| Methamidophos, one of the organophosphorus pesticides, was prohibited to manufacture and use in China in 2008. But, due to its low cost and high efficiency in killing a variety of pests, methamidophos is still manufactured and used in some aeras. Methamidophos has a relatively short half-life. However, due to its high application quantity, the problems of environmental pollution and food safety have attracted wide attentions. It is important to isolate high efficient methamidophos-degradating strains and use these strains to clean up the contaminated environments.Three methamidophos-degrading bacteria MAP-1, MAP-2 and JW-64-1 were isolated from the wastewater sludge of a pesticide factory which has produced organophosphorus pesticides for many years. According to the morphological observation, physiological biochemical tests,16S rRNA gene phylogenetic analysis, strains MAP-1, MAP-2 and JW-64-1 were identified as Hyphomicrobium sp., Methylobacterium sp., and Luteibacter sp., respectively.Strain JW-64-1 had the highest similarities with Luteibacter rhizovicinu DSM 16549T (98.6%), followed by L. yeojuensis DSM 17673T (98.4%), L. anthropi CCUG 25036T (98.2%), and showed less than 96.8% sequence similarities with other strains from the family of Xanthomonadaceae. Strain JW-64-1 contained iso-C15:0 (24.2%), iso-C17:0 (23.0%),17:1 iso co9c (20.0%) as the main cellular fatty acids. The predominant ubiquinone was Q-8 (98%). Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphoaminolipid (PN) were the predominant polar lipids. The DNA G+C content of the total DNA was 63.7±0.3 mol%. DNA-DNA hybridization results showed that strain JW-64-1 had low genomic relatedness with L. rhizovicinu (34.8%) and L. yeojuensis (25.6%), respectively. On the basis of the above results, we propose that the strain JW-64-1T represents a novel species in the genus Luteibacter, and designated as Luteibacter jiangsuensis sp. nov.The optimal medium for methamidophos degradation by MAP-1 or MAP-2 was MSM-3(5 mL CH3OH,1.0 g/L NH4NO3,1.5 g K2HPO4·H2O,0.5 g KH2PO4,0.5 g NaCl, 0.2 g MgSO4·7H2O, water 1, OOOmL, pH7.0-7.2)). The optimal medium for JW-64-1 was MSM-1(1.0 gNH4NO3,1.5 g K2HPO4·H2O,0.5 g KH2PO4,0.5 gNaCl,0.2 g MgSO4·7H2O, water 1, OOOmL, pH7.0-7.2)). Strain MAP-1 could utilize methamidophos as carbon, nitrogen or phosphous source, and it only needed 84 h to completely remove 3000 mg·L-1 methamidophs. Strain MAP-2 and JW-64-1 could use methamidophos as carbon source, and the two strains could degradate 69.7% and 49.5% of 1000 mg·L-1 methamidophos in 96h, respectively. The suitable temperature and pH for the growth and degradation of methamidophos by the three strains were all 25-30℃,6.0-8.0. Compared to that of strain MAP-1 or JW-64-1, that air condition grew worse had more effect on growth of strain MAP-2 and degradation of methamidophos. The addition of 1 mmol·L-1Ca2+ promoted the degradion of methamidophos, while, lmmol·l-1 Cu2+, Co2+ and Ni2+ inhibited the degradation. The growth of strain MAP-1 and degradation of methamidophos were promoted by adding 0.5% peptone or yeast. Contrast to other two strains, strain MAP-1 had a relatively wide spectrum of degrading substrate.Most of the methamidophos-degrading enzyme in strain MAP-1 was located in the cell membrane. It was the first time to find that methamidophos-degrading enzyme was located in cell membrane. The best reaction system was as follows:incubation 80μL crude enzyme in 5 mL total volum in pH 7.0 PBS for 10 min at 30℃. Ca2+ could promote the crude enzyme activity, whereas Co2+, Ni2+, surfactant Triton X-100, SDS, organic solvent ethyl acetate, dichloromethane and chloroform suppressed the enzyme activity significantly.By MS/MS and GC-MS, three metabolites (S-dimethyl thiophosphate, S-methyl thiophosphate and methyl phosphate) of methamidophos were identified, and the likely methamidophos biochemical degradation pathway of strain MAP-1 was proposed. The first step involved in methamidophos degradation is the scission of the P-N bond to form S-dimethyl thiophosphate and NH3. Then, S-dimethyl thiophosphate was subsequently hydrolyzed at the P-O bond to release CH3O- and form S-methyl thiophosphate. S-dimethyl thiophosphate could also be hydrolyzed at the P-S bond to release CH3S- and form methyl phosphate. Finally, S-methyl thiophosphate or methyl phosphate was likely transformed into phosphoric acid. It was found that 1,000 mg·kg-1 methamidophos and 500 mg·kg-1 acephate were degradated to non-detectable level within 9 days, and 40.0% of 100 mg·kg-1 isocarbophos was cleaned up within 15 days, when MAP-1 was inoculated into Magan soil at the concentration of 107 CFU per gram dry soil. The addiation of methanol or increase of inoculum of strain MAP-1 in soil could accelerate the degradation of the organophosphorus pesticides. Whether the soil was flooded or not, no notable effect on degradation was found. Strain MAP-1 showed the ability to eliminate methamidophos, acephate and isocarbophos at concentrations of 50,10 and 1 mg·kg-1. Strain MAP-1 showed the stronger degradation ability on isocarbophos at low concentration. When the concentration of isocarbophos was 1 mg·kg-1, the degradation rate was 94.6% in 7 days. Spray of 5×1011 cell·m-2 strain MAP-1 on pakchoi leaves could degrade methamidophos (three times of normal usage), acephate (normal usage) and isocarbophos (three times of normal usage) to non-detectable level in safety interval of 7 days, when using 5×1011cell·m-2 strain MAP-1. |
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