【作者】 苏丽君；

【导师】 周洪波； 顾帼华；

【作者基本信息】 中南大学 ， 微生物学， 2010， 硕士

【摘要】 嗜铁钩端螺旋菌(Leptospirillum ferriphilum)为革兰氏阴性菌,能够生长在极端酸性(pH1.5-1.8)环境中,只能通过氧化亚铁离子获得能量,是专性好氧微生物。在生物浸出研究中,嗜铁钩端螺旋菌被广泛报道,而且被认为是主要的铁氧化细菌。黄铁矿是分布最广的硫化矿,常与铜、钼、金、银、钴、镓等有色、稀贵金属元素伴生或共生。为了探讨细菌浸出黄铁矿的机理,提高工业实践中生物浸矿的效率,本文以不同能源培养的嗜铁钩端螺旋菌为研究对象,考察其生长特性的变化以及它们对黄铁矿的浸出效果；运用荧光实时定量PCR方法测定了它们在不同条件下在黄铁矿表面的吸附情况；通过吸附量、红外光谱、Zeta电位和接触角的测定以及原子力显微镜表面表征,考察黄铁矿与细菌作用后表面性质的变化,为揭示微生物与黄铁矿的作用机制奠定了基础。通过绘制L. ferriphilum YSK在不同能源物质(FeSO4·7H2O和黄铁矿)中的生长曲线,得知以黄铁矿为能源生长的L. ferriphilum YSK比以亚铁为能源生长的细菌的生长周期长。亚铁培养的细菌很快就能进入对数生长期,代时为22.1h；而黄铁矿培养的细菌进入对数生长期的时间较长,为12h左右,代时为33.2h,比以亚铁为能源培养的细菌大约长11个小时。黄铁矿的摇瓶浸出实验研究表明,黄铁矿的浸出是一个产酸的过程,浸出过程中pH值呈下降趋势,而氧化还原电位在浸出初期持续上升,形成较高的氧化还原电位有利于黄铁矿的浸出。黄铁矿培养的L. ferriphilum YSK比以亚铁为能源生长的细菌能先适应浸出体系的环境,浸出效果也较之好很多。浸出后期,溶液中积累了较高浓度的Fe3+,生成黄钾铁矾阻碍了黄铁矿的溶解,矿浆电位呈下降趋势,导致浸出速率减缓。应用实时荧光定量PCR研究不同能源培养的浸矿细菌L.ferriphilumYSK在黄铁矿上的吸附行为被证明是可行的,研究结果表明不同能源培养的L. ferriphilum在黄铁矿表面发生了明显的吸附现象,30分钟到40分钟之间两者都达到吸附平衡,但黄铁矿培养的L.ferriphilum在黄铁矿表面的吸附量比可溶性亚铁离子培养的高。在吸附过程中,细菌的表面性质起了至关重要的作用。溶液pH值的改变使得细菌的表面性质发生了变化,从而影响细菌在矿物表面的吸附行为,因此pH值被认为是影响浸矿细菌L. ferriphilum YSK在黄铁矿表面吸附的较为关键的因素,酸性条件下吸附量较高；而温度对浸矿细菌L. ferriphilumYSK在黄铁矿表面上的吸附行为有一定影响,在最佳生长温度时吸附量最高。对细菌和矿物的表面性质的研究表明,不同基质生长的L.ferriphilumYSK表面性质发生了一定的变化,黄铁矿培养的细菌比亚铁培养的细菌的接触角大,疏水性强,但是它们对黄铁矿表面性质的影响规律相似。透射电镜对浸矿细菌表面蛋白质的观察表明,固体基质(黄铁矿)中生长的细菌表面的蛋白质要比在可溶性亚铁离子中生长的细菌多,这也是导致固体基质培养的细菌疏水性更大的原因。细菌的吸附使黄铁矿的等电点朝细菌的等电点方向偏移。细菌在浸矿过程中的代谢使得黄铁矿中的铁元素在表面形成Fe2O3、FeOOH和Fe2(SO4)3等亲水物质导致黄铁矿表面亲水性增加。原子力显微镜测试表明L. ferriphilum菌对黄铁矿有较强的腐蚀能力。更多还原

【Abstract】 The gram-negative bacteria Leptospirillum ferriphilum is obligate aerobic microorganism, which habits in extremely acidic (pH1.5-1.8) environment and derives energy from the oxidation of ferrous ion. In biological leaching studies, L. ferriphilum has been widely reported and is considered the major iron-oxidizing bacteria. Pyrite is the most common sulfide mineral, and is often associated with various useful components such as copper, molybdenum, gold, silver, cobalt, gallium and other noble metals. In order to discuss the mechanism of bio-leaching of pyrite and improve the efficiency of bio-leaching in industrial practice, L. ferriphilumYSK grown on different energy sources were studied in this paper to investigate their growth characteristics and effect on pyrite bio-leaching; using fluorescence real-time quantitative polymerase chain reaction (PCR) method determined the adsorption quantity on pyrite surface under different conditions; investigate the surface properties after pyrite interaction with bacteria, application of adsorption, infrared spectroscopy, contact angle measurements and surface characterization by atomic force microscopy, and these research results are the basis for revealing the micro-organism and pyrite mechanism.By drawing the growth curves of L. ferriphilum YSK grown on different energy sources (FeSO4·7H2O and pyrite), it can be known that growth cycle of L. ferriphilumYSK grown on pyrite is longer than cells grown in ferrous ion solution. Ferrous ion cultured bacteria entered into period of logarithmic phase faster and the generation time was about 22.1h; however, time of pyrite grown cells into the period of logarithmic phase was about 12 hours and the generation time was about 33.2h, which was 11 hours longer than ferrous ion cultured bacteria. Flask bio-leaching experiments showed that, pyrite bio-leaching is an acid production process with pH value decreased while redox potential was ascend in initial bio-leaching stage and a relatively high potential was benefit for leaching of pyrite. Compared with ferrous ion grown bacteria, pyrite grown L. ferriphilum YSK can adapt to bio-leaching environment earlier and the leaching effect was much higher. In latter stage, solution was accumulated with high concentration of Fe3+ and generating jarosite hindered dissolution of pyrite, which leaded to the decline of slurry potential and low leaching velocity.Application of real-time quantitative PCR to research the adsorption behavior of different energy cultured L. ferriphilumYSK on pyrite surface was proved to be feasible. Results showed that different energy sources grown L. ferriphilum can adsorb onto pyrite surface obviously and reached equilibrium between 30 minutes and 40 minutes, but adsorption quantity of pyrite grown bacteria was higher than soluble ferrous ions grown bacteria. During the attachment process, surface properties of bacteria played a vital role. Due to the changes in pH value of solution, bacterial surface had been changed, which affected the adhesion of cells to mineral surface. Therefore, pH value was identified as the most crucial factor in L. ferriphilumYSK’s adsorption into the pyrite surface, and the adsorption amount was relatively high in acidic conditions. Temperature had some impact on the adsorption behavior of leaching bacteria L. ferriphilumYSK in pyrite surface and the optimum growth temperature, the maximum adsorption capacity.Surface properties of bacteria and mineral research suggested that the surface properties of different energy substrates grown L. ferriphilum YSK changed certainly, and pyrite cultured bacteria had bigger contact angle and stronger hydrophobic property than that cultivation in ferrous ion solution. Howerver, they shared a similar change trend of pyrite surface properties after conditioning with these cells. Study of outer membrane protein using transmission electron microscopy showed that solid matrix (pyrite) grown bacteria synthesized more surface protein than grown in soluble ion, which was the reason that solid matrix cultivated bacteria was more hydrophobic. Isoelectric points (IEPs) of pyrite after bacterial treatment moved towards IEPs of pure cells. The surface hydrophilicity of pyrite increased in leaching process for that the metabolization of bacteria made iron in pyrite formed into hydrophilic substances such as Fe2O3, FeOOH and Fe2(SO4)3. Images of atomic force microscopy showed that L. ferriphilum had strong corrosion ability for pyrite leaching.更多还原