多菌灵降解菌的分离、鉴定及其降解特性研究
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摘要
从长期受多菌灵污染的土壤和处理多菌灵废水的污泥中分离到3株高效多菌灵降解菌株，分别命名为djl-6、djl-6-2和CBDM-12。根据它们的培养特征、生理生化特征以及16s rDNA序列的同源性分析等，鉴定为红球菌属。
研究表明，这3株多菌灵降解菌都是革兰氏阳性菌，有球杆状变化，都能在添加多菌灵的无机盐平板上产生清晰可见的透明圈；其中djl-6在LB上培养时，菌落呈橙色；djl-6-2和CBDM-12菌落呈乳黄色；3株菌的最适生长温度为30℃、pH值为7.0；都能耐受一定浓度的NaCl和抗生素；对葡萄糖和果糖等单糖的利用要好于麦芽糖和淀粉等多糖，对蛋白胨、牛肉膏和酵母等有机氮源的利用明显的优于以硫酸铵等无机氮源。
降解菌株均能以多菌灵为唯一碳、唯一氮和唯一碳氮源进行生长。3株菌以多菌灵为唯一氮源时的生长和降解速度均好于以多菌灵为唯一碳和唯一碳氮源时的生长和降解速度。它们均能在2～3天时间内几乎完全降解100mg．L~(-1)的多菌灵，降解效果明显优于国内外已经报道的多菌灵降解菌株；降解菌株在中性稍微偏碱的环境中更有利于其对多菌灵的降解，它们降解多菌灵的最适温度都为30℃，其中菌株djl-6和djl-6-2降解多菌灵有比较宽的温度耐受范围；它们降解多菌灵的最适的Na~+浓度范围为20～30g．L~(-1)，通气量对3株菌降解多菌灵的影响不大；添加酵母粉和葡萄糖对多菌灵的降解有一定的促进作用，添加土壤浸液对多菌灵的降解没有影响；Cu~(2+)对3株菌降解多菌灵有一定的抑制作用，其他的金属离子对多菌灵的降解没有太大影响；3株菌都能有效的降解苯菌灵和苯酚，对速灭威和甲霜灵也有一定的降解效果；所有这些特性表明3株菌可作为多菌灵污染生物修复的理想材料。
系统发育分析和生理生化特征比较分析结果表明，djl-6和Rhodococcus baikonurensis、Rhodococcus erythropolsis亲缘关系较近，和Rhodococcus globerulus较远；多相分类结果表明，djl-6细胞中主要的脂肪酸组成为：C_(14∶0) 8.12％、C_(15∶0 1SO 2OH) 8.49％、C_(16∶0) 25.97％、C_(18∶0) 7.01％、C_(18∶1 w9c) 7.32％、C_(18∶0 TBSA 10Mc) 19.81％、C_(19∶0) 4.31％、C_(20∶0) 3.36％；氨基酸为：meso-DAP、Ala、Glu、Gly和Asp；糖为半乳糖，阿拉伯糖和葡萄糖；醌为MK-8(H2)；G+C mol％为59.1；djl-6和R．baikonurensis DSM44587、R．erythropolis DSM43066，R．globerulus DSM43954的DNA-DNA杂交同源性分别为23.8518％、31.8361％和-17.7155％(为0％)。所有这些特性表明djl-6是红球菌属中的一个新种，初步命名为庆笙红球菌Rhodococcus qingshengensis sp．nov．。
多菌灵降解酶是一种胞内非诱导组成型的酶。在提取该酶时，可把djl-6在LB培养基中培养到72～84h为宜，然后可采用添加溶菌酶、超声波破碎或French压榨等方法来提取。采用添加溶菌酶方法提取的降解酶酶活较高，而采用超声波和French压榨破碎虽然提取的酶量较大，所得总蛋白浓度也较高，但是酶活损失较大；多菌灵降解酶的最佳反应体系为在PH 7.0的缓冲系统中，控制温度为30℃，在5mL反应体系中添加30μL的粗酶液，反应时间控制在1小时之内，通常为30分钟；多菌灵降解酶在pH 7.0～8.0，温度4～45℃的范围内较稳定；金属离子Zn~(2+)和K~+对酶活有抑制作用。添加Tween 20对酶活测定没有影响，而添加Triton X-100和SDS，当浓度超过100mg．L~(-1)时就能对降解酶的活性产生一定地抑制作用；粗酶液的硫酸铵分级沉淀效果表明，当在上清液中添加硫酸铵浓度达到50％后，上清液中的酶活骤然降低。沉淀蛋白在硫酸铵的添加浓度达到40％以后，酶活就骤然上升，在50％以后达到最大。所以在用硫酸铵沉淀粗酶时可选择50％的硫酸铵沉淀浓度对降解酶进行粗分；同时降解酶能够在添加多菌灵的非变性胶上产生清楚可见的透明带。与此透明带对应的条带酯酶染色表明，该酶同时也是一种酯酶。把分离胶中的透明带切胶回收，可回收到一条分子量大小在97.4～66.2Kda之间的目的蛋白。
对多菌灵代谢过程进行跟踪研究，发现在djl-6接种24小时后有三种中间代谢物存在，分别是2-氨基苯并咪唑(m／z=134.3)、苯并咪唑(m／z=119.3)和一种分子离子峰为104.8和118.5的未知物质。而经过2～3天的培养后，HPLC分析表明100mg．L~(-1)的多菌灵几乎被完全降解。推测多菌灵可能首先被转化为2-氨基苯并咪唑，然后再进一步被转化，最后被完全降解。本研究也是首次发现2-氨基苯并咪唑和苯并咪唑在纯菌多菌灵代谢物中同时存在。
djl-6在红壤、潮土和高沙土中的降解实验表明，其在土壤中降解多菌灵需要一定的水分含量，稍高的水分含量有利于其对多菌灵的降解，但完全淹水的条件又会影响到其降解功能的发挥；与最适生长特点相符，在土壤中djl-6适宜的降解pH和温度分别为7.0和30℃；三种土壤中多菌灵的降解主要是由降解菌完成的，且接种量越大降解效果越好；添加葡萄糖对djl-6降解土壤中多菌灵有一定地促进作用。
投加多菌灵和djl-6对红壤、潮土和高沙土三种土壤酶活性的影响表明，对过氧化氢酶活性的影响都是加降解菌的酶活要高于未加菌的酶活。未加菌处理初期都表现出抑制现象，而后又缓慢得到恢复；对土壤脱氢酶活性的影响，高沙土和红壤、潮土的实验结果相反，在加菌处理和未加菌处理中脱氢酶活性一开始就表现出抑制现象。其后虽然中间得到了一定的恢复，但仍然低于起始时没有经过任何处理的脱氢酶活性；三种土壤的实验结果都表明，加菌处理和未加菌处理对蔗糖酶和脲酶活性的影响在初期时都表现出抑制作用，其后逐渐恢复。接种降解菌的土壤脲酶活性要高于未接菌土壤的脲酶活性。
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 Cu~(2+) 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_(14:0)8.12%, C_(15:0)ISO 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_(20:0)3.36%; 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 Zn~(2+) 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.
研究表明，这3株多菌灵降解菌都是革兰氏阳性菌，有球杆状变化，都能在添加多菌灵的无机盐平板上产生清晰可见的透明圈；其中djl-6在LB上培养时，菌落呈橙色；djl-6-2和CBDM-12菌落呈乳黄色；3株菌的最适生长温度为30℃、pH值为7.0；都能耐受一定浓度的NaCl和抗生素；对葡萄糖和果糖等单糖的利用要好于麦芽糖和淀粉等多糖，对蛋白胨、牛肉膏和酵母等有机氮源的利用明显的优于以硫酸铵等无机氮源。
降解菌株均能以多菌灵为唯一碳、唯一氮和唯一碳氮源进行生长。3株菌以多菌灵为唯一氮源时的生长和降解速度均好于以多菌灵为唯一碳和唯一碳氮源时的生长和降解速度。它们均能在2～3天时间内几乎完全降解100mg．L~(-1)的多菌灵，降解效果明显优于国内外已经报道的多菌灵降解菌株；降解菌株在中性稍微偏碱的环境中更有利于其对多菌灵的降解，它们降解多菌灵的最适温度都为30℃，其中菌株djl-6和djl-6-2降解多菌灵有比较宽的温度耐受范围；它们降解多菌灵的最适的Na~+浓度范围为20～30g．L~(-1)，通气量对3株菌降解多菌灵的影响不大；添加酵母粉和葡萄糖对多菌灵的降解有一定的促进作用，添加土壤浸液对多菌灵的降解没有影响；Cu~(2+)对3株菌降解多菌灵有一定的抑制作用，其他的金属离子对多菌灵的降解没有太大影响；3株菌都能有效的降解苯菌灵和苯酚，对速灭威和甲霜灵也有一定的降解效果；所有这些特性表明3株菌可作为多菌灵污染生物修复的理想材料。
系统发育分析和生理生化特征比较分析结果表明，djl-6和Rhodococcus baikonurensis、Rhodococcus erythropolsis亲缘关系较近，和Rhodococcus globerulus较远；多相分类结果表明，djl-6细胞中主要的脂肪酸组成为：C_(14∶0) 8.12％、C_(15∶0 1SO 2OH) 8.49％、C_(16∶0) 25.97％、C_(18∶0) 7.01％、C_(18∶1 w9c) 7.32％、C_(18∶0 TBSA 10Mc) 19.81％、C_(19∶0) 4.31％、C_(20∶0) 3.36％；氨基酸为：meso-DAP、Ala、Glu、Gly和Asp；糖为半乳糖，阿拉伯糖和葡萄糖；醌为MK-8(H2)；G+C mol％为59.1；djl-6和R．baikonurensis DSM44587、R．erythropolis DSM43066，R．globerulus DSM43954的DNA-DNA杂交同源性分别为23.8518％、31.8361％和-17.7155％(为0％)。所有这些特性表明djl-6是红球菌属中的一个新种，初步命名为庆笙红球菌Rhodococcus qingshengensis sp．nov．。
多菌灵降解酶是一种胞内非诱导组成型的酶。在提取该酶时，可把djl-6在LB培养基中培养到72～84h为宜，然后可采用添加溶菌酶、超声波破碎或French压榨等方法来提取。采用添加溶菌酶方法提取的降解酶酶活较高，而采用超声波和French压榨破碎虽然提取的酶量较大，所得总蛋白浓度也较高，但是酶活损失较大；多菌灵降解酶的最佳反应体系为在PH 7.0的缓冲系统中，控制温度为30℃，在5mL反应体系中添加30μL的粗酶液，反应时间控制在1小时之内，通常为30分钟；多菌灵降解酶在pH 7.0～8.0，温度4～45℃的范围内较稳定；金属离子Zn~(2+)和K~+对酶活有抑制作用。添加Tween 20对酶活测定没有影响，而添加Triton X-100和SDS，当浓度超过100mg．L~(-1)时就能对降解酶的活性产生一定地抑制作用；粗酶液的硫酸铵分级沉淀效果表明，当在上清液中添加硫酸铵浓度达到50％后，上清液中的酶活骤然降低。沉淀蛋白在硫酸铵的添加浓度达到40％以后，酶活就骤然上升，在50％以后达到最大。所以在用硫酸铵沉淀粗酶时可选择50％的硫酸铵沉淀浓度对降解酶进行粗分；同时降解酶能够在添加多菌灵的非变性胶上产生清楚可见的透明带。与此透明带对应的条带酯酶染色表明，该酶同时也是一种酯酶。把分离胶中的透明带切胶回收，可回收到一条分子量大小在97.4～66.2Kda之间的目的蛋白。
对多菌灵代谢过程进行跟踪研究，发现在djl-6接种24小时后有三种中间代谢物存在，分别是2-氨基苯并咪唑(m／z=134.3)、苯并咪唑(m／z=119.3)和一种分子离子峰为104.8和118.5的未知物质。而经过2～3天的培养后，HPLC分析表明100mg．L~(-1)的多菌灵几乎被完全降解。推测多菌灵可能首先被转化为2-氨基苯并咪唑，然后再进一步被转化，最后被完全降解。本研究也是首次发现2-氨基苯并咪唑和苯并咪唑在纯菌多菌灵代谢物中同时存在。
djl-6在红壤、潮土和高沙土中的降解实验表明，其在土壤中降解多菌灵需要一定的水分含量，稍高的水分含量有利于其对多菌灵的降解，但完全淹水的条件又会影响到其降解功能的发挥；与最适生长特点相符，在土壤中djl-6适宜的降解pH和温度分别为7.0和30℃；三种土壤中多菌灵的降解主要是由降解菌完成的，且接种量越大降解效果越好；添加葡萄糖对djl-6降解土壤中多菌灵有一定地促进作用。
投加多菌灵和djl-6对红壤、潮土和高沙土三种土壤酶活性的影响表明，对过氧化氢酶活性的影响都是加降解菌的酶活要高于未加菌的酶活。未加菌处理初期都表现出抑制现象，而后又缓慢得到恢复；对土壤脱氢酶活性的影响，高沙土和红壤、潮土的实验结果相反，在加菌处理和未加菌处理中脱氢酶活性一开始就表现出抑制现象。其后虽然中间得到了一定的恢复，但仍然低于起始时没有经过任何处理的脱氢酶活性；三种土壤的实验结果都表明，加菌处理和未加菌处理对蔗糖酶和脲酶活性的影响在初期时都表现出抑制作用，其后逐渐恢复。接种降解菌的土壤脲酶活性要高于未接菌土壤的脲酶活性。
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 Cu~(2+) 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_(14:0)8.12%, C_(15:0)ISO 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_(20:0)3.36%; 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 Zn~(2+) 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.
引文
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