【作者】 苏世鸣；

【导师】 曾希柏；

【作者基本信息】 中国农业科学院 ， 土壤学， 2010， 博士

【摘要】 利用微生物对砷(As)的累积与挥发来降低土壤中砷的环境风险已被认为是一种经济、有效且无二次污染的修复措施。近年来许多研究者在相关方面做了有益的探索,但从总体看,具有高累积与挥发砷能力真菌的报道还较少,真菌累积与挥发砷机理的研究还处于理论阶段。因此本研究首先采集了砷污染土壤,从中分离、筛选出耐砷真菌,经鉴定后研究了真菌对砷的累积与挥发能力,最后探讨了其可能机理。取得如下主要结果:1.稀释涂布法对耐砷真菌进行了分离。结果表明,6个土壤样品中共分离得到13株耐砷真菌,其中,菌株SM-12F1、SM-12F4和CZ-8F1分别在20000、30000、30000 mg·L-1砷胁迫下表现出更好的菌落生长状况;液态培养2d后,当As (V)浓度分别为50、80、50 mg·L-1时,SM-12F1、SM-12F4、CZ-8F1的生物量显著高于不加砷的对照。此外,As (V)并没有影响该三株真菌的产孢能力;扫描电镜(SEM)分析表明,As (V)并没有影响菌株SM-12F1、SM-12F4、CZ-8F1的菌丝生长,该三株真菌具有较高的耐砷能力。2.形态学与基于18S rRNA的分子生物学方法进行了菌株的鉴定。结果表明,真菌SM-12F1、SM-12F4和CZ-8F1的菌落形态、产孢结构等均分别与棘孢木霉(Trichoderma asperellum)、微紫青霉(Penicillin janthinellum)和尖孢镰刀菌(Fusarium oxysporum)的特征形态相同;系统发育树分析表明,SM-12F1、SM-12F4和CZ-8F1分别与T. asperellum、P. janthinellum和F. oxysporum在同一发育枝上,相似度分别为99%、96%和99%;序列递交GenBank后,该三株真菌获得登陆号分别为GU212867、GU212865、GU212866。3.室内条件下研究了真菌对砷的累积与挥发能力。结果表明,T. asperellum SM-12F1、P. janthinellum SM-12F4和F. oxysporum CZ-8F1均具有一定的累积与挥发砷能力,当培养液中As (V)含量为2500μg,浓度为50 mg·L-1时,SM-12F4在培养10d时表现出最高的砷累积能力,砷累积量为39.54μg,而CZ-8F1在培养时间为15d时表现出最高的砷挥发能力,砷挥发量为304.06μg;菌株SM-12F1和CZ-8F1细胞壁外吸附砷和胞内累积砷的含量分别占其总累积砷量的82.2%和63.4%,而SM-12F4在细胞内与细胞壁外累积的砷含量比例大约相同;浸有AgNO3的滤纸吸附砷实验表明,砷在处理(滤纸+砷+菌)中的含量均显著高于对照(滤纸+砷、滤纸+菌、滤纸),该结果进一步证实了三株真菌对砷的挥发能力。4.高效液相-氢化物发生-原子荧光(HPLC-HG-AFS)分析了真菌培养液及细胞内砷形态的变化。结果表明,培养2-3d后,T. asperellum SM-12F1、P. janthinellum SM-12F4和F. oxysporum CZ-8F1培养液中的As (V)全部转化为As (III);各培养时间下,三株真菌细胞内砷形态均以As (V)为主,当培养时间为15d时,SM-12F1细胞内可检测到少量的As (III)、MMA(一甲基砷)、DMA(二甲基砷),真菌细胞内存在着无机砷向甲基态砷的转化;培养液中pH、Eh随着培养时间的变化表明,As (V)向As (III)的转化主要发生于真菌细胞内,培养环境中Eh的变化并不是As (V)向As (III)转化的原因。培养7d后,SM-12F1和CZ-8F1培养液中pH分别为6.69和8.38,而SM-12F4培养液中pH为3.78,酸性的培养环境可能促进了无机砷在真菌细胞壁上的吸附。更多还原

【Abstract】 Arsenic (As) accumulation and volatilization by microorganism decreasing environmental risk of As in soil has been recognized as an economically efficient remediation method without secondary pollution. However, few fungi highly capable of As accumulation and volatilization have been identified, and the mechanisms of As accumulation and volatilization in fungi are unclear. In this study, three As resistant fungal strains were isolated and identificated from As contaminated soil samples, the abilities of As accumulation and volatilization of fungi were evaluated, and the possible mechanisms were investigated. The major findings were obtained as follows:1. Thirteen As resistant fungal strains were isolated from six soil samples. Among them, SM-12F1, SM-12F4, and CZ-8F1 performed better at As concentration of 20000, 30000, and 30000 mg·L-1 respectively. When incubated in liquid medium containing the As (V) concentration of 50, 80, 50 mg·L-1 for 2 days, the biomass for SM-12F1, SM-12F4, CZ-8F1 were significantly increased compared to CK, and the sporalation of SM-12F1, SM-12F4, CZ-8F1 was not affected by addition of As (V). Scanning Electron Microscope (SEM) analysis suggested that little impact on mycelium growth of the three fungal strains was found, the fungi SM-12F1, SM-12F4, CZ-8F1 showed higher ability of As resistance.2. The colony morphology, spore structure of SM-12F1, SM-12F4, and CZ-8F1 were respectively similar to those of Trichoderma asperellum, Penicillium janthinellum, and Fusarium oxysporum. Phylogenetic tree based on comparative analysis of 18S rRNA sequence data from the three isolates and their relatives indicated that SM-12F1, SM-12F4, and CZ-8F1 were homology with T. asperellum, P. janthinellum, and F. oxysporum, respectively, with 99%, 96%, and 99% of the similarity. Online sequences comparison to GenBank obtained accession number as follow: GU212867, GU212865, and GU212866.3. T. asperellum SM-12F1, P. janthinellum SM-12F4, and F. oxysporum CZ-8F1 were all capable of As accumulation and volatilization, the highest level of As was accumulated by P. janthinellum SM-12F4, with 39.54μg after 10 days in the culture system amended with 50 mg·L-1 As (V). F. oxysporum CZ-8F1 showed the highest amount of volatilized As, with 304.06μg after 15 days. T. asperellum SM-12F1 and F. oxysporum CZ-8F1 showed greater abilities for the absorption of extracellular As and the accumulation of intracellular As, which accounted for 82.2% and 63.4% of the total accumulated As, respectively. However, P. janthinellum SM-12F4 presented an approximately equipotent distribution of intracellular and extracellular As. The As content in the treated system (filter paper + arsenic + fungi) was significantly higher than that in the control (filter paper + arsenic; filter paper + fungi; filter paper), the abilities of As volatilization by three fungal strains were further certified.4. After cultivated for 2-3 days, As (V) was totally transferred into As (III) in medium of T. asperellum SM-12F1, P. janthinellum SM-12F4, and F. oxysporum CZ-8F1. During the whole cultivation period, As (V) was predominant in cells of three fungal strains, and after cultivated for 15 days, As (III), MMA (Monomethylarsonite), and DMA (Dimethylarsinite) were found in cells of T. asperellum SM-12F1. The change in pH and Eh in medium of three fungal strains showed that the transformation of As (V) into As (III) was conducted in fungal cells. After cultivated for 7 days, the pH in cultural medium of T. asperellum SM-12F1, F. oxysporum CZ-8F1, and P. janthinellum SM-12F1 was 6.69, 8.38, and 3.78 respectively. The adsorption of inorganic As on the cellular wall might be improved in the acidic culture environment.更多还原