EPS, metabolic activity, and community structure.It will help us to understand the potential risks of using PLA MPs in the wastewater treatment process and take appropriate regulatory measures in the future.

Reactor Setup
The sequencing batch reactor (SBR) was selected to simulate a typical secondary activated sludge treatment process in WWTPs.The seed sludge was obtained from the return sludge line at Xi'an No. 4 sewage plant (Xi'an, Shaanxi, China).Before the formal experiment, the sludge was inoculated into a lab-scale anaerobic-aerobic parent SBR, fed with the same synthetic wastewater used in this study, and operated for 1 week to achieve a steady state.The sludge was then divided into four SBRs (Figure S1) with a working volume of 1.4 L. The operating cycle of the SBR reactor was 6 h, including 5 min for filling, 90 min for the anaerobic phase, 210 min for the aerobic phase, 45 min for settling, and 10 min for draw and idle phases.

Operational Conditions
A mixture of acid-hydrolyzed casein, acetate (HAc), and propionate (HPr) was used as the carbon source in the synthetic wastewater with a COD ratio of 2:7:7 and a total COD of ~280 mg/L.Ammonium chloride (NH 4 Cl) and potassium dihydrogen phosphate (KH 2 PO 4 ) were used as inorganic N and P sources, respectively, resulting in 20 mg N/L of NH 4 + -N and 4.5 mg P/L of PO 4 3− -P.Trace elements were added, as previously described [21], and the detailed concentrations are listed in Table S1.
The SBR reactor was operated at a temperature of 20 ± 5 • C, a pH range of 7.0-8.5, a hydraulic retention time (HRT) of 12 h, and a sludge retention time (SRT) of 15 d.The mixed liquor suspended solids (MLSS) were maintained at approximately 3500 mg/L.After stabilizing the reactor performance, different concentrations of PLA MPs were added to the four reactors for comparative analysis, with an experiment duration of 30 d. Mirka et al. [22] investigated the particle size distribution ranges of MPs in several full-scale WTTPs worldwide and found that MPs with a diameter greater than 500 µm could account for over 70% of the influent.Liu et al. [23] found that the MP concentrations in sludge ranged from 4.4 × 10 3 to 2.4 × 10 5 particles/kg in 38 WWTPs in 11 countries worldwide.A similar study pointed out that the average MPs concentration in sludge from WTTPs in 12 countries was 12.8 × 10 3 particles/kg [24].Accordingly, the particle size of PLA MPs chosen in this study was ~580 µm, and the dosages in the four reactors were 0, 50, 100, and 200 particles/(g TS), respectively.The reactors were then named R0, R1, R2, and R3, respectively.

Biological Activity Batch Tests
To further evaluate the effects of different concentrations of PLA MPs on functionally relevant microbial activities for nitrogen and phosphorus removal, 800 mL of activated sludge was taken from each reactor at the end of the aerobic phase; washed using synthetic wastewater without carbon, nitrogen, and phosphorus sources [25,26]; and transferred to a reactor (1 L) for batch tests.

Enhanced Biological Phosphorus Removal (EBPR) Activity Batch Tests
Firstly, nitrogen gas was continuously introduced into the sludge to achieve anaerobic conditions with dissolved oxygen (DO) < 0.2 mg/L.Subsequently, HAc and KH 2 PO 4 were added to achieve COD and PO 4 3− -P concentrations of 300 mg/L and 10 mg P/L, respectively.The anaerobic condition was maintained for 1 h.Then, air was continuously introduced to ensure aerobic conditions (DO > 2 mg/L) for 3 h.During batch tests, pH was maintained at 7.0 ± 0.1 by adding NaOH or HCl.Samples were periodically collected for the determination of COD, PO 4 3− -P, MLSS, and mixed liquor volatile suspended solids (MLVSS).The contents of intracellular storage polymers, such as polyhydroxyalkanoates (PHAs) and glycogen, were also analyzed accordingly [21,27].

Nitrification Activity Batch Tests
Air was continuously introduced into the sludge to maintain aerobic conditions for 3 h.NH 4 Cl solution was added to achieve a NH 4 + -N concentration of 30 mg N/L.During the batch test, the samples were periodically collected for the determination of NH 4 + -N, NO 3 − -N, NO 2 − -N, MLSS, and MLVSS.In addition, the specific ammonia oxidation rate (AOR) and nitrite oxidation rate (NOR) were calculated accordingly [7].

Microbial Community Analysis
During the experiment, activated sludge samples were collected from the four reactors for DNA extraction and 16S rRNA gene amplicon sequencing.Genomic DNA was extracted using DNeasy PowerSoil Kit (QIAGEN, Inc., Venlo, The Netherlands).The primers, namely 341F (CCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT), were used for the PCR amplification of the bacterial 16S rRNA gene.The amplification products were purified via Agencourt AMPure Beads (Beckman Coulter, Brea, CA, USA), quantified via the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA), and sequenced using the Illumina MiSeq platform (Shanghai Paisano Biotechnology Co., Ltd., Shanghai, China).Finally, QIIME (v1.8.0) software was used for bioinformatics analysis of the sequencing data.The micro-organisms identified in the activated sludge system were linked to their functional information using the Activated Sludge Microbial Database (MiDAS: https:// www.midasfieldguide.org/guide/search(accessed on 31 June 2023)).

Chemical Analysis
To evaluate the pollutant removal performance during the experiment, the concentrations of COD, PO 4 3− -P, NH 4 + -N, NO 3 − -N, NO 2 − -N, TN, MLSS, and MLVSS were analyzed using the Standard Methods [28].EPS was extracted via thermal extraction [29].The protein and humic acid contents were determined using the modified Lowry method [30], and the polysaccharide content was determined via the anthrone-sulfuric acid method [31].PHAs were determined using a Shimadzu gas chromatograph coupled to triple quadrupole mass spectrometer (GCMS-TQ8040) [32].The glycogen content was analyzed using the glucose content kit (BC2505, Beijing Solarbio Science and Technology Co., Ltd., Beijing, China).

Statistical Analysis
All of the tests were performed in duplicate, and the results were expressed as mean ± standard deviation.Student's t-test was used to test the statistical significance between different SBRs using MATLAB R2014a.Non-metric multidimensional scaling (NMDS) analyses were performed to reveal the differences in microbial communities using R4.2.1 software.The relationship between effluent water quality and PLA MP concentration was analyzed via Pearson's correlation analysis using SPSS 22.

COD Removal Performance
As shown in Figure 1a, there was no significant difference (p > 0.05) in the COD removal performance between the four SBRs (R0, R1, R2 and R3), which had average removal efficiencies of 80.1%, 79.3%, 80.2% and 78.4%, respectively.Dai et al. [33] showed that PVC MPs (50 mg/L) had no negative effect on COD removal performance in an AGS system, probably due to a better tolerance of most heterotrophic micro-organisms in AGS to MPs.Similarly, a study by Zhang et al. [11] found that PS MPs (particle size 50-100 µm) had no significant effects on the organic matter removal performance in AGS systems.Moreover, compared to non-biodegradable MPs, PLA MPs can be readily degraded by micro-organisms through the secretion of proteases and lipases [34].The resulting small molecule organic matters can potentially serve as supplementary carbon sources for heterotrophic micro-organisms.Therefore, PLA MPs do not have a significant impact on COD removal.The average effluent COD concentration in R3 was 57.2 mg/L, slightly higher than in other SBRs, possibly due to the release of smaller PLA particles (e.g., nanoparticles (NPs)) and their degradation products during the biodegradation of PLA MPs.
moval performance between the four SBRs (R0, R1, R2 and R3), which had average removal efficiencies of 80.1%, 79.3%, 80.2% and 78.4%, respectively.Dai et al. [33] showed that PVC MPs (50 mg/L) had no negative effect on COD removal performance in an AGS system, probably due to a better tolerance of most heterotrophic micro-organisms in AGS to MPs.Similarly, a study by Zhang et al. [11] found that PS MPs (particle size 50-100 µm) had no significant effects on the organic matter removal performance in AGS systems.Moreover, compared to non-biodegradable MPs, PLA MPs can be readily degraded by micro-organisms through the secretion of proteases and lipases [34].The resulting small molecule organic matters can potentially serve as supplementary carbon sources for heterotrophic micro-organisms.Therefore, PLA MPs do not have a significant impact on COD removal.The average effluent COD concentration in R3 was 57.2 mg/L, slightly higher than in other SBRs, possibly due to the release of smaller PLA particles (e.g., nanoparticles (NPs)) and their degradation products during the biodegradation of PLA MPs.

Phosphorus Removal Performance
As shown in Figure 1b, the effluent PO4 3− -P concentration decreased with the increase in PLA MP dosage (p < 0.05) from 0.42 ± 0.35 mg/L in R0 to 0.32 ± 0.24 mg/L in R1 and 0.22 ± 0.16 mg/L in R3.Consequently, the range of the effluent PO4 3− -P concentration in R3 (0.05-0.61 mg/L) was lower than those in other SBRs (0.10-1.10 mg/L).Previous studies

Phosphorus Removal Performance
As shown in Figure 1b, the effluent PO 4 3− -P concentration decreased with the increase in PLA MP dosage (p < 0.05) from 0.42 ± 0.35 mg/L in R0 to 0.32 ± 0.24 mg/L in R1 and 0.22 ± 0.16 mg/L in R3.Consequently, the range of the effluent PO 4 3− -P concentration in R3 (0.05-0.61 mg/L) was lower than those in other SBRs (0.10-1.10 mg/L).Previous studies have shown that PE, PVC, and polyethersulfone (PES) MPs (50-10,000 particles/L) have no significant effects on PO 4 3− -P removal performance and the activity of polyphosphate (polyP)-accumulating organisms (PAOs) in activated sludge systems [35].He et al. [7] reported that PS MPs with a size range of 0-300 µm did not affect PAO activity.The results of this study showed that the dosing of PLA MPs may lead to improved biological P removal, while the causes of this phenomenon need further study.

Nitrogen Removal Performance
As shown in Figure 1c, with the increase in the PLA MP dosage, the average effluent NH 4 + -N concentration gradually increased from 0.25 mg/L in R0 to 1.35 mg/L in R2 (p < 0.05).It has been shown that PVC and PS MPs could reduce N removal performance in activated sludge systems by inhibiting the activities of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) [36].The average NH 4 + -N removal efficiencies in SBRs were above 97%, except in R2, which may be partially related to the decline in biological N removal activities (see Section 3.2.2).As shown in Figure 1d, the average effluent TN concentration gradually increased (p < 0.05) with the increase in the PLA MP dosage.The effluent TN concentration in R3 was 7.11 ± 1.68 mg/L, with an average removal efficiency of 66.1%, while the average TN removal efficiency reached 71.89% in R0.A previous study showed that 0.05 g/L of PS MPs with a diameter of 150-300 µm could reduce the NO 3 − -N reduction rate to 69-94% compared to the control [7].Caruso et al. [37] reported that tiny plastic debris resulting from weathering and fragmentation can break cellular structures and/or release hazardous chemicals, such as plastic additives, ultimately leading to reduced enzymatic activity and cell inactivation.It is possible that high concentrations of PLA MPs and their degradation products inhibited the activity and abundance of denitrifying bacteria (DNB), thus affecting the denitrification performance.

EBPR Activity
To evaluate the effects of biodegradable PLA MPs on P removal activity in the four SBRs, EBPR batch tests were conducted, and changes in COD and PO 4 3− -P concentrations during the tests are shown in Figure S2.As shown in Table 1, the specific average anaerobic P release rate (P rel ) was 37.7 mg P/(g VSS•h) after the introduction of low concentrations (50 particles/(g TS)) of PLA MPs, which was higher than that in R0 (29.1 mg P/(g VSS•h)).The specific average aerobic P uptake rate (P up ) in SBRs generally remained consistent at ~7.0 mg P/(g VSS•h).After exposure to high concentrations (200 particles/(g TS)) of PLA MPs, P rel and P up decreased to 23.1 and 6.3 mg P/(g VSS•h), respectively.This finding indicates that excessive accumulation of PLA MPs would potentially affect the P release/uptake capacity of PAOs, while it did not lead to deteriorated P removal performance in SBRs at a relatively low influent PO 4 3− -P concentration (4.5 mg/L).The P release-to-acetate uptake ratio (P/HAc) is commonly used as an indicator of changes in the activity and abundance of PAOs and their competitors, namely glycogenaccumulating organisms (GAOs) [38].As shown in Table 1, the P/Hac ratio increased to 0.60 P-mol/C-mol in R1, while it decreased to 0.49 and 0.48 P-mol/C-mol in R2 and R3, respectively.Compared to R0, the P removal performance in all SBRs dosed with PLA MPs increased to varying degrees.This finding may be because PLA MPs can not only act as supplementary carbon sources, but also biofilm carriers for the attachment and growth of heterotrophic micro-organisms, such as PAOs, which would promote the formation of biofilm and further enhance PAO activity by providing competitive advantages [39].When the density of PLA MPs increased to 200 particles/(g TS), the biofilm would fall off due to collision and friction between particles, which reduced the biofilm formation efficiency, thus reducing the PAO activity.However, the P/HAc value (0.48 P-mol/C-mol) remains high, indicating robust PAO activity and abundance, which is consistent with the microbial community results (see Section 3.4.3).
PAOs take up carbon sources such as acetate and store them in the form of PHAs during the anaerobic phase, subsequently using PHAs as the main energy source for excessive P uptake and polyP synthesis during the aerobic phase [40].The amount of PHAs stored by PAOs will directly affect their P uptake capacity in the aerobic phase [21].However, GAOs exhibit a metabolism similar to that of PAOs that compete with PAOs for VFA uptake and PHA synthesis without performing anaerobic P release or aerobic P uptake.Therefore, a higher PHA production-to-acetate uptake ratio (PHA/HAc) implies either a higher involvement of GAOs or a higher utilization of the glycolysis pathway by PAOs [41].In this study, the PHA/HAc ratio was 0.83 C-mol/C-mol in R0, which was higher than that in R2 and R3 (0.54 and 0.68 C-mol/C-mol, respectively).Considering the low GAO abundance, as detected via microbial community analysis, the result indicates that the large number of PLA MPs may prompt PAOs to use the TCA cycle over the glycolysis pathway to produce reducing equivalents.During the PHA synthesis process in the anaerobic phase, intracellularly stored glycogen could provide a significant portion of the energy for PAOs/GAOs through glycolysis [42].Therefore, a higher glycogen utilization-to-acetate uptake ratio (Glyc/HAc) would be related to an increased relative abundance of GAOs or increased use of glycolysis pathways [43].In this study, the Glyc/HAc ratio decreased from 0.21 C-mol/C-mol in R0 to 0.14 C-mol/C-mol in R3, indicating that excessive PLA MPs may reduce glycogen utilization by PAOs, which is correlated with the PHA/HAc ratio results, while the causes need further study.

Nitrification Activity
To reveal the effects of PLA MPs on nitrifying bacteria, the nitrification activity batch tests were conducted using sludge collected from the four SBRs, and changes in NH 4 + -N, NO 2 − -N, and NO 3 − -N concentrations during the tests are shown in Figure S3.As shown in Table 2, both AOR and NOR showed a decreasing trend with the increase in PLA MP dosage.Compared to R0, AOR in R1 decreased from 1.04 to 0.67 mg N/(g VSS•h), while NOR in R1 (1.06 mg N/(g VSS•h)) was comparable to R0 (1.15 mg N/(g VSS•h)).When the PLA MP dosage increased to 100 particles/(g TS), AOR significantly decreased to 0.31 mg N/(g VSS•h), indicating that AOB was more sensitive to PLA MP exposure.When the dosage of PLA MPs continued to increase to 200 particles/g TS, NOR decreased to 0.98 mg N/(g VSS•h), indicating that the addition of PLA MPs also had an impact on NOB activity.Similarly, Li et al. [36] found that 5000 particles/L of different MPs (PP, PE, PS, PES, and PVC) inhibited the activated sludge nitrification.In addition, a recent study found that LDH release increased by 40% after exposure to 10 mg/L of polytetrafluoroethylene (PTFE) NPs, which further affected microbial activity due to the destruction of cell integrity [47].The NPs produced during MP degradation could block cell channels and induce microorganisms to produce large amounts of ROS, potentially causing damage to proteins, cell structure, and DNA [48].Although no distinct changes in NH 4 + -N removal performance were detected in this study, nitrification activity tests showed that high concentrations (100-200 particles/(g TS)) of biodegradable PLA MPs would negatively affect the activities of autotrophic AOB and NOB, especially AOB. a : Other full-scale or lab-scale activated sludge systems without MP exposure.

Effects of PLA MPs on EPS
After 20 days of PLA MPs exposure, EPS was extracted from sludge flocs in the four SBRs to analyze the contents of protein, humic acid, and polysaccharide.As shown in Figure 2, with the increase in the PLA MP dosage, total EPS content showed a decreasing trend.Similarly, Wei et al. [52] found that high concentrations of PS MPs caused a reduction in cell viability, and the total EPS content was lower than that of the control group.In this study, humic acid content in EPS significantly decreased from 155.1 mg/L in R0 to 65.8 mg/L in R3.Polysaccharide content generally remained consistent at ~130 mg/L but decreased to 92.42 mg/L in R3, which may be related to the relatively larger particle size of MPs that are more likely to form biofilms on the surface.The high metabolic activity of micro-organisms in the biofilms would then consume the biodegradable components of EPS [53,54].In addition, it was observed that protein content slightly increased with PLA MPs at concentrations of 50 and 100 particles/(g TS).Changes in EPS components and microbial aggregates (e.g., flocs and biofilm) would have an impact on sludge hydrophilicity/hydrophobicity, potentially altering the interaction between MPs and sludge, which requires further study.Recent studies have revealed that EPS in biofilm systems tend to be enriched in proteins instead of carbohydrates, which have carboxylate and amine groups that can complex with NPs [55], and microbial aggregates would secrete more proteins to protect them from toxic chemicals [56].However, the EPS extraction method used in this study could not efficiently extract EPS from biofilms attached to the MP surface, and the components and changes in EPS in biofilms need further study.

Effects of PLA MPs on EPS
After 20 days of PLA MPs exposure, EPS was extracted from sludge flocs in the four SBRs to analyze the contents of protein, humic acid, and polysaccharide.As shown in Figure 2, with the increase in the PLA MP dosage, total EPS content showed a decreasing trend.Similarly, Wei et al. [52] found that high concentrations of PS MPs caused a reduction in cell viability, and the total EPS content was lower than that of the control group.In this study, humic acid content in EPS significantly decreased from 155.1 mg/L in R0 to 65.8 mg/L in R3.Polysaccharide content generally remained consistent at ~130 mg/L but decreased to 92.42 mg/L in R3, which may be related to the relatively larger particle size of MPs that are more likely to form biofilms on the surface.The high metabolic activity of micro-organisms in the biofilms would then consume the biodegradable components of EPS [53,54].In addition, it was observed that protein content slightly increased with PLA MPs at concentrations of 50 and 100 particles/(g TS).Changes in EPS components and microbial aggregates (e.g., flocs and biofilm) would have an impact on sludge hydrophilicity/hydrophobicity, potentially altering the interaction between MPs and sludge, which requires further study.Recent studies have revealed that EPS in biofilm systems tend to be enriched in proteins instead of carbohydrates, which have carboxylate and amine groups that can complex with NPs [55], and microbial aggregates would secrete more proteins to protect them from toxic chemicals [56].However, the EPS extraction method used in this study could not efficiently extract EPS from biofilms attached to the MP surface, and the components and changes in EPS in biofilms need further study.The effect of different concentrations of PLA MPs on the microbial community structure was analyzed via 16S rRNA gene amplicon sequencing.The amplicon sequence variants (ASVs) were obtained via clustering sequences with 100% similarity, and the Alpha diversity index was calculated for each sample based on the ASVs (Table S2).Good's coverage of the four samples was all above 0.99, indicating that the sequencing depth occupied more than 99% of the entire sequence and the sequencing depth was sufficient.The Shannon and Chao1 indices collectively reflect community richness, while the Gini-Simpson index reflects community diversity [57].The diversity results showed a generally upward trend in diversity indices for samples with different concentrations of PLA MPs.Compared to R0, Shannon and Chao1 indices in R3 increased by 70% and 67%, respectively.The Gini-Simpson index in R3 (0.961) is also higher than that in R0 (0.860), indicating that the presence of PLA MPs can potentially increase the richness and diversity of microbial communities.Non-degradable MPs may affect the microbial community diversity by releasing toxic additives used in the production process or their own toxic degradation products.For example, PET MPs release the toxic di-n-butyl phthalate (DBP) polymer that induces microbial ROS production, leading to the inactivity and even death of microbial cells, such as methanogenic bacteria [58].As PLA MPs in this study have relatively good biocompatibility and biodegradability and can act as carriers, this process would allow specific micro-organisms to attach to the surface to form a stable biofilm community structure, thereby increasing community diversity [59].Through the analysis of ASV data in Figure S4, it was found that 18-25% of the unique ASV existed in samples with PLA MP dosage, indicating that the community composition in SBRs gradually changed to one that was better adapted to the exposure to PLA MPs [60].

Microbial Composition
The microbial composition at the phylum level is shown in Figure 3a.Proteobacteria were the dominant phylum in R0, with a relative abundance of 97.79%.After the addition of PLA MPs, the relative abundance of Proteobacteria decreased to 90.16-94.24%,but it still dominated.Previous studies have shown that Proteobacteria are the main dominant phylum commonly found in activated sludge systems, containing a variety of micro-organisms that degrade organic pollutants and remove nutrients such as nitrogen (e.g., DNB, etc.) [61,62].Therefore, the decrease in the TN removal capacity with the addition of PLA MPs may be related to the decrease in the relative abundance of Proteobacteria.The presence of PLA MPs promoted the growth of Bacteroidetes, with a relative abundance of 4.63-7.80%.Some members of the phylum Bacteroidetes are capable of degrading complex organic matter [63] and potentially have the ability to cause denitrification [64].The microbial composition at the genus level is shown in Figure 3b.The dominant genus in R0 was Pseudomonas, with a relative abundance of 95.43%.Pseudomonas exhibited high sensitivity to PLA MPs, and its relative abundance gradually decreased to 58.98% in R3.Correspondingly, Acinetobacter, Acidovorax, and Azoarcus gradually gained advantages in community competition, with the relative abundances increasing from less than 0.3% in R0 to 12.57%, 8.47%, and 2.86% in R3, respectively.The results of non-metric multidimensional scanning (NMDS) analysis showed that the microbial community structure significantly evolved after the addition of PLA MPs for better adaptation (Figure 4).

Functionally Relevant Microbial Populations
To better investigate the driving factors of N and P removal performance in SBRs at different concentrations of PLA MPs, the MiDAS database [65] was used to further screen the functionally relevant microbial populations associated with EBPR, nitrification, and denitrification (Figure 5).Known PAOs (e.g., Accumulibacter, Dechloromonas, and Tetrasphaera) and GAOs (e.g., Competibacter and Defluviicoccus) involved in EBPR processes were all present at very low abundances (<0.5%) in the four SBRs.Notably, the relative abundance of potential PAO (i.e., Acinetobacter) increased from 0.08% to 5.81-12.57%with the addition of PLA MPs.Although Acinetobacter's contribution to EBPR remains controversial, many studies have shown that Acinetobacter has the ability to accumulate polyP and PHA [66][67][68], and it has previously been proposed to behave as PAOs in EBPR systems [69,70].The relative abundance of Competibacter increased from 0.05% in R0 to 0.16% in R1, but decreased to 0.03% in R3 as PLA MPs concentration continued to increase.The relative abundance of Defluviicoccus is 0.11% in R0, and the presence of PLA MPs increases it to 0.21-0.27%.Previous studies have shown that heterotrophic organisms such as PAOs are more tolerant of MPs and engineered NPs [35,71,72].In addition, Wu et al. [73] noted that the surface roughness of PLA/starch particles increased with the extension of retention time, which was conducive to microbial attachment and growth.It could, therefore, act as a biofilm carrier while releasing small-molecule organic matter as slow-release carbon sources for enriching specific heterotrophic populations, potentially influencing microbial dynamics.Some members of the genus Acinetobacter have been reported to be capable of decomposing refractory plastics, such as PS and low-density PE, and using decomposition products as carbon sources [74,75].Therefore, it is assumed that Acinetobacter may also be capable of utilizing biodegradable PLA as a carbon source.Further studies related to Acinetobacter's specific metabolic pathways or genes used for PLA degradation are warranted to better understand its increased abundance in the presence of PLA MPs.To sum up, the addition of PLA MPs may have changed the competition between PAOs and GAOs, ensuring that the potential PAOs (e.g., Acinetobacter) gradually gain advantages, which may be related to their specific carbon usage and metabolic characteristics.

Functionally Relevant Microbial Populations
To better investigate the driving factors of N and P removal performance in SBRs at different concentrations of PLA MPs, the MiDAS database [65] was used to further screen the functionally relevant microbial populations associated with EBPR, nitrification, and denitrification (Figure 5).Known PAOs (e.g., Accumulibacter, Dechloromonas, and Tetrasphaera) and GAOs (e.g., Competibacter and Defluviicoccus) involved in EBPR processes were all present at very low abundances (<0.5%) in the four SBRs.Notably, the relative abundance of potential PAO (i.e., Acinetobacter) increased from 0.08% to 5.81-12.57%with the addition of PLA MPs.Although Acinetobacter's contribution to EBPR remains controversial, many studies have shown that Acinetobacter has the ability to accumulate polyP and PHA [66][67][68], and it has previously been proposed to behave as PAOs in EBPR systems [69,70].The relative abundance of Competibacter increased from 0.05% in R0 to 0.16% in R1, but decreased to 0.03% in R3 as PLA MPs concentration continued to increase.The relative abundance of Defluviicoccus is 0.11% in R0, and the presence of PLA MPs increases it to 0.21-0.27%.Previous studies have shown that heterotrophic organisms such as PAOs are more tolerant of MPs and engineered NPs [35,71,72].In addition, Wu et al. [73] noted that the surface roughness of PLA/starch particles increased with the extension of retention time, which was conducive to microbial attachment and growth.It could, therefore, act as a biofilm carrier while releasing small-molecule organic matter as slow-release carbon sources for enriching specific heterotrophic populations, potentially influencing microbial dynamics.Some members of the genus Acinetobacter have been reported to be capable of decomposing refractory plastics, such as PS and low-density PE, and using decomposition products as carbon sources [74,75].Therefore, it is assumed that Acinetobacter may also be capable of utilizing biodegradable PLA as a carbon source.Further studies related to Acinetobacter's specific metabolic pathways or genes used for PLA degradation are warranted to better understand its increased abundance in the presence of PLA MPs.To sum up, the addition of PLA MPs may have changed the competition between PAOs and GAOs, ensuring that the potential PAOs (e.g., Acinetobacter) gradually gain advantages, which may be related to their specific carbon usage and metabolic characteristics.
The relative abundances of known AOB (i.e., Nitrosomonas) and NOB (i.e., Nitrospira) were less than 0.01% in all SBRs, meaning that there was no clear relationship between fluctuating nitrification performance (Section 3.1.3),decreased nitrification rates (Section 3.2.2),and AOB/NOB changes.The relative abundance of OLB12, which has been reported to have nitrification capacity [76], increased from 0.2% in R0 to 1.1% in R3, potentially contributing to nitrification performance in this study.Zhang et al. [77] found that a large number of heterotrophic DNB belong to the genus Pseudomonas.The relative abundance of Pseudomonas was 95.4% in R0, while it gradually decreased in SBRs with the addition of PLA MPs, reaching 59.0% in R3.The relative abundance of Acidovorax and Azoarcus, which also possess denitrification capacity [78,79], largely increased from 0.03% and 0.29% in R0, respectively, to 8.47% and 2.86% in R3, respectively.However, the increased involvement and contribution of Acidovorax and Azoarcus was not sufficient to compensate for the decrease in Pseudomonas abundance, ultimately leading to depressed denitrification performance in the SBRs with PLA MP exposure.Further studies with more tests (e.g., denitrification activity, ROS production and LDH release, morphology, etc.) and advanced tools (e.g., metagenomics) in the long term are still needed to establish the relationship between pollutant removal and functional activities/populations/genes, as well as to better understand the mechanisms of PLA MP exposure influencing system performance.

Conclusions
(1) The low concentration of PLA MPs (50 particles/(g TS)) had little effect on the pollutant removal performance in the activated sludge system.A high concentration of The relative abundances of known AOB (i.e., Nitrosomonas) and NOB (i.e., Nitrospira) were less than 0.01% in all SBRs, meaning that there was no clear relationship between fluctuating nitrification performance (Section 3.1.3),decreased nitrification rates (Section 3.2.2),

Figure 2 .
Figure 2. Variation in EPS components in the sludge flocs collected from different SBRs.Figure 2. Variation in EPS components in the sludge flocs collected from different SBRs.

Figure 2 .
Figure 2. Variation in EPS components in the sludge flocs collected from different SBRs.Figure 2. Variation in EPS components in the sludge flocs collected from different SBRs.

Figure 3 .
Figure 3. Microbial community compositions in different SBRs at the (a) phylum and (b) genus levels.

Figure 4 .
Figure 4. Non-metric multidimensional scaling (NMDS) plot showing microbial community difference in different samples.

Figure 4 .
Figure 4. Non-metric multidimensional scaling (NMDS) plot showing microbial community difference in different samples.

Figure 5 .
Figure 5.The relative abundances of functionally relevant microbial populations for N and P removal in different SBRs.

Figure 5 .
Figure 5.The relative abundances of functionally relevant microbial populations for N and P removal in different SBRs.

Table 1 .
Comparison of EBPR activities during the batch tests with the sludge collected from different SBRs.
a : Other full-scale or lab-scale EBPR systems without MP exposure.

Table 2 .
Comparison of nitrification activities during the batch tests with the sludge collected from different SBRs.