氯苯甲酸降解菌的分离筛选及降解特性研究
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摘要
氯苯甲酸(CBA)作为一种重要的氯代有机化合物,广泛应用于工农业生产,其在环境中的残留和危害也是不容忽视的。因此研究CBA的生物降解特性,具有理论和应用价值。论文的主要研究内容如下:
(1)比较了两个采自原始生境的土壤样品及处理氯代烃的活性污泥样品中含有的降解单氯苯甲酸的微生物数量。结果表明微生物降解CBA的能力具有广泛性并对不同取代氯苯甲酸降解有一定差异。
(2)从活性污泥中分离得到7株以3-CBA为唯一碳源生长的细菌。经鉴定3株为假单孢菌,其余4株为红球菌、节杆菌、产碱菌、伯克霍尔德氏菌。其中红球菌的降解能力明显高于其它菌,因此以该菌(命名为S-7)为出发菌株。通过形态学、生理生化反应以及16S rRNA基因序列测定,确定S-7为红平红球菌。
(3)菌株S-7能在10～30°C利用3-CBA。20°C时的降解速率较30°C快。这是首次报道红平红球菌在10°C低温条件下完全降解3-CBA。该菌广泛的温度适应性对于低温区域的生物修复有重要的意义。
(4)质粒提取和消除实验表明菌株S-7降解3-CBA的基因位于染色体上。在细胞破碎液中测定了(氯)邻苯二酚1,2-双加氧酶的活力而没有检测到2,3-双加氧酶的活性。HPLC检测到氯邻苯二酚的存在。全波长扫描发现产生了与氯-顺,顺-粘糠酸一样的紫外光谱。推断出菌株降解3-CBA途径:3-CBA首先转化为氯邻苯二酚,通过修饰邻位裂解途径降解氯邻苯二酚,形成的产物进入三羧酸循环。
(5)研究了在以葡萄糖为生长基质的条件下,菌株对2-CBA和4-CBA的降解能力。S-7可共代谢2-CBA,但是不能共代谢降解4-CBA。蛋白质电泳结果表明,葡萄糖为生长底物时,2-CBA的降解酶是由2-CBA本身所诱导,它们不同于细菌利用葡萄糖的酶。
(6)单独使用海藻酸钠或聚乙烯醇(PVA)为载体固定化菌株S-7时,不能形成稳定的微球。将海藻酸钠和聚乙烯醇混合使用可提高固定化微球的效果。海藻酸钠中加入壳聚糖后,固定化微球的性能有所提高,但降解3-CBA的效果并没有明显改善。
Chlorobenzoates (CBAs), as important chlorinated aromatic compounds, were widely used in agriculture and industry. However, the residue and risk of CBA in environment can not be neglected. Study the degradation characteristics exhibit theoretical and application importance. The detailed work of this thesis is as follows:
(1) The diversity of 2-, 3-, and 4-CBA degraders in two pristine soils and one contaminated sewage sludge was compared. Results showed that the ability of microbial populations in undisturbed soils to mineralize CBA was widespread. The result also indicated that the 3- and 4-CBA degraders were more diversity than the 2-CBA degraders.
(2) Seven strains, using 3-CBA as sole carbon sources, were isolated from the sewage sludge. Three strains were Pseudomonas sp., the other four were Rhodococcus sp., Arthrobacter sp., Alcaligenes sp. and Burkholderia sp. respectively. One of the strains, named S-7, can degrade 3-CBA effectively, so it was chosen as study strain. S-7 was identified by morphological, biochemical and 16S rDNA sequence analysis as Rhodococcus erythropolis.
(3) Strain S-7 can fully degrade 3-CBA in a range of temperatures from 10 to 30°C. Strain showed higher degradability at 20°C than at 30°C. The psychrotolerant ability is significant for bioremediation in low temperature regions.
(4) Plasmid purification and elimination showed strain S- 7 do not contain plasmid, the degrading function might be controlled by chromosome DNA. Catechol and chlorocatechol 1, 2-dioxygenase activities were present in cell free extracts, but no (chloro)catechol 2,3-dioxygenase activity was detected. HPLC detected the chlorocatechol in the fermentation broth. Spectral conversion assays with extracts from R. erythropolis S-7 showed accumulation of a compound with a similar UV spectrum as chloro-cis,cis-muconate from 3-CBA. Based on the results, a pathway for degradation of 3-CBA was proposed: 3-CBA was converted initially to chlorocatechol, and then degraded by modified ortho-pathway. Products entered the TCA cycle.
(5) When glucose was added as grow substrate, strain S - 7 can cometabolic degrade 2-CBA, but cannot biodegrade 4-CBA. Protein electrophoresis result showed that the unspecific enzymes for 2-CBA degradation were induced by 2-CBA itself when glucose was served as growth substrate.
(6) The methods of immobilization were studied. Using alginate or polyvinyl-alcohol (PVA) alone, the immobilized cell can not form steady beads. When added PVA to the sodium alginate, both strength and tenacity were enhanced greatly, and the biodegradation rate was faster than the free bacteria. Using sodium alginate and chitosan as carrier, although the properties of immobilized ball was better than using sodium alginate alone, the degradation rate was a little slower than the free bacteria.
(1)比较了两个采自原始生境的土壤样品及处理氯代烃的活性污泥样品中含有的降解单氯苯甲酸的微生物数量。结果表明微生物降解CBA的能力具有广泛性并对不同取代氯苯甲酸降解有一定差异。
(2)从活性污泥中分离得到7株以3-CBA为唯一碳源生长的细菌。经鉴定3株为假单孢菌,其余4株为红球菌、节杆菌、产碱菌、伯克霍尔德氏菌。其中红球菌的降解能力明显高于其它菌,因此以该菌(命名为S-7)为出发菌株。通过形态学、生理生化反应以及16S rRNA基因序列测定,确定S-7为红平红球菌。
(3)菌株S-7能在10～30°C利用3-CBA。20°C时的降解速率较30°C快。这是首次报道红平红球菌在10°C低温条件下完全降解3-CBA。该菌广泛的温度适应性对于低温区域的生物修复有重要的意义。
(4)质粒提取和消除实验表明菌株S-7降解3-CBA的基因位于染色体上。在细胞破碎液中测定了(氯)邻苯二酚1,2-双加氧酶的活力而没有检测到2,3-双加氧酶的活性。HPLC检测到氯邻苯二酚的存在。全波长扫描发现产生了与氯-顺,顺-粘糠酸一样的紫外光谱。推断出菌株降解3-CBA途径:3-CBA首先转化为氯邻苯二酚,通过修饰邻位裂解途径降解氯邻苯二酚,形成的产物进入三羧酸循环。
(5)研究了在以葡萄糖为生长基质的条件下,菌株对2-CBA和4-CBA的降解能力。S-7可共代谢2-CBA,但是不能共代谢降解4-CBA。蛋白质电泳结果表明,葡萄糖为生长底物时,2-CBA的降解酶是由2-CBA本身所诱导,它们不同于细菌利用葡萄糖的酶。
(6)单独使用海藻酸钠或聚乙烯醇(PVA)为载体固定化菌株S-7时,不能形成稳定的微球。将海藻酸钠和聚乙烯醇混合使用可提高固定化微球的效果。海藻酸钠中加入壳聚糖后,固定化微球的性能有所提高,但降解3-CBA的效果并没有明显改善。
Chlorobenzoates (CBAs), as important chlorinated aromatic compounds, were widely used in agriculture and industry. However, the residue and risk of CBA in environment can not be neglected. Study the degradation characteristics exhibit theoretical and application importance. The detailed work of this thesis is as follows:
(1) The diversity of 2-, 3-, and 4-CBA degraders in two pristine soils and one contaminated sewage sludge was compared. Results showed that the ability of microbial populations in undisturbed soils to mineralize CBA was widespread. The result also indicated that the 3- and 4-CBA degraders were more diversity than the 2-CBA degraders.
(2) Seven strains, using 3-CBA as sole carbon sources, were isolated from the sewage sludge. Three strains were Pseudomonas sp., the other four were Rhodococcus sp., Arthrobacter sp., Alcaligenes sp. and Burkholderia sp. respectively. One of the strains, named S-7, can degrade 3-CBA effectively, so it was chosen as study strain. S-7 was identified by morphological, biochemical and 16S rDNA sequence analysis as Rhodococcus erythropolis.
(3) Strain S-7 can fully degrade 3-CBA in a range of temperatures from 10 to 30°C. Strain showed higher degradability at 20°C than at 30°C. The psychrotolerant ability is significant for bioremediation in low temperature regions.
(4) Plasmid purification and elimination showed strain S- 7 do not contain plasmid, the degrading function might be controlled by chromosome DNA. Catechol and chlorocatechol 1, 2-dioxygenase activities were present in cell free extracts, but no (chloro)catechol 2,3-dioxygenase activity was detected. HPLC detected the chlorocatechol in the fermentation broth. Spectral conversion assays with extracts from R. erythropolis S-7 showed accumulation of a compound with a similar UV spectrum as chloro-cis,cis-muconate from 3-CBA. Based on the results, a pathway for degradation of 3-CBA was proposed: 3-CBA was converted initially to chlorocatechol, and then degraded by modified ortho-pathway. Products entered the TCA cycle.
(5) When glucose was added as grow substrate, strain S - 7 can cometabolic degrade 2-CBA, but cannot biodegrade 4-CBA. Protein electrophoresis result showed that the unspecific enzymes for 2-CBA degradation were induced by 2-CBA itself when glucose was served as growth substrate.
(6) The methods of immobilization were studied. Using alginate or polyvinyl-alcohol (PVA) alone, the immobilized cell can not form steady beads. When added PVA to the sodium alginate, both strength and tenacity were enhanced greatly, and the biodegradation rate was faster than the free bacteria. Using sodium alginate and chitosan as carrier, although the properties of immobilized ball was better than using sodium alginate alone, the degradation rate was a little slower than the free bacteria.
引文
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