Isolation And Characterization Of A Chlorothalonil-degrading Bacterium

In order to apply the biodegradation preparation to control the farmland pesticide pollution, the author isolated a chlorothalonil-degrading bacterial strain TP-D1 from the heavily TPN-polluted soil. Here is a brief summary in respect to its identification, biological characteristics, degrading spectrum, degradation efficient and metabolites.1. Thirty bacterial strains were isolated on NA plates containing chlorothalonil from the soils samples taken from around the wastewater outlets of chlorothalonil-produced factories in an AnYang city of Henan province. One strain made a clear zone around its colony on the mineral medium plate containing 50mg/L chlorothalonil. The strain could utilize chlorothalonil as its sole carbon source. By using GC-ECD, it was detected that 90.4% of TPN was degraded by the isolate after 4 days of incubation at 30℃, 180 rmin^-1 in liquid mineral medium.2. Biochemical and physiological tests in combination with analysis of its 16s rDNA sequence indicated that the strain is Ochrobactrum lupini. Growth determinations demonstrated that its optimal growth temperature and pH are at 30℃and 7.0 respectively. 1% glucose, 1% beef extract, and 1% ammonium oxalate and C: N ratio 4/1 was appropriate for its culture in a liquid medium.3. Degrading rates of chlorothalonil in soil by the strain was detected by using High Performance Liquid Chromatography (HPLC). Results showed that the recovery rate of TPN in soil was from 83.5% to 101.2%, acceptable for the the test of pesticide residue in soil. TP-D1 and TPN were co-cultured in the sterilized soil for 24hours at room temperature at the concentration of 10⁷cfu/g soil and 50mg/L respectively, the average degrading rate of TPN was 71.4%. And it could reach 95.01% and 99.7% after 3 days and one week respectively.4. Degradation spectrum of the strain TP-D1 indicated that it could not degrade Pentachloronitrobenzene,Benzenehexachloride,Dichloro-Diphenyl-Trichloroethane,Triazophos,Methyl Parathion,Aminobenzene and Hydroxybenzene.5. Metabolites of chlorothalonil cultured with TP-D1 at 30℃for 4 days were separated by thin layer chromatography (TLC) using the mixture of 5:3:2 hexane, Isopropyl, and alcohol-methanol as the developing solvent (v/v). The developed strip of the metabolites was collected under the ultraviolet radiation (254nm), dissolved in methanol, and naturally concentrated. Two biodegradation products were determined by the HPLC-MS method, one is l,3-dicarbamoyl-2,4,5,6-tetrachlorobenzene and another is 4-hydroxy-2,5,6-trichlorois ophthalonitril (TPN-OH). It was supposed that chlorothalonil was degraded by TP-D1 through the direct hydroxylation of chlorothalonil and the hydration of its cyano-groups.