Islation, Identification And Characteristics Of Cyperthroid-Degrading Strain JZL-3 And Methylotrophic Strain JZL-4

Pyrethroid insecticides have been used since the 1970s and represent an increasing proportion of world pesticide sales. In our country, pyrethroids have been widely used for insect control on both agricultural and home insecticides, replacing more toxic pesticides. Due to such long time and extensive use of synthetic pyrethroids, we have faced to the environment pollution and health risk problem. Biodegradation, as a security and cost-effctiveness means for environmental control, gets increasing concern and plays a crucial role. In this thesis, we study isolation, identification and degradation properties of a pyrethroid-degrading strain. And we compare to another highly efficient pyrethroid-degrading strain, hoping that the preliminary conclusions will be useful for future environment remediation. Because of the pythroid is dissolved in methanol, the condition of isolation degradation strain is also fit for isolation of the methylotrophic strain. We isolate and identify a facultatively methylotrophic strain.A cypermethrin-degrading enrichment culture was obtained through enriching sludge from wastewater-treating system of a pesticide manufacturer. Then a cypermethrin-degrading bacterium JZL-3 was isolated from this culture. This bacterium was identified as Arthrobacter sp. according to its physiological & biochemical characteristic and the similarity analysis of its 16S rRNA sequence. The optimal temperature and pH for the growth of strain JZL-3 were 20-35℃and pH 7-8 respectively. The strain JZL-3 can grew in the condition of 0.5%-10.0% NaCl concentration, and grew better in the LB medium with 0.5%-1.0% NaCl concentration.The optimal temperature and pH for the pyrethroid degradation by strain JZL-3 was 7.0 and 30℃, respectively. The degradation efficiency was related positively to initial inoculum size (1%-5%). When the initial inoculum size was more than 7%, the degradation efficiency was not been effected. The strain could degrade cypermethrin rapidly when initial concentration was 20-50mg/L, while the degradation rate was significantly reduced when initial concentration was more than 100mg/L. The strain JZL-3 were capable of degrading seven main pyrethroid pesticides with defferent degradation rates. The degradation rates of strain JZL-3 was descending as follows:permethrin, fenpropathrin, cypermethrin, fenvalerate, bifenthrin, cyhalothrin, deltamethrin. And no cis-trans isomer selectivity was observed to the biodegradation of permethrin and cypermethrin cis/trans isomers by Arthrobacter sp. JZL-3. Also, no plasmid was detected, which means the functional genes of degrading pythroids are on the genome DNA.In this chapter, strain JZL-3 was used to study factors affecting its degrading capacity of fenpropathrin in soil microcosms. The data shows that the degradation rates of cypermethrin by JZL-3 in sterilized soil were faster than those in unsterilized soil. It can degrade cypermethrin of varying concentrations (20-200mg/kg soil). was used to study The biodegradation effects of strain JZL-3 to low concentration cypermethrin(<100 mg/kg) in soil was higher than high concentration(200 mg/kg). Considering the cost and efficiency, 106 CFU/g inoculum size of strain JZL-3 was suitable for bio-remediation.Due to the pythroid is dissolved in methanol, we also isolated a facultatively methylotrophic strain JZL-4 from the same enrichment culture. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain JZL-4T formed a clade with Methylopila capsulata IM1T(97.2%), Albibacter methylovorans DM10T (94.9%)and Methylopila helvetica DM9T(94.1%). However, our isolate was distinguished clearly from Albibacter methylovorans in some essential phenotypic and genotypic characteristics. And strain JZL-4T had the highest similarity with Methylopila capsulata and shared several important characteristics with members of the genus Methylopila.Cells are gram-negative, strictly aerobic, non-sporulating, short rods (0.8-1.2×1.6-2.Oμm) with round ends, motile by lateral flagella, multiply by binary fussion. Granules of PHB are observed in the cells. Growth is observed over a temperature range of 10-37℃(optimum 30℃), a pH range of 5.0-10 (optimum 6.5-7.5) and NaCl concentration 0-2.5%(optimum 0.5%).The major cellular fatty acids are C18:1ω7c and C18:0.The dominant phospholipids are phosphatidylethanolamine, phosphatidylcholine, phosphatidyglycerol and phosphatidylmonomethylethanolamine. The major ubiquinone is Q-10. The G+C content of the DNA is 70.4 mol%. C1 compounds are assimilated via the isocitrate lyase-negative serine pathway.Based on phenotypic, genotypic and phylogenetic properties, strain JZL-4T(=ACCC 05406T=DSM 22718T=VKM B-2555T)was referred to as a novel species of the genus Methylopila, for which the name Methylopila jiangsuensis sp. nov. was proposed.