Water Research 226 (2022) 119273 Contents lists available at ScienceDirect Water Research journal homepage: www.elsevier.com/locate/watres Effects of trace PFOA on microbial community and metabolisms: Microbial selectivity, regulations and risks Congli Chen, Yuanping Fang, Xiaochun Cui, Dandan Zhou * Engineering Lab for Water Pollution Control and Resources Recovery of Jilin Province, School of Environment, Northeast Normal University, Changchun 130117, China A R T I C L E I N F O A B S T R A C T Keywords: Perfluorooctanoic acid (PFOA) Porous media Microbial community Antibiotic resistant genes (ARGS) Human bacterial pathogens (HBPs) Biological risks Perfluorooctanoic acid (PFOA), a "forever chemical", is continuously discharged and mitigated in the environ­ ment despite its production and use being severely restricted globally. Due to the transformation, attachment, and adsorption of PFOA in aquatic environments, PFOA accumulates in the porous media of sediments, soils, and vadose regions. However, the impact of trace PFOA in the porous media on interstitial 间隙水water and water safety is 渗流区 not clear. In this work, we simulated a porous media layer using a sand column and explored the effects of µglevel PFOA migration on microbial community alternation, microbial function regulation, and the generation and spread of microbial risks. After 60 days of PFOA stimulation, Proteobacteria became the dominant phylum 变形菌门 with an abundance of 91.8%, since it carried 71% of the antibiotic resistance genes (ARGs). Meanwhile, the halogen-related Dechloromonas abundance increased from 0.4% to 10.6%. In addition, PFOA significantly 脱氯单胞菌 stimulated protein (more than 1288%) and polysaccharides (more than 4417%) production by up-regulating amino acid metabolism (p< 0.001) and membrane transport (p < 0.001) to accelerate the microbial aggrega­ tion. More importantly, the rapidly forming biofilm immobilized and blocked PFOA. The more active antioxidant 固定和阻塞 system repaired the damaged cell membrane by significantly up-regulating glycerophospholipid metabolism and 甘油磷脂代谢和肽聚糖生物合成 peptidoglycan biosynthesis. It is worth noting that PFOA increased the abundance of antibiotic resistance genes 抗生素抗性基因 (ARGs) and human bacterial pathogens (HBPs) in porous media by 30% and 106%. PFOA increased the pro­ portion of vertical transmission ARGs (vARGs), and co-occurrence network analysis (r ≥ 0.8, p ≤ 0.01) verified that vARGs were mainly mediated by HBPs. A comprehensive understanding of PFOA interactions with its microecological environment is provided. 1. Introduction Perfluorooctanoic acid (PFOA, C7F15COOH) is a member of Per- and polyfluoroalkyl substances (PFASs), consisting of an ionizable hydro­ philic carboxylic acid (-CO−2 ) and a hydrophobic perfluorocarbon chain (-C7F15) (Gagliano et al., 2020; Hu et al., 2020). The strong carbon-fluorine bonds (bond energy 485 kJ mol− 1) give PFOA useful chemical properties for making various products and make PFOA extremely resistant to breakdown, earning it the moniker “the forever chemical” (Lindstrom et al., 2011; Wang et al., 2017). PFOA-induced bioaccumulation and health hazards are of considerable concern, mak­ ing PFOA one of the most researched pollutants in the last decade (Chen et al., 2021b; Xiang et al., 2020; Zareitalabad et al., 2013). PFOA has adverse impacts on the human body, such as congenital disabilities, growth retardation, kidney, and liver toxicity, and weakened immunity (Li et al., 2018; Vieira et al., 2013). However, it is frequently detected in aquatic environments (ng-μg L− 1), tap water (ng L− 1), and even human and wild animals’ blood (Li et al., 2020b, 2019b; Xiao et al., 2015). Many countries and organizations have adopted strict measures to restrict the production, use, and discharging of PFOA. In 2019, PFOA was added to the Stockholm Convention’s list of persistent organic pollutants (POPs). The United States Environmental Protection Agency established a health advisory level of 70 ng L− 1 for PFOA and PFOS individually or combined in drinking water. However, PFOA resists most conventional chemical and microbial treatment technologies (Espan et al., 2015; Trojanowicz et al., 2018). Inevitable, trace amounts of PFOA are continuously discharged into the environment. The PFOA migration process in the environment is very complex, accompanied by the mutual transformation of water, atmosphere, and soil (Li et al., 2019a). Due to PFOA’s transformation, attachment, and adsorption in the aquatic environment, PFOA accumulates in the porous media of sediments, soils, and vadose region, presenting much higher * Corresponding author. E-mail address: zhoudandan415@163.com (D. Zhou). https://doi.org/10.1016/j.watres.2022.119273 Received 5 July 2022; Received in revised form 19 September 2022; Accepted 17 October 2022 Available online 18 October 2022 0043-1354/© 2022 Published by Elsevier Ltd. C. Chen et al. Water Research 226 (2022) 119273 However, PFOA effects on porous interstitial water media and water safety are unclear. Porous media is a significant PFOA sink (Hou et al., 2022; Lyu et al., 2020). Simultaneously, it is a biofilm in the habitat. Therefore, the PFOA effects on the porous media microorganisms also require examination. The unique hydrophilic and lipophilic properties of PFOA make it easy to combine with membrane phospholipids and thereby prevent the 膜磷脂 membrane process, resulting in cell membrane disruption (Liao et al., 2010; Liu et al., 2016). And PFOA could break the balance of the anti­ oxidant system and induce oxidative damage, even apoptosis (Liu et al., 2016). Moreover, PFOA could damage DNA duo to its toxicity (Liu et al., 2014). Correspondingly, under environmental stress, microorganisms regulate their metabolic activities, such as producing a large amount of extracellular polymeric substances (EPS), to resist the damage of toxic substances, which promote biofilm formation. In addition, microor­ ganisms could activate the synthesis of antioxidant system functional enzymes to improve their resistance and gradually achieve self-repair of damaged functions (Glorieux et al., 2015; Page et al., 2009). More importantly, environmental stress and defense responses could cause the production and spread of antibiotic resistance genes (ARGs), a severe threat to ecology and public health, mainly in human bacterial patho­ 人体条件致病细菌 gens (HBPs) (Jiang et al., 2017). ARGs spread is divided into horizontal Fig. 1. Schematic diagram of experimental setups. The concentrations of nu­ gene transfer (HGT) (Feng et al., 2021; Von Wintersdorff et al., 2016) trients and minerals in the synthetic water of the two protocols were identical, and vertical gene transfer (VGT) (Li et al., 2022). Studies showed that except for the PFOA (0, 2.4 μM) levels. Synthetic water for each column was fed the ARGs generation and transmission characteristics are closely related at 0.2 mL min− 1 with a hydraulic retention time of 6.7 h. to environmental selection pressure (Guo et al., 2020; Lu et al., 2020; Shekhawat et al., 2021). Therefore, PFOA may play an important role in concentrations. For instance, in Diep and Plankenburg Rivers, South generating and spreading biological risks during migration in porous Africa, the maximum concentration of PFOA in sediments was 191.9 ng − 1 − 1 media. However, the PFOA effects on microbial risks production and g dw, and the PFOA content reached 2.4 µg g (average value) in the transferring are unclear. soil of an Air Force base training area in the United States (Bofa et al., Little is known about the PFOA effects on the porous media micro­ 2021; Hou et al., 2022). Proteobacteria and Acidobacteria became the 在PFOA的刺激下,变形菌门和酸杆菌门成为土壤中的优势门 ecology. However, we hypothesized that PFOA could cause considerable dominant phyla in the soil under PFOA stimulation (Cai et al., 2020), microbial risks due to its bioaccumulative and biotoxic nature. This and other pathways, such as carbohydrate metabolism and membrane work employed a series of columns (~200 mL) filled with quartz sands transport, were significantly affected. PFOA could also induce signifi­ to simulate porous media layers in aquatic environments, PFOA con­ cant suppression of microbial diversity in soils (Senevirathna et al., taining migration. The microbial communities’ evolution, microbial 2022), The relative abundance of several key microorganisms associated metabolic function changes, cell defense and repair capability regula­ with acidification and methanation was significantly reduced, resulting tion, and microbial risks production during PFOA migration were in a 19.2% reduction in methane production (Wang et al., 2021a). Fig. 2. Relative abundance of the bacterial community in biofilm at phylum (a) and genus (b) levels, with (inner circle) and without (outer circle) PFOA. . 属 门 2 C. Chen et al. Water Research 226 (2022) 119273 Fig. 3. (a) Biomass amount and EPSs contents ((b) polysaccharide, (c) protein) in the porous media. Asterisks (*, **, or ***) indicate significant difference (p < 0.05, 0.01, or 0.001); XPS analysis of the biomass in (d) Control and (e) PFOA. Control and PFOA represent the protocols without and with 2.4 µM PFOA in the influent. Fig. 4. Fisher’s exact test on KEGG pathway level 2. The bar graph represents the certain KEGG function abundance in control and PFOA samples (left); The proportion of differences in species abundance within a set confidence interval (right). Asterisks (*, **, or ***) indicate significant difference (p < 0.05, 0.01, or 0.001). investigated using chemical analysis, metagenomics, and metatran­ scriptomics. We explored the relationship between ARGs and HBPs and their transmission mechanism based on co-occurrence network analysis. This study provides a comprehensive understanding on the effects of PFOA migration on microecology and the potential microbial risks. 2. Materials and methods 2.1. Experimental setup The schematic diagram of the experimental setup is shown in Fig. 1. To simulate porous media layers, six plexiglass columns (height 100 有机玻璃列 3 C. Chen et al. Water Research 226 (2022) 119273 Fig. 5. Regulation of ROSs resistance by the antioxidant system under PFOA stress (PFOA vs. Control). mm; inner diameter 50 mm) were filled with 0.38 porosity quartz sands (0.25–0.35 mm). A stainless screen (60 mesh) was crimped and fixed at 卷曲固定 the bottom of the column to prevent leakage of quartz sand. Synthetic water was fed into the column with a peristaltic pump (L100–1S-2, Lange pump, China) at a 0.2 mL min− 1, resulting in a hydraulic retention time of 6.7 h. All columns were operated for 60 days at room temper­ ature (20 ◦ C) without light Fig. 1. The quality of synthetic water used for the experiment strictly referred to the standard of reclaimed water applied to agricultural irri­ gation (Masciopinto et al., 2020) and subsurface recharge (EPA, 2012). Synthetic water consisted of (mg L− 1) 42.52 CH3COONa•3H2O, 9.14 NH4NO3, 1.47 K2HPO4, corresponding to a chemical oxygen demand (COD) of ~ 20 mg L− 1. One milliliter of trace metal stock solution was added to the synthetic water, and the solutions composed by (g L− 1) 2 CaCl2, 0.5 MgSO4, 0.5 ZnSO4, 0.5 MnSO4, 0.1 (NH4)6Mo7O24, 0.1 H3BO3, 0.1 Al2O3, 0.1 FeSO4•7H2O. PFOA in the synthetic water was set to be 2.4 μM (equivalent to 1000 μg-PFOA L− 1), referring to the reports on real water environments (Han et al., 2017; Yuan et al., 2022). The porous medium used was quartz sand (99% SiO2, Sinopharm Chemical Reagent Co., Ltd, China). All the quartz sand was pretreated according to Cui et al. (2018). The microbes were obtained from soil leaching (h < 3 m) of an aquifer storage site in Jilin Province. The suspensions were centrifuged, washed with 0.02 mM phosphate-buffered saline (PBS) 3 times, and resuspended to a microbe concentration of ~ 108 cell mL− 1 (OD600 0.1). The bacteria suspension was mixed with the quartz sand according to a 10 mL g− 1 vol/mass ratio. A tubular rotary mixer (ROLLER 6 digital, 管式旋转搅拌机 IKA, Germany) was employed to attach microbes on the sand surface for 4 h. embedding in Spurr solution at 70 ◦ C for polymerization. The poly­ merized samples were cut into ultrathin sections (70 nm) using an ul­ tramicrotome (Leica, Reichert Ultracuts, Austria) and stained with uranyl acetate and lead citrate. Finally, the sections were observed in a JEM1230 transmission electron microscope coupled with energy dispersive spectroscopy. 2.2.2. Extracellular polymeric substance (EPS) quantification The biomass was washed with 0.02 M PBS after thorough stirring and centrifuged (TGL-16 K, Xiangyi, Laboratory Instrument Development Corp, China) at 4 ◦ C, 8000 rpm for 10 mins. The collected pellets were resuspended in deionized water. Then, the suspension was heated in a water bath for 30 mins at 60 ◦ C, and centrifuged at 12,000 rpm, 4 ◦ C for 30 mins; the obtained supernatant was the EPSs solution. The anthronesulfuric acid reagent method was used to determine polysaccharides, and the test results are expressed in glucose equivalent. Protein was determined by the modified Lowry method (Cui et al., 2018). 2.2.3. X-ray photoelectron spectroscopy spectrum analysis Before testing, the biomass was freeze-dried at − 80 ◦ C (Pilot 1–2, Boyikang, China). The X-ray photoelectron spectra (XPS) were recorded with a monochromatic aluminum potassium radiation (1486.71 eV) energy source, operating at 15 kV and 15 mA (ESCALAB 250, Thermo, USA). All binding energies were referenced to the neutral C1s peak of 284.6 eV to compensate for the surface charge effect. 2.2.4. Metagenomic and metatranscriptome analysis The samples were taken from 30 mm deep quartz sand and effluents in a stable reactor. All the samples were immediately frozen in liquid nitrogen, stored at − 80 ◦ C, and finally, the samples were sent to Novogene Bioinformatics Technology Co. Ltd (Beijing China) for meta­ genomic and metatranscriptome sequencing. For metagenomic sequencing, the details of DNA quality control, concentration determination, library construction, and data acquisition were referred to Zhang et al. (2021a). The unigenes containing ARGs and movable gene elements (MGEs) were aligned by CARD, INTE­ GRALL, the NCBI plasmid database, and ISfinder. Here, if the MGE sequence was identified on the scaftigs with the ARG sequence, the ARG was classified as horizontal transmission ARG (hARG). The remaining ARGs were classified as vertical transmission ARGs (vARGs). Further­ more, the identified HBPs were referred to the database, including 538 pathogenic species (Li et al., 2015). For metatranscriptome sequencing, RNA extraction and quality 2.2. Analytical methods 2.2.1. Electron microscopy observations Biomass was obtained from a port located at 30 mm from the column bottom. The sample was examined by a field-emission scanning electron microscope (SEM, XL-30 FE-SEM, FEI, USA) and a transmission electron microscope (TEM) coupled with energy dispersive spectroscopy (EDS) (TEM-EDS, JEM1230, JEOL Co., Ltd., Japan). Before observing, all the materials were soaked overnight in 2.5% glutaraldehyde and dehy­ drated for 30 mins with a gradient concentration of ethanol 10%, 30%, 50%, 70%, 90%, and 100% (Cui et al., 2018). For TEM observation, materials were pre-mixed with 1% osmium tetroxide at 4 ◦ C for 3 h and then dehydrated with the same method as SEM samples before 4 C. Chen et al. Water Research 226 (2022) 119273 甘油磷脂代谢 肽聚糖合成 Fig. 6. Proposed Peptidoglycan biosynthesis (blue frame), Glycerophospholipid metabolism, and TCA cycle (yellow frame) pathways based on transcriptomic an­ 转录组分析 alyses. The shade of red on the background of the enzyme represents the degree of up-regulation. 酶背景上的红色阴影表示上调的程度。 control, library construction and purification, data acquisition referred to Zhang et al. (2022b). The clean data calculated by Q20, Q30, and GC content were used for the downstream analyses. De novo assembly of the clean reads obtained by preprocessing each sample was performed with Trinity. The sequences were integrated, and repeated sequences (sequence identity threshold 0.95) were deleted using CD-HIT-EST to obtain a unigene dataset. Unigenes were mapped to the Kyoto Ency­ clopedia of Genes and Genomes (KEGG) functional database (blastp, e-value ≤ 1e-5) with DIAMOND software to obtain comprehensive gene function information. 2.2.5. Statistical analysis Data processing was performed using Microsoft Office 2016 and Origin 2021 student edition. TBtools was used to compare the abun­ dance of MGEs between different samples. Network analysis was per­ formed to understand the inter associations among HBPs and ARGs 5 C. Chen et al. Water Research 226 (2022) 119273 Fig. 7. (a) Double-circle diagrams of the bacterial com­ munity (outer circle), and possible distribution of bacterial species hosts of the total ARGs (inner circle) for each bio­ film sample with 0 (Control) and 2.4 µM PFOA (PFOA), respectively. (b) Relationships between antibiotic resis­ tance mechanisms and bacterial phyla in each sample. The 门 outmost two circles list the names of the resistance mech­ anisms and bacterial phyla. The third circle represents the read number of ARGs in this mechanism. The width of the bars between resistance mechanisms and bacteria phyla correlates to the percentages of respective mechanisms in these phyla. The different colors in the circle represent different resistance mechanisms and bacterial phyla. (strong and significant correlation Spearman’s r ≥ 0.8 and Pearson’s r ≥ 0.8, p-value ≤ 0.01) using the Comet in Cytoscape_v3.8.2. The results were visualized in Gephi_v0.9.2. 3.2. State of arts of PFOA in the biomass The SEM images (Fig. S2a) show that PFOA significantly promoted the aggregation of microorganisms on the medium porous surface, and consistently the biomass retained in the column increased by 119% (Fig. 3a). At the same time, PFOA stimulated the EPSs production, in which the contents of polysaccharides (Fig. 3b) and proteins (Fig. 3c) increased by nearly 4417% and 1288%, respectively. EPSs formed a natural barrier for microorganisms to resist the toxic effects of PFOA (Cao et al., 2022; Chen et al., 2021a), resulting in biofilms formation. However, TEM results (Fig. S2b) show that trace PFOA still caused cell membrane damage and intracellular leakage. A clear C-F bond peak appeared in the XPS F1s energy spectrum at bond energy of 688.8 eV (Fig. 3e) (Yang et al., 2020a), providing definite evidence of PFOA immobilization. In particular, the biofilm’s -NH2 basic group could provide adsorption sites for PFOA and thus promote PFOA immobili­ zation (Yan et al., 2021). In sum, PFOA could interact with the biofilm, destroy the cellular structures’ membranes, and thus reduce their integrity Fig. 3. 3. Results and discussion 3.1. Alternation of microbial community During the 60-day run, almost no PFOA was biodegraded, indicating that biodegradation performance was negligible for this hazard (Fig. S1). Interestingly, trace amounts of PFOA still exerted considerable selective pressure on the microbial community of the porous media. As shown in Fig. 2, Proteobacteria became the most dominant phylum (relative abundance 91%) in the PFOA-stimulated porous media, 56.9% greater than in the control. The enrichment of Proteobacteria was consistent with previous reports (Cai et al., 2020; Senevirathna et al., 2022), indicating that Proteobacteria was more advanced than others under PFOA pressure. The phosphate groups of Proteobacteria outer membrane phospholipids interact with PFOA, causing it to respond dramatically under the PFOA stress (Wojcik et al., 2018). The relative abundance at genus level of Pelomonas, Acinetobacter, Dechloromonas, Methylophilus, Acidovorax, Delftia, Methylobacterium, and Methyl­ oversatilis were all significantly increased by as much as 60–1200%. With a strong colonization ability, Acinetobacter was the most dominant genus in the porous media and could play an important role in micro­ organisms’ accumulation (Martinez-Campos et al., 2021). In addition, Dechloromonas abundance, which dechlorinate enzymes dcrA and cfrA could resist high concentration halogen compounds (Yang et al., 2020b; Zhang et al., 2022a; Zhao et al., 2018), increased by 1060% under PFOA stimuli. These results indicated that the microbial community in the porous media was altered to be more resistant, through a biofilm-forming and halogen-resistant community succession Fig. 2. 3.3. Microbial defense and repair 3.3.1. Stress-related functions and genes expression As shown in Fig. 4, considerable pathways of the functional traits of KEGG level 2 (14/20) increased significantly. PFOA significantly improved some microbial metabolic activities, the folding, sorting, and degradation pathway was the most significantly up-regulated (p < 0.001), indicating an advanced activity of genetic information processing. Besides, amino acid (p < 0.001) and lipid (p < 0.05) metabolisms belonging to metabolism function, membrane transport (p < 0.001) in charge of environmental information processing, and cell motility classified to cell processes (p < 0.001) were all enhanced. Such regulations contributed to the enhanced microbial resistance in response to PFOA in 6 C. Chen et al. Water Research 226 (2022) 119273 DNA damage (Liu et al., 2014). PFOA could induce oxidative stress due to the accumulation of reactive oxide species (ROSs) (Li et al., 2021), which then disrupt the balance of the antioxidant system and induce oxidative damage, even apoptosis (Liu et al., 2016). In this study, 细胞凋亡 intracellular ROSs concentrations increased by 69.8% at the early stage of the experiment (day 5) (see Fig. S4), indicating that the microor­ ganisms were subjected to oxidative damages. With biofilm formation and acclimation, microorganisms’ antioxidant capacity was signifi­ cantly increased. The metatranscriptome results supported that the microbial antioxidant systems responded positively to prevent oxidative damage from PFOA. As shown in Fig. 5, The excessed ROSs under PFOA exposure were successively removed by superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). First, SOD, in which the log2FC values of the encoding functional genes were 6.71–8.23 overexpressed (see Table S2), catalyzed the dismutation of superoxide anion (O−2 ) into hydrogen peroxide (H2O2) (Fabrello et al., 2021). Then, CAT, in which the log2FC values of the encoding functional genes were 4.86–6.10 overexpressed (see Table S2), catalyzed the conversion of hydrogen peroxide (H2O2) to water and molecular oxygen (Fabrello et al., 2021). ATP was synthesized on the mitochondria to provide the metabolite-required energy. Moreover, the glutathione antioxidant system is another primary enzymatic antioxidant system of organisms, which is crucial in numerous biological processes, such as DNA syn­ thesis, removal of oxidation (Jiao et al., 2020; Zhou et al., 2019). Our results showed that GSH played multiple roles in resisting oxidative damages from PFOA. On the one hand, the log2FC of genes encoding proteins GCLC and GSS, the rate-limiting enzymes for GSH synthesis, reached 8.53 and 9.49 (Deka et al., 2021). The generated GSH could reduce H2O2 at the expense of oxidizing GSH to GSSG by glutathione peroxidase (GPX) (Jiao et al., 2020). On the other hand, Glutathione S-transferase (GST) is a detoxifying enzyme of phase II of biotransfor­ mation, and the maximum value of log2FC reached 6.14. The increased GST expression catalyzes the conjugation of glutathione with PFOA (Fabrello et al., 2021) Fig. 5. 人类条件致病菌 Fig. 8. Variation in abundance of HBPs. (a) Top 10 abundance and species of HBPs in biofilm; (b) Network analysis of HBPs population relationship under PFOA or not. Positive relationship (pink line); negative relationship (green line). 3.3.3. Cell membrane repairing As shown in Fig. 6, the metatranscriptome results show significantly up-regulated expression of genes encoding essential proteins in the mi­ crobial glycerophospholipid metabolism pathway under PFOAinduction, including GPAT1_2 (log2FC 5.22), CDS1 (log2FC 5.81), PISD (log2FC 4.91), PLA2G16 (log2FC 5.75), aas (log2FC 7.41), GDE1 (log2FC 4.20) (Gonzalez-Baro and Coleman 2017; Jennings and Epand 2020; Simon and Cravatt 2008; Wang et al., 2021b), details are given in Table S4. This indicated that the phospholipid bilayer synthesis was accelerated to repair the damaged membranes (Teng et al., 2021). Acetyl-CoA is a kind of important cofactor involved in many metabolic pathways, such as carbohydrate, lipid, and amino acid catabolism (Zhang et al., 2021b). Acetyl-CoA reacts with oxaloacetate and enters the tricarboxylic acid (TCA) cycle catalyzed by gltA to form citric acid, further oxidized to generate energy. At the same time, acetyl-CoA was transformed into 1-Acyl-sn-glycerol-3P by GPAT1_2, contributing to the glycerophospholipids synthesis. In addition to the up-regulation of glycerophospholipid metabolic pathways, the KEGG pathway of peptidoglycan biosynthesis was also significantly enriched under the pressure of PFOA. Compared with the control group, the log2FC of MurA, MurB, MurC, and MurD were 7.73, 3.79, 7.96, and 3.93, respectively. These enzymes gradually catalyzed the formation of UDP-MurNAc-L-Ala-γ-D-Glu-from UDP-N-acetyl glucosamine (UDP-GlcNAc) (Hrast et al., 2014). Class A Multimodular penicillin-binding proteins (ClassA PBP) are membrane-bound enzymes, responsible for peptidoglycan polymerization and activity (Terrak et al., 2008). The log2FC of Class A PBP enriched to 7.21. indicating the syn­ thesis of peptidoglycan was accelerated, and the integrity of the cell wall could be improved Fig. 6. porous media (Padmanabhan et al., 2020). In addition, a small part of functional pathways was slightly inhibited, such as carbohydrate metabolism (p < 0.001), energy metabolism (p < 0.001), and xenobi­ otics biodegradation and metabolism (p < 0.001). It is worth noting that the abundance of functional traits related to human diseases, such as the traits encoding infectious diseases (p < 0.001) and endocrine and metabolic diseases (p < 0.001), was signifi­ cantly higher than the controls. Moreover, on pathway level 3, the overexpression of tuberculosis (p < 0.001), Legionellosis (p < 0.001), and Type 1 diabetes mellitus (p < 0.001) was also overexpressed (see Fig. S3). These functional traits implied that PFOA could promote the production and enrichment of human bacterial pathogens, posing a se­ vere biological risk (Xie et al., 2019) Fig. 4. As shown in Table S2, the expression of key genes related to cell repair, DNA repair, and transport regulation changed significantly under PFOA stress. The log2FoldChange (log2FC) values of the functional gene rpoS increased from 4.23 to 8.55, indicating the improvements of cells on self-recovering capability under PFOA stress (Fernandez-Gomez et al., 2020; Liu et al., 2018). Moreover, most of the critical genes regulating cell repair (cusC, mdtB, and motA), DNA repair (radA, recJ, recO, recA, rpoH, ruvB, and lexA), and transfer regulation (ftsY, gspE) were also significantly up-regulated (p < 0.001). ftsY and gspE were also played important roles in ARGs transfer and chromosome mutation, implying the potentiality of antibiotic resistance genes (ARGs) produc­ tion and transformation (Li et al., 2020a). 3.3.2. Antioxidants regulation The toxic effects of PFOA are ascribed to be mainly cell membrane damage (Fitzgerald et al., 2018), oxidative stress (Liu et al., 2016), and 7 C. Chen et al. Water Research 226 (2022) 119273 Fig. 9. (a) Relative abundance of ARGs encoding plasmids with PFOA press or not; (b) Network analysis depicts the co-occurrence patterns among vARG subtypes 编码质粒 (circle without border) and microbial communities (circle with border), including human bacterial pathogens (HBPs) and non-pathogenic bacteria (NPB) in the species level. 3.4. Microbial risks: resistance genes and pathogens main host of ARGs under PFOA stress. In addition, the hosts of ARGs analysis shows that there were 12 genera that carried ARGs in the top 20 abundance of bacteria (genus level) (see Fig. S5), and 9 of them increased significantly under the stimulation of PFOA, which plausibly explains the reason for the survival and significant increase of these bacteria is due to the fact that they carry ARGs with resistant to PFOA. And most of the hostable ARGs belong to efflux, reaching 81.5%. The result of circos-mech analysis further revealed the relationship between microbial community and the ARGs types (see Fig. 7b). The dominant Proteobacteria carried more than 65% of efflux ARGs. The top 20 abundance ARGs changes showed that PFOA stress could signifi­ cantly increase efflux ARGs. For example, emrB, AxyY, oqxB were newly 3.4.1. ARG patterns and hosts Studies have shown that pollutants, which exert selective biological pressure on microorganisms, could cause the production and spread of ARGs, such as heavy metals, pesticides, and disinfection by-products (DBPs) (Guo et al., 2020; Lu et al., 2020; Shekhawat et al., 2021). PFOA stress also changed the ARGs profiles. As shown in Fig. 7a, Pro­ teobacteria with the greatest abundance were the most attributable mi­ croorganisms of ARGs. Under PFOA influence, Proteobacteria abundance increased by 32%, and the ARGs carried by it increased by 13%, reaching 71% of the total ARGs. This indicated that Proteobacteria is the 8 C. Chen et al. Water Research 226 (2022) 119273 detected efflux ARGs, meanwhile, abeM, adeK, adeI, adeJ, and abeS, all belong to efflux ARGs, were increased 10-fold compared with the con­ trol group (Fig. S6). On the contrary, ARGs belong to inactivation, target-alteration, and target-replacement, such as sul1, dfrA7, and chrB, increased insignificantly or decreased. Efflux is a unique system used to expel any unnecessary or toxic substances out of the cells, providing binding sites for toxic substances and activating targeted exclusion (Dey et al., 2022) Fig. 7. 4. Conclusions This study was the first time to explore the microbial response be­ haviors and the biological risk characteristics and propagation laws caused by 2.4 µM PFOA during the migration of porous media. The re­ sults show that trace PFOA still impacted the microbial community structure and resistance mechanism in the solid medium and effluent. Proteobacteria and Acinetobacter, Dechloromonas had absolute biological 变形菌门和不动杆菌门,脱氯单胞菌 advantages under PFOA stress. The rapid formation of biofilm and the 3.4.2. Pathogens enhancement of antioxidant systems initiated the repair function of cell PFOA significantly increased the HBPs abundance in the porous membranes, which in turn led to the central resistance regulation of media by 106%, as shown in Fig. 8a. HBPs including Acinetobacter efflux ARGs. The bacteria carried ARGs were concentrated in the Pro­ 变形菌门 baumannii (from 1.1 to 30.6%), Delftia acidovorans (from 1.6 to 23.3%), teobacteria, and vARGs mediated by HBPs increased significantly, taking 鲍氏不动杆菌 食酸丛毛单胞菌 Ralstonia pickettii, Comamonas testosteroni, and Pseudomonas aeruginosa serious biological risks to the groundwater environment. Therefore, 皮氏罗尔斯顿菌 绿脓假单胞菌 铜绿假单胞菌 睾丸酮丛毛单胞菌 in porous media were all significantly greater than the control. Acine­ PFOA biological risks in the natural environment are worth concern. tobacter baumannii is associated with resistance to almost all available antibiotics, and it has been extensively reported in hospital-and deviceDeclaration of Competing Interest associated infections (Eze et al., 2021). Delftia acidovorans could cause catheter-related infection, infective endocarditis, urinary tract in­ The authors declare that they have no known competing financial fections, and nosocomial bacteremia (Patel et al., 2019). interests or personal relationships that could have appeared to influence As shown in Fig. 8b, the interaction network analysis reveals that the work reported in this paper. biofilm’s top 10 abundance HBPs significantly impacted HBPs in the effluent (16 nodes, 63 edges). Positive edges were more dominated in Acknowledgments the networks (Spearman’s r ≥ 0.8 and Pearson’s r ≥ 0.8, p-value ≤ 0.01), confirming that most HBPs have mutually beneficial survival relation­ This work was supported by the National Natural Science Key ships. For example, the most enriched HPB, Acinetobacter had a signifi­ Foundation of China (52230003). cant relationship with six pathogens, five of which were positively correlated, and only one was negatively correlated. PFOA stress also Supplementary materials significantly changed the effluent’s microbial community structure (see Fig. S7) and dominant bacterial species (see Fig. S8). The above results Supplementary material associated with this article can be found, in indicated PFOA migration aggravated the pathogenic risk of the water the online version, at doi:10.1016/j.watres.2022.119273. environment Fig. 8. References 3.4.3. ARGs transmission Amarasiri, M., Sano, D., Suzuki, S., 2020. 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