Bioremediation And Application Of Chlorimuron-Ethyl Degrading Strain

Chlorimuron-ethyl, a sulfonylurea herbicide produced by Dupond Co. in 1980s, was used to control broad-leaved weeds and grasses in soybean. Because of high phytotoxicity and transportation in surface runoff, Chlorimuron-ethyl was concerned about its possible impact on aquatic ecosystems. Because of its low cost and high efficiency, Chlorimuron-ethyl has been used worldwide, ultimately resulting into serious environment pollution. Microbial activity has been proved to be the most influential and significant cause of sulfonylurea herbicide removal and biotransformation. Microbial remediation has been deemed to be much more advantageous method than the others. So it has been an important research for us to use microbial resource to repair environment pollution.A bacterum strain LW4, capable of degrading Chlorimuron-ethyl, was phylogenetically analyzed. The metabolic pathway of Chlorumuron-ethyl by strain LW3 was detected and inferred basically. The effects of Chlorimuron-ethyl on soil microecosystem were studied in garden soil samples with a short-term treatment of Chlorumuron-ethyl at different concentrations. The culturable bacteria (plate counts), soil respireation, soil enzyme activities, and changes in microbial community structure were used to assess biological community in Chlorimuron-ethyl contaminated soil. The results will be valuable to build up alert index systems in contaminated soils.The main results of this study are as follows:One soil Chlorimuron-ethyl-degrading bacterium strain LW4 was obtained from contaminated soil. Based on physiological characteristics, biochemical tests and a partial analysis of the 16S rDNA gene sequence, the strain LW4 was identified as Pseudomonas sp. Together with another Chlorimuron-ethyl degrading bacterium LW3, the degrading characteristics of the two strains were analyzed. The optimal growth temperature of LW3 was 30℃, optimal initial pH was 7.0, and the optimal C/N was 8:1. The optimal growth temperature of LW4 was 30℃, optimal initial pH was 7.0, and the optimal C/N was 16:1, respectively. The two strains both could utilize Chlorimuron-ethyl as sole nitrogen resource and sole carbon resource. Addition of glucose and peptone could promote the degradation of Chlorimuron-ethyl respectively. Under low NaCl (0-20g·L-1NaCl) conditions, LW3 had higher degrading efficiency than LW4. When the concentration of NaCl increased (25-50g·L-1NaCl), the degrading efficiency of the two strains decreased, but LW4 decreased much lower than LW3. Different metal ion had different effects on degradation. Mg2+ could promote the degradation of LW3, Ca2+, Mn2+, Cu2+ could inhibit the degradation of LW3. Al3+ could promote the degradation of Chlorimuron-ethyl by LW4.The Chlorimuron-ethyl-degrading enzyme of LW3 and LW4 were extracted, and they both were intracellular enzyme and could not be induced. The degrading enzyme of LW3 had optimal pH of 7, optimal temperature of 30℃and was steady at pH 6-7, while the degrading enzyme of LW4 had optimal pH of 7, optimal temperature of 30℃and was steady at pH 6-8. Mg2+, Ni+ could promote the activity of LW3 and Zn2+, K+ could inhibit the activity, Al3+ could promote the activity of LW4 and Zn2+, K+ could inhibit the activity.The degradation products of Chlorimuron-ethyl in the culture medium extracts by LW3 were isolated and identified by HPLC and LC/MS. Two major metabolites of Chlorimuron-ethyl degradation were detected. Based on the identified products, the first step of Chlorimuron-ethy degrading by strain LW3 seemed to be ethyl ester saponification, followed the urea-bridge broke, forming N-formyl-benzosulfimide and 4-chloro-methoxy-pyrimidin-2-amine.The influences of Chlorimuron-ethyl on the cultural microorganisms in Yellow-brown soil were studied by traditional selective plating and direct viable counting methods in 50 days, soil respireation and enzymes activities were also investigated. The results showed that the bacteria differed markedly in their response on Chlorimuron-ethyl. The concentration of Chlorimuron-ethyl applied was an important factor affecting populations of various microorganisms. When the concentration of Chlorimuron-ethyl was higher than lOμg·kg-1 dry soil, the total number of bacteria in the soil samples polluted by Chlorimuron-ethyl was significantly lower than that in the control group. The resumed rates of bacterial number were significantly slower than that the control group during incubation. The fungi growth showed the similar result with bacteria, Actinomycete bacteria in the soil samples polluted by Chlorimuron-ethyl were inhibited during incubation. The soil respireations of every treatment were inhibited during two weeks of Chlorimuron-ethyl treatment and the effects were not obvious. The inhabitation could be recovered in 28 days. The results of enzymological studies indicated that the Chlorimuron-ethyl could inhibit the enzyme activities of polyphenol oxidase (21d), saccharase (7d), urase (7d) intensively. The higher the concentration was, the more obviously the inhibition was. The enzyme activities of acid phosphatase and dehydrogenase were lightly affected. The catalase activity was promoted at low Chlorimuron-ethyl concentration and inhibited at high concentration. Residual Chlorimuron-ethyl in soil could inhibit the growth of maize, in different soils, the inhibition ratio was sandy soil> brown soil> red soil.When LW3 was inoculated the contaminated soils, the degrading of chlorimuron-ethyl mainly occurred during the first 30 days. In the natural soil samples, the degradation efficiercy were higher than the sterilized soils. The strain could cooperate well with the indigenous microorganisms to metabolize Chlorimuron-ethyl in the soil. The indigenous microorganisms might dagrade the intermediate metabolites metabolized by LW3 and enhance the degrading effciency. When inoculated to different soils, the degrading efficiency was affected by the amount of strain, soil temperature, soil pH, soil moisture capacity, and so on. When inoculum size was 1.0×107CFU·g-1dry soil, pH was 7, temperature was 30℃, the degradation in soil had the highest efficiency. Use of LW3 to soak the seeds could relieve the inhibition to maize caused by the remained Chlorimuron-ethyl in soils. Adding LW3 strain directly to the contaminated soils could hardly relieve the inhibition, which maybe caused by the strain activities.