| The acetochlor; bensulfuron methyl and mixture herbicide have been widely applied in rice field for controlling weeds. However, the toxicity of its residues usually do harm to the sensitive next-season crops, and causes serious environmental pollution. Those herbicide residues in the soil generally could be eliminated through physical, chemical and biological processes, and the microbial degradation is the main pathway. Therefore,the degrading microorganisms used to removing environmental pollution is very important. In this studying, a bacterial mixture of acetochlor-degrading and bensulfuron methyl-resistance, named as 66C, were screened from contaminated rice field. Mixture 66C is composed of 66C-1, 66C-2, 66C-3, and 66C-4 which identified as Serratia marcescens, Pseudomonas hunanensis, Pseudomonas putida and Pseudomonas nitroreducens,respectively, which based on their morphology, physiology and 16S rRNA gene analysis. Anyone of them can not utilize acetochlor alone, and just the combination of four with equal proporition strain named HXDW can degrade acetochlor.The optional growth conditions of bacterial mixture HXDW were 100mg/L acetochlor concentration, pH9.0, 25 ?, and 2% NaCl concentration. Supplement of 10mg/L Pb2+ have improved the growth of HXDW. Whereas, 10mg/L Cd2+ and Mn2+have suppressed its' growth. Bacterial mixture HXDW can only use of acetochlor as the sole carbon source, and can't use butachlor, (S)-metolachlor and diflufenican. In MSM liquid medium contained 100 mg/L acetochlor, the degradation rate was 14% after 7days incubation using LC-MS. One acetochlor biodegradation products named 2-amino-3-Hydroxyanthranilic acid were identified by UHPLC-QTOF-MS based on positive patterns. The main plausible degradative pathways involved hydroxylation,carboxylation, dechlorination, deacetylation and deethoxymethyl. The result of whole cell protein SDS-PAGE showed that three special bands (58.0, 50.0 and 43.0kDa) were observed in bacterial mixture HXDW after induction of 100mg/L acetochlor or the mixture of 100 mg/L acetochlor and 30 mg/L BSM, while seven special bands (80.0,64.0, 58.0, 50.0, 43.0, 38.0 and 25.0 kDa) were observed after treatment with 30mg/L BSM. In addition, an extract method of osmotic shock was used to separate the endoenzyme, periplasmic space enzyme and exoenzyme from bacterial mixture HXDW,and the degrading rate on acetochlor was 17.43%, 18.34% and 44.95% in each of component after 24h treatment, respectively. The result demonstrate that the degrading-enzyme mainly derived from extracellular, and few exists in intracellular and periplasmic space.When sensitive strain Escherichia coli and Bacillus subtilis were exposed to the mixture of 60 mg/L acetochlor and 20mg/L BSM, the SOD activity decreased rapidly firstly up to two hours' treatment and then increased to maximum at twelve hours'treatment. At that time, the maximum SOD activity in E.coli and B.subtilis was 1.63 times and 1.08 times before treatment, respectively. Meanwhile, The CAT activity curve of E.coli showed two peaks, and the maximum activity was 1.23 times before treatment.Moreover, the CAT activity of B.subtilis changed slowly at the beginning of treatment,and the maximum activity was 4.20 times after eighteen hours' treatment. In general,Gram negetive bacteria-Escherichia coli, Gram positive bacteria-Bacillus subtilisresisted oxidant stress slowly through the increase of SOD and CAT enzymes after low concentration herbicide treatment. If exceeding this concentration, the CAT activity of the two strains were significantly decreased and SOD activity was not changed above this concentration, so it indicated that more than 60mg/L acetochlor and 20mg/L bensulfuron-methyl could damage to susceptible strains. |
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