Limited bisphenol A (BPA) degradation acceleration by pre-acclimating microplastic biofilms with BPA in natural lake water

Limited bisphenol A (BPA) degradation acceleration by pre-acclimating microplastic biofilms with BPA in natural lake water | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Limited bisphenol A (BPA) degradation acceleration by pre-acclimating microplastic biofilms with BPA in natural lake water Xiang Gao, Renxin Zhao, Jinhui Jiang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4349153/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Bisphenol A (BPA) and microplastics are prevalent in aquatic environments. Microplastic biofilms play a crucial role in the environmental degradation of BPA, but related research is lacking. We designed experiments to investigate the effect of BPA on microplastic biofilms and the effect of pre-acclimating biofilms on BPA degradation. Results : Even at low concentrations (0.1 mg L -1 ), BPA significantly reduced microplastic biofilm biomass ( P < 0.05). High-throughput 16S rRNA sequencing revealed that BPA altered biofilm diversity, as evidenced by changes in Chao-1 and Shannon indices. The primary phyla in the microplastic biofilm included Proteobacteria , Bacteroidetes , Actinobacteria, and Firmicutes . On the 7 th day of biofilm formation, the dominant bacterial genus shifted from Ohtaekwangia to Bdellovibrio in groups with BPA treatment, and the relative abundance of Bdellovibrio reached 4.32% ± 5.34%. On the 14 th day, Methylobacillus significantly increased in all treatments compared with the 7 th day ( P < 0.05). Adonis analysis demonstrated that the metabolic composition of the bacterial community also changed significantly ( P < 0.05). BPA (0.1 mg L -1 ) pre-acclimation of microplastic biofilms led to a significant increase in the amount of BPA-degrading bacteria with no significant effect on BPA degradation efficiency. After 7 days, the BPA removal rate in high-concentration microplastic treatments (1600 mg L -1 ) reached > 90%. Conclusions: Biofilms significantly increased the BPA degradation rate by 174.78% to 889.25% on the third day, indicating that the biofilm accelerates BPA degradation efficiency in the short term. Our findings provide a foundation for further understanding the environmental risks associated with the coexistence of bisphenols and microplastics. Bisphenol A Spherical polystyrene microplastics Co-pollution Biofilms Biodegradation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background Thin films, textile fibers, and plastic particles < 5 mm are defined as microplastics [ 1 ] . With the use of plastic products increasing each year and a lack of effective disposal methods, the high concentration of microplastics in the environment continues to increase. 320 million tons of plastic have been discarded, and 10% of these will persist in the environment [ 2 ] . As a major producer and manufacturer, China accounts for 23.9% of the world's total plastic production [ 3 ] . Microplastic pollution is widely distributed, with aquatic environments being primary areas of contamination. Lakes, as a relatively enclosed aquatic environment, can serve as an important pathway for the temporary storage and transport of microplastics. The content and pollution levels of microplastics in lakes are already much higher than those in the ocean [ 4 ] . In 2014, Chinese researchers first reported that the concentration of microplastics in the surface water of the Yangtze River Estuary reached 4137.3 n/m 3 and has been increasing annually [ 5 ] . Microplastics result from diverse sources, such as medical consumables, personal care products, and synthetic fiber products [ 6 ] . One of the main sources of microplastic pollution in aquatic environments is industrial waste, with a daily discharge of up to 2 million particles, corresponding to an average annual efflux of 50 million m 3 /years [ 7 ] . The concentration of microplastics in the environment is closely related to human activities [ 8 ] , and is an environmental issue that needs to be taken seriously. Because they are difficult to degrade, are light in weight, and have a large specific surface area, microplastics persist in aquatic environments for a long time. Their surfaces often have microorganisms attached that form unique biofilms [ 9 ] , which are sensitive to changes in environmental factors such as nutrient levels as well as ion and organic matter concentrations [ 10 ] . There are significant differences between the microbial community composition of microplastic biofilms and those found in the surrounding environment. The formation of biofilms enhances the metabolic capacity of microbial communities [ 11 , 12 ] . Microplastics have a strong adsorption and enrichment effect on organic pollutants, making their surfaces a hotspot for these compounds [ 13 ] . Relevant experiments have shown that the concentration of organic pollutants on the surface of microplastics was 106 times or greater than that in the surrounding environment [ 14 ] . The enrichment of organic pollutants by microplastics can lead to changes in the community structure of microplastic biofilms, further altering their composition and metabolism [ 15 , 16 ] . The influence of environmental factors, including the coexistence of organic pollutants, also acts as a feedback mechanism by increasing adsorption and altering degradation, affecting the environmental fate of organic pollutants [ 17 , 18 ] . A study found that the presence of microplastic biofilms can significantly enhance the degradation efficiency of debrominated diphenyl ethers [ 17 ] . The pollutant concentration on microplastic surfaces with biofilms is typically up to 3.8 times lower than on microplastics without biofilms, possibly because of biofilm enhancement of the degradation of pollutants adsorbed onto their surfaces [ 15 ] . In co-polluted environments, the interaction between microplastics and organic pollutants is significant, with microplastic biofilms playing an important role. Bisphenol A (BPA), a representative phenolic compound, is a pervasive endocrine disruptor found in various environmental matrices such as water, sediments, air, and biota [ 19 ] . The global demand for BPA is continuously increasing, with estimated consumption reaching 10.6 million tons by 2022 [ 20 ] . Widely used as a plasticizer crucial for various daily-life applications [ 21 ] , BPA is ubiquitously present alongside microplastics in the environment. However, research on microplastics and organic pollutants has primarily focused on persistent organic pollutants [ 22 ] , with insufficient attention paid to "pseudo-persistent organic pollutants" such as BPA. Unlike recalcitrant persistent organic pollutants, BPA-degrading bacteria are widely distributed in the environment [ 23 ] . BPA may further increase the relative abundance of bacterial species with degradative capacity in the community when it coexists with other pollutants, rather than primarily exhibiting toxic effects on bacterial communities such as those caused by persistent organic pollutants; however, this has yet to be studied. In addition, there is a lack of research regarding whether BPA acclimation of microplastic biofilms can enhance the subsequent degradation rate of BPA in aquatic environments. This research focused on the effect of BPA on the microbial community composition and metabolic pathways of microplastic biofilms in a co-contamination scenario. By acclimating microplastic biofilms to BPA co-contamination, its effect on the subsequent degradation of BPA in aquatic environments was examined. This study can lay a foundation for a deeper understanding of the environmental risks associated with the coexistence of bisphenol pollutants and microplastics. 2. Methods 2.1 Experimental setup 2.1.1 Effect of BPA on the development of microplastic biofilms Spherical polystyrene (PS) microplastics with a diameter of 1160 ± 559.962 µm were purchased from China Petroleum and Chemical Corporation (Beijing, China). To evaluate the effects of BPA on the formation of microplastic biofilms, lake water was collected from Nanhu Lake (an urban lake in Wuhan, China) and filtered through a 700 µm mesh. BPA purchased from Sigma-Aldrich (Darmstadt, Germany, purity ≥ 99%) was added to the filtered lake water at final concentrations of 0, 0.10, and 5.00 mg L − 1 , and all the groups were amended with 0.2 g of PS microplastics. Culture of microplastic biofilms was carried out in 250 mL flasks shaking at 120 rpm at 28°C. Three replicates were set up for each condition, and the biomass and bacterial community of biofilms were analyzed on the 7th and 14th day of the experiment. The biomass of microplastic biofilms was determined by the colorimetric method according to Lobelle [ 24 ] . The bacterial community of microplastic biofilms was analyzed by 16S rRNA high-throughput gene sequencing. Microbial DNA from microplastic biofilms was extracted using the MP Fast DNA™ SPIN Kit (CA, USA) according to the manufacturer’s instructions. The quality of DNA was verified by 1% agarose gel electrophoresis. The concentration and purity of DNA were determined using a Nanodrop One spectrophotometer (Thermo Fisher Scientific, USA). All isolated DNA samples were stored at -20°C and sent for sequencing on the Illumina MiSeq platform by Novogene Co., Ltd. (Tianjin, China). Primers 341 F (5′-CCTACGGGNGGCWGCAG-3′) and 806 R (5′-GGACTACVSGGGTATCTAAT-3′) were used to amplify the V3–V4 region with a 2 × 250 bp paired-end strategy. 2.1.2 Effect of BPA removal of BPA-pre-acclimation of microplastic biofilms in lake water To explore the influence of biofilms with and without BPA pre-acclimation on the degradation of ambient BPA from lake water, lake water was collected from three urban lakes (Nanhu Lake, Donghu Lake, and Yezhi Lake; Wuhan, China) (Table S1 ). PS microplastics were preincubated to form biofilms in lake water with or without 0.1 mg L − 1 BPA for 7 days. These microplastics were collected with meshes (425 µm in diameter) and rinsed with sterilized deionized water three times before use in the BPA degradation experiments. Two microplastic densities (50 and 1600 mg L − 1 ) with biofilms were treated with or without BPA acclimation, and microplastics without preincubated biofilms were used as controls. Groups without microplastics were used to estimate the removal by indigenous microorganisms and photodegradation. The BPA degradation experiment was performed in 250 mL flasks containing 120 mL lake water spiked with 5 mg L − 1 BPA, cultured at 28°C and 120 rpm. During the experiment, 1 mL of lake water was sampled and filtered through 0.22 µm membranes at 0, 10, and 30 min, 1, 3, 6, and 12 h, and 1, 3, and 7 d for determination of residual BPA. The BPA concentration was measured by a reverse-phase HPLC (LC40, Shimadzu, Kyoto, Japan) equipped with a UV detector at a wavelength of 278 nm using a C18 column (250 mm × 4.6 mm × 5 µm, Agilent, Santa Clara, CA, USA); the mobile phase was 65% acetonitrile and 35% ultrapure water (v/v) at a flow rate of 1 mL min − 1 , and the injection volume was 20 µL. The acetonitrile was HPLC grade and purchased from TEDIA (OH, USA). We used the following formula to calculate the degradation rate of BPA by microplastic biofilms (Eq. 1): BPA bdr−m = BPA removal – BPA ad−m – BPA photo, (1) where BPA removal (%) represents the overall rate of BPA removal, BPA ad−m (%) represents the rate of BPA adsorption by microplastics, and BPA photo (%) represents the rate of BPA removal by indigenous microorganisms or photodegradation. We estimated the adsorption of BPA by microplastics by assessing the removal dynamics of BPA (Fig. S2). At the initial stages of BPA removal, the contribution of degradation is minimal and can be neglected. The equilibrium point of removal was reached at 12 hours, and the removed amount at this time point was considered the adsorption of BPA by microplastics. We first calculated the average residual concentration at each time point in the CK group; next, we subtracted the average residual concentration of the CK group at each time point from the initial concentration of each treatment group and then subtracted the corresponding treatment group's residual concentration to obtain the adsorption of BPA by microplastics. 2.1.3 Bacterial counting relating to BPA degradation in microplastic biofilms Pre-incubated biofilm microplastics in low-density treatments (50 mg L − 1 ) were harvested on the 7th day with meshes (425 µm in diameter), rinsed with sterilized deionized water three times, and dried at room temperature for 30 minutes to determine BPA-degrading bacteria counts in biofilms; samples at the initial time of ambient BPA degradation set-up were also assessed. Microorganisms in biofilms were collected from microplastics in sterilized Milli-Q water by ultrasonic processing (60 W, 40 KHz) for 10 minutes and vortexing for 3 minutes. BPA-degrading bacteria were then counted before and after the experiment by spread plating on inorganic agar medium [ 25 ] with 10 mg L -1 of BPA as the sole carbon source. 2.2 Microbial community analysis The raw 16S rRNA sequences were subjected to quality filtering using Mothur software (v 1.41.1) [ 26 ] . To facilitate the comparison of bacterial community structures across samples at a consistent sequencing depth, each dataset was normalized. Operational taxonomic units (OTUs) were clustered at a 97% similarity level using the UPARSE pipeline in USEARCH (v 11.0.667) [ 27 ] . Chimeras were removed and singleton unique sequences were discarded before further analysis. Representative sequences from each OTU were taxonomically classified against the Ribosomal Database Project (RDP) database using the RDP classifier (v2.2) with a confidence threshold of 80% [ 28 ] . 2.3 Microbial functional prediction using PICRUSt2 The functional composition of microbial communities was predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) based on 16S rRNA marker gene profiles [ 29 ] . Functional annotations for PICRUSt2 predictions were derived from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [ 29 ] . KEGG Orthologs (KOs) table was generated for each dataset, and the relative abundance of predicted KOs was calculated. The differences in functional gene family composition across samples were visualized using principal coordinate analysis (PCoA) based on Bray-Curtis distance metrics. 2.4 Statistical Analysis Data including variations in biofilm biomassand BPA degradation were compared by one-way ANOVA, and variations in bacterial density were compared by independent t-test. Data including the BPA removal rate, BPA ad−m, and BPA bdr−m were compared by two-way ANOVA. When a significant interaction was found, one-way ANOVA with the Duncan test for multiple comparisons was performed. The significance level was set at 0.05. All analyses were performed in SPSS (version 18.0.0). Figures were drawn in OriginPro (version 8.5.1 SR2). The α-diversity indices, including Shannon, Simpson’s, and Chao-1 were calculated in Paleontological Statistics software (v 4.02). PCoA, heat maps, and Adonis analysis were performed using vegan, pheatmap, and ggplot2 packages in R. 3. Results 3.1 Biofilm biomass variation on PS microplastics in lake water influenced by BPA The biomass of biofilms on PS microplastics generally increased with time in all experiments (Fig. 1 ); significant differences were seen between that on the 7th day and the 14th day under different initial BPA concentrations. BPA in lake water had a significantly negative effect on the biomass of biofilms on PS microplastics ( P < 0.05), resulting in the lowest biomass for groups under higher BPA initial concentrations (5.00 mg L − 1 ) on both the 7th and 14th days. 3.2 Diversity comparison of the bacterial community on PS microplastics affected by BPA in lake water According to sequencing results, 7817 OTUs were obtained, 4385 of which (56.10%) were truncations clustered at the 97% similarity level. The total number of shared OTUs among all samples was 53. Alpha diversity indices including Chao-1 and Shannon were calculated and summarized in Supplementary Table S2, representing bacterial community richness and diversity, respectively. The Goods coverage was 99.7%, ensuring adequate sequencing depth. The Chao-1 index ranged from 516.77 ± 24.65 to 764.53 ± 49.41 for the groups with different initial BPA concentrations (Table S2); it decreased with initial BPA concentration on the 7th day but increased with initial BPA concentration by the 14th day. Higher initial BPA concentration had higher Chao-1 indices, significantly different compared with that of the CK without BPA on the 14th day (764.53 ± 49.41 vs 512.73 ± 32.29, P < 0.05). The Shannon index significantly increased with time for groups with higher initial BPA concentration ( P < 0.05). BPA also varied the Shannon index from 2.64 ± 0.30 to 3.58 ± 0.49 (Table S2), showing a similar trend to that of the Chao-1 index among treatments. PCoA analysis based on Bray-Curtis distance revealed the structural differences of bacterial communities in biofilms on PS microplastics between treatments. The two axes explained 48.79% of the total difference (Fig. 2 ). According to the results of Adonis analysis, initial BPA concentration significantly affected the structure of bacterial communities on microplastics, especially on the 14th day (Adonis: R 2 = 0.241, P < 0.05). For groups of each initial BPA concentration, the structure of bacterial communities was significantly different between experimental time points (Adonis: R 2 = 0.143, P < 0.01). 3.3. Microbial community composition of PS microplastics affected by BPA in lake water 3.3.1. Microbial community composition at the phylum level In total, 24 bacterial phyla were identified. Different relative abundances of bacteria in biofilm communities on PS microplastics were found under different experimental treatments (Fig. 3 ). Proteobacteria , Bacteroidetes , Actinobacteria , and Firmicutes were identified as the major bacteria phyla on microplastics under different initial BPA concentrations, accounting for 90.96%, 6.82%, 0.67%, and 0.59%, respectively, on the 7th day, and 83.01%, 13.10%, 1.48%, and 0.42%, respectively, on the 14th day. Their order of abundance was unchanged over time, although values varied. The relative abundance of Proteobacteria was significantly lower on the 14th day than on the 7th day in groups with 0.10 and 5.00 mg L − 1 BPA ( P < 0.05). Bacteroidetes relative abundance was greatest at lower initial BPA concentrations and lowest at higher BPA initial concentrations on both the 7th and 14th days, showing a significant difference on the 7th day ( P < 0.05). The relative abundance of Bacteroidetes was significantly greater on the 7th day than on the 14th day ( P 0.5% were chosen for further analysis. Different relative abundances of bacteria in biofilms on PS microplastics were found under different experimental treatments (Fig. 4 ). The number of shared genera across all samples was 33, with Methylophilus , Methylobacillus , and Methylovorus being predominant (all belonging to Methylophilaceae ). With different initial BPA concentrations, Methylophilus , Methylobacillus , and Methylovorus were the major genera, accounting for 55.93%, 2.59%, and 2.92%, respectively, on the 7th day; on the 14th day, Methylophilus and Methylobacillus were the major genera, accounting for 41.19% and 13.56%, respectively. Different bacterial community structures appeared over time. On the 7th day, the relative abundance of Methylophilus was the highest in all bacterial communities on PS under different initial BPA concentrations, and no significance was found between treatments ( P > 0.05). However, Bdellovibrio had a significantly larger relative abundance under high initial BPA concentration compared with under low initial BPA or the control ( P < 0.05). On the 14th day, Methylophilus abundance decreased while that of Methylobacillus and Ohtaekwangia increased. The relative abundances of Hydrogenophaga and Nevskia were significantly higher with 0.10 and 5.00 mg L − 1 BPA than that in group of CK ( P < 0.05). Compared with groups on the 7th day, treatments with both low and high initial BPA had significantly greater relative abundance of Methylobacillus on the 14th day ( P < 0.05). 3.4. Predicted metabolic functions of microbial communities To illustrate the metabolic function of bacterial communities on PS microplastics under different environmental conditions, PICRUSt2 was used to predict the metagenomic functional composition. Forty-five metabolic pathways (Level 2) from six KEGG metabolic pathway groups (Level 1) were predicted (Fig. 5 ). The relative abundances of metabolic pathway groups in different samples were similar, with the KEGG pathway Metabolism being the most abundant. Examining different initial BPA concentrations, the relative abundance of Metabolism varied from 66.71–68.29% on the 7th day, and from 68.30–71.17% on the 14th day; no significant differences were found ( P > 0.05). At Level 2, Metabolism of cofactors and vitamins , Amino acid metabolism , Carbohydrate metabolism , Cell motility , Energy metabolism , Xenobiotics biodegradation and metabolism , and Replication and repair were the major pathways predicted on the 7th day, accounting for 10.36%, 9.19%, 7.82%, 6.04%, 5.22%, 4.47%, and 4.43%, respectively. On the 14th day, this order remained the same, although their relative abundances changed slightly. PCoA analysis based on predicted relative KO abundances (Level 3) showed that samples were mainly separated by the first axis, which explained 88.02% of the variability (Fig. S1 ). Adonis analysis showed significant differences between samples under different experimental treatments (Adonis: R 2 = 0.052, P = 0.047). Initial BPA concentration showed no effect on the metabolic pathway structure of bacterial communities in biofilms on PS microplastics, as samples with different concentrations were not separated. 3.5. Density variation of bacteria relating to BPA degradation in preincubated biofilms at initial and harvest time in subsequent BPA degradation experiments After 7 days of preincubation with BPA (0.1 mg L − 1 ), the densities of degradation-related bacteria were significantly increased compared with the CK group in water from all three lakes ( P < 0.05, Fig. 6 ). However, after harvesting samples from the degradation experiment, significance remained only in Yezhi Lake water ( P < 0.05). The densities of BPA degradation-related bacteria showed a significant increase after 7 days in all treatment groups ( P < 0.05). 3.6. BPA biodegradation dynamics in lake water On the initial day, the degradation rate of BPA was not significantly different between treatments across the three lakes (Fig. 7 ). However, on the third day, the degradation rate of the groups with high-concentration microplastics addition and biofilms preincubated in Yezhi Lake and Nanhu Lake were significantly increased compared with the other treatment groups ( P < 0.05), indicating that the presence of a biofilm on microplastics has a significant effect on the degradation of BPA. Upon completion of the degradation experiment, the BPA degradation rate of the high-concentration microplastic addition groups was significantly increased compared with the low-concentration microplastics addition group ( P < 0.05). The removal rate with high-concentration microplastic treatment was 100%, indicating near-complete BPA breakdown. In contrast, there was no significant difference in the degradation rate between the low-concentration microplastics addition group and CK. 4. Discussion 4.1 Effect of BPA acclimation Microplastics are believed to be effective adhesion substrates for the development of biofilms made up of algae, bacteria, fungi, and protozoa, because of their recalcitrant nature and extensive specific surface area; this forms an ecological niche known as the “plastisphere” [ 9 , 10 , 30 , 31 ] . Biofilm formation takes time, suggesting a positive influence of exposure time on biofilm biomass [ 32 ] . Lobelle et al. [ 24 ] reported that 7 days was enough for the formation of biofilms on plastic debris in seawater, though the biomass significantly increased in the following week. Li et al [ 33 ] also found increasing biomass of biofilms on five types of microplastics with experimental time. Similar results were found in this study, with a significant increase in biomass of biofilms on PS microplastics occurring over time with different BPA initial concentrations. BPA had a negative effect on biofilm biomass, even when the initial concentration was low (0.10 mg L − 1 ); this effect was intensified when the initial concentration was high (5.00 mg L − 1 ). This was quite unusual, because many early reports found a positive effect of BPA on the growth of microorganisms at low initial concentrations, with negative effects at high initial concentrations [ 25 , 34 ] . BPA can enhance the growth of specific species when used as an additional carbon source [ 35 ] ; however, it can also suppress the development of organisms, mainly because of oxidative stress resulting from the overproduction and accumulation of reactive oxygen species [ 36 ] . Bisphenols can integrate into cell membranes, reducing the polycondensation and rigidity of lipid monomers as well as disrupting interactions between membrane-forming molecules [ 37 ] , Additionally, they can influence the expression of histone proteins and the methylation of genes, ultimately affecting the survival and reproduction of cells [ 38 ] . BPA may change the structure of the microbial community by suppressing the growth of some BPA-sensitive species while enhancing the growth of others, resulting in variation in community diversity. Zhao et al [ 39 ] found increased community diversity and decreased biofilm biomass with increased BPA concentration; they believed that increased competition caused by increased diversity was the reason for the decreased biofilm biomass [ 39 ] . This may be the main reason for the decreased biofilm biomass seen at low initial BPA concentration, because higher community diversity was also found in this study when compared with the control (Table S2). Another study showed that the adsorption capacity of microplastics coated with biofilms was significantly enhanced, resulting in higher concentrations of organic pollutants within these particles, which could potentially be the underlying cause behind this phenomenon [ 40 ] , though further experiments are needed to illustrate the mechanisms involved. High-throughput sequencing has been widely used to study microbiomes. Here, Proteobacteria and Bacteroidetes were the main phyla with high relative abundance in the biofilms of the groups with BPA treatment, followed by Actinobacteria and Firmicutes . Proteobacteria may be related to the degradation of BPA and its analogs, according to early reports that found that most bisphenol-degrading bacteria (including Pseudomonas [ 41 ] , Methylobacillus [ 42 ] and Hydrogenophaga [ 43 ] ) belonged to this phylum. Its increased abundance may result from the increased BPA concentration in the surrounding microenvironment [ 44 ] . Bacteroidetes , Actinobacteria , and Firmicutes have also been frequently isolated from surface water polluted with organic contaminants [ 45 – 48 ] , and have also been verified to have the potential for BPA degradation [ 49 – 51 ] . Methylophilus and Methylobacillus were the main genera in the biofilms on PS microplastics with BPA treatment. This finding was similar to the results from studies isolating BPA-degrading bacteria in BPA-polluted sediments [ 49 ] . Methylophilus can degrade organic chemicals with methyl groups [ 52 , 53 ] , which may indicate its involvement in BPA degradation (which has two methyl groups). Methylobacillus has also been shown to be involved in BPA degradation [ 35 , 54 , 55 ] . Bdellovibrio had significantly greater relative abundance in groups with high initial BPA concentration (5.0 mg L − 1 ); it can degrade tetracycline [ 56 ] , which may indicate its potential involvement in BPA degradation because of its similar benzene ring structure. Bdellovibrio may also be related to the formation of biofilms [ 57 ] ; microplastics are easily colonized by Bdellovibrio , which has a special predatory lifestyle. It is smaller than other microbes and it can parasitize other bacteria, acting as a predator, leading to the ultimate lysis and death of the host [ 58 ] . In our study, the relative abundance of Bdellovibrio increased significantly in the group treated with BPA, which may have led to the death of other bacteria and a decrease in the amount of biofilm. Hydrogenophaga has been shown to have a high organic matter utilization efficiency [ 59 ] ; this genus exists widely in polluted aquatic environments and can use many compounds for energy [ 60 , 61 ] . Because of its excellent adaptability, Hydrogenophaga can be used for the treatment of contaminated wastewater [ 62 ] . Relevant studies have shown that with sufficient nutrients, Hydrogenophaga and methylotrophs can coexist in aquatic environments [ 63 ] . In our study, the addition of BPA led to an increase in methylotrophs, which may have caused the rise in the relative abundance of Hydrogenophaga . In previous studies, Nevskia has been found to easily colonize the surface of microplastics and form stable microplastic biofilms with other bacteria [ 64 ] . This genus possesses strong organic pollutant degradation capabilities; when the concentration of organic pollutants in the environment increases, Nevskia significantly increases in relative abundance, which promotes their degradation [ 65 , 66 ] . In our study, the increase in the relative abundance of Bdellovibrio , Hydrogenophaga , and Nevskia in the group treated with BPA was also seen. Microbial community structure can change greatly with time [ 67 ] and environmental conditions [ 45 ] , as demonstrated by the results of Adonis analysis in this study, which showed significance in microbial community structure differences between samples from different initial BPA concentrations at different harvesting times. Amino acid metabolism and Metabolism of cofactors and vitamins were the most abundant metabolic pathways. This was similar to other reports analyzing the metabolic functions of biofilms on microplastics from the North Sea or aquaculture [ 68 , 69 ] . Although BPA is a type of organic pollutant, it can also serve as an additional carbon source, affecting microbial metabolic functions. According to early research, an increased abundance of Amino acid metabolism pathways were found with BPA addition [ 70 ] . Additionally, microbial tryptophan metabolism was found to be greatly enhanced when BPA was added to microplastic-polluted systems [ 71 ] . Vitamins can be used as cofactors of enzymes to participate in carboxylation, fatty acid metabolism, and amino acid catabolism; because of this, the Metabolism of cofactors and vitamins pathway may play an important role in maintaining metabolic homeostasis [ 68 ] . According to many reports, BPA can also induce oxidative stress in bacteria, resulting in membrane lipid peroxidation [ 36 – 38 ] . This correlates with the decreased abundance of the Membrane transport metabolic pathway found in this study. Based on the results from PICRUSt2 analysis, there was a significant correlation between microbial community structure and metabolic pathways, indicating that it was highly likely that the metabolic pathway group would change with the change of microbial community structure. Changes in the microbial community structure are one of the main reasons for the variations seen in the metabolic pathways between compared biofilms. 4.2 Contribution of BPA-acclimated microplastic biofilms to biodegradation of BPA The formation of biofilms constantly occurs in natural environments. Biofilms are believed to have a great influence on the metabolic functions in many ecosystems, including the degradation efficiency of organic pollutants [ 11 , 72 , 73 ] . Normally, more active metabolism is found for microorganisms in biofilms compared with those that are planktonic, indicating a greater pollutant degradation ability [ 74 ] . Additionally, the thickness of biofilms, the amounts of microorganisms, and the degradation of surrounding organic pollutants are significantly correlated. The disruption of the biofilm structure can destroy the cooperation among microorganisms and interfere with degradation efficiency [ 12 ] . Because of this, increased BPA degradation efficiency may occur when biofilms develop on microplastics, and efficiency may also vary with changes in biofilm biomass or community structure. Because of their unique properties, microplastics can serve as an excellent medium for microbial attachment and biofilm formation. A study on the effect of microplastic biofilms on nutrient cycling in simulated freshwater systems found that microplastic biofilms can promote the oxidation of ammonia nitrogen and nitrite as well as denitrification; the microplastic biofilms temporarily accumulate phosphorus and increase alkaline phosphatase activity in the system [ 16 ] . After culturing different biofilms attached to the surface of microplastics placed in tap water or contaminated wastewater, researchers found that the relative abundance of triclosan-degrading bacteria significantly increased in the biofilm community exposed to triclosan, enhancing degradation efficiency [ 18 ] . This indicates that environmental factors have a significant effect on microplastic biofilms, and that shaped biofilms influence the degradation of related substances. A recent study provided convincing evidence that the colonization of biofilms attached to the surface of microplastics in aquatic environments can affect the degradation efficiency of organic pollutants; using polystyrene, polyamide, and polyphenylene as materials, they found that in the presence of biofilms, the content of detected degradation products of debrominated diphenyl ethers significantly increased [ 17 ] . In an experiment exploring the interaction between microplastic biofilms and emerging contaminants, researchers found that the concentration of pollutants on the surface of microplastics with biofilms was generally 3.8 times lower than that of microplastics without biofilms. This may be because biofilms can promote the decomposition of pollutants adsorbed by microplastics [ 15 ] . In our study, the number of BPA-degrading bacteria and the abundance of related species in the microplastic biofilms in the group treated with BPA significantly increased, consistent with previous studies. The adsorption of BPA by microplastics is limited, and the presence of biofilms or BPA-acclimated biofilms has minimal effects [ 14 , 15 , 75 ] . BPA-acclimated biofilms initially boost the degradation of BPA by enhancing BPA-degrading bacteria such as Bdellovibrio , Hydrogenophaga , and Nevskia . However, the predominant core biofilm bacteria in all samples belonged to Methylophilaceae , potentially masking the theoretically expected acceleration of degradation. The results of BPA degradation after 7 days by BPA-degrading microplastic biofilms suggest that the presence of pre-acclimated microplastic biofilms can accelerate the BPA degradation rate in the environment in the short term (3 days); however, the metabolic activity of microorganisms was highly influenced by the environment, and this temporary acceleration may not be present in all aquatic environments. The initial acceleration was not seen after 7 days, possibly because of the higher concentration of BPA (5 mg L − 1 ) in the environment enhancing the BPA degradation capacity of the biofilms. The biodegradation activity of microplastic biofilms mainly depends on their biomass [ 12 , 74 ] . With prolonged degradation time, a higher concentration of microplastics led to the formation of more biofilm. By the 7th day of degradation, the BPA removal rate in the high-concentration microplastic treatment group approached 100%, while there was no significant difference between the rates in the low-concentration microplastic treatment group and the CK group. 5. Conclusions In aquatic environments, the presence of both BPA and microplastics can alter the community structure and metabolic functions of microplastic biofilms. Following BPA acclimation, there was a significant increase in the abundance of BPA-degrading bacteria in microplastic biofilms, but this did not lead to a significant improvement in BPA degradation efficiency. Within the short term (3rd day), biofilms significantly enhanced the degradation efficiency of BPA. However, as time progressed, microplastics served as favorable substrates for biofilm formation in all treatments, masking any biofilm-related acceleration of degradation. Higher concentrations of microplastics resulted in the formation of more biofilms, elevating the overall metabolic activity of the microplastic community in the aquatic environment and greatly enhancing BPA degradation efficiency. Declarations Competing interests: The authors declare that they have no competing interests. Funding: This work was financially supported by the Natural Science Foundation of China (32171624) Author’s contributions: Conceptualisation, X.G, JH.J, RX.Z; Methodology, X.G, JH.J; Investigation, X.G, JH.J; Data Curation, X.G, JH.J; Review and Editing, X.G, JH.J, RX.Z; Supervision, X.G, JH.J, RX.Z; Funding acquisition, JH.J. Acknowledgments: We thank Lisa Oberding, MSc, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript. 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Colonization, biofilm formation and biodegradation of polyethylene by a strain of Rhodococcus ruber [J]. Applied microbiology and biotechnology, 2004, 65(1): 97-104. JI H, WAN S, LIU Z, et al. Adsorption of antibiotics on microplastics (MPs) in aqueous environments: The impacts of aging and biofilms [J]. Journal of Environmental Chemical Engineering, 2024, 12(2): 111992. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4349153","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":298034559,"identity":"a19b7cb2-d949-4cf0-a495-7d41deb4f865","order_by":0,"name":"Xiang Gao","email":"","orcid":"","institution":"Central China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Gao","suffix":""},{"id":298034561,"identity":"dc81b39c-a1cf-4898-8b1e-b30b47dfcac5","order_by":1,"name":"Renxin Zhao","email":"","orcid":"","institution":"Inner Mongolia University","correspondingAuthor":false,"prefix":"","firstName":"Renxin","middleName":"","lastName":"Zhao","suffix":""},{"id":298034563,"identity":"6ca0e31b-53b7-48cc-b53b-e8572178e7fc","order_by":2,"name":"Jinhui Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBACxmYwxZbADxNoIFqLZAOxWmAgweAAsVqY25mfPfzCwJdnfPzws8c8DDayGw4wP3uA32Fs5sYyDGzFZmfSzI15GNKMNxxgMzfAr4XBTFqCgS1x2w0eNmkehsOJGw7wsEng18L+Daxl8wywlv/EaOExk/wA1LJBAqzlAFFayqSBgZw440yameQcg2TjmYfZzPBqMew/vk3yB8OxxP72w88k3lTYyfYdb36GX0sDMKB5/x2DckFBxYxPPRDIgxz3g6GGgLJRMApGwSgY0QAApCI/We2v2MUAAAAASUVORK5CYII=","orcid":"","institution":"Central China Normal University","correspondingAuthor":true,"prefix":"","firstName":"Jinhui","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-04-30 12:42:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4349153/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4349153/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56079339,"identity":"ec07ab9d-cc09-4d8f-a3e3-5e0fae65401f","added_by":"auto","created_at":"2024-05-08 09:21:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":36263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiomass variation of biofilms on PS microplastics with different BPA initial concentrations\u003c/strong\u003e. Mean ± SE, n = 3, BPA-CK, BPA-L, and BPA-H indicate PS microplastics in groups of control group (CK) and BPA initial concentrations of 0.10 and 5.00 mg L\u003csup\u003e-1\u003c/sup\u003e, respectively. Different uppercase and lowercase letters above the columns indicate significant differences between treatment groups at the same experimental time and between experimental times for the same treatment group, respectively, with significance set at 0.05.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4349153/v1/1b9c40a3e4c9e08d22190253.jpg"},{"id":56079340,"identity":"544d4518-ba92-42de-886d-3e19dfa54f0f","added_by":"auto","created_at":"2024-05-08 09:21:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePCoA analysis of the differences in bacterial communities in biofilms on PS microplastics among all treatments.\u003c/strong\u003e BPA-CK, BPA-L, and BPA-H indicate PS microplastics in groups of CK and BPA initial concentrations of 0.10 and 5.00 mg L\u003csup\u003e-1\u003c/sup\u003e, respectively. The numbers 7 and 14 indicate the time of incubation.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4349153/v1/93c2de1977e22ec1d8eb6411.jpg"},{"id":56079342,"identity":"414d6cfc-f9fd-42f3-9f97-135f69e14317","added_by":"auto","created_at":"2024-05-08 09:21:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative abundance of bacteria in the community of biofilms on PS microplastics at the phylum level based on high-throughput sequencing.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4349153/v1/75eebb8d234b8943a8574e22.jpg"},{"id":56079341,"identity":"49d26b29-ec17-4355-afdb-b0d28f902fe7","added_by":"auto","created_at":"2024-05-08 09:21:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative abundance of bacteria in the community of biofilms on PS microplastics at the genus level based on high-throughput sequencing.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4349153/v1/c6a5e667573915bc940652a7.jpg"},{"id":56079838,"identity":"ec8b922e-19c5-4313-aec2-0e5fed8fc012","added_by":"auto","created_at":"2024-05-08 09:29:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":121068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePredicted KEGG pathways metabolic at Levels 1 and 2 in the bacterial community in biofilms on PS microplastics under different environmental conditions\u003c/strong\u003e. Data underwent a logarithmic transformation.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4349153/v1/7c1ac973f6c41e57652d3243.jpg"},{"id":56079837,"identity":"c6c8bdff-99b1-4ea2-80d7-0967a53cef8b","added_by":"auto","created_at":"2024-05-08 09:29:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":43036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDensity of BPA-degrading bacteria in biofilms pre-incubated in lake water.\u003c/strong\u003e Mean ± S.E., n = 3, * and ** above the column indicate significance between pre-incubated biofilm with and without BPA at the 0.05 and 0.01 levels, respectively; an independent sample t-test was performed.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4349153/v1/b37e1c8eb2af687e33cb9ebd.jpg"},{"id":56079345,"identity":"3cc832cc-92d5-4318-8d29-d686c0190c80","added_by":"auto","created_at":"2024-05-08 09:21:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":64239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBPA biodegradation dynamics in lake water\u003c/strong\u003e. Water from Nanhu Lake (a), Donghu Lake (b), and Yezhi Lake (c); mean ± S.E., n = 3.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4349153/v1/7919b1b796bc90cbbb205457.jpg"},{"id":60012663,"identity":"be725401-0094-4606-9a34-ddbfef5c9285","added_by":"auto","created_at":"2024-07-10 13:45:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1452522,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4349153/v1/56e0d36c-e86b-4ea7-b160-10d43dfba453.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Limited bisphenol A (BPA) degradation acceleration by pre-acclimating microplastic biofilms with BPA in natural lake water","fulltext":[{"header":"1. Background","content":"\u003cp\u003eThin films, textile fibers, and plastic particles\u0026thinsp;\u0026lt;\u0026thinsp;5 mm are defined as microplastics\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. With the use of plastic products increasing each year and a lack of effective disposal methods, the high concentration of microplastics in the environment continues to increase. 320\u0026nbsp;million tons of plastic have been discarded, and 10% of these will persist in the environment\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. As a major producer and manufacturer, China accounts for 23.9% of the world's total plastic production\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Microplastic pollution is widely distributed, with aquatic environments being primary areas of contamination. Lakes, as a relatively enclosed aquatic environment, can serve as an important pathway for the temporary storage and transport of microplastics. The content and pollution levels of microplastics in lakes are already much higher than those in the ocean\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In 2014, Chinese researchers first reported that the concentration of microplastics in the surface water of the Yangtze River Estuary reached 4137.3 n/m\u003csup\u003e3\u003c/sup\u003e and has been increasing annually\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Microplastics result from diverse sources, such as medical consumables, personal care products, and synthetic fiber products\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. One of the main sources of microplastic pollution in aquatic environments is industrial waste, with a daily discharge of up to 2\u0026nbsp;million particles, corresponding to an average annual efflux of 50\u0026nbsp;million m\u003csup\u003e3\u003c/sup\u003e/years\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The concentration of microplastics in the environment is closely related to human activities\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, and is an environmental issue that needs to be taken seriously.\u003c/p\u003e \u003cp\u003eBecause they are difficult to degrade, are light in weight, and have a large specific surface area, microplastics persist in aquatic environments for a long time. Their surfaces often have microorganisms attached that form unique biofilms\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, which are sensitive to changes in environmental factors such as nutrient levels as well as ion and organic matter concentrations\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. There are significant differences between the microbial community composition of microplastic biofilms and those found in the surrounding environment. The formation of biofilms enhances the metabolic capacity of microbial communities\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Microplastics have a strong adsorption and enrichment effect on organic pollutants, making their surfaces a hotspot for these compounds\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Relevant experiments have shown that the concentration of organic pollutants on the surface of microplastics was 106 times or greater than that in the surrounding environment\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The enrichment of organic pollutants by microplastics can lead to changes in the community structure of microplastic biofilms, further altering their composition and metabolism\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The influence of environmental factors, including the coexistence of organic pollutants, also acts as a feedback mechanism by increasing adsorption and altering degradation, affecting the environmental fate of organic pollutants\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. A study found that the presence of microplastic biofilms can significantly enhance the degradation efficiency of debrominated diphenyl ethers\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The pollutant concentration on microplastic surfaces with biofilms is typically up to 3.8 times lower than on microplastics without biofilms, possibly because of biofilm enhancement of the degradation of pollutants adsorbed onto their surfaces\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In co-polluted environments, the interaction between microplastics and organic pollutants is significant, with microplastic biofilms playing an important role.\u003c/p\u003e \u003cp\u003eBisphenol A (BPA), a representative phenolic compound, is a pervasive endocrine disruptor found in various environmental matrices such as water, sediments, air, and biota\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The global demand for BPA is continuously increasing, with estimated consumption reaching 10.6\u0026nbsp;million tons by 2022\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Widely used as a plasticizer crucial for various daily-life applications\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, BPA is ubiquitously present alongside microplastics in the environment. However, research on microplastics and organic pollutants has primarily focused on persistent organic pollutants\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, with insufficient attention paid to \"pseudo-persistent organic pollutants\" such as BPA. Unlike recalcitrant persistent organic pollutants, BPA-degrading bacteria are widely distributed in the environment\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. BPA may further increase the relative abundance of bacterial species with degradative capacity in the community when it coexists with other pollutants, rather than primarily exhibiting toxic effects on bacterial communities such as those caused by persistent organic pollutants; however, this has yet to be studied. In addition, there is a lack of research regarding whether BPA acclimation of microplastic biofilms can enhance the subsequent degradation rate of BPA in aquatic environments.\u003c/p\u003e \u003cp\u003eThis research focused on the effect of BPA on the microbial community composition and metabolic pathways of microplastic biofilms in a co-contamination scenario. By acclimating microplastic biofilms to BPA co-contamination, its effect on the subsequent degradation of BPA in aquatic environments was examined. This study can lay a foundation for a deeper understanding of the environmental risks associated with the coexistence of bisphenol pollutants and microplastics.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental setup\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Effect of BPA on the development of microplastic biofilms\u003c/h2\u003e \u003cp\u003eSpherical polystyrene (PS) microplastics with a diameter of 1160\u0026thinsp;\u0026plusmn;\u0026thinsp;559.962 \u0026micro;m were purchased from China Petroleum and Chemical Corporation (Beijing, China). To evaluate the effects of BPA on the formation of microplastic biofilms, lake water was collected from Nanhu Lake (an urban lake in Wuhan, China) and filtered through a 700 \u0026micro;m mesh. BPA purchased from Sigma-Aldrich (Darmstadt, Germany, purity\u0026thinsp;\u0026ge;\u0026thinsp;99%) was added to the filtered lake water at final concentrations of 0, 0.10, and 5.00 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and all the groups were amended with 0.2 g of PS microplastics. Culture of microplastic biofilms was carried out in 250 mL flasks shaking at 120 rpm at 28\u0026deg;C. Three replicates were set up for each condition, and the biomass and bacterial community of biofilms were analyzed on the 7th and 14th day of the experiment.\u003c/p\u003e \u003cp\u003eThe biomass of microplastic biofilms was determined by the colorimetric method according to Lobelle\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. The bacterial community of microplastic biofilms was analyzed by 16S rRNA high-throughput gene sequencing. Microbial DNA from microplastic biofilms was extracted using the MP Fast DNA\u0026trade; SPIN Kit (CA, USA) according to the manufacturer\u0026rsquo;s instructions. The quality of DNA was verified by 1% agarose gel electrophoresis. The concentration and purity of DNA were determined using a Nanodrop One spectrophotometer (Thermo Fisher Scientific, USA). All isolated DNA samples were stored at -20\u0026deg;C and sent for sequencing on the Illumina MiSeq platform by Novogene Co., Ltd. (Tianjin, China). Primers 341 F (5\u0026prime;-CCTACGGGNGGCWGCAG-3\u0026prime;) and 806 R (5\u0026prime;-GGACTACVSGGGTATCTAAT-3\u0026prime;) were used to amplify the V3\u0026ndash;V4 region with a 2 \u0026times; 250 bp paired-end strategy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Effect of BPA removal of BPA-pre-acclimation of microplastic biofilms in lake water\u003c/h2\u003e \u003cp\u003eTo explore the influence of biofilms with and without BPA pre-acclimation on the degradation of ambient BPA from lake water, lake water was collected from three urban lakes (Nanhu Lake, Donghu Lake, and Yezhi Lake; Wuhan, China) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). PS microplastics were preincubated to form biofilms in lake water with or without 0.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BPA for 7 days. These microplastics were collected with meshes (425 \u0026micro;m in diameter) and rinsed with sterilized deionized water three times before use in the BPA degradation experiments. Two microplastic densities (50 and 1600 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with biofilms were treated with or without BPA acclimation, and microplastics without preincubated biofilms were used as controls. Groups without microplastics were used to estimate the removal by indigenous microorganisms and photodegradation.\u003c/p\u003e \u003cp\u003eThe BPA degradation experiment was performed in 250 mL flasks containing 120 mL lake water spiked with 5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BPA, cultured at 28\u0026deg;C and 120 rpm. During the experiment, 1 mL of lake water was sampled and filtered through 0.22 \u0026micro;m membranes at 0, 10, and 30 min, 1, 3, 6, and 12 h, and 1, 3, and 7 d for determination of residual BPA. The BPA concentration was measured by a reverse-phase HPLC (LC40, Shimadzu, Kyoto, Japan) equipped with a UV detector at a wavelength of 278 nm using a C18 column (250 mm \u0026times; 4.6 mm \u0026times; 5 \u0026micro;m, Agilent, Santa Clara, CA, USA); the mobile phase was 65% acetonitrile and 35% ultrapure water (v/v) at a flow rate of 1 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the injection volume was 20 \u0026micro;L. The acetonitrile was HPLC grade and purchased from TEDIA (OH, USA).\u003c/p\u003e \u003cp\u003eWe used the following formula to calculate the degradation rate of BPA by microplastic biofilms (Eq.\u0026nbsp;1):\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBPA\u003csub\u003ebdr\u0026minus;m\u003c/sub\u003e = BPA removal \u0026ndash; BPA\u003csub\u003ead\u0026minus;m\u003c/sub\u003e \u0026ndash; BPA\u003csub\u003ephoto,\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ewhere BPA removal (%) represents the overall rate of BPA removal, BPA\u003csub\u003ead\u0026minus;m\u003c/sub\u003e (%) represents the rate of BPA adsorption by microplastics, and BPA\u003csub\u003ephoto\u003c/sub\u003e (%) represents the rate of BPA removal by indigenous microorganisms or photodegradation.\u003c/p\u003e \u003cp\u003eWe estimated the adsorption of BPA by microplastics by assessing the removal dynamics of BPA (Fig. S2). At the initial stages of BPA removal, the contribution of degradation is minimal and can be neglected. The equilibrium point of removal was reached at 12 hours, and the removed amount at this time point was considered the adsorption of BPA by microplastics. We first calculated the average residual concentration at each time point in the CK group; next, we subtracted the average residual concentration of the CK group at each time point from the initial concentration of each treatment group and then subtracted the corresponding treatment group's residual concentration to obtain the adsorption of BPA by microplastics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3 Bacterial counting relating to BPA degradation in microplastic biofilms\u003c/h2\u003e \u003cp\u003ePre-incubated biofilm microplastics in low-density treatments (50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were harvested on the 7th day with meshes (425 \u0026micro;m in diameter), rinsed with sterilized deionized water three times, and dried at room temperature for 30 minutes to determine BPA-degrading bacteria counts in biofilms; samples at the initial time of ambient BPA degradation set-up were also assessed.\u003c/p\u003e \u003cp\u003eMicroorganisms in biofilms were collected from microplastics in sterilized Milli-Q water by ultrasonic processing (60 W, 40 KHz) for 10 minutes and vortexing for 3 minutes. BPA-degrading bacteria were then counted before and after the experiment by spread plating on inorganic agar medium\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e with 10 mg L\u003csup\u003e-1\u003c/sup\u003e of BPA as the sole carbon source.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Microbial community analysis\u003c/h2\u003e \u003cp\u003eThe raw 16S rRNA sequences were subjected to quality filtering using Mothur software (v 1.41.1)\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. To facilitate the comparison of bacterial community structures across samples at a consistent sequencing depth, each dataset was normalized. Operational taxonomic units (OTUs) were clustered at a 97% similarity level using the UPARSE pipeline in USEARCH (v 11.0.667)\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Chimeras were removed and singleton unique sequences were discarded before further analysis. Representative sequences from each OTU were taxonomically classified against the Ribosomal Database Project (RDP) database using the RDP classifier (v2.2) with a confidence threshold of 80%\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Microbial functional prediction using PICRUSt2\u003c/h2\u003e \u003cp\u003eThe functional composition of microbial communities was predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) based on 16S rRNA marker gene profiles\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Functional annotations for PICRUSt2 predictions were derived from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. KEGG Orthologs (KOs) table was generated for each dataset, and the relative abundance of predicted KOs was calculated. The differences in functional gene family composition across samples were visualized using principal coordinate analysis (PCoA) based on Bray-Curtis distance metrics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData including variations in biofilm biomassand BPA degradation were compared by one-way ANOVA, and variations in bacterial density were compared by independent t-test. Data including the BPA removal rate, BPA\u003csub\u003ead\u0026minus;m,\u003c/sub\u003e and BPA\u003csub\u003ebdr\u0026minus;m\u003c/sub\u003e were compared by two-way ANOVA. When a significant interaction was found, one-way ANOVA with the Duncan test for multiple comparisons was performed. The significance level was set at 0.05. All analyses were performed in SPSS (version 18.0.0). Figures were drawn in OriginPro (version 8.5.1 SR2). The α-diversity indices, including Shannon, Simpson\u0026rsquo;s, and Chao-1 were calculated in Paleontological Statistics software (v 4.02). PCoA, heat maps, and Adonis analysis were performed using vegan, pheatmap, and ggplot2 packages in R.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Biofilm biomass variation on PS microplastics in lake water influenced by BPA\u003c/h2\u003e \u003cp\u003eThe biomass of biofilms on PS microplastics generally increased with time in all experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003e); significant differences were seen between that on the 7th day and the 14th day under different initial BPA concentrations. BPA in lake water had a significantly negative effect on the biomass of biofilms on PS microplastics (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), resulting in the lowest biomass for groups under higher BPA initial concentrations (5.00 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on both the 7th and 14th days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Diversity comparison of the bacterial community on PS microplastics affected by BPA in lake water\u003c/h2\u003e \u003cp\u003eAccording to sequencing results, 7817 OTUs were obtained, 4385 of which (56.10%) were truncations clustered at the 97% similarity level. The total number of shared OTUs among all samples was 53. Alpha diversity indices including Chao-1 and Shannon were calculated and summarized in Supplementary Table S2, representing bacterial community richness and diversity, respectively. The Goods coverage was 99.7%, ensuring adequate sequencing depth.\u003c/p\u003e \u003cp\u003eThe Chao-1 index ranged from 516.77\u0026thinsp;\u0026plusmn;\u0026thinsp;24.65 to 764.53\u0026thinsp;\u0026plusmn;\u0026thinsp;49.41 for the groups with different initial BPA concentrations (Table S2); it decreased with initial BPA concentration on the 7th day but increased with initial BPA concentration by the 14th day. Higher initial BPA concentration had higher Chao-1 indices, significantly different compared with that of the CK without BPA on the 14th day (764.53\u0026thinsp;\u0026plusmn;\u0026thinsp;49.41 vs 512.73\u0026thinsp;\u0026plusmn;\u0026thinsp;32.29, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The Shannon index significantly increased with time for groups with higher initial BPA concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). BPA also varied the Shannon index from 2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 to 3.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 (Table S2), showing a similar trend to that of the Chao-1 index among treatments.\u003c/p\u003e \u003cp\u003ePCoA analysis based on Bray-Curtis distance revealed the structural differences of bacterial communities in biofilms on PS microplastics between treatments. The two axes explained 48.79% of the total difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e). According to the results of Adonis analysis, initial BPA concentration significantly affected the structure of bacterial communities on microplastics, especially on the 14th day (Adonis: R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.241, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For groups of each initial BPA concentration, the structure of bacterial communities was significantly different between experimental time points (Adonis: R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.143, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Microbial community composition of PS microplastics affected by BPA in lake water\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. Microbial community composition at the phylum level\u003c/h2\u003e \u003cp\u003eIn total, 24 bacterial phyla were identified. Different relative abundances of bacteria in biofilm communities on PS microplastics were found under different experimental treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eFirmicutes\u003c/em\u003e were identified as the major bacteria phyla on microplastics under different initial BPA concentrations, accounting for 90.96%, 6.82%, 0.67%, and 0.59%, respectively, on the 7th day, and 83.01%, 13.10%, 1.48%, and 0.42%, respectively, on the 14th day. Their order of abundance was unchanged over time, although values varied. The relative abundance of \u003cem\u003eProteobacteria\u003c/em\u003e was significantly lower on the 14th day than on the 7th day in groups with 0.10 and 5.00 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BPA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eBacteroidetes\u003c/em\u003e relative abundance was greatest at lower initial BPA concentrations and lowest at higher BPA initial concentrations on both the 7th and 14th days, showing a significant difference on the 7th day (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The relative abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e was significantly greater on the 7th day than on the 14th day (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Microbial community composition at the genus level\u003c/h2\u003e \u003cp\u003eIn total, 357 bacterial genera were identified. Those with relative abundances\u0026thinsp;\u0026gt;\u0026thinsp;0.5% were chosen for further analysis. Different relative abundances of bacteria in biofilms on PS microplastics were found under different experimental treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The number of shared genera across all samples was 33, with \u003cem\u003eMethylophilus\u003c/em\u003e, \u003cem\u003eMethylobacillus\u003c/em\u003e, and \u003cem\u003eMethylovorus\u003c/em\u003e being predominant (all belonging to \u003cem\u003eMethylophilaceae\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eWith different initial BPA concentrations, \u003cem\u003eMethylophilus\u003c/em\u003e, \u003cem\u003eMethylobacillus\u003c/em\u003e, and \u003cem\u003eMethylovorus\u003c/em\u003e were the major genera, accounting for 55.93%, 2.59%, and 2.92%, respectively, on the 7th day; on the 14th day, \u003cem\u003eMethylophilus\u003c/em\u003e and \u003cem\u003eMethylobacillus\u003c/em\u003e were the major genera, accounting for 41.19% and 13.56%, respectively.\u003c/p\u003e \u003cp\u003eDifferent bacterial community structures appeared over time. On the 7th day, the relative abundance of \u003cem\u003eMethylophilus\u003c/em\u003e was the highest in all bacterial communities on PS under different initial BPA concentrations, and no significance was found between treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, \u003cem\u003eBdellovibrio\u003c/em\u003e had a significantly larger relative abundance under high initial BPA concentration compared with under low initial BPA or the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the 14th day, \u003cem\u003eMethylophilus\u003c/em\u003e abundance decreased while that of \u003cem\u003eMethylobacillus\u003c/em\u003e and \u003cem\u003eOhtaekwangia\u003c/em\u003e increased. The relative abundances of \u003cem\u003eHydrogenophaga\u003c/em\u003e and \u003cem\u003eNevskia\u003c/em\u003e were significantly higher with 0.10 and 5.00 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BPA than that in group of CK (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with groups on the 7th day, treatments with both low and high initial BPA had significantly greater relative abundance of \u003cem\u003eMethylobacillus\u003c/em\u003e on the 14th day (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Predicted metabolic functions of microbial communities\u003c/h2\u003e \u003cp\u003eTo illustrate the metabolic function of bacterial communities on PS microplastics under different environmental conditions, PICRUSt2 was used to predict the metagenomic functional composition. Forty-five metabolic pathways (Level 2) from six KEGG metabolic pathway groups (Level 1) were predicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The relative abundances of metabolic pathway groups in different samples were similar, with the KEGG pathway \u003cem\u003eMetabolism\u003c/em\u003e being the most abundant.\u003c/p\u003e \u003cp\u003eExamining different initial BPA concentrations, the relative abundance of \u003cem\u003eMetabolism\u003c/em\u003e varied from 66.71\u0026ndash;68.29% on the 7th day, and from 68.30\u0026ndash;71.17% on the 14th day; no significant differences were found (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). At Level 2, \u003cem\u003eMetabolism of cofactors and vitamins\u003c/em\u003e, \u003cem\u003eAmino acid metabolism\u003c/em\u003e, \u003cem\u003eCarbohydrate metabolism\u003c/em\u003e, \u003cem\u003eCell motility\u003c/em\u003e, \u003cem\u003eEnergy metabolism\u003c/em\u003e, \u003cem\u003eXenobiotics biodegradation and metabolism\u003c/em\u003e, and \u003cem\u003eReplication and repair\u003c/em\u003e were the major pathways predicted on the 7th day, accounting for 10.36%, 9.19%, 7.82%, 6.04%, 5.22%, 4.47%, and 4.43%, respectively. On the 14th day, this order remained the same, although their relative abundances changed slightly.\u003c/p\u003e \u003cp\u003ePCoA analysis based on predicted relative KO abundances (Level 3) showed that samples were mainly separated by the first axis, which explained 88.02% of the variability (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Adonis analysis showed significant differences between samples under different experimental treatments (Adonis: R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.052, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047). Initial BPA concentration showed no effect on the metabolic pathway structure of bacterial communities in biofilms on PS microplastics, as samples with different concentrations were not separated.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5. Density variation of bacteria relating to BPA degradation in preincubated biofilms at initial and harvest time in subsequent BPA degradation experiments\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter 7 days of preincubation with BPA (0.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the densities of degradation-related bacteria were significantly increased compared with the CK group in water from all three lakes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However, after harvesting samples from the degradation experiment, significance remained only in Yezhi Lake water (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The densities of BPA degradation-related bacteria showed a significant increase after 7 days in all treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6. BPA biodegradation dynamics in lake water\u003c/h2\u003e \u003cp\u003eOn the initial day, the degradation rate of BPA was not significantly different between treatments across the three lakes (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, on the third day, the degradation rate of the groups with high-concentration microplastics addition and biofilms preincubated in Yezhi Lake and Nanhu Lake were significantly increased compared with the other treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the presence of a biofilm on microplastics has a significant effect on the degradation of BPA. Upon completion of the degradation experiment, the BPA degradation rate of the high-concentration microplastic addition groups was significantly increased compared with the low-concentration microplastics addition group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The removal rate with high-concentration microplastic treatment was 100%, indicating near-complete BPA breakdown. In contrast, there was no significant difference in the degradation rate between the low-concentration microplastics addition group and CK.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Effect of BPA acclimation\u003c/h2\u003e \u003cp\u003eMicroplastics are believed to be effective adhesion substrates for the development of biofilms made up of algae, bacteria, fungi, and protozoa, because of their recalcitrant nature and extensive specific surface area; this forms an ecological niche known as the \u0026ldquo;plastisphere\u0026rdquo; \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Biofilm formation takes time, suggesting a positive influence of exposure time on biofilm biomass\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Lobelle et al.\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e reported that 7 days was enough for the formation of biofilms on plastic debris in seawater, though the biomass significantly increased in the following week. Li et al\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e also found increasing biomass of biofilms on five types of microplastics with experimental time. Similar results were found in this study, with a significant increase in biomass of biofilms on PS microplastics occurring over time with different BPA initial concentrations.\u003c/p\u003e \u003cp\u003eBPA had a negative effect on biofilm biomass, even when the initial concentration was low (0.10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); this effect was intensified when the initial concentration was high (5.00 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This was quite unusual, because many early reports found a positive effect of BPA on the growth of microorganisms at low initial concentrations, with negative effects at high initial concentrations\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. BPA can enhance the growth of specific species when used as an additional carbon source \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e; however, it can also suppress the development of organisms, mainly because of oxidative stress resulting from the overproduction and accumulation of reactive oxygen species\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Bisphenols can integrate into cell membranes, reducing the polycondensation and rigidity of lipid monomers as well as disrupting interactions between membrane-forming molecules\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, Additionally, they can influence the expression of histone proteins and the methylation of genes, ultimately affecting the survival and reproduction of cells\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. BPA may change the structure of the microbial community by suppressing the growth of some BPA-sensitive species while enhancing the growth of others, resulting in variation in community diversity.\u003c/p\u003e \u003cp\u003eZhao et al\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e found increased community diversity and decreased biofilm biomass with increased BPA concentration; they believed that increased competition caused by increased diversity was the reason for the decreased biofilm biomass \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. This may be the main reason for the decreased biofilm biomass seen at low initial BPA concentration, because higher community diversity was also found in this study when compared with the control (Table S2). Another study showed that the adsorption capacity of microplastics coated with biofilms was significantly enhanced, resulting in higher concentrations of organic pollutants within these particles, which could potentially be the underlying cause behind this phenomenon\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, though further experiments are needed to illustrate the mechanisms involved.\u003c/p\u003e \u003cp\u003eHigh-throughput sequencing has been widely used to study microbiomes. Here, \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e were the main phyla with high relative abundance in the biofilms of the groups with BPA treatment, followed by \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e. \u003cem\u003eProteobacteria\u003c/em\u003e may be related to the degradation of BPA and its analogs, according to early reports that found that most bisphenol-degrading bacteria (including \u003cem\u003ePseudomonas\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eMethylobacillus\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e and \u003cem\u003eHydrogenophaga\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e) belonged to this phylum. Its increased abundance may result from the increased BPA concentration in the surrounding microenvironment \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eFirmicutes\u003c/em\u003e have also been frequently isolated from surface water polluted with organic contaminants \u003csup\u003e[\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e, and have also been verified to have the potential for BPA degradation \u003csup\u003e[\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMethylophilus\u003c/em\u003e and \u003cem\u003eMethylobacillus\u003c/em\u003e were the main genera in the biofilms on PS microplastics with BPA treatment. This finding was similar to the results from studies isolating BPA-degrading bacteria in BPA-polluted sediments \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eMethylophilus\u003c/em\u003e can degrade organic chemicals with methyl groups \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e, which may indicate its involvement in BPA degradation (which has two methyl groups). \u003cem\u003eMethylobacillus\u003c/em\u003e has also been shown to be involved in BPA degradation \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBdellovibrio\u003c/em\u003e had significantly greater relative abundance in groups with high initial BPA concentration (5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); it can degrade tetracycline \u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e, which may indicate its potential involvement in BPA degradation because of its similar benzene ring structure. \u003cem\u003eBdellovibrio\u003c/em\u003e may also be related to the formation of biofilms \u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e; microplastics are easily colonized by \u003cem\u003eBdellovibrio\u003c/em\u003e, which has a special predatory lifestyle. It is smaller than other microbes and it can parasitize other bacteria, acting as a predator, leading to the ultimate lysis and death of the host \u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. In our study, the relative abundance of \u003cem\u003eBdellovibrio\u003c/em\u003e increased significantly in the group treated with BPA, which may have led to the death of other bacteria and a decrease in the amount of biofilm. \u003cem\u003eHydrogenophaga\u003c/em\u003e has been shown to have a high organic matter utilization efficiency \u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e; this genus exists widely in polluted aquatic environments and can use many compounds for energy\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e. Because of its excellent adaptability, \u003cem\u003eHydrogenophaga\u003c/em\u003e can be used for the treatment of contaminated wastewater\u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e. Relevant studies have shown that with sufficient nutrients, \u003cem\u003eHydrogenophaga\u003c/em\u003e and methylotrophs can coexist in aquatic environments \u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. In our study, the addition of BPA led to an increase in methylotrophs, which may have caused the rise in the relative abundance of \u003cem\u003eHydrogenophaga\u003c/em\u003e. In previous studies, \u003cem\u003eNevskia\u003c/em\u003e has been found to easily colonize the surface of microplastics and form stable microplastic biofilms with other bacteria\u003csup\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e. This genus possesses strong organic pollutant degradation capabilities; when the concentration of organic pollutants in the environment increases, \u003cem\u003eNevskia\u003c/em\u003e significantly increases in relative abundance, which promotes their degradation \u003csup\u003e[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e. In our study, the increase in the relative abundance of \u003cem\u003eBdellovibrio\u003c/em\u003e, \u003cem\u003eHydrogenophaga\u003c/em\u003e, and \u003cem\u003eNevskia\u003c/em\u003e in the group treated with BPA was also seen. Microbial community structure can change greatly with time \u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e and environmental conditions \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e, as demonstrated by the results of Adonis analysis in this study, which showed significance in microbial community structure differences between samples from different initial BPA concentrations at different harvesting times.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmino acid metabolism\u003c/em\u003e and \u003cem\u003eMetabolism of cofactors and vitamins\u003c/em\u003e were the most abundant metabolic pathways. This was similar to other reports analyzing the metabolic functions of biofilms on microplastics from the North Sea or aquaculture \u003csup\u003e[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/sup\u003e. Although BPA is a type of organic pollutant, it can also serve as an additional carbon source, affecting microbial metabolic functions. According to early research, an increased abundance of \u003cem\u003eAmino acid metabolism\u003c/em\u003e pathways were found with BPA addition \u003csup\u003e[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]\u003c/sup\u003e. Additionally, microbial tryptophan metabolism was found to be greatly enhanced when BPA was added to microplastic-polluted systems \u003csup\u003e[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]\u003c/sup\u003e. Vitamins can be used as cofactors of enzymes to participate in carboxylation, fatty acid metabolism, and amino acid catabolism; because of this, the \u003cem\u003eMetabolism of cofactors and vitamins\u003c/em\u003e pathway may play an important role in maintaining metabolic homeostasis \u003csup\u003e[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/sup\u003e. According to many reports, BPA can also induce oxidative stress in bacteria, resulting in membrane lipid peroxidation \u003csup\u003e[\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. This correlates with the decreased abundance of the \u003cem\u003eMembrane transport metabolic\u003c/em\u003e pathway found in this study. Based on the results from PICRUSt2 analysis, there was a significant correlation between microbial community structure and metabolic pathways, indicating that it was highly likely that the metabolic pathway group would change with the change of microbial community structure. Changes in the microbial community structure are one of the main reasons for the variations seen in the metabolic pathways between compared biofilms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Contribution of BPA-acclimated microplastic biofilms to biodegradation of BPA\u003c/h2\u003e \u003cp\u003eThe formation of biofilms constantly occurs in natural environments. Biofilms are believed to have a great influence on the metabolic functions in many ecosystems, including the degradation efficiency of organic pollutants\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]\u003c/sup\u003e. Normally, more active metabolism is found for microorganisms in biofilms compared with those that are planktonic, indicating a greater pollutant degradation ability \u003csup\u003e[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/sup\u003e. Additionally, the thickness of biofilms, the amounts of microorganisms, and the degradation of surrounding organic pollutants are significantly correlated. The disruption of the biofilm structure can destroy the cooperation among microorganisms and interfere with degradation efficiency\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Because of this, increased BPA degradation efficiency may occur when biofilms develop on microplastics, and efficiency may also vary with changes in biofilm biomass or community structure.\u003c/p\u003e \u003cp\u003eBecause of their unique properties, microplastics can serve as an excellent medium for microbial attachment and biofilm formation. A study on the effect of microplastic biofilms on nutrient cycling in simulated freshwater systems found that microplastic biofilms can promote the oxidation of ammonia nitrogen and nitrite as well as denitrification; the microplastic biofilms temporarily accumulate phosphorus and increase alkaline phosphatase activity in the system\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. After culturing different biofilms attached to the surface of microplastics placed in tap water or contaminated wastewater, researchers found that the relative abundance of triclosan-degrading bacteria significantly increased in the biofilm community exposed to triclosan, enhancing degradation efficiency\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. This indicates that environmental factors have a significant effect on microplastic biofilms, and that shaped biofilms influence the degradation of related substances. A recent study provided convincing evidence that the colonization of biofilms attached to the surface of microplastics in aquatic environments can affect the degradation efficiency of organic pollutants; using polystyrene, polyamide, and polyphenylene as materials, they found that in the presence of biofilms, the content of detected degradation products of debrominated diphenyl ethers significantly increased\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In an experiment exploring the interaction between microplastic biofilms and emerging contaminants, researchers found that the concentration of pollutants on the surface of microplastics with biofilms was generally 3.8 times lower than that of microplastics without biofilms. This may be because biofilms can promote the decomposition of pollutants adsorbed by microplastics\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, the number of BPA-degrading bacteria and the abundance of related species in the microplastic biofilms in the group treated with BPA significantly increased, consistent with previous studies. The adsorption of BPA by microplastics is limited, and the presence of biofilms or BPA-acclimated biofilms has minimal effects\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/sup\u003e. BPA-acclimated biofilms initially boost the degradation of BPA by enhancing BPA-degrading bacteria such as \u003cem\u003eBdellovibrio\u003c/em\u003e, \u003cem\u003eHydrogenophaga\u003c/em\u003e, and \u003cem\u003eNevskia\u003c/em\u003e. However, the predominant core biofilm bacteria in all samples belonged to \u003cem\u003eMethylophilaceae\u003c/em\u003e, potentially masking the theoretically expected acceleration of degradation. The results of BPA degradation after 7 days by BPA-degrading microplastic biofilms suggest that the presence of pre-acclimated microplastic biofilms can accelerate the BPA degradation rate in the environment in the short term (3 days); however, the metabolic activity of microorganisms was highly influenced by the environment, and this temporary acceleration may not be present in all aquatic environments. The initial acceleration was not seen after 7 days, possibly because of the higher concentration of BPA (5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the environment enhancing the BPA degradation capacity of the biofilms.\u003c/p\u003e \u003cp\u003eThe biodegradation activity of microplastic biofilms mainly depends on their biomass\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/sup\u003e. With prolonged degradation time, a higher concentration of microplastics led to the formation of more biofilm. By the 7th day of degradation, the BPA removal rate in the high-concentration microplastic treatment group approached 100%, while there was no significant difference between the rates in the low-concentration microplastic treatment group and the CK group.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn aquatic environments, the presence of both BPA and microplastics can alter the community structure and metabolic functions of microplastic biofilms. Following BPA acclimation, there was a significant increase in the abundance of BPA-degrading bacteria in microplastic biofilms, but this did not lead to a significant improvement in BPA degradation efficiency. Within the short term (3rd day), biofilms significantly enhanced the degradation efficiency of BPA. However, as time progressed, microplastics served as favorable substrates for biofilm formation in all treatments, masking any biofilm-related acceleration of degradation. Higher concentrations of microplastics resulted in the formation of more biofilms, elevating the overall metabolic activity of the microplastic community in the aquatic environment and greatly enhancing BPA degradation efficiency.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding: This work was financially supported by the Natural Science Foundation of China (32171624)\u003c/p\u003e\n\u003cp\u003eAuthor\u0026rsquo;s contributions: Conceptualisation, X.G, JH.J, RX.Z; Methodology, X.G, JH.J; Investigation, X.G, JH.J; Data Curation, X.G, JH.J; Review and Editing, X.G, JH.J, RX.Z; Supervision, X.G, JH.J, RX.Z; Funding acquisition, JH.J.\u003c/p\u003e\n\u003cp\u003eAcknowledgments: We thank Lisa Oberding, MSc, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript.\u003c/p\u003e"},{"header":"Abbreviations","content":"bisphenol A (BPA); polystyrene (PS); operational taxonomic units (OTUs); Ribosomal Database Project (RDP); Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2); Kyoto Encyclopedia of Genes and Genomes (KEGG); KEGG Orthologs (KOs); principal coordinate analysis (PCoA)"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBAKIR A, ROWLAND S J, THOMPSON R C. 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Journal of Environmental Chemical Engineering, 2024, 12(2): 111992.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bisphenol A, Spherical polystyrene microplastics, Co-pollution, Biofilms, Biodegradation","lastPublishedDoi":"10.21203/rs.3.rs-4349153/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4349153/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Bisphenol A (BPA) and microplastics are prevalent in aquatic environments. Microplastic biofilms play a crucial role in the environmental degradation of BPA, but related research is lacking. We designed experiments to investigate the effect of BPA on microplastic biofilms and the effect of pre-acclimating biofilms on BPA degradation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Even at low concentrations (0.1 mg L\u003csup\u003e-1\u003c/sup\u003e), BPA significantly reduced microplastic biofilm biomass (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). High-throughput 16S rRNA sequencing revealed that BPA altered biofilm diversity, as evidenced by changes in Chao-1 and Shannon indices. The primary phyla in the microplastic biofilm included \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eActinobacteria,\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e. On the 7\u003csup\u003eth\u003c/sup\u003e day of biofilm formation, the dominant bacterial genus shifted from \u003cem\u003eOhtaekwangia\u003c/em\u003e to \u003cem\u003eBdellovibrio\u003c/em\u003e in groups with BPA treatment, and the relative abundance of \u003cem\u003eBdellovibrio\u003c/em\u003e reached 4.32% ± 5.34%. On the 14\u003csup\u003eth\u003c/sup\u003e day, \u003cem\u003eMethylobacillus\u003c/em\u003e significantly increased in all treatments compared with the 7\u003csup\u003eth\u003c/sup\u003e day (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Adonis analysis demonstrated that the metabolic composition of the bacterial community also changed significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). BPA (0.1 mg L\u003csup\u003e-1\u003c/sup\u003e) pre-acclimation of microplastic biofilms led to a significant increase in the amount of BPA-degrading bacteria with no significant effect on BPA degradation efficiency. After 7 days, the BPA removal rate in high-concentration microplastic treatments (1600 mg L\u003csup\u003e-1\u003c/sup\u003e) reached \u0026gt; 90%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eBiofilms significantly increased the BPA degradation rate by 174.78% to 889.25% on the third day, indicating that the biofilm accelerates BPA degradation efficiency in the short term. Our findings provide a foundation for further understanding the environmental risks associated with the coexistence of bisphenols and microplastics.\u003c/p\u003e","manuscriptTitle":"Limited bisphenol A (BPA) degradation acceleration by pre-acclimating microplastic biofilms with BPA in natural lake water","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-08 09:21:17","doi":"10.21203/rs.3.rs-4349153/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48704901-a4be-4de9-add3-fedc77c3c3ab","owner":[],"postedDate":"May 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-10T13:37:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-08 09:21:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4349153","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4349153","identity":"rs-4349153","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}