苏云金芽胞杆菌等微生物对环境中毒性金属的固定转化作用研究
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摘要
从亚微观角度研究并揭示微生物与毒性金属相互过程中的生物与化学等方面微观机理,有利于在更深刻地认识生物处理毒性金属污染的本质问题的基础上,发展出更为成熟高效的生物去除毒性金属污染及其回收利用技术。本研究以污染位点分离得到的非病原菌(苏云金芽胞杆菌、蜡状芽胞杆菌和枯草芽胞杆菌等细菌)为研究对象,探讨它们在与毒性金属相互作用过程中的微观变化及其对毒性金属固定转化机制,利用廉价的培养基对细菌进行培养并用于后续毒性金属处理。
通过研究发现：(1)从新疆铀矿污染位点分离得到的2株苏云金芽胞杆菌对毒性金属铀具有很好的耐受和吸附能力,最大的吸附容量大于400mg U/g干菌。X射线粉末衍射(XRD)、原子力显微镜(AFM)、扫描电镜(SEM)、透射电镜(TEM)和傅立叶红外变换光谱(FT-IR)等研究结果表明,在无外加营养源的条件下,细菌能够利用表面的磷酸、-CH2和酰胺类官能团介导U(Ⅵ)形成无定型的针状含铀化合物,并且在固定的过程中铀仍以U(Ⅵ)的形式存在。随着时间的延长,吸附在细胞上的无定型铀化合物将逐渐进入到细胞的内部,并且形成具有很好晶型的磷铵铀矿。此外,研究发现细胞质对铀的固定能力要强于细胞碎片,表明磷铵铀矿的形成可能是由于细胞质内丰富的磷酸或者其它的有机质导致的晶化；
(2)课题组成员前期的研究表明枯草芽胞杆菌的浮游细胞和生物膜对毒性金属铬的还原和固定能力具有很大的差异。进一步通过电镜观察及价态分析等探讨细菌与铬相互作用过程中的微观机制及造成差异的原因,TEM研究表明与100mg/L铬作用120h后,浮游细胞的细胞壁变得模糊不完整,并且在细胞的内部都有Cr信号,然而生物膜细胞的结构没有明显的变化,在细胞的内部无Cr信号。此外,X-射线光电子能谱(XPS)的结果则表明被浮游细胞和生物膜固定的铬均以三价铬的形式存在。基于以上的研究认识,提出联合利用浮游细胞和生物膜对10-L含铬电镀废水中的铬实现完全的还原和固定,处理后的铬浓度达到国家废水的排放标准(总铬浓度≦1.5mg/L)。同时被固定的铬能够以Cr2O3的形式被回收；
(3)以福建屏南毒性金属位点分离的蜡状芽胞杆菌为例,研究其对毒性金属镍的固定机理。结果发现该菌对镍的固定为快速的过程,在2h时吸附达到平衡,对镍的吸附量为17.7mg/g(干菌)。此外,电镜观察(AFM、SEM和TEM)、破碎实验、FT-IR和XRD分析结果表明,吸附以胞外固定为主,同时酰胺类和羧化类官能团参与了镍的固定,并且被固定在细菌上的镍是以无定型的形式存在；
(4)为了降低细菌在处理毒性金属中的成本以及增加实际应用的可能性,本文使用豆腐废水作为微生物的廉价培养基,并将培养好的微生物应用于实际电镀废水中铬、镍等毒性金属的去除。研究发现蜡状芽胞杆菌和人苍白杆菌在稀释的豆腐废水中就能很好的生长,表明豆腐水能够提供细菌生长的必需营养物质。此外,在添加了糖渣浸出液的豆腐废水且pH值为7.5时为细菌的最适宜生长条件。进一步的研究则表明在最佳条件下分别生长的蜡状芽胞杆菌和人苍白杆菌,对初始浓度分别为214mg/L和367mg/L的含铬、镍的电镀废水均具有较好的去除效率,并且细菌的菌量越大去除效率越高,去除效率可以达到80%以上。
Reveal the mechanism on biological and chemical changes between the reaction of microbial organisms and toxic metals from microcosmic perspective, is conducive to form deeply understanding of the nature of biological treatment for toxic metals pollution, and then develop more mature and efficient technologies on the removal and recycling of toxic metals. In this work, we chose the non-pathogenic soil bacteria (Bacillus thuringiensis, Bacillus subtilis, Bacillus cereus and other bacteria) as target strains, investigated the microscopic changes and transformation mechanism in the reaction process of toxic metals and bacteria, and utilized the bean curd wastewater for bacterial culture and applied it into the removal of toxic metals.
The main results were as follows:(1) Two B. thuringiensis strains, which were isolated from uranium-contaminated soil samples in Xinjiang, had highly resistant and possessed super accumulation ability to U(VI), and the maximum accumulation capacity was around400mg U/g biomass (dry weight). X-ray powder diffraction (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) analyses indicated that U(VI) was adsorbed on the bacterial surface firstly through coordinating with phosphate,-CH2and amide groups, then needle-like amorphous uranium compounds were formed, and the uranium immobilized on bacteria was still at the U(VI) state. With the extension of time, the adsorbed uranium nanoparticles entered into the intracellular region, and then the amorphous uranium compound was transformed into crystalline tetragonal-uramphite. Besides, the cytoplasm of B. thuringiensis had better uranium-immobilization ability than its cell debris, suggesting that the formation of uramphite was attributed to the rich phosphate or other organics in cytoplasm.
(2) Previous studies by our group member indicated that the planktonic cells and biofilms of B. subtilis had different effects on Cr(Ⅵ) reducution and Cr(Ⅲ) immobilization. Furthermore, electron microscope observation and valence analysis were applied in the reaction process betweeen chromium and bacteria, in order to investigate the mechanism and explore the reasons for the different ability of reduction and immobilization. TEM analysis indicating that the planktonic cells of B. subtilis became lean and rough after treated with100mg/L Cr(Ⅵ) for120h, and the chromium signal could be detected inside of bacteria by EDS analysis. However, after the Cr(Ⅵ) treatment, neither morphologic change nor chromium was detected inside of biofilm cells. Moreover, the result of X-ray photoelectron spectroscopy (XPS) indicating that the chromium immobilized on biofilms and planktonic cells was at Cr(Ⅲ) state. Additionally, a strategy combining the advantages of planktonic cells and biofilms was proposed, and the chromium removal from electroplating wastewater was successfully achieved in a10-L pilot-scale experiment, the remaining non-immobilized Cr was lower than the wastewater release standard of China (total Cr(?)1.5mg/L). Simultaneously, the immobilization Cr(Ⅲ) on bacteria can be further recycled in the form of Cr2O3.
(3) The biosorption mechanism of Ni(Ⅱ) by B. cereus was investigated in this study. This bacterium was isolated from a toxic metals contamination site in Pingnan, Fujian Province. It was found that the adsorption equilibrium reached rapidly in2h and the maximum nickel adsorption capability of B. cereus was17.7mg/g (dry weight). Additionally, electron microscopic (AFM, SEM and TEM), ultrasonic disrupted experiment, FT-IR and XRD analysis confirmed that the extracellular immobilization was the main biosorption process, the bacterial amido and carboxyl function groups had involved in nickel immobilized, and the Ni(Ⅱ) collected by the bacteria was amorphous.
(4) In order to reduce the cost and increase the feasibility of real application in removal of heavy metals by bacteria, the discharged soybean wastewater was used as cheap culture for bacterial cultivation, and the cultured bacteria were applied to remove toxic metals from electroplating wastewater. It was found that B. cereus and Ochrobactrum anthropi CTS-325could be well grown in dilute soybean wastewater, means that the soybean wastewater could ensure the necessary nutrition for bacterial growth. In addition, soybean wastewater with adding sucrose residue leach solution (pH7.5) was the optimum condition for bacterial growth. Further studies revealed that when the initial concentrations of chromium and nickel in electroplating wastewater were214mg/L and367mg/L respectively, the cultured bacteria in optimum medium had a strong capacity of Cr(VI) and Ni(II) removal, Meanwhile, the biomass of B. cereus and O. anthropi CTS-325could be greatly affected the removal efficiency, the more bacterial biomass, the better removal ability, and the removal efficiency could be higher than80%.
通过研究发现：(1)从新疆铀矿污染位点分离得到的2株苏云金芽胞杆菌对毒性金属铀具有很好的耐受和吸附能力,最大的吸附容量大于400mg U/g干菌。X射线粉末衍射(XRD)、原子力显微镜(AFM)、扫描电镜(SEM)、透射电镜(TEM)和傅立叶红外变换光谱(FT-IR)等研究结果表明,在无外加营养源的条件下,细菌能够利用表面的磷酸、-CH2和酰胺类官能团介导U(Ⅵ)形成无定型的针状含铀化合物,并且在固定的过程中铀仍以U(Ⅵ)的形式存在。随着时间的延长,吸附在细胞上的无定型铀化合物将逐渐进入到细胞的内部,并且形成具有很好晶型的磷铵铀矿。此外,研究发现细胞质对铀的固定能力要强于细胞碎片,表明磷铵铀矿的形成可能是由于细胞质内丰富的磷酸或者其它的有机质导致的晶化；
(2)课题组成员前期的研究表明枯草芽胞杆菌的浮游细胞和生物膜对毒性金属铬的还原和固定能力具有很大的差异。进一步通过电镜观察及价态分析等探讨细菌与铬相互作用过程中的微观机制及造成差异的原因,TEM研究表明与100mg/L铬作用120h后,浮游细胞的细胞壁变得模糊不完整,并且在细胞的内部都有Cr信号,然而生物膜细胞的结构没有明显的变化,在细胞的内部无Cr信号。此外,X-射线光电子能谱(XPS)的结果则表明被浮游细胞和生物膜固定的铬均以三价铬的形式存在。基于以上的研究认识,提出联合利用浮游细胞和生物膜对10-L含铬电镀废水中的铬实现完全的还原和固定,处理后的铬浓度达到国家废水的排放标准(总铬浓度≦1.5mg/L)。同时被固定的铬能够以Cr2O3的形式被回收；
(3)以福建屏南毒性金属位点分离的蜡状芽胞杆菌为例,研究其对毒性金属镍的固定机理。结果发现该菌对镍的固定为快速的过程,在2h时吸附达到平衡,对镍的吸附量为17.7mg/g(干菌)。此外,电镜观察(AFM、SEM和TEM)、破碎实验、FT-IR和XRD分析结果表明,吸附以胞外固定为主,同时酰胺类和羧化类官能团参与了镍的固定,并且被固定在细菌上的镍是以无定型的形式存在；
(4)为了降低细菌在处理毒性金属中的成本以及增加实际应用的可能性,本文使用豆腐废水作为微生物的廉价培养基,并将培养好的微生物应用于实际电镀废水中铬、镍等毒性金属的去除。研究发现蜡状芽胞杆菌和人苍白杆菌在稀释的豆腐废水中就能很好的生长,表明豆腐水能够提供细菌生长的必需营养物质。此外,在添加了糖渣浸出液的豆腐废水且pH值为7.5时为细菌的最适宜生长条件。进一步的研究则表明在最佳条件下分别生长的蜡状芽胞杆菌和人苍白杆菌,对初始浓度分别为214mg/L和367mg/L的含铬、镍的电镀废水均具有较好的去除效率,并且细菌的菌量越大去除效率越高,去除效率可以达到80%以上。
Reveal the mechanism on biological and chemical changes between the reaction of microbial organisms and toxic metals from microcosmic perspective, is conducive to form deeply understanding of the nature of biological treatment for toxic metals pollution, and then develop more mature and efficient technologies on the removal and recycling of toxic metals. In this work, we chose the non-pathogenic soil bacteria (Bacillus thuringiensis, Bacillus subtilis, Bacillus cereus and other bacteria) as target strains, investigated the microscopic changes and transformation mechanism in the reaction process of toxic metals and bacteria, and utilized the bean curd wastewater for bacterial culture and applied it into the removal of toxic metals.
The main results were as follows:(1) Two B. thuringiensis strains, which were isolated from uranium-contaminated soil samples in Xinjiang, had highly resistant and possessed super accumulation ability to U(VI), and the maximum accumulation capacity was around400mg U/g biomass (dry weight). X-ray powder diffraction (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) analyses indicated that U(VI) was adsorbed on the bacterial surface firstly through coordinating with phosphate,-CH2and amide groups, then needle-like amorphous uranium compounds were formed, and the uranium immobilized on bacteria was still at the U(VI) state. With the extension of time, the adsorbed uranium nanoparticles entered into the intracellular region, and then the amorphous uranium compound was transformed into crystalline tetragonal-uramphite. Besides, the cytoplasm of B. thuringiensis had better uranium-immobilization ability than its cell debris, suggesting that the formation of uramphite was attributed to the rich phosphate or other organics in cytoplasm.
(2) Previous studies by our group member indicated that the planktonic cells and biofilms of B. subtilis had different effects on Cr(Ⅵ) reducution and Cr(Ⅲ) immobilization. Furthermore, electron microscope observation and valence analysis were applied in the reaction process betweeen chromium and bacteria, in order to investigate the mechanism and explore the reasons for the different ability of reduction and immobilization. TEM analysis indicating that the planktonic cells of B. subtilis became lean and rough after treated with100mg/L Cr(Ⅵ) for120h, and the chromium signal could be detected inside of bacteria by EDS analysis. However, after the Cr(Ⅵ) treatment, neither morphologic change nor chromium was detected inside of biofilm cells. Moreover, the result of X-ray photoelectron spectroscopy (XPS) indicating that the chromium immobilized on biofilms and planktonic cells was at Cr(Ⅲ) state. Additionally, a strategy combining the advantages of planktonic cells and biofilms was proposed, and the chromium removal from electroplating wastewater was successfully achieved in a10-L pilot-scale experiment, the remaining non-immobilized Cr was lower than the wastewater release standard of China (total Cr(?)1.5mg/L). Simultaneously, the immobilization Cr(Ⅲ) on bacteria can be further recycled in the form of Cr2O3.
(3) The biosorption mechanism of Ni(Ⅱ) by B. cereus was investigated in this study. This bacterium was isolated from a toxic metals contamination site in Pingnan, Fujian Province. It was found that the adsorption equilibrium reached rapidly in2h and the maximum nickel adsorption capability of B. cereus was17.7mg/g (dry weight). Additionally, electron microscopic (AFM, SEM and TEM), ultrasonic disrupted experiment, FT-IR and XRD analysis confirmed that the extracellular immobilization was the main biosorption process, the bacterial amido and carboxyl function groups had involved in nickel immobilized, and the Ni(Ⅱ) collected by the bacteria was amorphous.
(4) In order to reduce the cost and increase the feasibility of real application in removal of heavy metals by bacteria, the discharged soybean wastewater was used as cheap culture for bacterial cultivation, and the cultured bacteria were applied to remove toxic metals from electroplating wastewater. It was found that B. cereus and Ochrobactrum anthropi CTS-325could be well grown in dilute soybean wastewater, means that the soybean wastewater could ensure the necessary nutrition for bacterial growth. In addition, soybean wastewater with adding sucrose residue leach solution (pH7.5) was the optimum condition for bacterial growth. Further studies revealed that when the initial concentrations of chromium and nickel in electroplating wastewater were214mg/L and367mg/L respectively, the cultured bacteria in optimum medium had a strong capacity of Cr(VI) and Ni(II) removal, Meanwhile, the biomass of B. cereus and O. anthropi CTS-325could be greatly affected the removal efficiency, the more bacterial biomass, the better removal ability, and the removal efficiency could be higher than80%.
引文
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