| Objective: The utility of reclaimed water plays an important role to alleviate the crisis of water shortage in the world. But the biological safety is one of the important factors hampering the promotion of reclaimed water. Chlorine disinfectant can inactivate effectively most bacteria which is already used in reclaimed water disinfection worldwide. However some bacteria which resist to chlorine become a kind of biological pollutants. There was little information on the influence of the disinfectant resistant bacteria on the biosafety of the reclaimed water in the related literatures home and abroad, and the mechanism and control of the disinfectant resistant bacteria was not clear. In this project, we will determine the occurrence of the chlorine resistant bacteria in the reclaimed water sampled from a reclamation plant via static disinfection, evaluate the inactivating effection and explore the mechanism of its chlorine resistance.Method: This study aimed at the reclaimed water from a reclamation plant of Tianjin. We determined the occurrence of chlorine resistant bacteria in the reclaimed water via static disinfection, and the standard strain of Esherichia coli 8099 was the control. We would find out the alive bacteria with high concentration of chlorine by using the minimum inhibitory concentration(MIC) experiment. After separated to single colony, the bacteria sample were identified by 16 s rDNA conservative district gene sequencing, and were analyzed the population structure of chlorine resistant bacteria. We evaluated the efficacy of the disinfection of chlorine resistant bacteria and standard bacteria with the same species, and the influence by environmental factors. The differences between the isolated bacteria and the standard bacteria before and after disinfection were compared by the optical microscope and the election microscope. And fluorescent staining analysis would be used to detected spore damage. Electrophoretic patterns of spore coat and spore cell protein were detected by SDS-PAGE.Result: Some bacteria survived in 700mg/L chlorine concentration in the MIC experiment which beyond E.coli 8099 group. The alived bacteria in group 700mg/L chlorine were purified and indentified by 16 srDNA sequence detection and results in Bacillus subtilis. Chlorine inactivate experiment was performed on one of the separated B.subtilis(BS4) and standard strains of B. subtilis ATCC9372. The inactivate rate of vegetative BS4 by 0.3mg/L and 1.0mg/L chlorine, the inactivate rate of BS4 spore by 3.0mg/L, 6.0mg/L and 9.0mg/L chlorine were lower than that of each ATCC9372 group. The condition of the higher temperature and lower pH was of good benefit to disinfection. We found that both spores had clear ultra-structure and smooth surface transmission electron microscope. BS4 has thinner outer coat and thicker inner coat than ATCC9372. After chlorine disinfection, spore coats of BS4 were fold and broken lightly, while spore coats of ATCC9372 were damaged heavily and the protoplasm inside the spores became turbid or overflown from the spores. By fluorescent staining, the spore coat and membrane of BS4 showed lighter broken than ATCC9372 after chlorine disinfected. The protein of spore coat and spore cell between ATCC9372 and BS4 were different. The changes of spore coat and spore cell after disinfection were one of the reasons which lead to the inactivation difference.Conclusion: The high concentration chlorine cannot inactivate all the bacteria in the reclaimed water. Therefore chlorine resistant bacteria exist in the reclaimed water. These bacteria could be screened by MIC test, and were identified as Bacillus. Under same disinfection condition, separated strain BS4 had a lower inactivation rate than standard strain ATCC9372. BS4 was more tolerant to chlorine than ATCC9372. The mechanism of BS4 spore's resistance to chlorine may due to the different structure and protein of the spore. This research separate chlorine resistant B.subtilis from reclaimed water for the first time. The chlorine resistant mechanism of the B.subtilis was preliminary discussed. The results will provide theoretical bases to guarantee the biological safety of reclaimed water and promote the application of the reclaimed water. |
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