Isolation Identification And Degradation Characters Of Carbendazim-Degrading Strains

Three efficient carbendazim-degrading bacteria djl-6, djl-6-2 and CBDM-12 were isolated by continuous enrichment, screened from long carbendazim-treated soils and the sludge of the wastewater of carbendazim treatment They were all identified as Rhodococcus sp. according to their morphological observation, physiological biochemical test, comparison sequences of 16S rDNA and phylogenetic analysis.The isolates were all gram-positive, had spherical and bacilliform growth cycle, could produce obvious clearing zones around the colonies on the minimum medium amended with carbendazim as sole carbon sources. And among them, djl-6 could form orange colony on LB agar medium after 2～3 days of incubation, whereas djl-6-2 and CBDM-12 could form creamcolored colony. The optimum temperature and pH value for their growth were all 30℃and 7.0 respectively. Still they could all sustain on high salt concentration, and some antibiotics such as ceftazidime. They could grow well when using glucose or fructose as sole carbon sources, and peptone, beef cream or yeast extract as sole nitrogen sources.Studies indicate that the isolates could use carbendazim as the sole carbon, sole nitrogen and sole carbon and nitrogen sources, they grew and degraded better when carbendazim was used as sole nitrogen source than as sole carbon and sole carbon and nitrogen sources. Carbendazim, 100mg.L^-1, could almost be degraded completely within 2～3 days incubation of the isolates, exceeded the biodegradability of carbendazim-degrading strains reported so far. A little alkaline condition benefit the biodegradation of carbendazim. The optimum temperature for their biodegradability was all 30℃, and the isolates djl-6 and djl-6-2 could still sustain a broad range of temperature. The feasible salt concentration for the carbendazim degradation was 20～30mg.L^-1, the influence of aeration on the biodegradability of the isolates was trivial. Adding yeast extract and glucose could accelerate the biodegradation of carbendazim, while the soil extract could not accelerate just because of the poor nutriment in the soils. The ion except Cu²⁺ had no definite inhibition on the degradation of carbendazim by the isolates. What’s more, the isolates still can degrade benomyl and phenol efficiently, and degrade MTMC and metalaxyl in some degree. In conclusion, all the characteristics shown above that the three isolates were the optical candidates for bioremediation of carbendazim-contaminated conditions.Phylogenetic analysis and comparison of physiological biochemical test shown that, djl-6 was analogous to Rhodococcus baikonurensis, Rhodococcus erythropolsis, but far from Rhodococcus globerulus and other species of Rhodococcus. Polyphasic taxonomy shown that the main fatty acid composition of djl-6 were C_14:08.12%, C_15:0ISO 2OH 8.49%, C_16:0 25.97%, C_18:0 7.01%, C_18:1 w9c 7.32%, C_18:0 TBSA 10Me 19.81%, C_19:0 4.31% and C_20:03.36%; the main amino acid in djl-6 were meso-DAP, Ala, Glu, Gly and Asp; the main sugar in djl-6 were galactose, glucose and arabinose; the main menaquinone was MK-8（H2）; the G+C mol% was 59.1%; the hybridization value of djl-6 with R. baikonurensis DSM44587, R. erythropolis DSM43066, R. globerulus DSM43954 were 23.8518%, 31.8361% and -17.7155% （approximate to 0%） respectively. Drawn from the above characteristics, the djl-6 represented a novel Rhodococcus species, for which the name Rhodococcus qingshengensis sp. nov. was proposed.Studies also found that the carbendazim-degrading enzyme was a constitutive and not inductive enzyme. To extract the enzyme, culture djl-6 in LB liquid medium for 72～84 hours, then break it up with lysozyme, ultrasonic and French pressure cell press. The enzyme activity could save a lot with adding lysozyme method, lose a lot with ultrasonic and French pressure cell press, but could get more crude enzyme with the latter two methods. The best reaction system as following: incubation 30μL crude enzyme in pH7.0 PBS for 30～60min at 30℃. The enzyme was jarless among pH 7.0～8.0, and temperature 4～45℃, the ion Zn²⁺ and K⁺ could inhibit the enzyme activity, adding Tween 20 would not inhibit the enzyme activity, but Triton X-100 and SDS would inhibit enzyme activity when the adding concentration exceed 100mg.L^-1. When the concentration of ammonium sulfate precipitation attained to 50%, the enzyme activity in the liquid phase lost a lot, while the enzyme activity of sediment would flare up when the concentration of ammonium sulfate precipitation attained to 40%. Studies still found that the carbendazim-degrading enzyme was an ester enzyme according to the analysis of enzyme activity. An object protein with a molecular weight between 97.4～66.2 KDa was harvested through recycling the Clear strap in PAGE amended with carbendazim.After 24 hours incubation of djl-6 in the medium amended with carbendazim as sole carbon source, high pressure liquid chromatography-mass spectrometry （HPLC-MS） analysis showed the presence of 2-aminobenzimidazole （rn/z=134.3）, benzimidazole （m/z=119.3）, and an unknown metabolite with molecular ions （M⁺） of m/z 104.8 and 118.5 Through the analysis of HPLC, 100mg.L^-1 of carbendazim could almost, be degraded completely after two to three days incubation of djl-6. The biodegradation in the isolate djl-6 seems to be initiated with the cleavage of the methyl carbemate side chain, resulting in the formation of 2-aminobenzimidazole and 2-aminobenzimidazole is further converted to benzimidazole and so on in succession. This is the first report of the intermediates 2-aminobenzimidazole and benzimidazole were found together in the culture filtrate of pure bacterium.Bioremediation of djl-6 in red soil, fluvo-aquic soil and high-sandy soil showed that the degradation of carbendazim in the soils need a definite soil humidity, a little high humidity benefited the biodegradation of carbendazim, but the biodegradation could be inhibited when flooding the soils completely. Matched to the growth characters, the optimum pH and temperature for biodegradation of djl-6 in the soils were pH 7.0 and 30℃respectively. Studies also found that the biodegradation of carbendazim mainly was completed by the isolate djl-6 through the analysis of incubation amount of djl-6 and sterilizing the soils or not, a little of incubation benefit the biodegradation of carbendazim. The addition of glucose accelerated biodegradation of carbendazim in the soils.Effects of adding carbendazim and carbendazim-degrading strain djl-6 on the peroxidase, dehydrogenase, saccharase and urease activity in red soils, fluvo-aquic soil and high-sandy soil indicated that the activity of peroxidase of adding strain djl-6 and carbendazim exceed that of adding carbendazim only, and the activity of peroxidase would be inhibited at the beginning when adding carbendazim only, but would be resumed when the microbes adapt to the condition of adding carbendazim. Because of the different composition of microbes in high-sandy soil, the results of dehydrogenase activity change in high-sandy soil were different from that of red soils and fluvo-aquic soil, the activity of dehydrogenase was inhibited at the beginning with or without incubation of djl-6, and could not resume to the beginning level without adding djl-6 and carbendazim with the time go on. The influence of adding djl-6 and carbendazim on the saccharase and urease activity in red soils, fluvo-aquic soil and high-sandy all showed that the activity could be inhibited at the beginning, but would be resumed with time go on, Still the activity of urease of incubation of djl-6 exceeded that of without incubation of djl-6.