【作者】 田林双；

【导师】 戴传超；

【作者基本信息】 南京师范大学 ， 微生物学， 2007， 硕士

【摘要】 菲具有典型的PAHs结构，是构成多种致癌、致畸、致突变PAHs的基本结构单元，常作为微生物降解PAHs的研究模型。本研究尝试从能够降解木质素的内生真菌中筛选菲降解菌株，并将筛选的内生真菌与其共生植物联合培养进行菲降解实验，且对内生真菌降解菲的机理进行研究。本实验室前期研究分离出多株能够降解木质素的内生真菌，在此研究工作的基础上，本研究从中筛选菲降解菌株，筛选结果表明内生真菌B3（Phomopsis sp．）、Y17（Ceratobasidumi sp．）能够降解菲，降解效率分别达到2.95mg／d和8.23mg／d，目前无该属真菌降解PAHs的报道。与最近十年文献报道的菲降解真菌比较，菌株B3和Y17显示出较强的竞争力，有望用于PAHs污染土壤的微生物修复。实验结果同时也证明了我们起初的推测，即内生真菌中的确存在着可降解PAHs的菌株。对植物内生真菌B3降解菲的条件进行研究。结果表明乳糖为碳源、蛋白胨为氮源、二者浓度均为10g／L、愈创木酚作为诱导物时，B3菌株10天内对初始浓度为100mg／L菲的降解率达52.54％。菌株B3无LiP和MnP活力，只有Lac活力，但其与B3降解菲无明显关系。在菲浓度为500mg／L的情况下，B3生长未受太大影响，显示出较高的菲耐受能力。进一步利用内生真菌B3与水稻的共生关系，将菌株B3与宿主植物水稻联合培养，进行菲降解实验，结果显示B3和水稻对菲的降解起协同促进作用。实验过程中还发现水稻感染内生真菌B3后，对菲胁迫的反应不同。水稻生长状况、根长抑制实验、水稻幼根SOD酶活力变化等实验结果说明菲对水稻有毒害作用，而当水稻感染内生真菌B3后，对菲胁迫起一定的缓解作用。为了进一步探讨内生真菌降解菲的机理，对菲高效降解菌株—Y17（Ceratobasidumi sp．）降解菲的机理进行研究。实验结果表明菌株Y17具有较高的MnP酶活力和极低的LiP酶活力，但无Lac酶活力，MnP是菌株Y17主要的木质素降解酶。污染物菲的添加，极大的促进了Y17分泌LiP和MnP。Mn²⁺显著的促进Y17分泌MnP，而NaN₃抑制了Y17分泌MnP，但在MnP促进和抑制这两种情况下，Y17对菲的降解未受太大影响，说明MnP并不是Y17降解菲的关键因素。研究还表明菌丝和新鲜培养基是Y17降解菲的关键因素，利用Y17进行生物修复必须满足这两个前提条件。借助于GC-MS，对Y17降解菲的中间代谢产物进行了初步的鉴定，结果认为Y17降解菲产生多种中间代谢产物，推测Y17降解菲的过程复杂，Y17可能具有多种菲代谢途径。更多还原

【Abstract】 Phenanthrene is a three-ring polycyclic aromatic hydrocarbon. Among PAHs phenanthrene was selected because it is one of the most abundant PAHs in the environment, there have been many studies used phenanthrene as model PAHs for the investigation of degradation by different fungi and bacteria. This study screened phenanthrene-degrading filamentous fungi from endophytic fungus which utilized lignin as their carbon source. Then endophytic fungus showed phenanthrene degradation ability was co-cultured with its host plant, Monitoring the phenanthrene degradation and plant reaction to the phenanthrene. At last, the phenanthrene degradation mechanism by endophytic fungus was discussed.Our previous study isolated some endophytic fungi which utilized lignin as their carbon source. Based on this research, the present study screened phenanthrene-degrading filamentous fungi from the isolated endophytic fungi. The results showed that endophytic fungus B3 （Phomopsis sp.） and Y17 （Ceratobasidumi sp.） had phenanthrene degradation ability. The degradation rate reached 2.95 mg/d and 8.23 mg/d, respectively. Compared with the other screened phenanthrene degrading fungi and some white rot fungi which had been used in the PAHs biodegradation, the degradation rate of phenanthrene by B3 and Y17 were relatively high. This result indicated that B3 and Y17 might be a potential candidate for an effective bioremediation of PAHs contamination. To our knowledge, this is the first report on PAHs degradation by endophytic fungus. In addition, this is also the first report of Phomopsis sp. and Ceratobasidumi sp. that showed ability in degrading PAHs. The present results confirmed our hypothesis that some endophytic fungi might have the ability to degrade PAHs.Degradation of phenanthrene by B3 （Phomopsis sp.） in different conditions were studied. The results showed that lactose as the carbon source, peptone as nitrogen source, 10g/L respectively facilitated the degradation of phenanthrene. Guaiacol acted as inducer on the phenanthrene degradation. After 10 days rotary culture, the degradation rate of phenanthrene （100mg/L） reached 52.54%. No apparent correlation between degradation percentage and the tested ligninolytic enzyme. In addition, B3 was able to tolerate higher concentrations of phenanthrene at the concentration of 500 mg/L.Previous results showed that the endophytic fungus B3 had a reciprocal relationship with the paddy rice. In this paper, when B3 was co-culture with paddy rice, synergetic effect was occurred on the degradation of phenanthrene. Meanwhile, B3 decreased the injury of paddy rice under the condition of phenanthrene stress.The mechanism of phenanthrene degradation by endophytic fungus Y17 was studied. Among extracellular ligninolytic enzyme activities tested, MnP was the predominant ligninolytic enzyme and its production was greatly induced in the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, the promotion and inhibition studies on MnP by different concentration of Mn²⁺ and NaN₃ were performed. Additionally, fungi mycelium free and resuspended experiments were carried out. The obtained results showed no apparently connection between MnP activity and phenanthrene degradation. While the mycelium and fresh medium were the crucial factors affecting the oxidation of phenanthrene. After phenanthrene was degraded by Y17, the products were analyzed by GC-MS. The result showed that Y17 may have two or more pathways for phenanthrene degradation. However, more work is needed to get the further information of phenanthrene degradation by endophytic fungus.更多还原