Studies On Biodegradation Of Nicosulfuron And Thifensulfuron-methyl By Microorganism

Sulfonylurea herbicides are novel herbicides with extremely high biological activities, and are widely used around the world. It has been widely used in a variety of crops to control broadleaf weeds and grasses. Its residues in soil and water may have potential impacts on the environment. In many areas, phytotoxicity to succession crops caused by sulfonylurea herbicides have been reported, which led to severe economic loss in agriculture. Therefore, excessive use of sulfonylurea herbicides has raised concerns for environmental contamination and phytotoxicity to crops. Pesticides degradation by microbe has the characteristics of quick, safe and environment friendly, which is an important way for bioremediation of pesticides pollution. In this study, the biodegradation of nicosulfuron and thifensulfuron-methyl by microbial degradation, and its metabolic pathways were investigated. The main results were summarized as follows:1、Mixed flora capable of degrading nicosulfuron were obtained from sulfonylurea herbicides contaminated farmland soil. A bacterial strain ZWS11was isolated by streaking plate method from mixed flora and identified as potential nicosulfuron-degrading bacterium. Based on morphological, physicochemical of the bacterium and phylogenetic analysis of16S rRNA sequence, strain ZWS11was identified as Alcaligenes faecalis. Strain ZWS11could degrade more than80%of initial nicosulfuron supplemented with500.0mg/L under the conditions of pH7.0,180rpm and30℃after incubation for6days. Additionally, strain ZWS11could degrade nicosulfuron over a broad range of initial concentrations, and good degradation was found when the initial pH value was6.0-8.0, inoculation volume was1%-10%. Strain ZWS11was also capable of degrading rimsulfuron, tribenuron-methyl and thifensulfuron-methyl, but not ethametsulfuron-ethyl and bensulfuron-methyl. Four metabolites were detected and identified by HRLC-MS, among them4,6-Dihydroxypyrimidine is the first reported in the degradation of nicosulfuron by microbial degradation. On the basis of these metabolites, the primary degradation pathway of nicosulfuron were proposed, and the degradation might be proceeded mainly via cleavage of sulfonylurea bridge, o-dealkylation of heterocyclic, and contraction of sulfonylurea bridge by elimination of sulfur dioxide group.2、The degradation enzyme of strain ZWS11was found mainly in the extracellular. The optimal pH value, temperature and reaction time of extracellular crude enzyme solution were5.0-7.0,35℃and30min, respectively. High degradation activities were still observed when extracellular crude enzyme solution was pretreated for2h at high temperature or extreme pH conditions. The degradation efficiency of extracellular crude enzyme to sulfonylurea herbicides descended as follows:nicosulfuron> thifensulfuron-methyl> metsulfuron-methyl> rimsulfuron> Triasulfuron. Meanwhile, the enzyme activities were inhibited when supplemented with SDS, PMSF, phoxim, malathion, etc. in the reaction system, whereas no inhibition was observed when EDTA, TPP, PBO, chlorpyrifos etc. was supplemented. In addition, homologous genes related with degrading or metabolizing sulfonylurea herbicides, such as SulE, Tsme, suaC and subC were not found in strain ZWS11. Different protein bands were obtained from extracellular crude enzyme solution by SDS-PAGE. The protein bands were analyzed by peptide mass fingerprinting, but no enzyme related with degrading or metabolizing sulfonylurea herbicide was found.3、Three thifensulfuron-methyl-degrading bacteria, named ZWS13, ZWS16and ZWS18, were isolated from sulfonylurea herbicides contaminated farmland soil and identified as Staphylococcus sp., Ochrobactrum sp. and Stenotrophomonas sp., respectively, by morphological and sequence analysis of16S rRNA. The effects of different initial concentrations of thifensulfuron-methyl, initial pH levels, temperatures and inoculum sizes on the degradation were examined. Different strains could degrade thifensulfuron-methyl over a broad range of initial concentrations. At the same initial concentration, the degradation efficiency of strain ZWS18was higher than that of strain ZWS13and ZWS16. At a concentration of50.0mg/L, the degradation rates of thifensulfuron-methyl were approximately99%by different strains after incubation for10d at40℃. The degradation of thifensulfuron-methyl by three strains was different at different inoculum sizes (1%,3%and5%). For strain ZWS16, the degradation efficiency of thifensulfuron-methyl at pH6.0was faster than that at pH7.0and8.0; for strain ZWS13, the degradation rate enhanced with the increase of pH levels; and for strain ZWS18, significant differences were not detected on the degradation rate when pH was6.0-8.0. Three strains of bacteria could also degrade nicosulfuron and tribenuron-methyl.4、Degradation products of thifensulfuron-methyl by strains ZWS13, ZWS16and ZWS18were identified through HPLC-MS. Thifensulfuron acid was detected. On the basis of metabolites, the degradation pathways of thifensulfuron-methyl were proposed, which might proceed via cleavage of the sulfonylurea bridge, o-demethylation, de-esterification and cleavage of the triazine ring.5、After35days of incubation, only approximately18.9%of thifensulfuron-ethyl was degraded in sterile soil without inoculation of bacteria. However, when strain ZWS16and ZWS18were inoculated into the sterile soil, the degradation rates of thifensulfuron-methyl were increased to58.1%and52.4%, respectively. These data clearly demonstrated that the inoculation of strain ZWS16and ZWS18significantly accelerated the degradation of thifensulfuron-methyl in sterile soil.