| In this dissertation, first take atrazine degrading bacteria HB-5 as the main object , studied the degradate capacity and the degradate pathway of HB-5; isolated another atrazine degrading bacteria HB-6,which can mineralizate atrazine .studied the degradate capability and pathwany of HB-6, Furthermore, the degrading genes have been cloned, sequenced and compared with sequences of other atrazine degrading strains in Genebank. The results could be summarized as follows:1.Introduced the physical and chemical characteristics, general situations of application, ecological toxicological characteristics and pollutant management of Atrazine.Described a variety of detection methods for atrazine and its degradation products in encironmental media. On the based of summing up the previous work ,make a comprehensive analysis of atrazine degradate patheway ,degradate genes and enzymes accordingly the issues that would be investigated was presented by the author.2.Studied the degradate pathway and degradate capability of degrading Bacteria HB-5 that we had isolated.We have already known that HB-5 can degradate atrazine of 100mg·L-1 completely in 24h,and HB-5 containing the degradate gene TrzN, AtzB, AtzC.Adding HB-5 to the inorganic salt medium that atrazine as the sole nitrogen source, 30℃shake culture, at 0h, 12h, 24h, 48h, 72h, 96h, 120h sampling,analyzed the degradate products by HPLC and HPLC-MS.The results show that HB-5 can degradate atrazine (100mg·L-1) completely in 24h,meanwhile the major metabolite was cyanuric acid, and a small amount of hydroxyatrazine was detected. Hydroxy-atrazine reach its maximum amount approximately 20 mg·L-1 and then gradually disappear,at the same time, the concentration of cyanuric acid is about 96 mg·L-1,and gradually increasd to 100 mg·L-1. Atrazine metabolites were detected and identified by HPLC-MS,at different time 24h and 96h, we detected two metabolits,their ion size were 198 (M/Z) and 129 (M/Z),we can know that they are Hydroxy-atrazine and cyanuric acid,and we found that HB-5 can degradate atrazine to cyanuric ,but can not dagradate the next step.3.Isolated a strain from the laboratory strains HB-1, HB-3, HB-6, HB-7, HW-23, WB-1, cyanuric acid as the sole nitrogen source and determinate their degradate capability of cyanuric acid . and found that HB-6 has the highest cyanuric acid degradation rate within 24h.we can observing clearing zones on indicator agar plates containing 1000mg·L-1 of Atrazine,which shows that HB-6 can live on and degrade 1000mg·L-1 of atrazine, So HB-6 was selected to make further study.4.Adding HB-6 to the inorganic salt medium that atrazine as the sole nitrogen source, 30℃shake culture, at 0h, 12h, 24h, 48h, 72h, 96h, 120h 144h,sampling,analyzed the degradate products by HPLC and HPLC-MS.The results show that HB-6 can degradate atrazine (100mg·L-1) completely in 24h,meanwhile the metabolite were cyanuric acid and hydroxyatrazine. Hydroxy-atrazine reach its maximum amount approximately 10mg/L and then gradually disappear,at the same time, the concentration of cyanuric acid is about 60mg·L-1,and gradually disappeared. we detected three metabolites in the culture medium, the size of their ions were 198(M/Z), 129(M/Z) and 60 (M/Z).we can know that they are Hydroxy-atrazine , cyanuric acid and urea. there is only urea in 144h,it is proved that HB-6 can mineralize atrazine.5.The characterization of Atrazine metabolism by HB-6 was studied. The result suggested: culture temperature and pH conditions has a great influence on the growth of bacteria and biodegradation of Atrazine, The optimal conditions for HB-6 growth and Atrazine biodegradation as following the temperature between 20℃ 40℃, pH 6.0 9.0; high concentration of atrazine inhibit the growth and degradation.6.Identification of the isolates was performed by conventional methods based on biochemical reactions and confirmed by 16SrRNA sequencing.Comparing the 16S rRNA gene sequence of strain HB-6 with the sequence available in the Genebank database revealed 99% similarity with a Bacillus subtilis.Therefore, we will hereafter refer to it as Bacillus subtilis.7.The degrading genes in the atrazine high-effeciency degrading strain HB-6 have been cloned. Primers of atzA, atzB, atzC and trzN, atzD, atzE, atzF were designed referring to former literatures. The total DNA of HB-6 acted as the DNA model to carry on PCR (polymerase chain reaction) and atzB,atzC and trzN genes in HB-6 were cloned, sequenced and compared with former atrazine degrading genes in Genebank, and the similarity were almost all over 98%, respectively.
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