A Butachlor-degrading Bacterium And Its Application In Wastewater Treatment And Soil Pollution Remediation

Butachlor is one of the most widely used herbicides in China. As a result of massproduction and extensive use, it has caused considerable pollution to the water and soil andposes a threat to ecosystem and human health. Therefore, there is an increasing concern onthe cleanup of water and soil contaminated by butachlor. One bacterial strain capable ofutilizing butachlor as sole carbon and energy source was isolated. The biodegradationcharacteristics and mechanisms of butachlor and pretilachlor were investigated, and theapplication of the bacterium strain in butachlor wastewater treatment and soil pollutionremediation were also investigated. The results are summarized as follows:A bacterial strain BD-1 capable of utilizing butachlor as sole carbon and energysources was isolated from the sludge of a membrane bioreactor after successive enrichmentcultures. BD-1 was identified as Pseudomonas stutzeri based on morphology, physiologicaland biochemical characteristics, Biolog GN2, and the results of the 16S rDNA homologuesequence analysis.The degradation characteristics of butachlor by bacterial strain BD-1 was investigatedin pure cultures. The degradation of butachlor by BD-1 was fitted to the fisr-order function.Degradation half-lives of butachlor at concentration of 1.0,10.0 and 100.0 mg L^-1 by BD-1were measured to be 0.11 d, 0.60 d and 0.96 d, under conditions of pH 7.0, 25℃andbiomass of 0.2 (OD₄₁₅). The degradation rates of butachlor by bacterial strain BD-1 wereaffected by pH and temperature following an order of pH 7.0＞pH 6.0＞pH8.0, and of 30℃＞20℃＞40℃, respectively. Seven metabolites were identified by GC/MS based onmass spectra data and fragmentation patterns. The main biodegradation products ofbutachlor were 2-chloro-2',6'-diethylacetanilide and 2,6-diethylacetanilide. The metabolismof butachlor by strain BD-1 involved dechlorination, dealkylation, hydrolyzation andhydroxylation. According to the capability of the bacterial strain BD-1 to utilize butachloras sole carbon and energy sources, it is reasonable to propose that butachlor is mineralizedby the BD-1.Bacterial strain BD-1 was able to use pretilachlor as sole carbon and energy source.The ability of BD-1 to degrade pretilachlor in pure cultures was affected by initialconcentration, pH and temperature. The degradation half-lives of pretilachlor weremeasured to be 0.10 d, 0.23 d and 0.46 d at concentrations of 1, 10 and 100 mg L^-1,respectively. The optimal conditions for biodegradation of pretilachlor were pH 7.0 and 30 ℃. Five metabolites were detected and identified by GC/MS. 2-chloro-2',6'-diethylacetanilidewas the major biodegradation products of pretilachlor by BD-1. The mainmetabolisms of pretilachlor by BD-1 included dechlorination, dealkylation, hydrolyzationand hydroxylation.One membrane bioreactor (MBR) was constructed with the bacterial strain BD-1. TheMBR was used for the treatment of pesticide wastewater containing butachlor and otherchloroacetanilide herbicides. The results showed that MBR had high removal efficiency forCOD_Cr, pesticide, turbidity and odor in the pesticide wastewater. The pollutant removalefficiency of MBR was significantly affected by influent pH and COD_Cr. The optimaloperating conditions were pH 8.0 and 1500 mg L^-1 of influent COD_Cr. The addition ofnutrient salts had little effects on the removal efficiency of MBR. About 81%, 95 %, 99 %and 100 % of COD_Cr, total pesticide, butachlor and turbidity in pesticide wastewater wereremoved under the optimal operating conditions. Continuous operation of MBR for morethan 12 months showed stable performance for butachlor wastewater treatment.Pseudomonas stutzeri BD-1 was immobilized with calcium-alginate. Degradation ofbutachlor by immobilized BD-1 proved to be more efficient than free BD-1. Thedegradation half lives of butachlor by the immobilized and free BD-1 at concentrations100.0 mg L^-1 were measured to be 1.55 h and 1.93 h, respectively, under conditions of pH7.0 and 30℃. Immobilized BD-1 showed ideal performance for butachlor removing in testpH and temperature. Compared to the free BD-1, immobilized BD-1 could withstand a widerange of pH and temperature change. Butachlor degradation using immobilized BD-1 wasreused for five times, and enhancing degradation rate was observed. The degradationhalf-life of butachlor was shorten from 1.91 h to 0.91 h after 5th reuse.Immobilized microorganism reactor was set up to determine the feasibility of usingimmobilized strain BD-1 to remove of butachlor from simulated wastewater. The butachlorremoval efficiency of the immobilized microorganism reactor was significantly affected bypH and flow rate. A butachlor removal efficiency of greater than 99 % was achieved underthe condition of pH 7.0 and flow rate of below 50 mL min^-1.A membrane bioreactor-immobilized microorganism combined system was constructedby connecting immobilized column to the effluent port of MBR and its use in butachlorwastewater treatment was investigated. At the optimal operating conditions, the COD_Cr,BOD₅, butachlor, turbidity and odor removal efficiency of this system were 97.68 %, 98.90% 100 %, 100 %, 100 %, respectively, Other chemicals in the butachlor wastewater were also effectively removed.Degradation of butachlor at levels of 2.0, 4.0, and 10.0 mg kg^-1 in laboratory soil wereall fitted to the first-order kinetics, and the corresponding degradation half-lives weredetermined to be 6.53 d, 8.81 d, and 11.27 d, respectively. The degradation half-lives ofbutachlor in soil were extended significantly with the increasing concentration of butachlor.The soil microbial diversity was inhibited significantly 1d after butachlor treatment. Theinhibitory effects were enhanced with the increasing concentration of butachlor.The degradation of butachlor in laboratory soil was significantly enhanced by theinoculation of bacterial strain BD-1. Degradation of butachlor in strain BD-1 inoculatedtreatments (2.0 mg kg^-1+BD-1, 4.0 mg kg^-1+BD-1, and 10.0 mg kg^-1 mg kg^-1+BD-1) werefitted to fist-order kinetics and the corresponding degradation half-lives were measured tobe 6.53 d, 8.81 d, and 11.27 d, respectively. Compared to the un-inoculated controls, thedegradation half-lives of butachlor were shorted by 94.33 %, 94.78 % and 91.22 %,respectively. The addition of bacterial strain BD-1 could eliminate the inhibitive effects ofbutachlor on soil microbes and enhance soil microbial diversity.Under the field conditions, bacterial strain BD-1 could effectively enhance thedegradation of butachlor in soil. Degradation of butachlor in all treatments was fitted to thefirst-order kinetics. The degradation half-lives of butachlor in control soil and strain BD-1inoculated soil were 11.17 d, 4.95 d at 1.26 kg a.i. ha^-1, 11.67 d, 4.95 d at 2.52 kg a.i. ha^-1and 15.71 d, 5.99 d at 6.30 kg a.i. ha^-1, respectively. Biolog analysis showed that butachlorcould reduce the microbial diversity in the soil during initial time of experiment, while theaddition of BD-1 could restore and enhance the soil microbial diversity. The resultsindicated that bacterial strain BD-1 inoculation is a promising method for the remediation ofbutachlor contaminated soil.