Evaluation of biofouling characteristics on reverse osmosis membranes of disinfection-residual-bacteria (DRB) after seven kinds of disinfection

Evaluation of biofouling characteristics on reverse osmosis membranes of disinfection-residual-bacteria (DRB) after seven kinds of disinfection | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Article Evaluation of biofouling characteristics on reverse osmosis membranes of disinfection-residual-bacteria (DRB) after seven kinds of disinfection Yin-Hu Wu, Haobin Wang, Wen-Long Wang, Li-Wei Luo, Gen-Qiang Chen, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1811963/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2023 Read the published version in npj Clean Water → Version 1 posted 12 You are reading this latest preprint version Abstract Biofouling is a critical defect of the reverse osmosis (RO) system. It has been reported that disinfection processes tend to select certain undesirable disinfection-residual bacteria (DRB), leading to severe long-term biofouling potential. To provide constructive guidance on biofouling prevention in RO systems, this study evaluated the biofouling characteristics of RO membranes of DRB after the application of five mature disinfection methods (NaClO, NH 2 Cl, ClO 2 , UV, and O 3 ) and two novel disinfection methods (K 2 FeO 4 and flow-through electrode system (FES)). After a 32-day biofilm cultivation on the RO membranes, the DRB biofilm of K 2 FeO 4 and O 3 caused a slight normalised flux drop (22.4 ± 2.4% and 23.9 ± 1.7%, respectively) of RO membrane compared with the control group (non-disinfected, ~ 27% normalised flux drop). Moreover, the biofouling degree of the NH 2 Cl-DRB biofilm was similar to that of the control group. The remaining disinfection types aggravated membrane biofouling. The biofouling behaviour of DRB showed no relationship with bacterial concentration or activity. The thickness and density of the biofilms as well as the organics/bacterial number ratio in the DRB biofilm, helped explain the difference in the fouling degree between each group. Moreover, microbial community analysis showed that the relative abundance of typical highly secretory and biofouling-related genera, such as Pseudomonas , Sphingomonas , Acinetobacter , Methylobacterium , Sphingobium , and Ralstonia , were the main reasons for the difference in biofouling degree. All types of disinfection effectively prevented pathogen reproduction in the DRB biofilm. However, the relative abundance of (opportunistic) pathogens increased significantly after NaClO and ClO 2 disinfection. Disinfection-residual-bacteria (DRB) Biofilm Membrane fouling Microbial community Disinfection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Water scarcity is a pressing global challenge 1 . The accelerated carbon neutrality process worsens water shortages 2 , 3 . Meanwhile, countries and regions continue to face water contamination 4 . Water reclamation is a win-win strategy for increasing freshwater supply and shortening the water footprint of human beings 5 , 6 . Reverse osmosis (RO) is one of the most applicable and stable units for high-quality reclaimed water production for industrial reuse, potable reuse, and groundwater replenishment 7 – 10 . Many large-scale water reclamation plants have been successively operated 11 – 13 . However, RO system has a fatal defect, that is, the unmanageable membrane fouling 14 . Membrane fouling of RO mainly includes scaling, colloidal fouling, organic fouling, and biofouling 15 . Among these, biofouling is the most complicated and uncontrollable, although numerous studies have made significant efforts to reduce it 16 – 19 . Disinfection is a widely applied pretreatment process used to deal with biofouling in RO systems. However, it may lead to undesirable effects 18 . After reducing the number of bacteria in the feed water, the disinfection process exerts a salient selection effect on the bacterial community and the extracellular polymeric substance (EPS) secreting ability of the bacteria. Some unwanted bacteria, that are resistant to disinfection or can adapt to adverse environments, might survive disinfection processes and become disinfection-residual-bacteria (DRB) 20 , 21 . DRB might possess a higher proportion of bacteria with higher EPS secreting ability, leading to a more severe biofouling of RO membranes, especially in the long-term operation of RO systems 22 . Research in laboratory and full-scale water treatment plants has shown the probability of aggravated biofouling after disinfection 11,23−25 . However, most of these studies are limited to a single type of disinfection process 26 . To date, there is still a lack of systematic and broad comparison of the biofouling-control effects of various disinfection methods. To provide more constructive and reliable guidance on the prevention of RO biofouling, this study compared the DRB characteristics of seven different disinfection methods, including five widely used disinfection methods (NaClO, NH 2 Cl, ClO 2 , UV, and O 3 ) and two novel disinfection methods (K 2 FeO 4 and flow-through electrode system (FES) 27 ) via a long-term (32 days) biofilm cultivation experiment on RO membranes. Furthermore, the bacteria and organic matter in the biofilm on the RO membranes were analysed using heterotrophic plate count (HPC), adenosine triphosphate (ATP), dissolved organic matter (DOM), and fluorescence excitation-emission matrix (EEM) to determine the primary reasons for the differences in biofouling in each group. The microbial community structure of DRB biofilms was analysed using high-throughput sequencing. Based on these results, the correlations between the fouling characteristics of DRB biofilms and bacterial density, organic density, biofilm morphology, and the microbial community of DRB were analysed. 2. Materials And Methods 2.1 Water samples Reclaimed water was sampled from a large-scale water reclaimed plant in Beijing, China. A schematic of the advanced treatment process is shown in Fig. S1. The effluent from the denitrification filter was chosen as the sample because subsequent treatment units had a bacterial removal effect 28 . The reclaimed water samples were transported to the laboratory within 1 h, then filtered by filter papers to remove particles, and kept at 4°C before disinfection. Water quality parameters of the sampled water were measured as soon as they arrived at the laboratory, and are listed in Table 1 . Table 1 Chemical and physical properties of water samples before disinfection. pH TDS (mg/L) TN (mg/L) NO 3 − (mg/L) NH 4 − (mg/L) TP (mg/L) TOC (mg/L) 7.8 ± 0.1 579 ± 27 13.2 ± 1.2 12.6 ± 1.4 0.38 ± 0.12 0.16 ± 0.02 5.84 ± 0.04 2.2 Disinfection and biofilm culture Five commonly used disinfection methods, including NaClO, NH 2 Cl, ClO 2 , UV, and O 3 , and two novel disinfection technologies, namely K 2 FeO 4 and a flow-through electrode system (FES), were compared in this study. The steps followed in the experiment are shown in Fig. 1 . Briefly, water samples were filtered using filter paper (medium speed, Newstar, Hangzhou, China) to remove large flocs before disinfection. Seven types of disinfection processes were conducted by following the technical parameters described in preliminary experiments (Table 2 ) to achieve similar bacterial log removal, except for two novel disinfection methods as they could not achieve a high disinfection effect in actual wastewater. Square-wave alternating pulse current FES devices were set up based on a previous study 27 . The voltage amplitude and hydraulic retention time were set at 4 V and 27.7 s, respectively, to achieve the best disinfection performance of the system. UV irradiation was performed using a laboratory-scale collimated light-beam apparatus 24 , 29 . Other oxidizing disinfection processes were performed in sterilised glass bottles at 25°C and 150 rpm. The reaction was quenched with Na 2 S 2 O 3 solution to avoid oxidative damage to the RO membrane. Table 2 Dosage of each disinfection method. Disinfection method Free chlorine Chloramine Chlorine dioxide UV Dosage 5 mg/L 30 min 5 mg/L 30 min 1 mg/L 30 min 30 mJ/cm 2 Disinfection method Ozone Ferret FES Dosage 3 mg/L 10 min 5 mg/L 10 min E = 4 V, T = 27.7 s Aromatic polyamide composite LP100 RO membrane (Vorton, China) was cut into round coupons (d = 32 mm, S = 804 mm 2 ), and the pretreatment procedure was conducted based on a previous study 30 . For biofilm culturing, the membrane coupon was soaked in 18 mL of disinfected water or control samples in a 5 cm sterilised round plastic Petri dish at 25°C. Water samples were refreshed daily. After 32 days of culture, the membrane was gently rinsed twice with phosphate buffer saline (PBS) to remove suspending bacteria for sequencing analysis. The experiments were conducted in triplicates for each group. 2.3 Evaluation of the disinfection effect The disinfection effect was evaluated based on the concentration of culturable bacteria; ATP was tested before the biofilm culture experiments with triplicate experiments immediately after the disinfection processes were completed. Culturable bacteria were measured by HPC via colony-forming unit (CFU) counting 28 . ATP was tested using luminescence analysis. For total ATP measurement, 100 µL of the bacterial suspension and 100 µL of CellTiter-Glo Luminescent Cell Viability Assay (Promega, USA) were mixed in 96-well plates. After incubation at 25°C and at 150 rpm in the dark for 1 min, luminescence intensity was measured using a microplate reader (SpectraMax M5, Molecular Devices, USA). For the extracellular ATP test, water samples were filtered through a 0.1 µm membrane (Millipore) to remove bacteria; the subsequent steps were the same as those for total ATP. Intracellular ATP concentration was calculated by subtracting the extracellular ATP concentration from the total ATP concentration. 2.4 RO cross-flow unit and membrane performance tests A laboratory-scale cross-flow RO system was used to evaluate the performance of RO membranes before and after biofilm growth 31 . Briefly, the membrane compaction phase was performed with a 30 min rinse of ultra-pure water at 1.2 MPa until the permeate flux was stable. The flow rate of the influent was set at 1.0 mL/min via a constant flow pump (NPL-100). The feed water was then switched to a 500 mg/L NaCl solution (conductivity of approximately 1000 µs/cm). The permeate flux was recorded after reaching a constant value. The normalised flux was calculated by dividing the flux of the fouled membrane by the clean membrane before biofilm growth. The resistance of the RO membrane was calculated, as follows: \({R}_{b}={R}_{T}-{R}_{m}-{R}_{P}=\frac{\varDelta P}{\sigma J}-{R}_{m}-{R}_{P}\) (1) Where, Δ P is the transmembrane pressure, σ is the kinetic viscosity of water, and J is the flux. The resistance of the biofilm R b is calculated by subtracting the resistance of the virgin membrane R m and the resistance of the concentration polarisation R p from the resistance of the fouled membrane R T . 2.5 Biofilm analysis 2.5.1 Heterotrophic plate counts (HPC) and microbial activity Microbial amount and activity were determined using HPC and ATP concentrations. A piece of 10 mm×10 mm fouled membrane was cut off and vortexed in 0.5 mL normal saline for 30 s. HPC and ATP concentrations were tested using the same procedure, as described in Section 2.3 . 2.5.2 Organic matter analysis DOM and EEM were applied to reflect the characteristics of organic matter in DRB biofilms 23 , 32 . Briefly, a piece of 10 mm×10 mm fouled membrane was cut and shaken in 5 mL NaOH solution (pH 12) for 24 h at 25°C and 150 rpm. Then, HCl solution (pH 2) was added to adjust the pH of the solution to 7.0 ± 0.2. After neutralisation, the volume of the solution was adjusted to 15 mL by adding ultrapure water. The solution was filtered through a 0.45-µm nylon membrane (Whatman, England) before total organic carbon (TOC) measurement on a TOC-5000A analyser. EEM spectra were recorded using a fluorescence spectrophotometre (F-7100, Hitachi). The EEM spectrum was divided into six zones for integration. The types of fluorescent substances in each zone are shown in Table S1 24,33 . 2.5.3 Surface morphology of the fouled membrane A laser scanning confocal microscope (LSCM, LSM710META) was used to measure the thickness of the biofilm on the RO membrane. A 5 ×10 mm membrane with the DRB biofilm of each sample was cut for LSCM observation. The staining groups of the fluorescent dyes and their targets are listed in Table S2. The surface morphology of the DRB biofilm was examined using field-emission scanning electron microscopy (FESEM, SU8220, Hitachi, Japan) and the accelerating voltage was set to 5 kV. A piece of 5 × 5 mm membrane coupons was cut for observation. 2.5.4 Microbial community analysis Three pieces of 40 mm 2 membrane coupons were cut for microbial community analysis. Microbial community analysis was conducted as previously described 23 . Briefly, DNA was extracted using the E.Z.N.A.® soil DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.). Furthermore, 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were used as primer pairs for amplification. Sequencing was performed using an Illumina MiSeq PE300 platform. All analysed sequences were submitted to the NCBI SRA database under accession number PRJNA803872. Pathogen identification was based on the Virulence Factor Database (VFDB) from the Institute of Pathogen Biology, CAMS & PUMC. 3. Results And Discussion 3.1 Effects of DRB biofilm on the RO membrane performance The effects of different disinfection methods on the RO membrane flux of the DRB biofilms were investigated. Not all disinfection methods can control biofouling. These methods were divided into three groups, according to the normalised flux (Fig. 2 a). The first group, classified as “alleviated”, included K 2 FeO 4 and O 3 . Their DRB performed light normalised flux drop (22.4 ± 2.4% and 23.9 ± 1.17%, respectively) compared to the control group (without disinfection pre-treatment), thereby indicating alleviated biofouling potentials. Meanwhile, the DRB of NH 2 Cl had a similar biofouling degree as the control group (~ 27%), and was classified as “equal”. Finally, the DRB of the “aggravated” group, which included FES, UV, NaClO, and ClO 2 , caused more biofouling of the RO membrane than the control group. Notably, the biofouling of ClO 2 -DRB was the most severe, with a flux drop percentage of 35.5 ± 4.0%. The two most frequently used disinfection methods, namely UV and NaClO significantly aggravated the biofouling of RO membranes, matching the results of long-term studies in previous research 23 , 24 . The hydraulic resistance of the DRB biofilms ( Rm ) is shown in Fig. 2 b. The resistance of the K 2 FeO 4 -DRB biofilm (2.48×10 13 m − 1 ) was the lowest as it had the least impact on the RO membrane flux drop. Also, the resistance of the O 3 -DRB biofilm (2.71×10 13 m − 1 ) in the “alleviated” group was lower than that of the control group (3.07×10 13 m − 1 ). The resistance of the NH 2 Cl-DRB biofilm (3.48×10 13 m − 1 ) was slightly higher than that of the control group. The DRB biofilm resistance of all the “aggravated” groups was much higher than that of the control group. Among them, the average resistance of NaClO was the highest, at 5.18×10 13 m − 1 . However, it had a relatively high within-group error, corresponding to the membrane flux drop shown in Fig. 2 a. 3.2 Disinfection effect of seven kinds of disinfection methods Alleviation of biofouling after disinfection might be associated with lower bacterial concentrations in the feed water after disinfection 23 . Therefore, variations in the concentrations of HPC and ATP in the water samples during the disinfection processes were tested (Table 3 ). The five conventional disinfection methods achieved a bacterial inactivation rate of 3-lg, while K 2 FeO 4 and FES exhibited a relatively low inactivation effect (~ 1-lg). Overall, HPC had no monotonous correlation with biofouling potential. The DRB biofilm of K 2 FeO 4 caused only a 22% decrease in membrane flux, although the inactivation effect of K 2 FeO 4 was the lowest (< 1-lg). NaClO was the most effective method for bacterial inactivation. However, its DRB aggravated biofouling at an average relative flux drop of ~ 35%. Table 3 Disinfection effect measured by HPC and ATP content. Disinfection methods HPC (CFU/mL) Inactivation rate (lg) ATP (ng/mL) (Total) ATP (ng/mL) (Intracellular) Control 2.7 ± 0.1*10^5 -- 2.99 ± 0.08 2.90 ± 0.08 K 2 FeO 4 2.4 ± 0.2*10^4 1.06 ± 0.05 0.65 ± 0.01 0.61 ± 0.00 O 3 1.5 ± 0.5*10^2 3.28 ± 0.14 2.93 ± 0.18 0.11 ± 0.01 NH 2 Cl 4.3 ± 2.3*10^1 3.88 ± 0.25 0.24 ± 0.02 0.18 ± 0.02 FES 1.6 ± 0.1*10^4 1.23 ± 0.04 0.61 ± 0.02 0.35 ± 0.00 UV 2.5 ± 0.8*10^2 3.06 ± 0.15 5.11 ± 0.24 4.92 ± 0.23 NaClO 3.0 ± 1.0*10^1 3.98 ± 0.14 4.03 ± 0.12 0.21 ± 0.01 ClO 2 1.5 ± 0.1*10^2 3.27 ± 0.01 0.59 ± 0.02 0.45 ± 0.02 Previous studies have reported that ATP can be an effective indicator for predicting biofouling potentials in the short- or medium-term operation of RO systems (less than 16 days) 15 , 34 . However, this conclusion is not valid in long-term experiments (Table 3 ). The intracellular ATP in the control group was 2.90 ng/mL, accounting for 97% of the total ATP. Total and intracellular ATP concentrations increased after UV treatment, partly because of the rapid DNA repair mechanism triggered by UV radiation. No correlation was found between the DRB biofouling potentials (membrane flux drop) and the ATP concentration of DRB. Evaluation of the disinfection effect suggested that the amount and the activity of DRB were not the decisive factors of biofouling potential during long-term operation. 3.3 Characteristics of the DRB biofilm on RO membranes To determine the reasons for varying performances of RO membranes fouled by different DRB biofilms, this study dissected the fouled RO membranes and performed a series of tests. The number of bacteria and organics in the DRB biofilms, the morphological characteristics of DRB biofilms, and the bacterial community structure were analysed. 3.3.1 Bacteria and dissolved organic matters (DOM) in the DRB biofilms The live bacterial density of the DRB biofilm was measured using HPC, as shown in Fig. 3 a. DRB biofilms of NH 2 Cl, FES, and UV possessed the maximum live bacteria density (approximately 10 6 CFU/cm 2 ). The live bacterial density of NaClO- and ClO 2 -DRB biofilms were lower than that of the control group (approximately 10 5 CFU/cm 2 ), but they led to the worst flux drop in the RO membrane. Hence, the live bacterial density of the DRB biofilm was not a key factor leading to differences in biofouling degrees. This conclusion is consistent with previous studies 24 . Unlike bacterial cells, the extracellular polymeric substances (EPS) on RO membranes have a more direct relationship with flux drop 31 , 35 . Hence, the total amount of organics in the DRB biofilms was measured by DOM (Fig. 4 a), and the different component of DOM was tested using EEM (Fig. S2). The amount of DOM did not show a clear correlation with the biofouling degree. The DOM of K 2 FeO 4 , which was in the “alleviated” group, was much higher than that of the “aggravated” group. Therefore, the total amount of organics did not play a key factor in the degree of DRB biofilm fouling. Considering that the ratio of EPS to bacteria could affect the biofouling degree 35 , this study calculated the ratio of DOM to HPC in the DRB biofilms (Fig. 4 b). The DOM/HPC ratio in the control group was approximately 4 mg/10 7 live cells. The DRB biofilm of K 2 FeO 4 , O 3 , NH 2 Cl, FES, and UV possessed a relatively lower DOM/HPC ratio, while those of NaClO and ClO 2 (5.4 and 7.6 mg/10 7 live cell, respectively) were significantly higher than the control group. This implied that these two disinfectants could aggravate biofouling of the RO membrane by reshaping the microbial community and changing the EPS production capacity of the residual bacteria. The proportions of DOM components among all the groups were similar (Fig. S2). Tyrosine/tryptophan amide (Zone Ⅰ) and protein-containing tyrosine/tryptophan (Zone II) were the dominant components of fluorescent organic matter in each DRB biofilm, indicating that amino acids and proteins were predominant in the DRB biofilms, compared with polysaccharides, fulvic acids, or humid acids. 3.3.2 Morphological characteristics of DRB biofilms Compared to the bacterial and organic densities in the biofilm, the degree of biofouling was more closely correlated with the thickness and consecutiveness of the biofilm on RO membranes. The thickness of the DRB biofilm was tested using z-stack images of the LSCM. The surface image of the DRB biofilm showed that bacteria, proteins, and α-/β-polysaccharides were evenly distributed in the DRB biofilm (Fig. S3). The DRB biofilm of K 2 FeO 4 was relatively loose in section view, while the others were consecutive (Fig. 5 ). The average thickness of the DRB biofilm was measured via cross-section (Fig. S4) and is shown in Fig. 5 . As a result, the DRB biofilm of the UV group (55 ± 1µm) was significantly thicker than that of the control group (33 ± 1µm). The DRB biofilm thickness of the “alleviated” group was approximately 22 µm, which was the lowest of all the groups. The biofilm thickness partly illustrated the difference in the biofouling degree, supplemented by the DOM/HPC ratio. The two disinfection methods in the “alleviated” group caused a low DOM/HPC ratio and thinner biofilm on the RO membrane, leading to a marginal flux drop of the fouled membrane. The UV DRB developed a thick biofilm and led to severe biofouling of the RO membrane. The biofilms of NaClO and ClO 2 were not very thick. However, the high DOM/HPC ratio in the biofilm could narrow the water channels between bacterial cells 35 , leading to the highest flux drop in the RO membrane. The FESEM images of the DRB biofilm are shown in Fig. 6 . The DRB biofilm fully covered all the RO membranes. However, the compactness of the biofilms was different. The UV and NaClO-DRB biofilms were compact and continuous, partly illustrating their severe biofouling performance. In contrast, there were gaps between the bacteria and the EPS matrix in the DRB biofilm of the remaining groups. Regular crystals containing Fe were observed in the K 2 FeO 4 group (Fig. S5), indicating that K 2 FeO 4 could cause scaling of the RO membrane. 3.4 Microbial community analysis of DRB biofilm on RO membranes Disinfection processes can exert three levels of change on the bacteria: metabolic change of a single bacteria, shift in the microbial community, and variation of nutrient conditions 20 . Among them, a shift in the microbial community is most likely to affect the biofouling degree during long-term operations 36 . Therefore, we analysed the alpha and beta diversities of the DRB microbial community. The alpha diversity indices of the observed species as well as the ACE, Chao1, and Shannon indices of bacteria in the DRB biofilm are listed in Table 4 . The community richness and evenness of the control group were the highest, followed by K 2 FeO 4 (“alleviated” group), which has been reported to have minimal impact on the bacterial community 37 . The community evenness of the ClO 2 -DRB biofilm (“aggravated” group) was the lowest, indicating a significant selection effect of ClO 2 . Overall, community richness and evenness did not show a monotonous correlation with the biofouling behaviour of DRB biofilms. Table 4 Statistical table of Alpha diversity index. Disinfection methods Observed species ACE Chao1 Shannon Simpson Control 864.7 ± 4.9 1004.9 ± 22.7 1004.1 ± 34.8 4.19 ± 0.08 0.052 ± 0.005 K 2 FeO 4 445.7 ± 4.5 539.9 ± 18.4 544.0 ± 29.4 3.75 ± 0.05 0.066 ± 0.003 O 3 135.3 ± 0.5 184.3 ± 9.7 166.0 ± 9.6 2.69 ± 0.00 0.146 ± 0.002 NH 2 Cl 164.3 ± 26.7 307.8 ± 119.2 230.6 ± 66.0 2.46 ± 0.04 0.132 ± 0.013 FES 296.0 ± 3.7 459.3 ± 65.0 399.8 ± 22.8 2.45 ± 0.08 0.286 ± 0.019 UV 221.3 ± 1.7 464.1 ± 63.7 327.5 ± 29.1 2.82 ± 0.05 0.119 ± 0.006 NaClO 261.3 ± 25.0 283.1 ± 20.6 283.7 ± 17.7 2.39 ± 0.15 0.240 ± 0.010 ClO 2 94.3 ± 6.9 167.2 ± 82.9 125.2 ± 37.3 2.00 ± 0.04 0.191 ± 0.009 A Venn diagram of the OTUs that appeared in each group is shown in Fig. S6. Only 36 OTUs were shared by all the groups. The control group had the highest OTUs, indicating that each disinfection method had a selection effect. Besides the control group, the number of OTUs in the K 2 FeO 4 -DRB biofilm was the highest. High numbers of OTUs caused fierce competition among the species, and inhibited biofilm growth and EPS secreting, resulting in the least biofouling of the K 2 FeO 4 -DRB biofilm. The DRB biofilm with ClO 2 and O 3 disinfection possessed the lowest OTUs, indicating that the two aforementioned oxidising disinfection methods had strong selectivity for the bacteria 38 . However, as their biofouling performance differed, ClO 2 selected more biofilm formation and EPS-secreting species than O 3 . The microbial community structure at the phylum level is shown in Fig. S7a; class and genus levels are shown in Fig. S8. α-Proteobacteria and γ-Proteobacteria were the dominant classes in all the DRB biofilms. Actinobacteria and Bacteroidetes were the second and third most abundant phyla, respectively. The top three phyla accounted for 90% of all bacteria. A heat map at the genus level is shown in Fig. S7b. Previously reported biofouling-related genera had significantly higher relative abundances in the “aggravated” group, namely, ClO 2 , NaClO, UV, and FES. For instance, Methylobacterium , which is a typical disinfection-resistant and biofouling-related bacteria genus 23 , 35 , was dominant in the DRB biofilm of FES and ClO 2 with a relative abundance of 54.8 ± 2.3% and 28.6 ± 1.7%, respectively. Sphingobium , a highly secretory genus 24 , 39 , was found to dominate the DRB biofilm under UV (30.0 ± 0.8%). In addition, the relative abundance of Pseudomonas was significantly higher in the NaClO-DRB biofilm than in the other groups (45.9 ± 1.8%). Pseudomonas , which is a typical DRB of chlorine 20 causes biofouling of RO membranes 23,40−42 . In contrast, the relative abundance of these genera was significantly lower in the K 2 FeO 4 -and O 3 -DRB biofilms (< 10%). Thus, the relative abundance of highly secretory or biofouling-related genera plays a decisive role in the biofouling potential of DRB during long-term operation. A community structure similarity analysis was performed using the PCA algorithm (Fig. 7 ). The community structures of all the disinfection groups were significantly different from those of the control group. The bacterial community structures of UV-, O 3 -, NH 2 Cl-, NaClO-, and K 2 FeO 4 -DRB biofilms were similar, whereas the community structures of ClO 2 and FES DRB biofilms were similar. This may be the cause of the inability of these two disinfectants to control the highly secretory genus Methylobacterium . Apart from biofouling of RO membranes, the DRB biofilm can act as a shelter for pathogenic bacteria, leading to health risks of RO concentrate 43 . Thus, the cumulative abundance of pathogens and opportunistic pathogens in the DRB biofilm was analysed and shown in Fig. S9. The relative abundance of pathogenic bacteria in the DRB biofilm increased significantly after disinfection with NaClO and ClO 2 , indicating that these two chlorine-containing oxidative disinfectants selected (opportunistic) pathogens. K 2 FeO 4 , FES, and NH 2 Cl partially reduced the relative abundance of (opportunistic) pathogens. Furthermore, their abundance in DRB biofilms of O 3 and UV were the lowest, demonstrating that they controlled the spread of (opportunistic) pathogens in biofilms. 3.5 Correlation between microbial community structure and RO membrane flux To identify the key genera in the microbial community of DRB affecting the RO membrane flux, correlation coefficients between the normalised flux and relative abundance of the top 50 genera in all the experimental groups and the control group are shown in Fig. 8 . The relative abundance of Methylobacterium (a typical disinfection-resistance and biofouling-related genus 23 , 35 ) was negatively correlated with the normalised flux of the fouled RO membrane (p < 0.05). The relative abundances of two kinds of high EPS-secreting bacteria, Microbacterium and Pseudomonas , were also negatively correlated with the normalised flux. These two genera consist of typical chlorine-resistant and highly secretory bacteria. Hence, Methylobacterium , Microbacterium , and Pseudomonas deserve special attention as they play an essential role in the aggravated biofouling potential after disinfection. Additionally, we calculated the accumulative relative abundance of typical highly secretory or biofouling-related genera reported in the literature, including Pseudomonas , Sphingomonas , Acinetobacter , Methylobacterium , Sphingobium , and Ralstonia 23 – 25 , 39 , 40 , 44 (Fig. 9 ). The cumulative relative abundance of these bacteria in the “aggravated” group, namely the FES, UV-, NaClO-, and ClO 2 - DRB biofilms was significantly higher than that in the control group (~ 5%). Remarkably, the highly secretory bacteria accounted for over half of the total bacteria in the DRB biofilms of FES and NaClO, that is 56.4% and 53.0%, respectively. In contrast, the DRB biofilm of the “alleviated” group possessed a similar proportion of highly secretory bacteria as that of the control group. The decrease in bacterial numbers after disinfection could explain biofouling alleviation. Therefore, variation in the relative abundance of typical highly secretory and biofouling-related genera was the main reason for the change in biofouling potentials after different disinfection processes. 4. Conclusions In the long-term experiment (32 days), K 2 FeO 4 and O 3 alleviated biofouling of the DRB biofilm. Biofouling degree of the NH 2 Cl-DRB biofilm was similar to that of the control group, whereas the other four types of disinfection aggravated membrane biofouling. Furthermore, as ferrate introduced iron flocs and aggravated inorganic scaling in RO systems, O 3 was recommended as a practical approach to prevent biofouling in RO systems. Morphological analysis combined with the DOM/HPC ratio explained the difference in the fouling behaviour of the DRB biofilms. A high DOM/HPC ratio along with denser and thicker DRB biofilms led to severe biofouling. Community analysis revealed that the selection effect of disinfection on typical highly secretory and biofouling-related genera was the primary reason for biofouling aggravation. Typical genera included Pseudomonas , Sphingomonas , Acinetobacter , Methylobacterium , Sphingobium , and Ralstonia . Declarations Acknowledgements This study was supported by the Key Program of the National Natural Science Foundation of China (No. 51738005) and the National Natural Science Foundation of China (No. 52000114). Competing interests We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Author Contributions Hao-Bin Wang : Conceptualization, Methodology, Original draft preparation. Yin-Hu Wu : Writing- Reviewing and Editing, Project administration, Funding acquisition, Supervision. Wen-Long Wang : Writing- Reviewing and Editing. Li-Wei Luo : Conceptualization. Gen-Qiang Chen : Conceptualization. Zhuo Chen : Writing- Reviewing and Editing Writing. Song Xue : Formal analysis. 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1","display":"","copyAsset":false,"role":"figure","size":83358,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the experimental procedure.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1811963/v1/ea3496bfe9c1489999af2b35.png"},{"id":23830352,"identity":"ce361a05-254e-4a11-a6d6-c1d17a86863b","added_by":"auto","created_at":"2022-07-13 19:08:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94289,"visible":true,"origin":"","legend":"\u003cp\u003eNormalised flux (a) and biofouling layer resistance (b) of RO membranes with DRB biofilm after seven different kinds of disinfection 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DRB biofilms.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1811963/v1/b883bda5d73e4e9a2da4f761.png"},{"id":23830353,"identity":"a8cafd30-4256-4552-ada9-d2a78a75d6b8","added_by":"auto","created_at":"2022-07-13 19:08:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":80599,"visible":true,"origin":"","legend":"\u003cp\u003eThickness of DRB biofilms measured with laser scanning confocal microscope (LSCM).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1811963/v1/5d5d188c84941990a09e8362.png"},{"id":23829747,"identity":"673da142-3313-4097-b50d-a84c7b7dc93b","added_by":"auto","created_at":"2022-07-13 19:03:37","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1054335,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscope (SEM) images of DRB biofilms developed on the surface of RO membranes.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1811963/v1/866080d9d51aa8925734e651.jpeg"},{"id":23829743,"identity":"bb978848-5755-412e-b7e8-1acd44c0dbe0","added_by":"auto","created_at":"2022-07-13 19:03:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":112880,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of microbial communities of the biofilm on the RO membranes\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-1811963/v1/71d729478cc4b82486ba18df.png"},{"id":23830354,"identity":"c78a911c-7962-46f0-ab06-b06e49590d4a","added_by":"auto","created_at":"2022-07-13 19:08:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":276607,"visible":true,"origin":"","legend":"\u003cp\u003eHeat maps of the correlation coefficient between normalised flux and relative abundance of the top 50 genera. ***, **, and * represent p\u0026lt;0.01, p\u0026lt;0.05, and p\u0026lt;0.1, respectively.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-1811963/v1/60c5f7ea6aec905fbcb158e1.png"},{"id":23829748,"identity":"f8f5a3c9-0910-430b-b8fd-b7c82551eb22","added_by":"auto","created_at":"2022-07-13 19:03:38","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":152314,"visible":true,"origin":"","legend":"\u003cp\u003eAccumulative relative abundance of typical highly secretory or biofouling-related genera reported by previous research (a) and its correlation with normalized flux drop of RO membranes (b).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-1811963/v1/0c52a3813f54cfef2237ceca.png"},{"id":34628840,"identity":"c936ef4c-f854-4f96-a124-0e39d4dc7ee7","added_by":"auto","created_at":"2023-03-22 07:08:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1747258,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1811963/v1/a46ed1cd-39f3-43c6-86fb-9b6a8bc978f5.pdf"},{"id":23829744,"identity":"666eba11-fc9e-47a8-b2a3-1dd650d0512c","added_by":"auto","created_at":"2022-07-13 19:03:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4007989,"visible":true,"origin":"","legend":"SUPPLEMENTAL MATERIAL","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-1811963/v1/f382e25bf60ef4b07177e840.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Evaluation of biofouling characteristics on reverse osmosis membranes of disinfection-residual-bacteria (DRB) after seven kinds of disinfection","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWater scarcity is a pressing global challenge \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The accelerated carbon neutrality process worsens water shortages \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Meanwhile, countries and regions continue to face water contamination \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Water reclamation is a win-win strategy for increasing freshwater supply and shortening the water footprint of human beings \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eReverse osmosis (RO) is one of the most applicable and stable units for high-quality reclaimed water production for industrial reuse, potable reuse, and groundwater replenishment \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Many large-scale water reclamation plants have been successively operated \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, RO system has a fatal defect, that is, the unmanageable membrane fouling \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Membrane fouling of RO mainly includes scaling, colloidal fouling, organic fouling, and biofouling \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Among these, biofouling is the most complicated and uncontrollable, although numerous studies have made significant efforts to reduce it \u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDisinfection is a widely applied pretreatment process used to deal with biofouling in RO systems. However, it may lead to undesirable effects \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. After reducing the number of bacteria in the feed water, the disinfection process exerts a salient selection effect on the bacterial community and the extracellular polymeric substance (EPS) secreting ability of the bacteria. Some unwanted bacteria, that are resistant to disinfection or can adapt to adverse environments, might survive disinfection processes and become disinfection-residual-bacteria (DRB) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. DRB might possess a higher proportion of bacteria with higher EPS secreting ability, leading to a more severe biofouling of RO membranes, especially in the long-term operation of RO systems \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Research in laboratory and full-scale water treatment plants has shown the probability of aggravated biofouling after disinfection \u003csup\u003e11,23\u0026minus;25\u003c/sup\u003e. However, most of these studies are limited to a single type of disinfection process \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. To date, there is still a lack of systematic and broad comparison of the biofouling-control effects of various disinfection methods.\u003c/p\u003e \u003cp\u003eTo provide more constructive and reliable guidance on the prevention of RO biofouling, this study compared the DRB characteristics of seven different disinfection methods, including five widely used disinfection methods (NaClO, NH\u003csub\u003e2\u003c/sub\u003eCl, ClO\u003csub\u003e2\u003c/sub\u003e, UV, and O\u003csub\u003e3\u003c/sub\u003e) and two novel disinfection methods (K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e and flow-through electrode system (FES) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e) via a long-term (32 days) biofilm cultivation experiment on RO membranes. Furthermore, the bacteria and organic matter in the biofilm on the RO membranes were analysed using heterotrophic plate count (HPC), adenosine triphosphate (ATP), dissolved organic matter (DOM), and fluorescence excitation-emission matrix (EEM) to determine the primary reasons for the differences in biofouling in each group. The microbial community structure of DRB biofilms was analysed using high-throughput sequencing. Based on these results, the correlations between the fouling characteristics of DRB biofilms and bacterial density, organic density, biofilm morphology, and the microbial community of DRB were analysed.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Water samples\u003c/h2\u003e \u003cp\u003eReclaimed water was sampled from a large-scale water reclaimed plant in Beijing, China. A schematic of the advanced treatment process is shown in Fig. S1. The effluent from the denitrification filter was chosen as the sample because subsequent treatment units had a bacterial removal effect \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The reclaimed water samples were transported to the laboratory within 1 h, then filtered by filter papers to remove particles, and kept at 4\u0026deg;C before disinfection. Water quality parameters of the sampled water were measured as soon as they arrived at the laboratory, and are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical and physical properties of water samples before disinfection.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTDS (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTN (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTP (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTOC (mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e579\u0026thinsp;\u0026plusmn;\u0026thinsp;27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Disinfection and biofilm culture\u003c/h2\u003e \u003cp\u003eFive commonly used disinfection methods, including NaClO, NH\u003csub\u003e2\u003c/sub\u003eCl, ClO\u003csub\u003e2\u003c/sub\u003e, UV, and O\u003csub\u003e3\u003c/sub\u003e, and two novel disinfection technologies, namely K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e and a flow-through electrode system (FES), were compared in this study. The steps followed in the experiment are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Briefly, water samples were filtered using filter paper (medium speed, Newstar, Hangzhou, China) to remove large flocs before disinfection. Seven types of disinfection processes were conducted by following the technical parameters described in preliminary experiments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) to achieve similar bacterial log removal, except for two novel disinfection methods as they could not achieve a high disinfection effect in actual wastewater. Square-wave alternating pulse current FES devices were set up based on a previous study \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The voltage amplitude and hydraulic retention time were set at 4 V and 27.7 s, respectively, to achieve the best disinfection performance of the system. UV irradiation was performed using a laboratory-scale collimated light-beam apparatus \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Other oxidizing disinfection processes were performed in sterilised glass bottles at 25\u0026deg;C and 150 rpm. The reaction was quenched with Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e solution to avoid oxidative damage to the RO membrane.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDosage of each disinfection method.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisinfection method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFree chlorine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChloramine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChlorine dioxide\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUV\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDosage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 mg/L 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 mg/L 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 mg/L 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 mJ/cm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisinfection method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOzone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFerret\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDosage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 mg/L 10 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 mg/L 10 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eE\u0026thinsp;=\u0026thinsp;4 V, T\u0026thinsp;=\u0026thinsp;27.7 s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAromatic polyamide composite LP100 RO membrane (Vorton, China) was cut into round coupons (d\u0026thinsp;=\u0026thinsp;32 mm, S\u0026thinsp;=\u0026thinsp;804 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e), and the pretreatment procedure was conducted based on a previous study \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. For biofilm culturing, the membrane coupon was soaked in 18 mL of disinfected water or control samples in a 5 cm sterilised round plastic Petri dish at 25\u0026deg;C. Water samples were refreshed daily. After 32 days of culture, the membrane was gently rinsed twice with phosphate buffer saline (PBS) to remove suspending bacteria for sequencing analysis. The experiments were conducted in triplicates for each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Evaluation of the disinfection effect\u003c/h2\u003e \u003cp\u003eThe disinfection effect was evaluated based on the concentration of culturable bacteria; ATP was tested before the biofilm culture experiments with triplicate experiments immediately after the disinfection processes were completed. Culturable bacteria were measured by HPC via colony-forming unit (CFU) counting \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. ATP was tested using luminescence analysis. For total ATP measurement, 100 \u0026micro;L of the bacterial suspension and 100 \u0026micro;L of CellTiter-Glo Luminescent Cell Viability Assay (Promega, USA) were mixed in 96-well plates. After incubation at 25\u0026deg;C and at 150 rpm in the dark for 1 min, luminescence intensity was measured using a microplate reader (SpectraMax M5, Molecular Devices, USA). For the extracellular ATP test, water samples were filtered through a 0.1 \u0026micro;m membrane (Millipore) to remove bacteria; the subsequent steps were the same as those for total ATP. Intracellular ATP concentration was calculated by subtracting the extracellular ATP concentration from the total ATP concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 RO cross-flow unit and membrane performance tests\u003c/h2\u003e \u003cp\u003eA laboratory-scale cross-flow RO system was used to evaluate the performance of RO membranes before and after biofilm growth \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Briefly, the membrane compaction phase was performed with a 30 min rinse of ultra-pure water at 1.2 MPa until the permeate flux was stable. The flow rate of the influent was set at 1.0 mL/min via a constant flow pump (NPL-100). The feed water was then switched to a 500 mg/L NaCl solution (conductivity of approximately 1000 \u0026micro;s/cm). The permeate flux was recorded after reaching a constant value. The normalised flux was calculated by dividing the flux of the fouled membrane by the clean membrane before biofilm growth.\u003c/p\u003e \u003cp\u003eThe resistance of the RO membrane was calculated, as follows:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({R}_{b}={R}_{T}-{R}_{m}-{R}_{P}=\\frac{\\varDelta P}{\\sigma J}-{R}_{m}-{R}_{P}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhere, Δ\u003cem\u003eP\u003c/em\u003e is the transmembrane pressure, \u003cem\u003eσ\u003c/em\u003e is the kinetic viscosity of water, and \u003cem\u003eJ\u003c/em\u003e is the flux. The resistance of the biofilm \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e is calculated by subtracting the resistance of the virgin membrane \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and the resistance of the concentration polarisation \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e from the resistance of the fouled membrane \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Biofilm analysis\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Heterotrophic plate counts (HPC) and microbial activity\u003c/h2\u003e \u003cp\u003eMicrobial amount and activity were determined using HPC and ATP concentrations. A piece of 10 mm\u0026times;10 mm fouled membrane was cut off and vortexed in 0.5 mL normal saline for 30 s. HPC and ATP concentrations were tested using the same procedure, as described in Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Organic matter analysis\u003c/h2\u003e \u003cp\u003eDOM and EEM were applied to reflect the characteristics of organic matter in DRB biofilms \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Briefly, a piece of 10 mm\u0026times;10 mm fouled membrane was cut and shaken in 5 mL NaOH solution (pH 12) for 24 h at 25\u0026deg;C and 150 rpm. Then, HCl solution (pH 2) was added to adjust the pH of the solution to 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2. After neutralisation, the volume of the solution was adjusted to 15 mL by adding ultrapure water. The solution was filtered through a 0.45-\u0026micro;m nylon membrane (Whatman, England) before total organic carbon (TOC) measurement on a TOC-5000A analyser. EEM spectra were recorded using a fluorescence spectrophotometre (F-7100, Hitachi). The EEM spectrum was divided into six zones for integration. The types of fluorescent substances in each zone are shown in Table S1 \u003csup\u003e24,33\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Surface morphology of the fouled membrane\u003c/h2\u003e \u003cp\u003eA laser scanning confocal microscope (LSCM, LSM710META) was used to measure the thickness of the biofilm on the RO membrane. A 5 \u0026times;10 mm membrane with the DRB biofilm of each sample was cut for LSCM observation. The staining groups of the fluorescent dyes and their targets are listed in Table S2.\u003c/p\u003e \u003cp\u003eThe surface morphology of the DRB biofilm was examined using field-emission scanning electron microscopy (FESEM, SU8220, Hitachi, Japan) and the accelerating voltage was set to 5 kV. A piece of 5 \u0026times; 5 mm membrane coupons was cut for observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4 Microbial community analysis\u003c/h2\u003e \u003cp\u003eThree pieces of 40 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e membrane coupons were cut for microbial community analysis. Microbial community analysis was conducted as previously described \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Briefly, DNA was extracted using the E.Z.N.A.\u0026reg; soil DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.). Furthermore, 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were used as primer pairs for amplification. Sequencing was performed using an Illumina MiSeq PE300 platform. All analysed sequences were submitted to the NCBI SRA database under accession number PRJNA803872. Pathogen identification was based on the Virulence Factor Database (VFDB) from the Institute of Pathogen Biology, CAMS \u0026amp; PUMC.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effects of DRB biofilm on the RO membrane performance\u003c/h2\u003e \u003cp\u003eThe effects of different disinfection methods on the RO membrane flux of the DRB biofilms were investigated. Not all disinfection methods can control biofouling. These methods were divided into three groups, according to the normalised flux (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The first group, classified as \u0026ldquo;alleviated\u0026rdquo;, included K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e and O\u003csub\u003e3\u003c/sub\u003e. Their DRB performed light normalised flux drop (22.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% and 23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17%, respectively) compared to the control group (without disinfection pre-treatment), thereby indicating alleviated biofouling potentials. Meanwhile, the DRB of NH\u003csub\u003e2\u003c/sub\u003eCl had a similar biofouling degree as the control group (~\u0026thinsp;27%), and was classified as \u0026ldquo;equal\u0026rdquo;. Finally, the DRB of the \u0026ldquo;aggravated\u0026rdquo; group, which included FES, UV, NaClO, and ClO\u003csub\u003e2\u003c/sub\u003e, caused more biofouling of the RO membrane than the control group. Notably, the biofouling of ClO\u003csub\u003e2\u003c/sub\u003e-DRB was the most severe, with a flux drop percentage of 35.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0%. The two most frequently used disinfection methods, namely UV and NaClO significantly aggravated the biofouling of RO membranes, matching the results of long-term studies in previous research \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe hydraulic resistance of the DRB biofilms (\u003cem\u003eRm\u003c/em\u003e) is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. The resistance of the K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e-DRB biofilm (2.48\u0026times;10\u003csup\u003e13\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was the lowest as it had the least impact on the RO membrane flux drop. Also, the resistance of the O\u003csub\u003e3\u003c/sub\u003e-DRB biofilm (2.71\u0026times;10\u003csup\u003e13\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the \u0026ldquo;alleviated\u0026rdquo; group was lower than that of the control group (3.07\u0026times;10\u003csup\u003e13\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The resistance of the NH\u003csub\u003e2\u003c/sub\u003eCl-DRB biofilm (3.48\u0026times;10\u003csup\u003e13\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was slightly higher than that of the control group. The DRB biofilm resistance of all the \u0026ldquo;aggravated\u0026rdquo; groups was much higher than that of the control group. Among them, the average resistance of NaClO was the highest, at 5.18\u0026times;10\u003csup\u003e13\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. However, it had a relatively high within-group error, corresponding to the membrane flux drop shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Disinfection effect of seven kinds of disinfection methods\u003c/h2\u003e \u003cp\u003eAlleviation of biofouling after disinfection might be associated with lower bacterial concentrations in the feed water after disinfection \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, variations in the concentrations of HPC and ATP in the water samples during the disinfection processes were tested (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The five conventional disinfection methods achieved a bacterial inactivation rate of 3-lg, while K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e and FES exhibited a relatively low inactivation effect (~\u0026thinsp;1-lg). Overall, HPC had no monotonous correlation with biofouling potential. The DRB biofilm of K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e caused only a 22% decrease in membrane flux, although the inactivation effect of K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e was the lowest (\u0026lt;\u0026thinsp;1-lg). NaClO was the most effective method for bacterial inactivation. However, its DRB aggravated biofouling at an average relative flux drop of ~\u0026thinsp;35%.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDisinfection effect measured by HPC and ATP content.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisinfection methods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPC (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInactivation rate (lg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eATP (ng/mL)\u003c/p\u003e \u003cp\u003e(Total)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eATP (ng/mL)\u003c/p\u003e \u003cp\u003e(Intracellular)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1*10^5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2*10^4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5*10^2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e2\u003c/sub\u003eCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3*10^1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1*10^4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8*10^2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaClO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0*10^1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1*10^2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\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\u003ePrevious studies have reported that ATP can be an effective indicator for predicting biofouling potentials in the short- or medium-term operation of RO systems (less than 16 days) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, this conclusion is not valid in long-term experiments (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The intracellular ATP in the control group was 2.90 ng/mL, accounting for 97% of the total ATP. Total and intracellular ATP concentrations increased after UV treatment, partly because of the rapid DNA repair mechanism triggered by UV radiation. No correlation was found between the DRB biofouling potentials (membrane flux drop) and the ATP concentration of DRB. Evaluation of the disinfection effect suggested that the amount and the activity of DRB were not the decisive factors of biofouling potential during long-term operation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Characteristics of the DRB biofilm on RO membranes\u003c/h2\u003e \u003cp\u003eTo determine the reasons for varying performances of RO membranes fouled by different DRB biofilms, this study dissected the fouled RO membranes and performed a series of tests. The number of bacteria and organics in the DRB biofilms, the morphological characteristics of DRB biofilms, and the bacterial community structure were analysed.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Bacteria and dissolved organic matters (DOM) in the DRB biofilms\u003c/h2\u003e \u003cp\u003eThe live bacterial density of the DRB biofilm was measured using HPC, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. DRB biofilms of NH\u003csub\u003e2\u003c/sub\u003eCl, FES, and UV possessed the maximum live bacteria density (approximately 10\u003csup\u003e6\u003c/sup\u003e CFU/cm\u003csup\u003e2\u003c/sup\u003e). The live bacterial density of NaClO- and ClO\u003csub\u003e2\u003c/sub\u003e-DRB biofilms were lower than that of the control group (approximately 10\u003csup\u003e5\u003c/sup\u003e CFU/cm\u003csup\u003e2\u003c/sup\u003e), but they led to the worst flux drop in the RO membrane. Hence, the live bacterial density of the DRB biofilm was not a key factor leading to differences in biofouling degrees. This conclusion is consistent with previous studies \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnlike bacterial cells, the extracellular polymeric substances (EPS) on RO membranes have a more direct relationship with flux drop \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Hence, the total amount of organics in the DRB biofilms was measured by DOM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), and the different component of DOM was tested using EEM (Fig. S2). The amount of DOM did not show a clear correlation with the biofouling degree. The DOM of K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e, which was in the \u0026ldquo;alleviated\u0026rdquo; group, was much higher than that of the \u0026ldquo;aggravated\u0026rdquo; group. Therefore, the total amount of organics did not play a key factor in the degree of DRB biofilm fouling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering that the ratio of EPS to bacteria could affect the biofouling degree \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, this study calculated the ratio of DOM to HPC in the DRB biofilms (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The DOM/HPC ratio in the control group was approximately 4 mg/10\u003csup\u003e7\u003c/sup\u003e live cells. The DRB biofilm of K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e, O\u003csub\u003e3\u003c/sub\u003e, NH\u003csub\u003e2\u003c/sub\u003eCl, FES, and UV possessed a relatively lower DOM/HPC ratio, while those of NaClO and ClO\u003csub\u003e2\u003c/sub\u003e (5.4 and 7.6 mg/10\u003csup\u003e7\u003c/sup\u003e live cell, respectively) were significantly higher than the control group. This implied that these two disinfectants could aggravate biofouling of the RO membrane by reshaping the microbial community and changing the EPS production capacity of the residual bacteria.\u003c/p\u003e \u003cp\u003eThe proportions of DOM components among all the groups were similar (Fig. S2). Tyrosine/tryptophan amide (Zone Ⅰ) and protein-containing tyrosine/tryptophan (Zone II) were the dominant components of fluorescent organic matter in each DRB biofilm, indicating that amino acids and proteins were predominant in the DRB biofilms, compared with polysaccharides, fulvic acids, or humid acids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Morphological characteristics of DRB biofilms\u003c/h2\u003e \u003cp\u003eCompared to the bacterial and organic densities in the biofilm, the degree of biofouling was more closely correlated with the thickness and consecutiveness of the biofilm on RO membranes. The thickness of the DRB biofilm was tested using z-stack images of the LSCM. The surface image of the DRB biofilm showed that bacteria, proteins, and α-/β-polysaccharides were evenly distributed in the DRB biofilm (Fig. S3). The DRB biofilm of K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e was relatively loose in section view, while the others were consecutive (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The average thickness of the DRB biofilm was measured via cross-section (Fig. S4) and is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. As a result, the DRB biofilm of the UV group (55\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026micro;m) was significantly thicker than that of the control group (33\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026micro;m). The DRB biofilm thickness of the \u0026ldquo;alleviated\u0026rdquo; group was approximately 22 \u0026micro;m, which was the lowest of all the groups. The biofilm thickness partly illustrated the difference in the biofouling degree, supplemented by the DOM/HPC ratio. The two disinfection methods in the \u0026ldquo;alleviated\u0026rdquo; group caused a low DOM/HPC ratio and thinner biofilm on the RO membrane, leading to a marginal flux drop of the fouled membrane. The UV DRB developed a thick biofilm and led to severe biofouling of the RO membrane. The biofilms of NaClO and ClO\u003csub\u003e2\u003c/sub\u003e were not very thick. However, the high DOM/HPC ratio in the biofilm could narrow the water channels between bacterial cells \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, leading to the highest flux drop in the RO membrane.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe FESEM images of the DRB biofilm are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The DRB biofilm fully covered all the RO membranes. However, the compactness of the biofilms was different. The UV and NaClO-DRB biofilms were compact and continuous, partly illustrating their severe biofouling performance. In contrast, there were gaps between the bacteria and the EPS matrix in the DRB biofilm of the remaining groups. Regular crystals containing Fe were observed in the K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e group (Fig. S5), indicating that K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e could cause scaling of the RO membrane.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Microbial community analysis of DRB biofilm on RO membranes\u003c/h2\u003e \u003cp\u003eDisinfection processes can exert three levels of change on the bacteria: metabolic change of a single bacteria, shift in the microbial community, and variation of nutrient conditions \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Among them, a shift in the microbial community is most likely to affect the biofouling degree during long-term operations \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Therefore, we analysed the alpha and beta diversities of the DRB microbial community.\u003c/p\u003e \u003cp\u003eThe alpha diversity indices of the observed species as well as the ACE, Chao1, and Shannon indices of bacteria in the DRB biofilm are listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The community richness and evenness of the control group were the highest, followed by K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e (\u0026ldquo;alleviated\u0026rdquo; group), which has been reported to have minimal impact on the bacterial community \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The community evenness of the ClO\u003csub\u003e2\u003c/sub\u003e-DRB biofilm (\u0026ldquo;aggravated\u0026rdquo; group) was the lowest, indicating a significant selection effect of ClO\u003csub\u003e2\u003c/sub\u003e. Overall, community richness and evenness did not show a monotonous correlation with the biofouling behaviour of DRB biofilms.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical table of Alpha diversity index.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisinfection methods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eObserved species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChao1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShannon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSimpson\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e864.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1004.9\u0026thinsp;\u0026plusmn;\u0026thinsp;22.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1004.1\u0026thinsp;\u0026plusmn;\u0026thinsp;34.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.052\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e445.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e539.9\u0026thinsp;\u0026plusmn;\u0026thinsp;18.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e544.0\u0026thinsp;\u0026plusmn;\u0026thinsp;29.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.066\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e135.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e184.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e166.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.146\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e2\u003c/sub\u003eCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e164.3\u0026thinsp;\u0026plusmn;\u0026thinsp;26.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e307.8\u0026thinsp;\u0026plusmn;\u0026thinsp;119.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e230.6\u0026thinsp;\u0026plusmn;\u0026thinsp;66.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.132\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e296.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e459.3\u0026thinsp;\u0026plusmn;\u0026thinsp;65.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e399.8\u0026thinsp;\u0026plusmn;\u0026thinsp;22.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.286\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e221.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e464.1\u0026thinsp;\u0026plusmn;\u0026thinsp;63.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e327.5\u0026thinsp;\u0026plusmn;\u0026thinsp;29.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.119\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaClO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e261.3\u0026thinsp;\u0026plusmn;\u0026thinsp;25.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e283.1\u0026thinsp;\u0026plusmn;\u0026thinsp;20.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e283.7\u0026thinsp;\u0026plusmn;\u0026thinsp;17.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.240\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e94.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e167.2\u0026thinsp;\u0026plusmn;\u0026thinsp;82.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e125.2\u0026thinsp;\u0026plusmn;\u0026thinsp;37.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.191\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\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\u003eA Venn diagram of the OTUs that appeared in each group is shown in Fig. S6. Only 36 OTUs were shared by all the groups. The control group had the highest OTUs, indicating that each disinfection method had a selection effect. Besides the control group, the number of OTUs in the K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e-DRB biofilm was the highest. High numbers of OTUs caused fierce competition among the species, and inhibited biofilm growth and EPS secreting, resulting in the least biofouling of the K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e-DRB biofilm. The DRB biofilm with ClO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e3\u003c/sub\u003e disinfection possessed the lowest OTUs, indicating that the two aforementioned oxidising disinfection methods had strong selectivity for the bacteria \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, as their biofouling performance differed, ClO\u003csub\u003e2\u003c/sub\u003e selected more biofilm formation and EPS-secreting species than O\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe microbial community structure at the phylum level is shown in Fig. S7a; class and genus levels are shown in Fig. S8. α-Proteobacteria and γ-Proteobacteria were the dominant classes in all the DRB biofilms. Actinobacteria and Bacteroidetes were the second and third most abundant phyla, respectively. The top three phyla accounted for 90% of all bacteria. A heat map at the genus level is shown in Fig. S7b.\u003c/p\u003e \u003cp\u003ePreviously reported biofouling-related genera had significantly higher relative abundances in the \u0026ldquo;aggravated\u0026rdquo; group, namely, ClO\u003csub\u003e2\u003c/sub\u003e, NaClO, UV, and FES. For instance, \u003cem\u003eMethylobacterium\u003c/em\u003e, which is a typical disinfection-resistant and biofouling-related bacteria genus \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, was dominant in the DRB biofilm of FES and ClO\u003csub\u003e2\u003c/sub\u003e with a relative abundance of 54.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3% and 28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7%, respectively. \u003cem\u003eSphingobium\u003c/em\u003e, a highly secretory genus \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, was found to dominate the DRB biofilm under UV (30.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%). In addition, the relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003e was significantly higher in the NaClO-DRB biofilm than in the other groups (45.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%). \u003cem\u003ePseudomonas\u003c/em\u003e, which is a typical DRB of chlorine \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e causes biofouling of RO membranes \u003csup\u003e23,40\u0026minus;42\u003c/sup\u003e. In contrast, the relative abundance of these genera was significantly lower in the K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e-and O\u003csub\u003e3\u003c/sub\u003e-DRB biofilms (\u0026lt;\u0026thinsp;10%). Thus, the relative abundance of highly secretory or biofouling-related genera plays a decisive role in the biofouling potential of DRB during long-term operation.\u003c/p\u003e \u003cp\u003eA community structure similarity analysis was performed using the PCA algorithm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The community structures of all the disinfection groups were significantly different from those of the control group. The bacterial community structures of UV-, O\u003csub\u003e3\u003c/sub\u003e-, NH\u003csub\u003e2\u003c/sub\u003eCl-, NaClO-, and K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e-DRB biofilms were similar, whereas the community structures of ClO\u003csub\u003e2\u003c/sub\u003e and FES DRB biofilms were similar. This may be the cause of the inability of these two disinfectants to control the highly secretory genus \u003cem\u003eMethylobacterium\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eApart from biofouling of RO membranes, the DRB biofilm can act as a shelter for pathogenic bacteria, leading to health risks of RO concentrate \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Thus, the cumulative abundance of pathogens and opportunistic pathogens in the DRB biofilm was analysed and shown in Fig. S9. The relative abundance of pathogenic bacteria in the DRB biofilm increased significantly after disinfection with NaClO and ClO\u003csub\u003e2\u003c/sub\u003e, indicating that these two chlorine-containing oxidative disinfectants selected (opportunistic) pathogens. K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e, FES, and NH\u003csub\u003e2\u003c/sub\u003eCl partially reduced the relative abundance of (opportunistic) pathogens. Furthermore, their abundance in DRB biofilms of O\u003csub\u003e3\u003c/sub\u003e and UV were the lowest, demonstrating that they controlled the spread of (opportunistic) pathogens in biofilms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Correlation between microbial community structure and RO membrane flux\u003c/h2\u003e \u003cp\u003eTo identify the key genera in the microbial community of DRB affecting the RO membrane flux, correlation coefficients between the normalised flux and relative abundance of the top 50 genera in all the experimental groups and the control group are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The relative abundance of \u003cem\u003eMethylobacterium\u003c/em\u003e (a typical disinfection-resistance and biofouling-related genus \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e) was negatively correlated with the normalised flux of the fouled RO membrane (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The relative abundances of two kinds of high EPS-secreting bacteria, \u003cem\u003eMicrobacterium\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, were also negatively correlated with the normalised flux. These two genera consist of typical chlorine-resistant and highly secretory bacteria. Hence, \u003cem\u003eMethylobacterium\u003c/em\u003e, \u003cem\u003eMicrobacterium\u003c/em\u003e, and \u003cem\u003ePseudomonas\u003c/em\u003e deserve special attention as they play an essential role in the aggravated biofouling potential after disinfection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, we calculated the accumulative relative abundance of typical highly secretory or biofouling-related genera reported in the literature, including \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e, \u003cem\u003eMethylobacterium\u003c/em\u003e, \u003cem\u003eSphingobium\u003c/em\u003e, and \u003cem\u003eRalstonia\u003c/em\u003e \u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The cumulative relative abundance of these bacteria in the \u0026ldquo;aggravated\u0026rdquo; group, namely the FES, UV-, NaClO-, and ClO\u003csub\u003e2\u003c/sub\u003e- DRB biofilms was significantly higher than that in the control group (~\u0026thinsp;5%). Remarkably, the highly secretory bacteria accounted for over half of the total bacteria in the DRB biofilms of FES and NaClO, that is 56.4% and 53.0%, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, the DRB biofilm of the \u0026ldquo;alleviated\u0026rdquo; group possessed a similar proportion of highly secretory bacteria as that of the control group. The decrease in bacterial numbers after disinfection could explain biofouling alleviation. Therefore, variation in the relative abundance of typical highly secretory and biofouling-related genera was the main reason for the change in biofouling potentials after different disinfection processes.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn the long-term experiment (32 days), K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e and O\u003csub\u003e3\u003c/sub\u003e alleviated biofouling of the DRB biofilm. Biofouling degree of the NH\u003csub\u003e2\u003c/sub\u003eCl-DRB biofilm was similar to that of the control group, whereas the other four types of disinfection aggravated membrane biofouling. Furthermore, as ferrate introduced iron flocs and aggravated inorganic scaling in RO systems, O\u003csub\u003e3\u003c/sub\u003e was recommended as a practical approach to prevent biofouling in RO systems.\u003c/p\u003e \u003cp\u003eMorphological analysis combined with the DOM/HPC ratio explained the difference in the fouling behaviour of the DRB biofilms. A high DOM/HPC ratio along with denser and thicker DRB biofilms led to severe biofouling.\u003c/p\u003e \u003cp\u003eCommunity analysis revealed that the selection effect of disinfection on typical highly secretory and biofouling-related genera was the primary reason for biofouling aggravation. Typical genera included \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e, \u003cem\u003eMethylobacterium\u003c/em\u003e, \u003cem\u003eSphingobium\u003c/em\u003e, and \u003cem\u003eRalstonia\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis study was supported by the Key Program of the National Natural Science Foundation of China (No. 51738005) and\u0026nbsp;the National Natural Science Foundation of China (No. 52000114).\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eWe declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eHao-Bin Wang\u003c/strong\u003e: Conceptualization, Methodology, Original draft preparation. \u003cstrong\u003eYin-Hu Wu\u003c/strong\u003e: Writing- Reviewing and Editing, Project administration, Funding acquisition, Supervision. \u003cstrong\u003eWen-Long Wang\u003c/strong\u003e: Writing- Reviewing and Editing. \u003cstrong\u003eLi-Wei Luo\u003c/strong\u003e: Conceptualization. \u003cstrong\u003eGen-Qiang Chen\u003c/strong\u003e: Conceptualization. \u003cstrong\u003eZhuo Chen\u003c/strong\u003e: Writing- Reviewing and Editing Writing. \u003cstrong\u003eSong Xue\u003c/strong\u003e: Formal analysis. \u003cstrong\u003eAo Xu\u003c/strong\u003e: Writing- Reviewing. \u003cstrong\u003eYu-Qing Xu\u003c/strong\u003e: Investigation. \u003cstrong\u003eNozomu Ikuno\u003c/strong\u003e: Writing- Reviewing and Resource. \u003cstrong\u003eKazuki Ishii\u003c/strong\u003e: Resource. \u003cstrong\u003eHong-Ying Hu\u003c/strong\u003e: Project administration, Supervision.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eDNA sequences are available at the NCBI Sequence Read Archive, accession number: PRJNA803872.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGleick, P. 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Biotechnol.\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 2533-2548, doi:10.1007/s00253-015-6439-y (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-clean-water","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjcleanwater","sideBox":"Learn more about [npj Clean Water](http://www.nature.com/npjcleanwater/)","snPcode":"41545","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"npj Clean Water","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Disinfection-residual-bacteria (DRB), Biofilm, Membrane fouling, Microbial community, Disinfection","lastPublishedDoi":"10.21203/rs.3.rs-1811963/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1811963/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiofouling is a critical defect of the reverse osmosis (RO) system. It has been reported that disinfection processes tend to select certain undesirable disinfection-residual bacteria (DRB), leading to severe long-term biofouling potential. To provide constructive guidance on biofouling prevention in RO systems, this study evaluated the biofouling characteristics of RO membranes of DRB after the application of five mature disinfection methods (NaClO, NH\u003csub\u003e2\u003c/sub\u003eCl, ClO\u003csub\u003e2\u003c/sub\u003e, UV, and O\u003csub\u003e3\u003c/sub\u003e) and two novel disinfection methods (K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e and flow-through electrode system (FES)). After a 32-day biofilm cultivation on the RO membranes, the DRB biofilm of K\u003csub\u003e2\u003c/sub\u003eFeO\u003csub\u003e4\u003c/sub\u003e and O\u003csub\u003e3\u003c/sub\u003e caused a slight normalised flux drop (22.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% and 23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7%, respectively) of RO membrane compared with the control group (non-disinfected, ~\u0026thinsp;27% normalised flux drop). Moreover, the biofouling degree of the NH\u003csub\u003e2\u003c/sub\u003eCl-DRB biofilm was similar to that of the control group. The remaining disinfection types aggravated membrane biofouling. The biofouling behaviour of DRB showed no relationship with bacterial concentration or activity. The thickness and density of the biofilms as well as the organics/bacterial number ratio in the DRB biofilm, helped explain the difference in the fouling degree between each group. Moreover, microbial community analysis showed that the relative abundance of typical highly secretory and biofouling-related genera, such as \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e, \u003cem\u003eMethylobacterium\u003c/em\u003e, \u003cem\u003eSphingobium\u003c/em\u003e, and \u003cem\u003eRalstonia\u003c/em\u003e, were the main reasons for the difference in biofouling degree. All types of disinfection effectively prevented pathogen reproduction in the DRB biofilm. However, the relative abundance of (opportunistic) pathogens increased significantly after NaClO and ClO\u003csub\u003e2\u003c/sub\u003e disinfection.\u003c/p\u003e","manuscriptTitle":"Evaluation of biofouling characteristics on reverse osmosis membranes of disinfection-residual-bacteria (DRB) after seven kinds of disinfection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-13 19:03:35","doi":"10.21203/rs.3.rs-1811963/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2022-07-28T14:11:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-07-26T18:33:37+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-07-20T08:46:53+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-07-20T06:28:43+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-07-15T18:46:45+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-07-06T17:45:00+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-07-06T12:05:31+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2022-07-05T20:49:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-04T11:26:36+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2022-07-01T06:00:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-06-30T13:26:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Clean Water","date":"2022-06-30T13:26:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-clean-water","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjcleanwater","sideBox":"Learn more about [npj Clean Water](http://www.nature.com/npjcleanwater/)","snPcode":"41545","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"npj Clean Water","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9dc461af-f4c1-45ac-b091-2b8662a1e494","owner":[],"postedDate":"July 13th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-03-22T07:08:03+00:00","versionOfRecord":{"articleIdentity":"rs-1811963","link":"https://doi.org/10.1038/s41545-023-00240-2","journal":{"identity":"npj-clean-water","isVorOnly":false,"title":"npj Clean Water"},"publishedOn":"2023-03-21 04:00:00","publishedOnDateReadable":"March 21st, 2023"},"versionCreatedAt":"2022-07-13 19:03:35","video":"","vorDoi":"10.1038/s41545-023-00240-2","vorDoiUrl":"https://doi.org/10.1038/s41545-023-00240-2","workflowStages":[]},"version":"v1","identity":"rs-1811963","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1811963","identity":"rs-1811963","version":["v1"]},"buildId":"qQ7_6M8ijIrYJ9CiyUnPg","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}