【作者】 彭安安；

【导师】 夏金兰；

【作者基本信息】 中南大学 ， 生物学， 2012， 博士

【摘要】 在微生物冶金过程中,浸矿微生物的主要作用是通过对亚铁和/或还原型无机硫化物的氧化为浸出体系提供/再生Fe3+(和H+),消解浸出过程中的元素硫层,降低含硫化合物的生成,保证硫化矿溶出过程的持续进行。目前,对于微生物的亚铁氧化模型已经研究得比较清楚,而元素硫的生物氧化过程十分复杂,代谢机制仍然不清楚。本文综合运用微生物学、生物化学、分子生物学研究方法和基于同步辐射的X射线荧光原位分析技术,对嗜酸硫氧化细菌Acidithiobacillus ferrooxidans的元素硫活化氧化作用机制和影响因素进行了初步探讨,加深了对硫生物氧化过程的认识。主要工作包括以下几个方面：1.建立了以表面活性剂Tween-80为稳定剂的硫酸钡比浊法,并证明了该方法在浸矿液样品的硫酸根离子浓度测定中的实用性和可靠性。采用热水浴法结合Triton X-114相分离法,连续分步分离提取了未经机械破碎的A. ferrooxidans ATCC23270细胞胞外、外膜和周质空间蛋白质,建立和优化了适用于A. ferrooxidans特化空间蛋白质的双向电泳实验体系。2.筛选鉴定了3种17株嗜酸硫氧化细菌,丰富了硫生物氧化菌种资源。对其中代表性菌株的超微结构观察结果表明,元素硫能在这三种菌体内以聚集的颗粒存在,可能为体内能源的储存方式。筛选到的Acidithiobacillus albertensis BY-05硫氧化活性较高,为国内首次报道,该菌与A. ferrooxidans协同作用可提高黄铜矿和闪锌矿的浸出率。3.运用XRD、基于同步辐射的硫的K边XANES等分析手段比较研究了三种不同硫氧化特点的浸矿细菌在单独/混合浸出黄铜矿过程中矿物组成和含硫中间产物的形态变化,结果表明黄铜矿溶出过程中,初期低电位下有辉铜矿生成,而元素硫和黄钾铁矾的累积是引起钝化现象的主要因素。混合菌浸矿体系可以提供更多的Fe3+氧化剂和更低的pH值,有效的促进矿物分解、消解矿物表面积累的元素硫层、抑制黄钾铁矾的生成,从而提高铜离子浸出率。浸出初期电位低于400mV (vs SCE)时,黄铜矿的浸出速率较快,电位迅速升高至540mV (vs SCE)后,浸出速率明显变慢。4.采用2D-PAGE结合MALDI-TOF MS/MS鉴定,筛选到A.ferrooxidans ATCC23270不同特化空间与硫氧化密切相关的蛋白质39个(其中胞外13个,外膜9个,周质空间17个),并用RT-qPCR方法从转录水平对结果进行了验证。生物信息学分析结果表明,筛选到的蛋白质近一半为功能未知蛋白,其余的与能量代谢、转运结合和转录翻译等细胞功能有关,约70%含有半胱氨酸残基,其中8个半胱氨酸残基丰度较高的蛋白质还含有1-2个可能与硫代谢密切相关的-CXXC-结构域,是巯基在元素硫活化、氧化过程中扮演重要角色的证据。采用基于同步辐射的Micro-XRF mapping原位检测了用金属Ca2+选择性标记的A. ferrooxidans细胞表面的巯基含量,发现以单质硫作为能源生长的A. ferrooxidans表面蛋白质巯基的含量约为以亚铁为能源生长时的5倍。5.研究了表面活性剂Tween-80对A. ferrooxidans硫氧化及其分子机制的影响,结果表明Tween-80的加入使细菌对硫吸附和代谢发生了变化,影响了细菌细胞EPS组成和细菌胞外蛋白质表达。10-2g/L的Tween-80能改善细菌对硫的吸附-活化过程,促进A. ferrooxidans在不溶性能源底物(So和CuFeS2)中的生长和代谢,24天可提高黄铜矿浸出率约16%。6.比较研究了A. ferrooxidans对不同形态的硫(环状a-S与线状μ-S)的利用差异及相关的分子机制,发现无定形的μ-S比晶形的a-S更容易被A. ferrooxidans吸附和氧化利用。XRD检测到a-S从特定部位开始被破坏,a-S在被细菌氧化过程中可能部分转化成了μ-S。DRIFTS分析结果表明以不同形态的硫为能源生长的细菌细胞表面EPS的组成差异较大。RT-qPCR结果显示,当A. ferrooxidans以μ-S为能源生长时,与吸附和活化有关的胞外蛋白质的表达水平明显下调,转运疏水性物质进入细胞周质空间的蛋白质和能量代谢相关的酶表达水平则上调。更多还原

【Abstract】 The sulfur/ferrous iron-oxidizing microorganisms contribute to the transformation of sulfur and other intermediary sulfur compounds to sulfuric acid, regenerating protons and Fe3+for the bioleaching process. The microbial ferrous oxidation model has been described clearly, but the sulfur oxidation mechanism remains unclear because of the complexity of the sulfur bio-oxidation process. To clarify the molecular mechanism of microbial sulfur activation and oxidation in the acidophilic sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans, techniques of microbiology, biochemistry and molecular biology, and the in situ synchrotron radiation X-Ray fluorescence analysis were used. The main contents and results are as follows.1. Establishment of the experimental methods was mentioned. By choosing surfactant Tween-80as the stabilizing agent, the barium sulfate turbidimetry for determining sulfate concentration in the complex bioleaching samples was improved. Compared with the national standard method GB/T5750.5-2006, it is faster and easier for operation, indicating its wide applicability. Extracellular, outer membrane and periplasimic proteins of A. ferrooxidans were separated by an innovative continuous treatment using hot water bathe followed by a one-step Triton X-114phase-partitioning. The two dimensional polyacrylamide gel electrophoresis for the extracted compartment-specific proteins was also optimized.2. Isolation and identification of three species of acidophilic sulfur-oxidizing bacteria were described and their sulfur oxidation related physiological characteristics were compared. Ultrastructure of the three strains Acidithiobacillus albertensis BY-05, A. ferrooxidans DXS and Acidithiobacillus thiooxidans BY-02showed that inside the cells there were highly refractile sulfur granules, which may play the role of energy storage. The new strain A. albertensis BY-05had high sulfur oxidation capacity, and could enhance the leaching capacity of chalcopyrite and sphalerite when mixed with A. ferrooxidans. 3. The relevant sulfur speciation on the surface of chalcopyrite leached by three typical mesophilic bacteria A. ferrooxidans, Leptospirillum ferriphilum and A. thiooxidans and their mixture was investigated using X-ray diffraction (XRD) and X-ray absorption near edge structure (XANES). The results showed that the mixed culture had a higher sulfur/iron oxidation activity than the pure cultures of the bacteria. The fitting results of XANES spectra indicated that the mixed culture restrained the sulfur and jarosite passivation layer obviously. Additionally, the dissolution rate of chalcopyrite was higher when the redox potential was less than400mV (vs SCE), and above540mV (vs SCE) the rate decreased apparently.4. Comparative proteomics strategy was used to screen the sulfur activation and oxidation relative proteins from specific cellular compartments of A. ferrooxidans ATCC23270,39expression upregulated proteins (including13extracellular proteins,9outer membrane proteins and17periplasmic proteins) selected in the2-DE profiles were identified by MALDI-TOF MS/MS and verified at transcriptional level by RT-qPCR. About half of the selected proteins were function-unknown, others were annotated to categories of energy metabolism, transport and binding, cell structure, cellular processes, etc.. Bioinformatics prediction showed that70%of them contain cysteine residues in sequence. Eight proteins which contain abundant of the cysteine residues even have one or two functional motifs such as-CXXC-The thiol groups on the A. ferrooxidans cell surface were selectively marked by Ca2+and observed by in situ micro synchrotron radiation X-Ray Fluorescence (Micro-SR-XRF) mapping analysis, the result revealed that the number of the thiols on the surface of the cells grown on elemental sulfur was about five times as that grown on ferrous substrate. It indicates that the thiol-rich proteins played important roles in sulfur activation and oxidation process.5. Effects of the surfactant Tween-80on the growth, sulfur oxidation, and expression of selected typical sulfur metabolism relevant genes of A. ferrooxidans ATCC23270were investigated. The results showed that in the presence of10-2g/L Tween-80the growth of A. ferrooxidans and its metabolism on the insoluble substrate S0and CuFeS2was promoted. After24days of bioleaching, the copper extraction yield of chalcopyrite at10-2g/L of Tween-80increased by16%compared to the bioleaching experiment without Tween-80. FT-IR spectra analysis revealed that this was probably caused by the extracellular polymeric substances whose composition could be changed by the surfactant addition. RT-qPCR was used to analyze the differential expressions of17selected sulfur metabolism relevant genes in response to the addition of Tween-80. Down-regulation of the extracellular protein genes indicated the influence of Tween-80on bacteria-sulfur adsorption. Variation of the expression level of the enzymes provided a supplement to sulfur metabolism investigation.6. The differential utilization of α-and μ-sulfur by A. ferrooxidans was also investigated in this thesis. The growth and sulfur oxidation of A. ferrooxidans on μ-S showed shorter lag phase. The initial adsorption capacity of the cells was higher on μ-S than that on α-S. The results of SEM, DRIFTS and XRD analyses indicate that the surface of sulfur was modified by cells and the orthorhombic α-S was destroyed by A. ferrooxidans from special region of the crystal. Differential expression of11selected sulfur adsorption-activation and metabolism relevant genes was detected by RT-qPCR. The results show that the expression of the hydrophobic substrate transport proteins and the sulfur metabolism related proteins was up-regulated, and the adsorption and activation related proteins were down-regulated when the cells were grown on μ-S.更多还原