Collaborative removal of microplastics, bacteria, antibiotic resistance genes, and heavy metals in a full-scale wastewater treatment plant

The unique advantages of plastics (Chu et al. 2022) make them widely used in the world (Lievens et al. 2022). Plastics decompose into small particles or flakes (Chu et al. 2019) in the environment. Particles smaller than 5 mm are defined as microplastics (MPs) (Thompson et al. 2004). MPs’ pollution is a growing global concern (Pasquier et al. 2022) due to its potential risk to different ecosystems, and its impacts on the health of living organisms (Jiang et al. 2023). MPs are widely distributed, affecting rivers, lakes, soil, and air within the environment. They are ingested by organisms, and lead to toxic effects on growth, reproduction, and inflammation (Xiong et al. 2022). MPs are also easily enriched in organisms and cause persistent biological toxicity by disrupting the ecological balance (Golwala et al. 2021; Khan et al. 2022). MPs in air samples could be transported to the lung tissue and lead to the fibrotic type of disease (Özgen Alpaydin et al. 2024). They have been found in many foods (e.g., seafood, fruits, vegetables, honey, and beer) which can be taken via ingestion and lead to damage to the immune system and gut inflammation (Ansar et al. 2024). MPs also adsorb different contaminants. Heavy metals such as Cr, Cu, Hg, Ni, and Pb are designated as potentially toxic elements (PTEs) (Cai & Wang 2022). MPs migrate with PTEs (Chai et al. 2020) through flakes and cracks on the surface (Haque et al. 2023), increasing the risk of heavy metal contamination to the soil and agriculture. Besides heavy metals, MPs carry antibiotic resistance genes (ARGs), which induce potential threats to public health (Wang et al. 2024) and promote the transmission of ARGs. Selectively combined pollution formed by MPs with ARGs resistant to rifamycin and vancomycin has been reported (Peng et al. 2024).

Most of the MPs’ pollution originates from industrial waste, urban, and stormwater runoff, the degradation of larger plastics released into the environment, and wastewater treatment plants (WWTPs). MP particles and fibers enter the WWTPs through domestic wastewater. The distribution of MPs in the environment, both in terms of quantity and type, is uneven. For example, the concentrations of MPs in surface water, groundwater, and sediment vary with an abundance of 1.6 ng/L, 2.7 ng/L, and 33.8 ng/kg, respectively (Al Nahian et al. 2023). Polyethylene (PE), polyethylene glycol terephthalate (PET), polypropylene (PP), polyvinylchloride (PVC), polyformaldehyde (POM), nylon, and polystyrene (PS) are common materials of MPs found in the marine and freshwater environment (Islam et al. 2022). The top materials contributing to MPs’ pollution in urban water through atmospheric deposition are PE, PP, PET, and polyamide (PA) (Sun et al. 2022) in laboratory research. PE, PET, PP, and PE-PP copolymers were the most abundant polymer types in a WWTP with a treatment capacity of 2.45 million m³/day (Zhang et al. 2022).

The traditional WWTPs can remove most of the MPs; however, they are difficult to be completely removed (Pan et al. 2022). The removal rates vary from 50 to 96.8% at the centralized WWTPs and from 14.2 to 53.6% in decentralized WWTPs (Maw et al. 2024). MPs may enter rivers and accumulate in sediments for decades with the effluent of WWTPs (Cole et al. 2011). The removal efficiency of MPs in the primary treatment of WWTPs ranged from 16.5 to 98.4%, the removal efficiency of the secondary treatment improves the ratio to 78.1 to 100% (Tang & Hadibarata 2021), and that of which for tertiary treatment is 87.3 to above 99.9% (Tang & Hadibarata 2021). Biological approaches were continually adopted to dispose of the pollution of MPs (Amjad et al. 2022) in experiments. Laboratory investigations have found that some bacteria degraded MPs including PE, PS, and PP (Hossain et al. 2023). The degradation of PE was related to Candidatus_Cloacimonas and Candidatus_Caldatribacterium, those of PS, polyamide (PA), and PVC were related to Candidatus_Contubernalis and Candidatus_Caldatribacterium in the disposal of municipal solid waste (Li et al. 2022). Adsorption, filtration, and coagulation were also used in the removal of MPs in WWTPs (Shen et al. 2022). The membrane filtration process in WWTPs also had a good performance on MPs’ removal (Sadia et al. 2022). WWTPs were effective in removing MPs in sewage; however, some MP particles were usually enriched in the waste-activated sludge (Repinc et al. 2022).

MPs’ distribution in sewage reflects the pollution characteristics of MPs in the region and wastewater-activated sludge acts as a vector of ARGs, heavy metals, and bacteria by concentrating MPs. Greater attention should be given to the MPs’ removal capacity of WWTPs and their migration through WWTPs. In the study, the distribution characteristics and removal efficiencies of MPs in a full-scale WWTP were investigated. Additionally, in-depth exploration was performed into the co-removal properties and correlations between MPs and microorganisms, ARGs, as well as PTEs. It is anticipated that the results will provide an evaluation of the WWTP system's capability to remove MPs and offer a deeper understanding of the co-removal performance of MPs along with ARGs and PTEs in WWTP.

The largest full-scale WWTP of the Beijing Municipal Administrative Center (Tongzhou District, Beijing, China) with a treatment capacity of 180,000 m³/day was selected to investigate the disposal of MPs and associated pollutants (ARGs and PTEs). The studied WWTP consists of three-level treatment processes, and implements higher local emission standards (COD < 30 mg/L, ⁠, total nitrogen <15 mg/L, total phosphorus <0.5 mg/L), which has a typical scale, process, and emission standard in China. The primary process is composed of coarse screens, fine screens, and superfine screens with grid distances of 15, 3, and 1 mm, respectively, as well as a grit chamber. The secondary process is a step-feed anoxic/oxic-activated sludge process, and the tertiary processes include coagulation, submerged ultrafiltration, and ultraviolet disinfection. No industrial wastewater but only sewage was collected into the studied WWTP.

Samples of municipal wastewater (MW) and sludge were collected from the WWTP in Section 2.1 in July 2023 at six process sections. Samples were taken from the total influent of WWTP (TI), the biological process influent (BI), the biological process effluent (BE), the coagulation effluent (CE), the final effluent (FE), and the sludge. A tap water (TAP) sample from the same location of WWTP was also collected. All samples were 6 h mixed samples and collected in triplicate. Samples of around 1,000 mL of wastewater or tap water were collected into bottles and stored at 4 °C in the shade before pretreatment.

Samples of 0.15 L water were filtered in triplicate with nitrocellulose filters with a pore size of 0.22 μm to capture the MPs and any associated microorganisms. The total DNA was then extracted from these filters using the PowerWater DNA Isolation Kit (Mo Bio Laboratories Inc., USA). After genomic DNA extraction, the quality and quantity of the extracted DNA were assessed by 1% agarose gel electrophoresis to ensure that they met the requirements for library preparation. The DNA samples were then stored at −20 °C until further analysis (Gajdoš et al. 2023).

The preparation of PE libraries followed the guidelines of the NEXTFLEX™ Rapid DNA-Seq Kit (Bioo Scientific, USA). Fragments with approximately 400 bp in size were obtained by shearing using a Covaris M220 ultrasonicator (Stewart et al. 2024). The amplified fragments were constructed into a PE library according to the protocol of the Illumina MiSeq platform. The ‘Y’-shaped joint was connected, and then the self-ligating fragment was removed using magnetic beads. PCR amplification was used to enrich the library template. The final library was obtained by recovering PCR products from magnetic beads (Zhou et al. 2020).

The paired-end sequencing process was conducted on an Illumina NovaSeq/HiSeq Xten platform (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd (Shanghai, China). Sequencing was performed using NovaSeq Reagent Kits/HiSeq X Reagent Kits (Chen et al. 2022). The sequencing was based on fluorescence-labeled nucleotide bases.

During the sequencing process, one end of each library molecule served as the primer base, and the template information was immobilized on the sequencing surface after an initial round of amplification. The opposite end of the molecule was then immobilized near another primer, creating a ‘bridge’ structure. PCR amplification was subsequently performed to generate clusters of DNA molecules, each representing a single-template sequence.

DNA polymerase and reversibly terminated, fluorescently labeled deoxyribonucleotide triphosphates (dNTPs) were added to the reaction. These dNTPs allow for the incorporation of only one base per cycle, and the type of nucleotide incorporated is determined by scanning the surface of the reaction plate. The fluorescence signals emitted by the incorporated bases were collected and counted, allowing for the reconstruction of the template DNA sequence (Wang et al. 2016). The use of reversible terminators minimizes sequencing bias by ensuring that each base is added in a controlled and sequential manner.

Multiple samples were pooled and sequenced in parallel, with a unique 500 bp tag sequence introduced into each sample's sequence to facilitate sample identification and analysis. Paired-end reads of 150 bp in length were generated for each sample in the experiment.

Following sequencing, open reading frame (ORF) prediction was performed using the MetaGene tool (available at http://metagene.cb.k.u-tokyo.ac.jp/). Genes with predicted ORFs longer than 100 bp were selected and translated into amino acid sequences for further analysis.

The data analysis was done over the Majorbio Cloud platform (https://cloud.majorbio.com/).

About 200 mL of the water sample was taken for vacuum filtration. Then, the immerse filter membrane was treated by ultrasonic in an ethanol solution in order to disperse substances adsorbed on the filter membrane into the ethanol solution. The filter membrane was then taken out from the ethanol solution, and washed with ethanol three times. 30% H₂O₂ was added into the ethanol solution to remove the organic matter and the solution was left to stand for 24 h. The ethanol solution was concentrated and dropped onto the high-reflection glass. The samples were prepared following the methods described by another study (Cheng et al. 2022). After ethanol was completely volatilized, the laser direct infrared (LDIR) test was performed by the Agilent 8700 Laser Direct Infrared Imaging system.

A saturated ZnCl₂ (Sigma-Aldrich, 99.999% trace metals basis) solution was prepared. The sludge sample was placed in a 100 mL beaker, and then 60 mL of the ZnCl₂ solution was added, the mixture was fully stirred for 2 min before overnight for 12 h. Then, the suspension was transferred and another 60 mL of 30% H₂O₂ was added to remove organics. The solution was then treated as the same method as the water samples. During the LDIR test, the particle analysis mode and the automatic test method (matching degree >0.65, particle size range 20–500 μm) were selected.

All reagents were of analytical reagent grade. Deionized water used in experiments was obtained from a Milli-Q water system (Millipore, USA). Stock standard solutions of Cr (III), Cu (II), Hg (II), Pb (II), Tl (I), and Zn (II) (1,000 mg L⁻¹) were from Sigma-Aldrich (USA). Nitric acid (HNO₃, 99%, pure) and perchloric acid (70 wt%) were supplied by Sigma-Aldrich (USA). Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis was carried on by iCAP™ 7600 (Thermo, USA).

The sample was filtrated by 0.45 μm membrane immediately after collection. The initial 50–100 mL solution was discarded, and the required volume of filtrate was collected. The pH of the solution was adjusted to pH < 2 with HNO₃. Five millilitres of HNO₃ (1 : 1) was mixed to the 50 mL sample. The mixture was then put on a hot plate and evaporated to near the boiling state. After cooling down, 5 mL of HNO₃ (1:1) was added again. Then, HNO₃ (1:1) or perchloric acid was added for digestion. Some HNO₃ (1:9) was added to dissolve the residue. The solution was reheated at 45 °C for 1 h to get further digestion. The solution was then cooled down and filtered for ICP-OES analysis (Erdiwansyah et al. 2024). Two hundred milligrams of the sludge sample was taken into poly(tetrafluoroethylene) (PTFE) digestion vessels, which was dissolved in 5 mL HNO₃ (1:1). The digested samples were placed on an electric heating plate, heated for acid removal, and diluted to a volume of 100 mL HNO₃ for ICP-OES. The experiments were carried out three times in parallel to evaluate the contents of Cr, Cu, Hg, Ni, and Pb. ICP-OES calibration standard solutions in 5% HNO₃ were prepared by single standards. Three independent quality control (QC) standards in 5% HNO₃ were incorporated in each analytical run. Independent analytical QC solutions were all from Sigma-Aldrich (Cr, Cu, Hg, Ni, and Pb).

Data were expressed as the mean values. Statistical tests and figure plotting were all executed using OriginPro 2024 (OriginLab Corporation, USA). Pearson correlation coefficient and principal component analysis (PCA) were used to explore the potential relationships between MPs, microbial communities, PTEs, and ARGs. Pearson's correlation coefficient (r) was used to determine the extent of linear correlations between parameters. Differences between samples were considered significant if p < 0.05 at a 95% confidence interval of the mean, and a one-way analysis of the variance (ANOVA) and paired t-tests were performed. To investigate correlations between MPs and ARGs, correlation networks with Spearman coefficient |r| > 0.6 and p-value < 0.05 were established.

Silicone and polybutadiene fragments in sludge were more than that in water samples. Besides, PP, EVA, PTFE, PE, PET, PS, PVC, and phenolic epoxy resin were found in sludge samples. The MPs in sludge mainly enriched from the MPs of sewage, and the difference showed the enrichment selectivity of sludge to MPs. The MPs in tap water in this area were also investigated. Seven different MPs were found in tap water. They were PU, POM, acrylonitrile butadiene styrene copolymer (ABS), and a small amount of ACR, PET, PS, and PVC. The dominated MPs were PU and POM, which further affected the quality of drinking water in this area.

The total removal efficiencies of ACR, EVA, polybutadiene, PTFE, PS, and PP reached 100% in the studied WWTP. PU had a removal efficiency of 93.41%, with the highest content in the influent. Fluororubber and PET achieved total removal efficiencies of 50%. In the biological process, fluororubber had the highest removal efficiency of 100%, followed by PP and ACR with removal efficiencies of 66.67 and 50%, respectively. The removal efficiencies of PLA and PET by coagulation, flocculation, and sedimentation reached 100%, and that of PU was 58.33%. Overall, the WWTP removed MPs with an efficiency of 76%, irrespective of silicone release, which is basically consistent with previous research findings. The removal efficiency of MPs in WWTPs varied across different processes, generally ranging from 70 to 95% (Bayo et al. 2023; Fauzi et al. 2024), with some specific processes achieving even higher efficiencies (Xu et al. 2019).

The studied WWTP could remove MPs significantly considering both counts and types. The effluent of the coagulation process contains lower MPs than tap water; however, the final effluent of WWTP still contains MPs which may pose health risks during reclaimed water reuse. Besides, MPs in sludge are a more concerned problem for the environment.

The influent microorganisms, Dechromonases, were adsorbed and removed during the primary processes, exhibiting synergistic effects with MPs’ removal. It was speculated that polar molecules such as PU could adsorb microorganisms in the influent to form larger polymers, increasing the removal efficiency. Moreover, the microbial Dechloromonas and Thauera showed similar removal characteristics with MPs during the coagulation process. Dechromonas is an important biological phosphorus removal microorganism, and it is easy to remove during the adsorption and coagulation process in water treatment.

The biological process further reduced tetQ. In the final effluent, tetQ was further reduced after the tertiary processes. The concentrations of qnrS and tetQ in the sludge were much lower than in sewage, which indicated that these ARGs did not transfer to the sludge in concentrations comparable to those found in the sewage. The concentration variation of tetQ was similar to the rule of MPs removal, which certificated potential relevance between certain ARGs and MPs.

Heavy metals such as Cr, Cu, Hg, Ni, and Pb, which are usually present in high concentrations in sewage, were selected for association evaluation with MPs. The heavy metals with the highest concentration in the influent were Cr and Cu (Table 1). No heavy metals in the final effluent exceed the discharge standard. Cr and Cu were the main heavy metals in domestic sewage in this area. Cr and Cu metal ions were well removed in the primary processes. High concentrations of heavy metals were detected in the sludge, in which the concentrations of Cr and Cu were more than 10 times higher than those in the influent of WWTP. Pb was found in the sludge but was not detected in the influent of WWTP, which indicated that sludge had an adsorption effect on heavy metals.

Most heavy metals are removed during the pretreatment stage, which is consistent with the removal characteristics of MPs, indicating that heavy metals are highly likely to adhere to large particles formed by MPs and settle due to blocking effects. At the same time, the content of heavy metals in the sludge significantly increases, and they can migrate with sludge treatment and utilization, causing further ecological risks. The adsorption capacity of MPs to heavy metals is related to the degree of carbonylation, and the action decreases with dichlorination (Jiang et al. 2022). MPs could enter the roots of plants, and these plants were found to contain higher heavy metals, such as Cd.

The biplot indicated that the ARGs sul1, sul2, tecT, and tetQ obtained a high positive loading value in the direction of PC1, which accounted for 83.18% of the total data variance. Furthermore, polybutadiene, PP, PTFE, ACR, and PU earned a moderately high positive value in PC1. Cr was found clustered in the same quadrant, suggesting that polybutadiene, PP, PTFE, ACR, and PU were correlated with sul1, sul2, tecT, tetQ, and Cr in the right quadrant. A relatively closer distance between tetQ and PU to the origin suggested that the selective adsorption of PU had an impact on the migration of the ARGs tetQ. In contrast, PLA, PVC, CPE, PET, fluororubber, Cu, and qnrS were clustered in the left quadrant, indicating that they shared a similar migration with each other. A relatively closer distance between qnrS and CPE to the origin suggested a potential co-migration by adsorption.

The interactions between MPs, heavy metals, microorganisms, and ARGs were complex. MPs interacted with polar proteins on the surface of microbial through functional groups, resulting in consistent migration. Microorganisms can transmit ARGs via plasmids. ARGs further migrate, transmit, and diffuse through the interactions between microorganisms and MPs. The removal of heavy metals was mainly concentrated in the primary treatment process, which was consistent with the removal of MPs. Heavy metals and MPs were prone to forming larger aggregated particles, resulting in coprecipitation. The heavy metals were enriched in sludge, which may migrate with MPs and be adsorbed by the sludge. The interaction between MPs and heavy metals should be given attention, especially during sludge treatment and disposal.

The main MPs detected in the studied WWTP were PU, ACR, fluororubber, PE, CPE, and silicone. The primary treatment could remove some MPs; however, coagulation demonstrated a significant removal efficiency for MPs. PU contains more polar groups, making it prone to forming larger polymers with other molecules via hydrogen bonding interactions, which are subsequently removed in the primary treatment processes. Microorganism species and ARG variation are associated with MPs. The similarity in removal characteristics between Dechloromonas and MPs may be attributed to the formation of large particles through adsorption and condensation. Dechloromonas was found to be easily adsorbed by ACR and fluororubber. PP and polybutadiene were co-removed with Nitrospira. PVC was selectively degraded by Nitrospirota. Silicone was selectively removed by Bacteroidetes, Anaerolineae, and Nitrospira. Further attention should be given to the potential adsorption and removal mechanisms of biological phosphorus removal bacteria at different process stages of WWTPs. A relative correlation between qnrS and CPE was found. The migration of bacA was correlated with PE, fluororubber, and silicone. The adhesive used for component connection during membrane filtration might produce a significant amount of silicone MPs in the final effluent. PTEs and ARGs can migrate with MPs, leading to their accumulation in organisms, which may affect the safety of the entire food chain and promote the transmission of ARGs in the environment. The release of MPs in the sludge of WWTPs poses potential threats to soil and the entire ecosystem during landfill or disposal as fertilizer. The samples for this study were collected in summer, and microbial community characteristics may vary with temperature. Based on the study's findings, the adhesive used in the membrane filter should be further optimized to minimize the release of silicone.

All authors have read, understood, and have complied as applicable with the statement on ‘Ethical responsibilities of Authors’ as found in the Instructions for Authors.

The authors thank key projects of Beijing Polytechnic (2024X007-KXZ) and the funding program for scientific and technological innovation talents in Tongzhou District, Beijing (JCQN2023002) for the financial support.

All authors contributed to the study's conception and design. Methodology and supervision were performed by R.S. Material preparation, data collection, and analysis were performed by P.L. and R.Y. The first draft of the manuscript was written by P.L. and all authors commented on previous versions of the manuscript. All authors read and approved of the final manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.