Inactivation of chlorine-resistant bacteria (CRB) via various disinfection methods: Resistance mechanism and relation with carbon source metabolism.

With the widespread use of chlorine disinfection, chlorine-resistant bacteria (CRB) in water treatment systems have gained public attention. Bacterial chlorine resistance has been found positively correlated with extracellular polymeric substance (EPS) secretion. In this study, we selected the most suitable CRB controlling method against eight bacterial strains with different chlorine resistance among chloramine, ozone, and ultraviolet (UV) disinfection, analyzed the resistance mechanisms, clarified the contribution of EPS to disinfection resistance, and explored the role of carbon source metabolism capacity. Among all the disinfectants, UV disinfection showed the highest disinfection capacity by achieving the highest average and median log inactivation rates for the tested strains. For Bacillus cereus CR19, the strain with the highest chlorine resistance, 40 mJ/cm2 UV showed a 1.90 log inactivation, which was much higher than that of 2 mg-Cl2/L chlorine (0.67 log), 2 mg-Cl2/L chloramine (1.68 log), and 2 mg/L ozone (0.19 log). Meanwhile, the UV resistance of the bacteria did not correlate with EPS secretion. These characteristics render UV irradiation the best CRB controlling disinfection method. Chloramine was found to have a generally high inactivation efficiency for bacteria with high chlorine-resistance, but a low inactivation efficiency for low chlorine-resistant ones. Although EPS consumed up to 56.7% of chloramine which an intact bacterial cell consumed, EPS secretion could not explain chloramine resistance. Thus, chloramine is an acceptable CRB control method. Similar to chlorine, ozone generally selected high EPS-secreting bacteria, with EPS consuming up to 100% ozone. Therefore, ozone is not an appropriate method for controlling CRB with high EPS secretion. EPS played an important role in all types of disinfection resistance, and can be considered the main mechanism for bacterial chlorine and ozone disinfection resistance. However, as EPS was not the main resistance mechanism in UV and chloramine disinfection, CRB with high EPS secretion were inactivated more effectively. Furthermore, carbon source metabolism was found related to the multiple resistance of bacteria. Those with low carbon source metabolism capacity tended to have higher multiple resistance, especially to chlorine, ozone, and UV light. Distinctively, among the tested gram-negative bacteria, in contrast to other disinfectants, chloramine resistance was negatively correlated with EPS secretion and positively correlated with carbon source metabolism capacity, suggesting a special disinfection mechanism.

Comparison of disinfection-residual-bacteria (DRB) after seven different kinds of disinfection: Biofilm formation, membrane fouling and mechanisms.

Membrane fouling is the Achilles' heel of the reverse osmosis (RO) system for high-quality reclaimed water production. Previous studies have found that after the significant selection effect of traditional disinfection, the remaining disinfection-residual bacteria (DRB) may possess more severe biofouling potentials. To provide more constructive advice for the prevention of biofouling, we compared the RO membrane fouling characteristics of DRB after using five commonly used disinfection methods (NaClO, NH2Cl, ClO2, UV, and O3) and two novel disinfection methods (K2FeO4 and the flow-through electrode system (FES)). Compared with the control group (undisinfected, 21.1 % flux drop), the UV-DRB biofilm aggravated biofouling of the RO membrane (23.4 % flux drop), while the FES, K2FeO4, and NH2Cl treatments showed less severe biofouling, with final flux drops of 6.9 %, 8.1 %, and 8.1 %, respectively. Adenosine triphosphate (ATP) was found to be a capable indicator for predicting the biofouling potential of DRB. Systematic analysis showed that the thickness and density of the DRB biofilms were most closely related to the different fouling degree of RO membranes. Moreover, the relative abundance of bacteria with higher extracellular polymeric substance (EPS) secretion levels, such as Pseudomonas and Sphingomonas, was found closely related with the biofouling degree of RO membranes.

Increased risks of antibiotic resistant genes (ARGs) induced by chlorine disinfection in the reverse osmosis system for potable reuse of reclaimed water.

The reverse osmosis (RO) process has been applied extensively in wastewater reclamation for industrial and potable reuse. To prevent biofouling, chlorine disinfection was usually used in pretreatment. However, this study found that chlorine disinfection could significantly increase risks of antibiotic resistant genes (ARGs) in the RO system. With the increase of chlorine concentration from 0 to 5 mg/L, the accumulative relative abundance of 14 common ARGs in the membrane foulants increased by 49.6%. Among these ARGs, tolC, acrA and acrB (resistance to multiple drugs) showed the highest increament after chlorine disinfection. Especially, the relative abundance of tolC in the group with 5 mg/L chlorine increased by 113.3% compared with the control group. These ARGs tended to be enriched in a few bacterial genus, including Candidatus, Thiomonas, Silanimonas, Xanthomonas and Pseudomonas. These results indicated that the foulants on RO membranes might become a potential sink of ARGs. Considering the possibility of membrane breach, the ARGs may contaminate the permeate and bring great biological risks.

Pretreatment for alleviation of RO membrane fouling in dyeing wastewater reclamation.

Adsorption and coagulation were commonly used to alleviate reverse osmosis (RO) membrane fouling caused by dissolved organic matters (DOM), but the effects of changed composition and structure of DOM in dyeing wastewater after adsorption and coagulation on RO membrane fouling have seldom been studied. This study aimed at resolving the mechanism how the RO membrane fouling during dyeing wastewater treatment was alleviated by using adsorption and coagulation. The dyeing wastewater caused serious RO membrane fouling. Pretreatment with granular activated carbon (GAC), polyferric sulfate (PFS) and polyaluminum chloride (PACl) were conducted. It was shown that GAC could remove most of the DOM (95%) and preferred to adsorb protein, hydrophobic neutrals and fluorescent compounds. Both coagulants of PFS and PACl preferred to remove polysaccharides (the removal rate was 9-19% higher than that of DOM), high-MW compounds and these compounds with high fouling potential. Afterwards, the RO membrane fouling potential of the dyeing wastewater was tested. The GAC and PFS performed well to alleviate fouling. After GAC treatment, the decline rate of RO flux was similar to that of raw wastewater after 6-fold dilution. With pretreatment by PFS or PACl, the fouling potential of dyeing wastewater was much lower than that of raw wastewater after diluted to the same DOM content. Changes in polysaccharides content in the DOM had more effects on RO membrane fouling than that of proteins after these pretreatment. Although the DOM changed significantly after pretreatment, the fouling type was still intermediate blocking.

Chlorine-resistant bacteria (CRB) in the reverse osmosis system for wastewater reclamation: Isolation, identification and membrane fouling mechanisms.

Chlorine disinfection is often used as a pretreatment technology to control biofouling of reverse osmosis (RO) membranes. However, previous studies showed that biofouling of the RO system was aggravated after chlorine disinfection. Chlorine-resistant bacteria (CRB) were presumed to be closely related to the aggravation of fouling caused by chlorine disinfection. In order to analyze the membrane fouling mechanisms of CRB, 5 CRB strains were isolated from the surface of fouled RO membranes for wastewater reclamation, and 3 reference bacterial strains, Sphingopyxis soli BM1-1, Pseudomonas aeruginosa PAO1 and Escherichia coli CGMCC1.3373, were selected for comparative study. The chlorine resistance, membrane fouling potential, secretion and adhesion characteristics of these strains were evaluated. Among these isolated strains, 3 strains showed much higher chlorine resistance than PAO1 under the condition of 0.5, 2, 5 mg/L-Cl2, especially Bacillus CR19 and Bacillus CR2. Furthermore, a significant positive correlation was found between membrane fouling potential and chlorine resistance of all the strains in this study. The membrane fouling potential of the above 8 strains increased monotonically with the increase of chlorine resistance (under the condition of 0.5 mg/L-Cl2). Serious fouling caused by extracellular substances was observed in biofouling layers of the strains with high chlorine resistance, which lead to more severe flux decline. Extracellular polymeric substances (EPS) amount per cell was found to be the main factor related to the chlorine resistance as well as the fouling potential. Computational fluid dynamics (CFD) simulation was used to demonstrate the filtration resistance induced by the secretion of EPS. However, CRB with higher EPS amount may not show higher membrane adhesion potential, and thus may not be the dominant strain on the RO membranes before chlorine disinfection. These CRB with high fouling potential but low membrane adhesion potential, such as Bacillus CR19 and Bacillus CR2, may become the dominant bacteria on the membrane surface after chlorine disinfection and thus aggravate membrane fouling significantly.

Risks, characteristics, and control strategies of disinfection-residual-bacteria (DRB) from the perspective of microbial community structure.

The epidemic of COVID-19 has aroused people's particular attention to biosafety. A growing number of disinfection products have been consumed during this period. However, the flaw of disinfection has not received enough attention, especially in water treatment processes. While cutting down the quantity of microorganisms, disinfection processes exert a considerable selection effect on bacteria and thus reshape the microbial community structure to a great extent, causing the problem of disinfection-residual-bacteria (DRB). These systematic and profound changes could lead to the shift in regrowth potential, bio fouling potential, as well as antibiotic resistance level and might cause a series of potential risks. In this review, we collected and summarized the data from the literature in recent 10 years about the microbial community structure shifting of natural water or wastewater in full-scale treatment plants caused by disinfection. Based on these data, typical DRB with the most reporting frequency after disinfection by chlorine-containing disinfectants, ozone disinfection, and ultraviolet disinfection were identified and summarized, which were the bacteria with a relative abundance of over 5% in the residual bacteria community and the bacteria with an increasing rate of relative abundance over 100% after disinfection. Furthermore, the phylogenic relationship and potential risks of these typical DRB were also analyzed. Twelve out of fifteen typical DRB genera contain pathogenic strains, and many were reported of great secretion ability. Pseudomonas and Acinetobacter possess multiple disinfection resistance and could be considered as model bacteria in future studies of disinfection. We also discussed the growth, secretion, and antibiotic resistance characteristics of DRB, as well as possible control strategies. The DRB phenomenon is not limited to water treatment but also exists in the air and solid disinfection processes, which need more attention and more profound research, especially in the period of COVID-19.

Effect of ultraviolet disinfection on the fouling of reverse osmosis membranes for municipal wastewater reclamation.

Membrane fouling is a prominent problem that hinders the stable and efficient operation of the reverse osmosis (RO) system for wastewater reclamation. Previous studies showed that chlorine disinfection, which was commonly used in industrial RO systems as pretreatment, could lead to significant change in microbial community structure and resulted in serious biofouling. In order to prevent biofouling during wastewater reclamation, the effect of ultraviolet (UV) disinfection on RO membrane fouling was investigated and the mechanism was also revealed in this study. With the disinfection pretreatment by UV of 20, 40 and 80 mJ/cm2, the bacteria in the feed water were inactivated significantly with a log reduction of 1.11, 2.55 and 3.61-log, respectively. However, RO membrane fouling aggravated with higher UV dosage. Especially, in the group with the UV dosage of 80 mJ/cm2, the normalized RO membrane flux decreased by 15% compared with the control group after 19-day operation. The morphology of the fouled RO membranes indicated serious biofouling in all groups. The analysis on the microbial amount of the foulants showed that the heterotrophic plate counts (HPC) and ATP content on the fouled RO membranes with and without UV disinfection were at the same level. However, the total organic carbon content of the foulants with the UV dosage of 40 and 80 mJ/cm2 was significantly higher than the control group, with higher content of proteins and polysaccharides as indicated by EEM and FTIR spectrum. Microbial community structure analysis showed that some typical UV-resistant bacteria were selected and remained on the RO membrane after disinfection with high UV dosage, including. These residual bacteria after disinfection with high UV dosage showed higher extracellular polymeric substances (EPS) secretion compared with those without UV disinfection, and thus aggravated RO membrane fouling. Thicker EPS could decrease the transmission of UV rays, and thus bacteria with higher EPS secretion might be selected after UV disinfection.

Effects of microbial inactivation approaches on quantity and properties of extracellular polymeric substances in the process of wastewater treatment and reclamation: A review.

Microbial extracellular polymeric substances (EPS) have a profound role in various wastewater treatment and reclamation processes, in which a variety of technologies are used for disinfection and microbial growth inhibition. These treatment processes can induce significant changes in the quantity and properties of EPS, and altered EPS could further adversely affect the wastewater treatment and reclamation system, including membrane filtration, disinfection, and water distribution. To clarify the effects of microbial inactivation approaches on EPS, these effects were classified into four categories: (1) chemical reactions, (2) cell lysis, (3) changing EPS-producing metabolic processes, and (4) altering microbial community. Across these different effects, treatments with free chlorine, methylisothiazolone, TiO2, and UV irradiation typically enhance EPS production. Among the residual microorganisms in EPS matrices after various microbial inactivation treatments, one of the most prominent is Mycobacterium. With respect to EPS properties, proteins and humic acids in EPS are usually more susceptible to treatment processes than polysaccharides. The affected EPS properties include changes in molecular weight, hydrophobicity, and adhesion ability. All of these changes can undermine wastewater treatment and reclamation processes. Therefore, effects on EPS quantity and properties should be considered during the application of microbial inactivation and growth inhibition techniques.

Metagenomics analysis of the key functional genes related to biofouling aggravation of reverse osmosis membranes after chlorine disinfection.

Chlorine disinfection is a common technology to control biofouling in the pretreatment of the reverse osmosis (RO) system for wastewater reclamation. However, chlorine disinfection could even aggravate the RO membrane biofouling because of the changes of microbial community structure. In this study, the mechanism of biofilm formation and EPS secretion after chlorine disinfection was investigated by analyzing the genes coding quorum sensing, exopolysaccharide biosynthesis, and amino acid biosynthesis. After 1, 5, and 15 mg-Cl2/L chlorine disinfection, the relative abundances of the functional genes all increased significantly. Compared with the control group, chlorine-resistant bacteria (Acidovorax, Arenimonas, and Pseudomonas) also harbored higher relative abundances of these functional genes. The high relative abundances of these genes might provide the bacterial community after chlorine disinfection with high potential of biofilm formation and EPS secretion and then cause severe RO membrane biofouling. In the sample with 5 mg-Cl2/L chlorine disinfection, the correlation coefficients (r) between each two of the three kinds of functional genes were more than 0.9 and much stronger than that in the control group. These results indicated that the bacterial community selected by chlorine disinfection could build more stable biofilm to resist chlorine but also could cause more severe RO membrane biofouling.

Evaluating method and potential risks of chlorine-resistant bacteria (CRB): A review.

Chlorine-resistant bacteria (CRB) are commonly defined as bacteria with high resistance to chlorine disinfection or bacteria which can survive or even regrow in the residual chlorine. Chlorine disinfection cannot completely control the risks of CRB, such as risks of pathogenicity, antibiotic resistance and microbial growth. Currently, researchers pay more attention to CRB with pathogenicity or antibiotic resistance. The microbial growth risks of non-pathogenic CRB in water treatment and reclamation systems have been neglected to some extent. In this review, these three kinds of risks are all analyzed, and the last one is also highlighted. In order to study CRB, various methods are used to evaluate chlorine resistance. This review summarizes the evaluating methods for chlorine resistance reported in the literatures, and collects the important information about the typical isolated CRB strains including their genera, sources and levels of chlorine resistance. To our knowledge, few review papers have provided such systematic information about CRB. Among 44 typical CRB strains from 17 genera isolated by researchers, Mycobacterium, Bacillus, Legionella, Pseudomonas and Sphingomonas were the five genera with the highest frequency of occurrence in literatures. They are all pathogenic or opportunistic pathogenic bacteria. In addition, although there are many studies on CRB, information about chlorine resistance level is still limited to specie level or strain level. The difference in chlorine resistance level among different bacterial genera is less well understood. An inconvenient truth is that there is still no widely-accepted method to evaluate chlorine resistance and to identify CRB. Due to the lack of a unified method, it is difficult to compare the results about chlorine resistance level of bacterial strains in different literatures. A recommended evaluating method using logarithmic removal rate as an index and E. coli as a reference strain is proposed in this review based on the summary of the current evaluating methods. This method can provide common range of chlorine resistance of each genus and it is conducive to analyzing the distribution and abundance of CRB in the environment.

Simulating and predicting the flux change of reverse osmosis membranes over time during wastewater reclamation caused by organic fouling.

During the operation of the RO system, it's significant to predict the flux change over time. Previous research conducted detailed exploration on the dynamics of RO membrane fouling, and provided a solid database for modelling. In this study, a modified intermediate blocking model with two parameters was proposed to describe the flux change of RO membranes under a huge variety of conditions. Raw data reported by over 20 research groups from 11 different countries was used to validate the feasibility of this model. It proved applicable to describe the flux change of RO membranes fouled by pure organic matter or mixture and tertiary treated wastewater. In order to reveal the relationship between model parameters and foulant concentrations, RO membrane fouling behaviors of typical foulants (sodium alginate (SA), bovine serum albumin (BSA) and mixture) were further investigated. We found that the change of model parameters with SA concentrations was in accordance with Langmuir adsorption isotherm model. Therefore, the model parameters could be calculated by SA concentrations under certain optional conditions, and then the flux change could be predicted by this model. In this way, a novel time-course model was established, which could predict the flux change of RO membranes over time only with SA concentrations. Besides, the synergic effect between SA and BSA on RO membrane fouling was directly quantified.