硫化铜矿浸矿细菌超微结构与吸附机理及SFORase的纯化
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摘要
为了探讨细菌浸矿机理，促进提高工业实践中生物浸矿的效率，本文对城门山难浸铜矿石的细菌浸出、主要浸矿细菌的形态和超微结构、细菌吸附机理以及T.f中的硫化氢-三价铁氧化还原酶(SFORase)的分离与纯化进行了研究。
在对城门山难浸铜矿的细菌浸出可行性研究中，对含铜褐铁矿，氧化铜-硫化铜混合矿和含泥氧化矿进行了物相分析、多元素分析后，又进行了酸耗试验，摇瓶试验，柱浸试验，并比较了不同的工艺条件对浸出过程的影响。结果表明含铜褐铁矿和含泥氧化矿宜于酸浸，而氧化铜一硫化铜混合矿(次生硫化铜占铜品位的94.97％)适宜于细菌浸出。此外还运用紫外诱变育种方法，将经适当紫外诱变处理的T，f正突变菌株用于浸矿，可提高细菌的亚铁氧化能力，使铜浸出率提高10％左右。
运用透射电镜(TEM)和扫描电镜(SEM)对最常见的浸矿细菌如T.f(Thiobacillus ferrooxidans)、T.t(Thiobacillusferrooxidans)和L.f(Leptosprillum ferrooxidans)等的形态和超微结构进行了研究。结果表明，亚铁培养基培养或硫培养基培养细菌的排列形式有单生，对生、链状和单层细菌成群排列。L.f单个存在，T.f和T.t大多以单个形式存在；可见到T.t的对生；T.t比T.f更多的以链状形式存在，T.f和T.t都能见到单层细菌成群排列的形式。在外形大小上，T.t略大于T.f，且T.t的两端比T.f略为尖锐；
用磷钨酸染色，大t两端一般各有一脂粒存在。在超薄切片观察中，分别在德兴大f和大宝山大f中观察到了具有较宽胞周隙的菌株，这些菌株在样品中所占比例小，它们应具有较强的贮存和分泌蛋白质(酶)的能力。在德兴Tf中观察到了PHB颗粒，它们是碳源和能源的储存体。在江西德兴、城门山、广东大宝山等矿区的厂f中均观察到球状体，尽管目前还不明了其生理功能。 在研究不同培养基培养的浸矿细菌的形态差别时，发现以元素硫为能源的培养基培养的细菌可观察到外膜泡，而用亚铁培养基培养的细菌却观察不到这种结构，外膜泡的形成与否与培养基质中能源物质的存在状态(固态或液态)密切相关。另外还观察到外膜泡的形成具有方向性，即仅在细菌的吸附面才形成外膜泡，外膜泡中含有丰富的与吸附功能等有关的大分子物质。在大宝山T.f观察中，还观察到了接合过程的早期现象，一个大f通过性菌毛与另一个大f相连接;在另一观察中还发现，在两个端部靠近的大f之间有一管状物从一个细菌端部穿入相邻细菌的端部，这种现象是否与细菌的DNA转移有关还有待研究。在观察从大宝山堆浸场积液库中原始菌(未经分离，直接用gK培养)中，还观察到了真菌、异养菌等，有些细菌存在荚膜或粘膜，表明了浸矿微生物的生态多样性。 浸矿细菌的吸附是一个研究得较多的领域，细菌与矿物表面的作用涉及许多物理的和生物一化学的参数。运用扫描电镜(带能谱)、透射电镜、FTIR透射光谱、FTIR反射光谱、以及电镜细胞化学等技术，对浸矿细菌吸附至矿物的复杂界面作用进行了研究。细菌与矿物相互 作用后既改变了细菌表面的化学性质，也改变了矿物表面的化学性 质。 黄铜矿和黄铁矿细菌浸出的扫描电镜及能谱分析表明，与无菌对照相比较，黄铜矿细菌浸出的金属溶解率更高且铜浸出率随着吸附菌数的增加而增加。细菌吸附至黄铜矿和黄铁矿都表现出首先吸附至矿物的缺陷位点及裂缝处。从黄铜矿、黄铁矿的细菌浸出过程的能谱分析可以看出，黄铁矿的溶解速率明显慢于黄铜矿，说明黄铁矿的电化学高电位对细菌浸出产生不利影响。 采用电镜细胞化学方法证实了大f表面存在的脂类，多糖及蛋白质等物质。浸矿细菌表面的多聚物(EPS)介导了细菌与硫化矿物的接触，在它们的界面作用中起着重要作用。由细菌表面的多糖一蛋白质复合物构成的多糖纤维大部分带负电，能与二价阳离子形成极性键，促进细菌吸附至矿物。EPS对细菌还有保护作用，在营养缺乏时可供给细菌营养。EPS在细菌吸附至矿物过程中以及细菌生物膜形成过程中起着关键作用。 铁离子电镜细胞化学实验证明厂f外膜上存在Fe2+，为铁离子在细菌及细菌生物膜中的循环提供了实验证据。 黄铜矿浸出过程的扫描电镜观察表明，浸出3天时，大f还是零星地分布于矿物表面’;浸出10天时，大部分细菌成群聚集，分泌物增多，形成的微菌落逐渐向周边扩展;浸出第17天时，大面积的矿物表面已初步形成生物膜。浸出20天时，试样表面基本被细菌生物膜覆盖，己形成较完整的细菌生物膜。细菌在生物膜内可以创造它们自己的微环境，在这个微环境中亚铁和三价铁离子的浓度都得以增加，铁细菌通过自身的铁氧化系统氧化更多的亚铁而获得更多的能量，致使细菌的生长率和硫化矿的溶解率都得以增加。 0 }} 傅立叶变换红外光谱实验证明，浸矿细菌表面存在一CeeNH一、0H、CH3、CHZ等基团。硫培养细菌和铁培养细菌的红外透射光谱在指纹区存在差异，表明同种细菌生长在不同环境时，由于环境胁迫作用使其表面成分有所不同。红外反射光谱也证实了以上主要表面基团的存在。这些表面基团的存在以及随不同环境而变化的事实反映了浸矿细菌吸附至矿物过程中界面作用的复杂性。多经基肚键、C一O键和氢键等也在吸附过程中起着重要作用。 硫化氢一三价铁氧化还原酶是浸矿过程中硫氧化的关键酶。在从大?
In order to investigate the bioleaching mechanism and increase the bioleaching rate in the process of production, the following studies have been worked on: 1. Bioleaching of copper sulfide ores of Chengmen Mountain, Jiangxi; 2.The shapes and the microstructures of the bioldaching bacteria; 3. The attachment of bioleaching bacteria; and 4.The purification of Hydrogen SulfiderFerric ion Oxidoreductase from Thiobacillus ferrooxidans .
The shape and the ultrastructure of major organisms in bioleaching field such as T.f(Thiobacillus ferrooxidans), T.t(Thiobacillus ihiooxidans) and L.j[Leptospirillum ferrooxidans} have been studied by Transmission Electronic Microscope(TEM) and Scanning Electronic Microscope(SEM). The results show that the bacteria raised in ferrous ion medium or sulfur medium have the form of solitary, oppositeness, chain and thick growth. L.f exists in solitary; most of T.fand T.t exist in solitary; some of T.t in oppositeness; the number of T.t in the form of chain is more than that of T.f; both T.f and T.t can be seen in thick growth. The size of T.t is a slightly bigger than that of T.f, the ends of T.f is somewhat blunt, while the ends of T.t is sharp-pointed. Both ends of T.t can be seen a lipid granule respectively when dyed with phosphotungstic acid. Observing the super-cut section of T.f of Dexing and Dabao Mountain, there are a few of bacteria with the widen periplasmic space. General speaking, they would
have a stonger ability of storing and secreting proteins. In the ultrastructures of Dexing's T.f, some of PHB granule can be seen. They are the storing bodies of Carbon resource and energy. The bacteria raised in sulfur medium possess globular and/or tubular outer-membrane vesicles, but the bacteria raised in iron medium have not. The formation of outer-membrane vesicles are closely be related to the state S(solid) /L(liquid) of energy resource substance. Moreover, there is an orientation during its forming, the vesicles can be formed only on the attachment side of the cell, and it contains plenty macromolecular substances that are related to attachment. The early phase's conjugation of Dabao Mountain T.f was observed, a T.f is in touch with another by sex pili. Also, a tube-like object on one cell's end pierced through another near cell's end. If this phenomenon be related to diverting the genetic factor? It isn't clear now. There are fungus and heterotrophic bacteria and so on in the accumulating solution pool of Dabao Mountain dump leaching site, some of bacteria possess capsule or the layer of mucus. They exhibit the diversity of leaching organisms in ecological system.
In the research of bioleaching feasibility on difficultly dissolved copper ores of Chengmen Mountain, mineralogic analysis and multi-element analysis have been carried out, and then a series of tests such as acid-consume, leaching by rocking bottles, column leaching were also carried out. The leaching rate under different technological
conditions were checked respectively. The results demonstrate that coppery limonite and mud-bearing copper oxide ore are suitable for acid leaching while copper oxide and copper sulfide mixed ores are suitable for bioleaching. In addition, an UV-mutagenesis T.f-cms (Chengmenshan T.f) for bioleaching , by which copper leaching rate has raised about 10%, has been ultilized in this paper.
The attachment of bioleaching microorganisms is a field that has been studied extensively. The author has studied on the attachment of bacteria by SEM(with EDAX),TEM, FTIR, especially by the method of electronic microscope cytochemistry. The interactions between the bacteria and the mineral involve a lot of physical, biological and chemical parameter. Both the bacteria and the mineral surface' chemical character were changed after their interactions.
The results of SEM and ED AX show that, comparing with asepsis condition, the metal dissolved rate of bioleaching for chalcopyrite is higher than that of pyrite, and leaching rate of copper increases as the number of bacteria of attachment increases. When
在对城门山难浸铜矿的细菌浸出可行性研究中，对含铜褐铁矿，氧化铜-硫化铜混合矿和含泥氧化矿进行了物相分析、多元素分析后，又进行了酸耗试验，摇瓶试验，柱浸试验，并比较了不同的工艺条件对浸出过程的影响。结果表明含铜褐铁矿和含泥氧化矿宜于酸浸，而氧化铜一硫化铜混合矿(次生硫化铜占铜品位的94.97％)适宜于细菌浸出。此外还运用紫外诱变育种方法，将经适当紫外诱变处理的T，f正突变菌株用于浸矿，可提高细菌的亚铁氧化能力，使铜浸出率提高10％左右。
运用透射电镜(TEM)和扫描电镜(SEM)对最常见的浸矿细菌如T.f(Thiobacillus ferrooxidans)、T.t(Thiobacillusferrooxidans)和L.f(Leptosprillum ferrooxidans)等的形态和超微结构进行了研究。结果表明，亚铁培养基培养或硫培养基培养细菌的排列形式有单生，对生、链状和单层细菌成群排列。L.f单个存在，T.f和T.t大多以单个形式存在；可见到T.t的对生；T.t比T.f更多的以链状形式存在，T.f和T.t都能见到单层细菌成群排列的形式。在外形大小上，T.t略大于T.f，且T.t的两端比T.f略为尖锐；
用磷钨酸染色，大t两端一般各有一脂粒存在。在超薄切片观察中，分别在德兴大f和大宝山大f中观察到了具有较宽胞周隙的菌株，这些菌株在样品中所占比例小，它们应具有较强的贮存和分泌蛋白质(酶)的能力。在德兴Tf中观察到了PHB颗粒，它们是碳源和能源的储存体。在江西德兴、城门山、广东大宝山等矿区的厂f中均观察到球状体，尽管目前还不明了其生理功能。 在研究不同培养基培养的浸矿细菌的形态差别时，发现以元素硫为能源的培养基培养的细菌可观察到外膜泡，而用亚铁培养基培养的细菌却观察不到这种结构，外膜泡的形成与否与培养基质中能源物质的存在状态(固态或液态)密切相关。另外还观察到外膜泡的形成具有方向性，即仅在细菌的吸附面才形成外膜泡，外膜泡中含有丰富的与吸附功能等有关的大分子物质。在大宝山T.f观察中，还观察到了接合过程的早期现象，一个大f通过性菌毛与另一个大f相连接;在另一观察中还发现，在两个端部靠近的大f之间有一管状物从一个细菌端部穿入相邻细菌的端部，这种现象是否与细菌的DNA转移有关还有待研究。在观察从大宝山堆浸场积液库中原始菌(未经分离，直接用gK培养)中，还观察到了真菌、异养菌等，有些细菌存在荚膜或粘膜，表明了浸矿微生物的生态多样性。 浸矿细菌的吸附是一个研究得较多的领域，细菌与矿物表面的作用涉及许多物理的和生物一化学的参数。运用扫描电镜(带能谱)、透射电镜、FTIR透射光谱、FTIR反射光谱、以及电镜细胞化学等技术，对浸矿细菌吸附至矿物的复杂界面作用进行了研究。细菌与矿物相互 作用后既改变了细菌表面的化学性质，也改变了矿物表面的化学性 质。 黄铜矿和黄铁矿细菌浸出的扫描电镜及能谱分析表明，与无菌对照相比较，黄铜矿细菌浸出的金属溶解率更高且铜浸出率随着吸附菌数的增加而增加。细菌吸附至黄铜矿和黄铁矿都表现出首先吸附至矿物的缺陷位点及裂缝处。从黄铜矿、黄铁矿的细菌浸出过程的能谱分析可以看出，黄铁矿的溶解速率明显慢于黄铜矿，说明黄铁矿的电化学高电位对细菌浸出产生不利影响。 采用电镜细胞化学方法证实了大f表面存在的脂类，多糖及蛋白质等物质。浸矿细菌表面的多聚物(EPS)介导了细菌与硫化矿物的接触，在它们的界面作用中起着重要作用。由细菌表面的多糖一蛋白质复合物构成的多糖纤维大部分带负电，能与二价阳离子形成极性键，促进细菌吸附至矿物。EPS对细菌还有保护作用，在营养缺乏时可供给细菌营养。EPS在细菌吸附至矿物过程中以及细菌生物膜形成过程中起着关键作用。 铁离子电镜细胞化学实验证明厂f外膜上存在Fe2+，为铁离子在细菌及细菌生物膜中的循环提供了实验证据。 黄铜矿浸出过程的扫描电镜观察表明，浸出3天时，大f还是零星地分布于矿物表面’;浸出10天时，大部分细菌成群聚集，分泌物增多，形成的微菌落逐渐向周边扩展;浸出第17天时，大面积的矿物表面已初步形成生物膜。浸出20天时，试样表面基本被细菌生物膜覆盖，己形成较完整的细菌生物膜。细菌在生物膜内可以创造它们自己的微环境，在这个微环境中亚铁和三价铁离子的浓度都得以增加，铁细菌通过自身的铁氧化系统氧化更多的亚铁而获得更多的能量，致使细菌的生长率和硫化矿的溶解率都得以增加。 0 }} 傅立叶变换红外光谱实验证明，浸矿细菌表面存在一CeeNH一、0H、CH3、CHZ等基团。硫培养细菌和铁培养细菌的红外透射光谱在指纹区存在差异，表明同种细菌生长在不同环境时，由于环境胁迫作用使其表面成分有所不同。红外反射光谱也证实了以上主要表面基团的存在。这些表面基团的存在以及随不同环境而变化的事实反映了浸矿细菌吸附至矿物过程中界面作用的复杂性。多经基肚键、C一O键和氢键等也在吸附过程中起着重要作用。 硫化氢一三价铁氧化还原酶是浸矿过程中硫氧化的关键酶。在从大?
In order to investigate the bioleaching mechanism and increase the bioleaching rate in the process of production, the following studies have been worked on: 1. Bioleaching of copper sulfide ores of Chengmen Mountain, Jiangxi; 2.The shapes and the microstructures of the bioldaching bacteria; 3. The attachment of bioleaching bacteria; and 4.The purification of Hydrogen SulfiderFerric ion Oxidoreductase from Thiobacillus ferrooxidans .
The shape and the ultrastructure of major organisms in bioleaching field such as T.f(Thiobacillus ferrooxidans), T.t(Thiobacillus ihiooxidans) and L.j[Leptospirillum ferrooxidans} have been studied by Transmission Electronic Microscope(TEM) and Scanning Electronic Microscope(SEM). The results show that the bacteria raised in ferrous ion medium or sulfur medium have the form of solitary, oppositeness, chain and thick growth. L.f exists in solitary; most of T.fand T.t exist in solitary; some of T.t in oppositeness; the number of T.t in the form of chain is more than that of T.f; both T.f and T.t can be seen in thick growth. The size of T.t is a slightly bigger than that of T.f, the ends of T.f is somewhat blunt, while the ends of T.t is sharp-pointed. Both ends of T.t can be seen a lipid granule respectively when dyed with phosphotungstic acid. Observing the super-cut section of T.f of Dexing and Dabao Mountain, there are a few of bacteria with the widen periplasmic space. General speaking, they would
have a stonger ability of storing and secreting proteins. In the ultrastructures of Dexing's T.f, some of PHB granule can be seen. They are the storing bodies of Carbon resource and energy. The bacteria raised in sulfur medium possess globular and/or tubular outer-membrane vesicles, but the bacteria raised in iron medium have not. The formation of outer-membrane vesicles are closely be related to the state S(solid) /L(liquid) of energy resource substance. Moreover, there is an orientation during its forming, the vesicles can be formed only on the attachment side of the cell, and it contains plenty macromolecular substances that are related to attachment. The early phase's conjugation of Dabao Mountain T.f was observed, a T.f is in touch with another by sex pili. Also, a tube-like object on one cell's end pierced through another near cell's end. If this phenomenon be related to diverting the genetic factor? It isn't clear now. There are fungus and heterotrophic bacteria and so on in the accumulating solution pool of Dabao Mountain dump leaching site, some of bacteria possess capsule or the layer of mucus. They exhibit the diversity of leaching organisms in ecological system.
In the research of bioleaching feasibility on difficultly dissolved copper ores of Chengmen Mountain, mineralogic analysis and multi-element analysis have been carried out, and then a series of tests such as acid-consume, leaching by rocking bottles, column leaching were also carried out. The leaching rate under different technological
conditions were checked respectively. The results demonstrate that coppery limonite and mud-bearing copper oxide ore are suitable for acid leaching while copper oxide and copper sulfide mixed ores are suitable for bioleaching. In addition, an UV-mutagenesis T.f-cms (Chengmenshan T.f) for bioleaching , by which copper leaching rate has raised about 10%, has been ultilized in this paper.
The attachment of bioleaching microorganisms is a field that has been studied extensively. The author has studied on the attachment of bacteria by SEM(with EDAX),TEM, FTIR, especially by the method of electronic microscope cytochemistry. The interactions between the bacteria and the mineral involve a lot of physical, biological and chemical parameter. Both the bacteria and the mineral surface' chemical character were changed after their interactions.
The results of SEM and ED AX show that, comparing with asepsis condition, the metal dissolved rate of bioleaching for chalcopyrite is higher than that of pyrite, and leaching rate of copper increases as the number of bacteria of attachment increases. When
引文
[1] 魏鹏，龚文琪，雷绍民．微生物技术在低品位矿物资源开发与综合回收中的应用[J]，有色金属，2000，52(4)：188-190．
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