一株氧化亚铁硫杆菌的分离鉴定及表面钝化剂三乙烯四胺（TETA）抗生物氧化机理的研究
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摘要
嗜酸氧化亚铁硫杆菌(Acidthiobacillus ferrooxidans,以下简称A. f菌)大量存在于酸性矿山废水及尾矿中,是目前研究得最为深入的浸矿细菌之一。我国尾矿存有量超过百亿吨,且每年以几亿吨的速度增长。A. f菌的存在能大大加速尾矿风化及重金属元素的溶出,会对邻近区域的环境构成严重威胁。
本研究中拟在不需要进行预处理的情况下直接对尾矿进行钝化,从微观水平上研究尾矿中A. f菌的生长特性以及钝化条件下硫化物矿物的化学和生物氧化情况,从不同方面为寻找新的方法抑制尾矿的氧化及减缓重金属的释放提供理论依据,主要研究内容包括:氧化亚铁硫杆菌DBS-8的分离鉴定和表面钝化剂三乙烯四胺(TETA)钝化尾矿的抗生物氧化性能研究。
本论文选用9K培养基并以Fe~(2+)作为能源,从尾矿样品中分离纯化得到对Fe~(2+)及元素硫氧化活性均较好的菌株DBS-8。通过16S rRNA扩增和DNA鉴定DBS-8为氧化亚铁硫杆菌并构建其系统发育树。另外,通过设计细菌培养条件的正交试验,重点考察了Fe~(2+)初始浓度、接种量、pH、温度及硫酸铵浓度这五个环境因素对细菌生长的影响,得出各初始条件对细菌生长影响的显著性顺序为:pH>接种量>温度>硫酸铵浓度>初始亚铁浓度。这表明pH对A. f菌生长影响最大,通过破坏其酸性环境可以最大程度上抑制细菌生长。
此外,运用TETA对尾矿进行表面钝化处理,研究钝化前后尾矿抗化学氧化和生物氧化的能力。结果表明,TETA对尾矿具有一定的钝化效果,但随着时间的延长,钝化效果逐渐减弱。浸矿60d后,除了铁离子溶出的抑制率高达73%-79%外,浸矿体系中的Cu~(2+)、Zn~(2+)、Cd~(2+)、Mn~(2+)溶出的抑制效果均较差。这可能因为TETA形成的钝化膜并不稳定,经过长期的侵蚀作用而逐渐脱落。同时,细菌的存在并没有促进溶液中金属离子的溶出,反而使其离子浓度降低。这可能与细菌对金属离子的吸收作用以及金属配合物的形成有关。溶液中因缺乏细菌生长所需的Fe~(2+)而导致细菌生长缓慢,生物浸矿作用远小于细菌对金属离子的吸收强度,从而导致溶液中金属离子浓度的降低。由于尾矿成分复杂,浸矿体系中影响重金属溶出的因素较多,本论文只对TETA表面钝化效果和尾矿的生物浸出机理作了初步的探讨,有关钝化处理工艺的优化和生物浸出机理尚需更深入的研究。
Acidthiobacillus ferrooxidans (A. f) which exists in acid mine drainage and tailings is one of the most important and lucubrated bacteria in bioleaching process at the present. The capacity of tailings in China is more than one hundred million tons with several million tons of growth rates every year. A. f strain can accelerate the weathering of tailings and leaching process of heavy metals which threaten the environment of neighboring region.
This research intended to get a new passivating program to passivate the mine tailings without pre-oxidation. It studied on the growth and characteristic of A. f strain and chemical and biological situation of sulfide minerals under passivation at the micro level. It aimed to find new methods of inhibiting the oxidation of tailings and slowing the release of heavy metals in different ways. The main content of the research includes: the isolation and identification of an A. f strain named by DBS-8 and researches on anti-biooxidation mechanism by surface passivation agent triethlenetetramine (TETA).
Taking 9K medium with Fe~(2+) as energy source, a strain DBS-8 which had high Fe~(2+) and elemental sulfur oxidation activity was obtained. After PCR amplification of 16S rRNA and DNA testing, DBS-8 was identified as an A. f strain and its phylogeny was constructed. Then, an orthogonal experiment was designed to investigate the impact of inoculums volume﹑initial pH﹑concentration of ferrous﹑temperature and concentration of ammonium sulfate on the growth of the strain. The order of the above factors which affected the growth of strain DBS-8 most is: pH> inoculum> temperature> ammonium sulfate concentration > the initial ferrous concentration. This showed that pH impacted the growth of A. f strain most. Through destroying the acid environment, it can inhibit the growth of the bacteria to the maximum extent.
In addition, through using TETA to passivate the tailing surface, the capability of its anti-chemical oxidation and anti-biological oxidation both before and after passivating was studied. Results showed that TETA had certain effect on the leaching process. However, as the time went, its effect became weak. After 60 days of leaching process, the effects of inhabitation of the leaching rate of Cu~(2+)、Zn~(2+)、Cd~(2+)、Mn~(2+) ions were weak while its effect of inhabitation of the leaching rate of Fe3+ was high to range from 73% to 79%. This was possibly due to the instability of TETA. Trough long-term leaching, TETA fell out for erosion. In the meantime, the bacteria decreased the leaching concentration of heavy metals in contrast. It is possibly related to the absorption to the metals or the formation of metal chelates. It slowed down the growth of the bacteria for lack of Fe~(2+) for energy resource. As a result, the effect of bioleaching to promote the metal leaching rate is less than the absorption by bacteria to lead to a related low concentration of metal ions. For the complicated composition of the tailings and the diversity of effects to the leaching concentration of heavy metals in the leaching solution, the thesis made a preliminary study on the effects of surface passivator TETA and bioleaching mechanism of mine tailings. However, optimization of the passivating technology and the detailed mechanism of bioleaching need further research.
本研究中拟在不需要进行预处理的情况下直接对尾矿进行钝化,从微观水平上研究尾矿中A. f菌的生长特性以及钝化条件下硫化物矿物的化学和生物氧化情况,从不同方面为寻找新的方法抑制尾矿的氧化及减缓重金属的释放提供理论依据,主要研究内容包括:氧化亚铁硫杆菌DBS-8的分离鉴定和表面钝化剂三乙烯四胺(TETA)钝化尾矿的抗生物氧化性能研究。
本论文选用9K培养基并以Fe~(2+)作为能源,从尾矿样品中分离纯化得到对Fe~(2+)及元素硫氧化活性均较好的菌株DBS-8。通过16S rRNA扩增和DNA鉴定DBS-8为氧化亚铁硫杆菌并构建其系统发育树。另外,通过设计细菌培养条件的正交试验,重点考察了Fe~(2+)初始浓度、接种量、pH、温度及硫酸铵浓度这五个环境因素对细菌生长的影响,得出各初始条件对细菌生长影响的显著性顺序为:pH>接种量>温度>硫酸铵浓度>初始亚铁浓度。这表明pH对A. f菌生长影响最大,通过破坏其酸性环境可以最大程度上抑制细菌生长。
此外,运用TETA对尾矿进行表面钝化处理,研究钝化前后尾矿抗化学氧化和生物氧化的能力。结果表明,TETA对尾矿具有一定的钝化效果,但随着时间的延长,钝化效果逐渐减弱。浸矿60d后,除了铁离子溶出的抑制率高达73%-79%外,浸矿体系中的Cu~(2+)、Zn~(2+)、Cd~(2+)、Mn~(2+)溶出的抑制效果均较差。这可能因为TETA形成的钝化膜并不稳定,经过长期的侵蚀作用而逐渐脱落。同时,细菌的存在并没有促进溶液中金属离子的溶出,反而使其离子浓度降低。这可能与细菌对金属离子的吸收作用以及金属配合物的形成有关。溶液中因缺乏细菌生长所需的Fe~(2+)而导致细菌生长缓慢,生物浸矿作用远小于细菌对金属离子的吸收强度,从而导致溶液中金属离子浓度的降低。由于尾矿成分复杂,浸矿体系中影响重金属溶出的因素较多,本论文只对TETA表面钝化效果和尾矿的生物浸出机理作了初步的探讨,有关钝化处理工艺的优化和生物浸出机理尚需更深入的研究。
Acidthiobacillus ferrooxidans (A. f) which exists in acid mine drainage and tailings is one of the most important and lucubrated bacteria in bioleaching process at the present. The capacity of tailings in China is more than one hundred million tons with several million tons of growth rates every year. A. f strain can accelerate the weathering of tailings and leaching process of heavy metals which threaten the environment of neighboring region.
This research intended to get a new passivating program to passivate the mine tailings without pre-oxidation. It studied on the growth and characteristic of A. f strain and chemical and biological situation of sulfide minerals under passivation at the micro level. It aimed to find new methods of inhibiting the oxidation of tailings and slowing the release of heavy metals in different ways. The main content of the research includes: the isolation and identification of an A. f strain named by DBS-8 and researches on anti-biooxidation mechanism by surface passivation agent triethlenetetramine (TETA).
Taking 9K medium with Fe~(2+) as energy source, a strain DBS-8 which had high Fe~(2+) and elemental sulfur oxidation activity was obtained. After PCR amplification of 16S rRNA and DNA testing, DBS-8 was identified as an A. f strain and its phylogeny was constructed. Then, an orthogonal experiment was designed to investigate the impact of inoculums volume﹑initial pH﹑concentration of ferrous﹑temperature and concentration of ammonium sulfate on the growth of the strain. The order of the above factors which affected the growth of strain DBS-8 most is: pH> inoculum> temperature> ammonium sulfate concentration > the initial ferrous concentration. This showed that pH impacted the growth of A. f strain most. Through destroying the acid environment, it can inhibit the growth of the bacteria to the maximum extent.
In addition, through using TETA to passivate the tailing surface, the capability of its anti-chemical oxidation and anti-biological oxidation both before and after passivating was studied. Results showed that TETA had certain effect on the leaching process. However, as the time went, its effect became weak. After 60 days of leaching process, the effects of inhabitation of the leaching rate of Cu~(2+)、Zn~(2+)、Cd~(2+)、Mn~(2+) ions were weak while its effect of inhabitation of the leaching rate of Fe3+ was high to range from 73% to 79%. This was possibly due to the instability of TETA. Trough long-term leaching, TETA fell out for erosion. In the meantime, the bacteria decreased the leaching concentration of heavy metals in contrast. It is possibly related to the absorption to the metals or the formation of metal chelates. It slowed down the growth of the bacteria for lack of Fe~(2+) for energy resource. As a result, the effect of bioleaching to promote the metal leaching rate is less than the absorption by bacteria to lead to a related low concentration of metal ions. For the complicated composition of the tailings and the diversity of effects to the leaching concentration of heavy metals in the leaching solution, the thesis made a preliminary study on the effects of surface passivator TETA and bioleaching mechanism of mine tailings. However, optimization of the passivating technology and the detailed mechanism of bioleaching need further research.
引文
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