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题名: 抗砷菌的筛选及工业污泥中砷生物转化行为的研究
作者: 田海霞
学位类别: 博士
答辩日期: 2014-11
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 景传勇
关键词: 好氧砷还原菌,厌氧砷还原菌,铁还原菌,工业污泥,迁移转化,aerobic, anaerobic As(V) reduction, iron reducation, industry sludge, mobilization
其他题名: Isolation of Arsenic Resistant Bacteria and Arsenic Biotransformation in Industry Sludge
学位专业: 环境科学
中文摘要:     微生物在砷的生物地球化学循环过程中起着至关重要的作用,它们可以直接或者间接调控砷的赋存形态及价态,进而影响其迁移转化。研究表明砷还原菌的厌氧砷呼吸作用可以增强砷的移动性及毒性,但这种促进作用是否由吸附态砷的原位还原引起的,还有待进一步研究。另一方面,铁还原菌的铁还原作用与砷的还原过程相耦合,可间接调控砷的迁移转化。在目前的众多报道中,研究者们往往借助人工合成铁氧化物这种简化的体系作为砷的载体来探讨微生物对吸附态砷迁移转化的调控,但其结果难以直接外推至实际高砷环境介质中。工业污泥是一种典型高砷环境介质,它们不仅含有高浓度砷,而且含有其它多种重金属,堆放处理的工业污泥已对环境构成了潜在的威胁。微生物在此典型环境中如何调控砷的迁移转化已经成为一个重要的科学问题。本文旨在:1)从典型高砷污染地区土壤及地下水样品中分离抗砷菌株;2)探讨抗砷菌株的抗砷机制;3)考察不同砷代谢机制的菌株对工业污泥中砷迁移转化的规律。
    首先,本研究在好氧条件下从山西大同盆地及内蒙古河套平原砷污染区的土壤及地下水样品中筛选获得了八株高抗砷菌株,它们分别属于Arthrobacter, Pseudomonas,Sphingomonas 与Acinetobacter 四个属。砷抗性试验(MIC)表明菌株的As(III)抗性范围为10~40 mM;As(V)的抗性范围为160~500 mM。表面增强拉曼(SERS)研究结果表明,大部分As(V)并没有进入细胞质内,而是和细胞壁上多种物质包括糖、脂类、蛋白质等相互作用从而缓解其对细胞的毒性。该研究推进了原位光谱技术在微生物细胞结构研究中的应用。研究结果不仅为山西及内蒙砷污染区的抗砷菌多样性提供了理论依据,而且为砷抗性机制研究中细胞壁截留所发挥的作用提供了有力证据。
    其次, 本研究在厌氧条件下, 筛选到嗜碱属厌氧砷呼吸还原菌群(Alkaliphilus)。该菌群中包括三个OTU,单菌株分离并未获得具有砷还原能力的菌株。从菌群基因组中扩增得到了arrA 基因片段,表明砷呼吸还原酶调控了菌群的砷还原过程。同步辐射的扫描透射X 射线显微(STXM)研究显示As(V)主要紧密地分布在菌群细胞周围,还原的As(III)均匀分布在细胞周围及间隙。同时利用传统酶活性测定方法得出细胞质中酶活性为36 μM/min,细胞膜碎片中酶活性为68 μM/min。结合两种方法我们推论,菌群介导的砷还原作用主要发生在细胞膜上。该研究结果一方面拓宽了人们对嗜碱属砷还原调控作用的认识,另一方面拓展了基于同步辐射的STXM 技术在微生物-金属界面反应的应用。
    最后,本研究分别探讨了好氧砷还原菌Pantoea sp. IMH(含arsC gene)、厌氧砷还原菌Alkaliphilus oremlandii OhILAs(含arrA gene)、铁还原菌Shewanella oneidensis MR-1 三株不同砷代谢机制的细菌对工业污泥中砷的转化及脱附释放影响。研究结果表明砷还原菌株IMH 及OhILAs 可以分别在好氧及厌氧条件下,20 小时内将溶解态As(V)全部还原为As(III),但菌株细胞的存在及这种砷的还原作用并不能促进进一步吸附态砷的脱附释放。X 射线近边吸收结构(XANES)结果表明这种还原作用可以提高固相上As(III)的比例,从23.7%增加到33.2%(IMH)和36.5%(OhILAs);而铁还原菌株MR-1 不可以将溶解态As(V)还原为As(III),但其可通过还原污泥中的铁氧化物进而促进大量吸附态砷的释放。XANES 结果表明MR-1 接种处理可提高固相上As(III)的比例至56.6%,同时伴有黄铁矿(FeS2)的生成,但没有显著影响砷的再吸附。该研究结果诠释了直接或间接砷调控菌株对工业污泥中砷迁移转化的影响,一方面强调了好氧与厌氧环境中,砷还原菌对砷的形态转化的控制作用,揭示了在工业污泥这种特殊的复合重金属污染体系中,含ars 操纵子菌株具有更高抗性,发挥着更重要作用;另一方面也强调了铁还原菌对工业污泥中砷的促进脱附作用。
英文摘要:     Microorganisms play an important role in the As biogeochemical cycle in the environment by directly or indirectly regulating As speciation and species. It has been proved that As(V) respiratory reduction could enhance As mobility and toxicity.However, it is unclear that whether As(V) respiratory reduction of the adsorbed arsenic could occur in-situ on the solid phase. Moreover, iron reduction coupled with As(V) reduction could indirectly affect the As mobility. In the previous studies,synthesized iron-oxides was always used as As carrier to monitor the As biotransformation. However, the obtained conclusion may be not applied to other environment system, such as industry sludge, which has extremely high As and other
heavy metals. Casually disposing massive amount of industry sludge threatens the environment health. How microbe regulates As transformation in the industry sludge becomes an important issue. The aims of the present work were (i) to isolate arsenic resistant bacteria from the As-polluted soil and underground water samples; (ii) to investigate the arsenic resistant mechanism; (iii) to compare the extent of As redox
transformation and release in the industry sludge affected by different arsenic metabolizing bacteria.
    Firstly, eight As-resistant bacteria were isolated from soil and groundwater samples collected in Shanxi Province and Inner Mongolia. The results indicated that the isolates are representatives of Arthrobacter, Pseudomonas, Sphingomonas, and Acinetobacter. The MIC of As(III) and As(V) for each bacteria was determined to be the range 10 to 40 mM and 160 to 500 mM, respectively. SERS analysis results showed that the bacteria in our study primarily resist As(V) through sequestration of As(V) by the cell wall. The change in SERS peaks and their relationships with cell wall suggested that As(V) mainly interacts with components on the cell wall including polysaccharides, lipids, and proteins. This work propels the application of in-situ SERS spectra technology in investigating As resistant mechanism. Furthermore, the conculsion not only supplies the theoretical evidence for As resistant bacteria diversity in the As-polluted area in Shanxi province and Inner Mongolia but also emphasizes the important role of cell wall in the As resistant mechanism.
    Secondly, mixed bacterial consortium was isolated from soil in Hetao Plain of Inner Mongolia. The 16S rRNA gene library analysis showed that there are three OTU.No single isolate with As(V) reducing ability was isolated. A partial arrA gene sequence was amplified from genome of mixed bacterial consortium, indicating that the Arr reductase regulate the As(V) reduction. STXM analysis showed most As(V) were tightly bound with the cell wall, whereas As(III) was distributed homogeneously around the cell. Therefore, it is a reasonable hypothesis that As(V) reduction may occur outside the cell membrane. To verify this surmise, the location of As(V) reductase was analyzed using the traditional colorimetric approach. The results showed that As(V) reductase activity was 36 μM/min in the cytoplasm and 68 μM/min in the membrane fraction, confirming the As(V) reduction occurring mostly in the membrane fraction. The conclusions extend our knowledge of key role of Alkaliphilus genus in the As(V) transformation and the STXM application into the
microbe-metal interface reaction study.
    Finally, As biotransformation and mobilization mediated by two As(V) reducing bacteria Pantoea sp. IMH (harbors arsC gene), Alkaliphilus oremlandii OhILAs (arbors arrA gene) and a Fe(III)-reducing bacterium Shewanella oneidensis MR-1 in the industry sludge was explored. The incubation results showed that Pantoea sp. IMH and strain OhILAs could aerobically or anaerobically reduced 100% As(V) released from waste residues, though total As release was not enhanced. XANES results showed that this As(V) reduction enhanced the As(III) ratio on the solid phase,from 23.7% to 33.2% for IMH and to 36.5% for OhILAs incubation. In contrast,strain MR-1 substantially enhanced the As mobilization due to iron oxides reductive dissolution without changing the As speciation. XANES result indicated that strain MR-1 induced the higher As(III) ratio (from 23.7% to 56.6%) and the formation of
secondary iron mineral pyrite, which contributed little to the As re-adsorption and immobilization. This study could serve as an example to interpret the As biotransformation in industry sludge regulated directly and indirectly by microbes. The conclusions indicate that the aerobic As(V) reducing bacteria with ars operon have higher metal resistance ability and play an important role in the As transformation. As mobility enhancement in the industry sludge induced by iron reducing bacteria need to be reiterated.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34374
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
田海霞. 抗砷菌的筛选及工业污泥中砷生物转化行为的研究[D]. 北京. 中国科学院研究生院. 2014.
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