Study On The Distribution Of Opportunistic Pathogens And Virulence Genes In Biofilm In Drinking Water Distribution System

Biofilm in drinking water distribution system(DWDS)is the key factor to explore and control the microbiological risk of drinking water,and the pathogenic potential of biofilm is getting more and more attention from the public.At present,there are few studies on the pathogenic potential of biofilm based on the two aspects of opportunistic pathogens and microbial functions.On the other hand,most of the microbes in the environment are not culturable,the microbial DNA and molecular biotechnologies are needed for deeper resaearch,so it is important to obtain enough and representive microbes from oligtrophic environment,such as DWDS,for the study of microbial communities and functions.This study first explored the pretreatment methods for biofilm sample from actual DWDS,which can increase the amount of microbial biomass obtained from environmental substances.Then the differences in biofilm growth among different drinking water simulation devices were compared and their applicable conditions were put forward.At last,relying on the pilot platform built in SX City and JX City,and taking different water quality and pipe as the research object,the impact of water quality and pipe on the pathogenic potential of biofilm was compared by metagenomic sequencing technology and suggestions for actual water supply operation were provided.The major results are as follows:(1)At 20 kHz ultrasonic frequency,for biofilm samples in actual DWDS,the optimal sonication method is to ultrasound the collected microbial attachment medium(such as sediment and tube scale)15 times at 13 W for 1 min,then transfer the medium to fresh PBS,and allow 20 min between each ultrasound.This optimal ultrasound treatment can increase the amount of DNA by at least 4.78 times without degrading its quality.For the biofilm samples in actual DWDS,the optimal method of concentration is filtration with a 0.22 ?m filter.(2)The period of the biofilm in non-circulating(NC)pipeline is longer than that in the half-circulating(HC)pipeline and the bench-top reactors.Compared with NC pipelines,other simulation devices enhance the potential pathogenicity of biofilms,but weaken the coexistence relationship between the genera.The growth of biofilm on the in-situ coupons is obviously affected by the flow pattern of the water flow.The installation of the PWG coupons can reduce or avoid the influence of the flow state change,and the instalation of the Robbins coupons will reduce the Reynolds number and cause the change of flow state,which in turn affects the growth of biofilms.(3)The difference in opportunistic pathogens in the two typical urban DWDS is mainly reflected in abundance rather than species.In the actual DWDS,most of opportunistic pathogens are from Gamma Proteobacteria in biofilm.The temperature mainly affects the opportunistic pathogens by influencing the microbial activity of the biofilm.High temperature will reduce the microbial activity of the biofilm,while the low microbial activity and the number of opportunistic pathogens have a strong correlation,which is positive on the ductile iron pipes and negative on the HDPE pipes.The correlation between the microbial density of the biofilm and the number of opportunistic pathogens is weak.(4)Water quality and pipe materials affect the basic metabolic activities of biofilm microorganisms,such as metabolism of carbohydrates,nitrogen and sulfur.However,these two factors do not have significant influence on metabolism of phosphorus.In addition,pipe materials affect the correlation between opportunistic pathogens and aggressive virulence genes in biofilms.The aromatic compounds released from plastic pipes such as HDPE pipes are degraded and metabolized by biofilm microorganisms.There is a relationship between the metabolite intermediates benzoate and lipopolysaccharide synthesis in biofilm on HDPE pipe,which makes the biofilm have stronger pathogenic potential than that on the ductile iron pipes.(5)The high residual chlorine environment can enhance the metabolism of glutathione in water and biofilm microorganisms,but the mechanism of disfectant resistance mainly depends on the non-redox reaction involved in glutathione S-transferase,rather than the redox reaction involved in glutathione peroxidase and glutathione reductase.In addition,a high residual chlorine environment can increase the resistance of bacteria to specific antibiotics.