Mechanisms Of Waterborne Pathogens Inactivation Under Ultraviolet Disinfection And Chlorination

In the process of water treatment,disinfection processes can inactivate pathogenic microorganisms and reduce the risk of waterborne diseases.Ultraviolet（UV）disinfection and chlorination are the most mainstream of sewage disinfection technologies.There are obvious differences in mechanisms of pathogens inactivation between UV disinfection and chlorination.Pathogenic bacteria and viruses are two main types of pathogenic microorganisms.There are significant differences in the morphology,size,physiological characteristics,modes of occurrence and tolerances of severe environment between pathogenic bacteria and viruses.These differences in characteristics of pathogenic microorganisms and mechanisms of pathogens inactivation could lead to the differences in different inactivation efficiencies between pathogenic bacteria and viruses under disinfection.Hence,it is important to correct evaluation of the damage of pathogens under disinfection for controlling the spread of waterborne diseases.The detection methods are the basis of evaluation of inactivation efficiency.However,due to limitations of the detection methods,there is not yet a detection method to accurately reflect the inactivating efficiencies of pathogenic bacteria and viruses.The main goal of this paper was to reveal the different effects of UV light and chlorine on bacteria and viruses.We chose Escherichia coli（E.coli）and Poliovirus as models for aquatic pathogenic bacteria and virus,respectively.UV disinfection and chlorination experiments were carried out.The applicability of various detection methods in evaluating disinfection efficiencies of pathogenic microorganisms were analyzed.The main researching contents were as follows:1)The tolerance of pathogens to UV light was as follows:poliovirus(inactivated rate k=0.16 cm²·mJ^-1)>multiple-antibiotic-resistant E.coli(k=0.27⁰.34 cm²·mJ^-1)>sensitive E.coli ATCC25922(k=0.53 cm²·mJ^-1).UV disinfection could induce some microorganisms into a viable but non-culturable state.The photo-reactivation and dark repair percentages of E.coli were 0.018%and 0.00042%after 24 h light exposure and dark repair following UV disinfection at 20 mJ/cm² UV dose,respectively.When the UV dose increased to 40 and 80 mJ/cm²,photo-reactivation and dark repair percentages of E.coli decreased in the range of 0-0.0013%.Similarly,poliovirus is appreciably more resistant to chlorine than E.coli for microbial culture methods.The predicted concentrations of chlorine required for the reduction of 4-log poliovirus and E.coli were<1.5 and 2.56 mg/L,respectively.2)Mechanism of bacteria inactivation under UV disinfection.UV light could go through the cell membrane and induce a direct injury to DNA at low doses.More apparent damage in mRNA was observed during UV disinfection.The transcriptional levels of chromosomal replication initiator protein（dnaA）gene and single-stranded DNA-binding protein（ssb）gene decreased,leading to cell replication dysfunction.The RecA mRNA reached the detection limit at 50 mJ/cm² and resulted in the loss of DNA damage repair function.UV disinfection cause a slight inhibition in the expression of glutamic acid decarboxylase（gadA）mRNA.After treatment with high UV doses（>100mJ/cm²）,bacterial surface become wrinkles,deboss and holes observed by scanning electron microscopy（SEM）.Adenosine triphosphate（ATP）related to microbial metabolism lost gradually.Flow cytometry found that membrane permeability of only9.9%E.coli changed at 400 mJ/cm².UV disinfection caused a selective change in the inhibition zone diameters.The inhibition zone diameters of the strains resistant to antibiotics were more difficult to alter than those susceptible to antibiotics.Hence,the number of change in inhibition zone diameters was lower for the MAR SER6-1 strain（3:GEN,CHL,and NOR）than the double antibiotic-resistant SER2 strain（7:AMP,STP,GEN,CTX,CHL,CIP,and NOR）.UV disinfection caused a slight reduction in antibiotic resistance genes（ARGs）.The relative abundance of ARGs increased at high UV doses.3)Mechanism of bacteria inactivation under chlorination.The primary inactivation mechanism at low concentrations of chlorines（≤5 mg/L）is the damage to cellular membrane.The membrane permeability of E.coli cells was broken quickly within 2min.Cellular ATP was outflow.At a chlorine concentration of 5 mg/L,intracellular ATP of E.coli almost completely released.E.coli lost the ability to perform cell respiration,metabolism,and transport.Meanwhile,HClO could penetrate into the cell membrane and induce an injury to mRNA.The functions of DNA damage repair（RecA）,chromosomal replication initiator protein（dnaA）,single-stranded DNA-binding protein（ssb）,and glutamic acid decarboxylase（gadA）were gradually disturbed,leading to the loss of culturability.The functions of dnaA and RecA were completely inhibited at 5mg/L chlorine for 30 min（at which E.coli lost culturability）.The ssb and gadA genes were non-detectable at 8 mg/L chlorine for 30 min.Excessive chlorine（>8 mg/L）could induce DNA lesion,leading gradual loss of replication function.The oxidation or hydrolysis of extracellular ATP occurred.4)Considerable discrepancies among different detection methods in evaluating bacterial disinfection efficiency.FCM and ATP analysis can be used to assess chlorination efficiency but cannot determine cell survival during UV disinfection.Due to the limit of amplicon lengths and the damage of nucleic acid at high disinfectant doses,the detection of bacterial or viral genome by qPCR is conservative in the assessment of bacterial inactivation.m RNA could potentially indicate bacterial viability.However,the degradation level of mRNA depended on the target genes.The RNA involved in the DNA damage repair function can be a potential indicator of bacterial inactivation.5)Considerable discrepancies among RT-qPCR,TCID₅₀ and ICC-RT-qPCR methods in evaluating viral disinfection efficiency.The sensitivity of three detection methods was as follows:ICC-RT-qPCR（0.44 TCID50）>RT-qPCR（4.4 TCID50）>TCID50（44 TCID50）.The fragment size was the longer,and the damage in genes was more seriously.The untranslated terminal regions（5’UTR）fragment disappeared significantly more rapidly than the structural protein（VP1）fragment during UV disinfection.However,the degradation level of 5’UTR fragment approximately equal to that of VP1 during chlorination.The TCID₅₀ and ICC-RT-qPCR methods were able to detect poliovirus survival at UV doses up to 20 and 100 mJ/cm²,respectively.The infectious of poliovirus was undetectable by TCID₅₀ assay with chlorine dose more than2 mg/L,while the dose chlorine dose was 5 mg/L detected by ICC-RT-qPCR.RT-PCR showed that nucleic acid could still be detected after the disappearance of virus infectious.RT-qPCR methods can lead to an underestimation of viral inactivation.6)The damage of RNA in Poliovirus is more seriously than that of DNA in E.coli.The result evaluated with the quantitative PCR method is opposite with that of the traditional culture method in which virus is more tolerant to disinfectants than bacteria.The differences between bacterial and viral inactivation did not depend on the differences in microbial nucleic acid.It is probably attributed to the resistance of viral capsid protein to disinfectants,which might be higher than that of the outer cell structure of the bacteria.