| Chlorpyrifos is one of the most widely used insecticides belonging to the organophosphorus group; its wide contamination to the environment has given rise to much public concern. Several studies have been conducted on the microbial degradation of chlorpyrifos; its major pathway of degradation has been prelimimarily demonstrated. However, no microorganisms that could completely mineralize chlorpyrifos efficiently have been reported to date.In this study, several chlorpyrifos-degrading strains were isolated by enrichment culture and screening. Their growth characteristics, degrading abilities, and mineralizing mechanisms were also investigated. Furthermore, the co-culture and mixed cultures of degrading bacteria and fungus, and their degrading enzymes were also studies. The main results are as follows:1. From three activated sludge samples, several bacterial strains DM, TRP and TCR, and one fungal strain TCF were isolated. Based on the results of phenotypic features, physiological-biochemical properties, and phylogenetic similarity of 16S/18S rRNA gene sequences, these isolates were designated as Paracoccus sp. DM/TRP, Serratia sp. TCR and Trichosporon sp. TCF. Their 16S/18S rDNA partial sequences were deposited into the GenBank under accession No. EF070123, EF070124, EF070125, and EF091819, respectively.2. The growth and degrading abilities of each pure culture for the chlorpyrifos and TCP degradation was investigated by UV-Vis, GC-FPD, and HPLC. Under the condition of 30?C and pH 7, both strain DM and TRP could degrade 50mg/L chlorpyrifos or TCP within 5 days; strain TCR could transform chlorpyrifos to TCP without further metabolism; strain TCF could completely mineralize 50mg/L chlorpyrifos within 7 days, as well as 50mg/L TCP within 4 days. Effects of different initial chlorpyrifos concentration, additional carbon source, total inocula biomass amount, incubation temperatures, and initial pH were investigated to determine the optimal biodegradation conditions. Results showed that these isolates could effectively degrade chlorpyrifos when provided as the sole carbon source, with total inocula 0.15~0.3g/L, at 15~35℃, pH 7~9, and initial chlorpyrifos 25~200mg/L; Addition of extra carbon source, such as glucose or sucrose, could greatly enhance the biodegradation of chlorpyrifos.3. Co-cultures of bacterial strain TCR and fungal strain TCF could significantly improve their degrading ability, which were able to mineralize 50mg/L chlorpyrifos within 18 hours completely. When mixed cultures of Serratia sp. TCR, Trichosporon sp. TCF and Paracoccus sp. TRP were used, 50mg/L chlorpyrifos could be completely mineralized within 12 hours. The optimum conditions for chlorpyrifos degradation by the mixed culture were with total inocula biomass 0.15g/L, sucrose 0.5%, at 30?C and pH 8.4. Possible intermediate metabolites of chlorpyrifos biodegradation were analyzed by GC-MS, HPLC, and TLC. 3,5,6-trichloro-2-pyridinol (TCP) was detected as the major intermediate metabolites. Paracoccus sp. DM/TRP and Trichosporon sp. TCF all could utilize TCP or pyridine as the sole carbon or nitrogen sources, suggesting that the chlorpyrifos degradation involve the pyridine ring cleavage, and finally mineralized into CO2 and H2O. These strains could also degrade TCP, pyridine, methyl parathion, and carbofuran, showing a board substrate range, which indicated their high potential use in bioremediation of contaminated environment.5. SDS-PAGE and Native-PAGE analysis of the total proteins of strains under induced and noninduced cultures indicated that the degrading enzymes in Paracoccus sp. DM/TRP might be inducible, while those in Serratia sp. TCR and Trichosporon sp. TCF might be constitutively expressed. Enzymetic degradation assays indicated that crude enzyme extracts could rapidly degrade chlorpyrifos into nontoxic substances; they were effective at 20~40℃and pH 7~9, with the optimum pH 8 and temperature at 30℃for bacteria and 35℃for fungus. These enzyme extracts might be useful in rapid detoxification and cleanup of pesticide residues on the surface of fruits and vegetables. Keywords: Biodegradation, Chlorpyrifos, 3,5,6-trichloro-2-pyridinol, Degrading microbes, Complete mineralization...
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