Isolation And Characterization Of Carbofuran-degrading Bacteria

Carbofuran(C₁₂H₁₅NO₃)is a carbamate insecticide and nematocide used for the control of a variety of soildwelling and foliar-feeding pests on many fruit and vegetable crops. Carbofuran is known to be a more persistent insecticide than other carbamate or organophosphorus insecticides.Continued use of the insecticide at single field sites has led to the problem of enhanced degradation Hence,the experiment of carbofuran-degrading by using microorganisms was conducted in our lab.1.Carbofuran-degrading bacteria were isolated from activated sludge samples of the Agri-chemistry factory in Shandong province through the batch culture enrichment technique.About 200 carbofuran-degrading organisms were isolated for their ability to degrade pesticide.Bacterial strains CYW-32 and CYW-44 were selected due to their effcient degardation of carbofuran.The 16S rRNA analysis results showed that the isolates belong to the genus of Methylobacterium and Bacillus respectivelly.Combined physiological and biochemical tests,strain CYW-32 was identified as a Methylobacterium organophilium.,and strain CYW-44 was identified as a Bacillus subitilis,by API strip test. Additionally,this is the first report that carbofuran was degraded by Methylobacterium organophiliurn,and Bacillus subitilis.100mg/L carbofuran was degraded by strain CYW-44 up to 97.72%within 5 days.When time-course degradation of carbofuran was examined by liquid chromatographic analysis,almost no accumulation of carbofuran was observed within 6 days.5.8mg/L carbofuran in contaminated soil was completely degraded by strain CYW-44 while strain CYW-32 cast a pall on degradation.100mg/L carbofuran in simulated wastewater was degraded up to 53.73%and 41.38%by strain CYW-32 and strain CYW-44 respectively.2.HPLC analysis results demonstrated that there was no accumulation of carbofuran phenol and the other metabolic intermediate during the degradation of carbofuran by strain CYW-44,while carbofuran phenol was found accumulated during degradation by strain CYW-32.After 7 days growth and degradation in carbofuran,the optimal carbofuran-degradation by strain CYW-32 was obtained at pH 7.0,temperature 30-32℃, and inoculation quantity had no significant influence on growth of and degradation of carbofuran respectively.Growth of strain CYW-44 and degradation of carbofuran were not affected significantly by pH value,temperature and inoculation quantity.89.6% carbofuran was degraded even at pH 5.Preliminary mechanism of degradation was investigated.Both ectoenzyme and endoenzyme of strain CYW-44 played significant roles during carbofuran degradation.SDS-PAGE analysis showed that a new protein band was found when strain CYW-44 growed on the carbofuran medium.It indicated that degradation activities of some enzymes could be induced by carbofuran.3.By using single-factor test and orthogonal combination experiment,fermentation conditions was optimized:optimal conditions of strain CYW-32 as the medium was composed of Methanol 2.0g,NH₄NO₃ 2.0g,K₂HPO₄ 7.5g,KH2PO4 2.0g,MgSO4.7H2O 0.2g,CaCl2.2H2O 15mg,NaCl 0.5g,H2O 1000mL;The optimum fermentation condition for the strainCYW-32:pH 7.0;temperature 32℃;rotation speed 150r/min;volume 50ml/250ml.Optimal conditions of strain CYW-44 as the medium(%)was composed of glucose 2%,soybean powder 2%,NaCl 2%,KH₂PO₄ 3%,MgSO₄·7H₂O 0.03%.The relative parameters including:culture pH 6.0,temperature 28℃,rotation speed 180r/min, volume 25ml/250ml.4.Both strains exhibited extra biological functions besides degrading of carbofuran. 500mg/L methanol was efficiently degraded within 20h in mineral salts medium by strain CYW-32.Single cell protein was produced with a high yield of 89.26%by utilization of methanol as source of carbon and energy.The bacterium was also rich in various kinds of amino acid.It was found that the bacterial strain CYW-44 provided an inhibit effect against...