重离子辐照诱变选育嗜酸氧化亚铁硫杆菌及其分子机理研究
发布时间：2019-03-18 18:50
【摘要】：易处理金矿的储量正在逐年急剧减少,开采利用难处理金矿迫在眉睫。生物氧化法因其对环境友好和成本低等优点在对难处理金矿的开采冶炼方面有广阔的应用前景。嗜酸氧化亚铁硫杆菌作为该法中最主要的细菌,存在着氧化能力弱、不耐砷、对各种离子不耐受等缺点,严重限制了生物氧化的工业化进程,所以选育高氧化活性和优良性能菌种已成为重中之重。本论文通过利用碳离子和X射线辐照诱变嗜酸氧化亚铁硫杆菌并选育得到具有氧化活性增高且稳定遗传的突变菌株,同时通过分子生物学方法检测原始菌株和高氧化活性突变菌株的铁氧化通路相关酶基因的表达差异,探讨诱变引起该细菌氧化活性提高的分子机理。1、嗜酸氧化亚铁硫杆菌野生型菌株的分离鉴定从阳山金矿矿坑水中纯化获得一株细菌,经形态特征生理生化和分子鉴定表明其属于嗜酸氧化亚铁硫杆菌,取名YS-1。该菌长约1.2-1.5μm,宽约0.4-0.7μm,可以氧化利用二价铁和单质硫或硫化合物作为其生长的能量来源,是典型的无机化能自养型微生物。菌株YS-1的最佳生长条件为:pH值2.0,摇床转速150rpm,接种量10%,温度35℃。值得注意的是该菌株最适生长温度比其标准株ATCC23270高5℃,因为金矿的生物预氧化过程会散发热量,所以菌株YS-1适合用于矿石的生物预氧化。2、碳离子和X射线诱变菌株YS-1及优良性能菌株选育通过不同剂量的碳离子和X射线辐照对数期菌液,统计其致死率。结果显示重离子辐照剂量是320Gy时,菌株的致死率达95%;在X射线辐照剂量为360Gy时致死率是82.78%。辐照之后,一方面通过耐温和耐砷驯化分别得到可以在39℃条件下和含砷量为90mmol/L的条件下生长的突变菌株;另一方面通过选育得到两株氧化活性提高的突变体,一株是在剂量为320Gy的碳离子辐照后筛选得到的,命名为SJ-1,其氧化速率提高非常显著,在第48h时就可以将9K培养基中的二价铁离子全部氧化,比原始菌株YS-1缩短12h。另一株是在剂量为360Gy的X射线诱变后筛选所得,命名为JW-1,其氧化活性略低于菌株SJ-1。对突变菌株SJ-1和JW-1进行传代培养,结果表明两者均遗传稳定。3、金矿的生物预氧化和化学浸出为了检测菌株的氧化活性对难处理金矿氧化效果的影响,将所有诱变前后的菌株用于对金矿进行生物预氧化。结果显示突变菌株SJ-1使矿石的铁浸出率比原始菌株YS-1增高28.12%,达到73.15%。而菌株JW-1使矿石的铁浸出率增加到66.19%,比原始菌株提高21.16%,表明氧化活性越高的菌株对难处理金矿的作用效果越显著。将生物氧化后的矿石用于化学浸出,结果表明经过菌株SJ-1氧化后的矿石其金浸出率高达91.73%,比直接氰化高43.87%;而经过菌株JW-1氧化的矿石其金浸出率达到89.89%,高于直接氰化时金浸出率42.03%。4、菌株二价铁氧化通路相关酶基因表达的研究通过实时定量PCR技术测定突变菌株SJ-1及JW-1和原始菌株YS-1的二价铁氧化通路相关基因在mRNA水平上的表达,发现碳离子和X射线辐照对菌株YS-1的铁氧化通路相关基因表达的影响极其显著。在突变菌株SJ-1和JW-1的各个生长阶段,几乎所有被检测基因的表达量都高于原始菌株YS-1,且菌株SJ-1的基因上调表达更加显著。该结果表明菌株氧化活性的提高和铁氧化通路关键基因表达密切相关。
[Abstract]:The reserves of the easy-to-treat gold deposits are decreasing year by year, and it is urgent to exploit the hard-to-treat gold deposits. The biological oxidation method has wide application prospect in the mining and smelting of the refractory gold ore because of the advantages of environment-friendly and low cost and the like. Acidithiobacillus ferrooxidans, as the main bacteria in the method, has the defects of weak oxidation capacity, no arsenic, and intolerance to various ions, and severely limits the industrialization process of biological oxidation, so the breeding of high oxidation activity and excellent performance strain has become the most important. by using carbon ion and X-ray irradiation to induce the Acidithiobacillus ferrooxidans and breeding to obtain the mutant strain with increased oxidation activity and stable inheritance, at the same time, the expression difference of the related enzyme gene of the iron oxide pathway of the original strain and the high-oxidation active mutant strain is detected by a molecular biological method, the molecular mechanism of the improvement of the oxidation activity of the bacteria is explored, Isolation and identification of wild-type strain of Acidithiobacillus ferrooxidans were obtained from the pit water of Yangshan gold mine to obtain a bacterium. The morphological characteristics, physiological and biochemical characteristics and molecular identification showed that it belonged to the Acidithiobacillus ferrooxidans, named YS-1. The bacterium has a length of about 1.2 to 1.5. m u.m and a width of about 0.4 to about 0.7. m u.m, and it is possible to oxidize and utilize a divalent iron and elemental sulfur or a sulfur compound as an energy source for its growth, and is a typical autotrophic microorganism. The optimal growth conditions of strain YS-1 were: pH 2.0, shaking speed 150 rpm, inoculum 10%, temperature 35 鈩，
本文编号：2443136
[Abstract]:The reserves of the easy-to-treat gold deposits are decreasing year by year, and it is urgent to exploit the hard-to-treat gold deposits. The biological oxidation method has wide application prospect in the mining and smelting of the refractory gold ore because of the advantages of environment-friendly and low cost and the like. Acidithiobacillus ferrooxidans, as the main bacteria in the method, has the defects of weak oxidation capacity, no arsenic, and intolerance to various ions, and severely limits the industrialization process of biological oxidation, so the breeding of high oxidation activity and excellent performance strain has become the most important. by using carbon ion and X-ray irradiation to induce the Acidithiobacillus ferrooxidans and breeding to obtain the mutant strain with increased oxidation activity and stable inheritance, at the same time, the expression difference of the related enzyme gene of the iron oxide pathway of the original strain and the high-oxidation active mutant strain is detected by a molecular biological method, the molecular mechanism of the improvement of the oxidation activity of the bacteria is explored, Isolation and identification of wild-type strain of Acidithiobacillus ferrooxidans were obtained from the pit water of Yangshan gold mine to obtain a bacterium. The morphological characteristics, physiological and biochemical characteristics and molecular identification showed that it belonged to the Acidithiobacillus ferrooxidans, named YS-1. The bacterium has a length of about 1.2 to 1.5. m u.m and a width of about 0.4 to about 0.7. m u.m, and it is possible to oxidize and utilize a divalent iron and elemental sulfur or a sulfur compound as an energy source for its growth, and is a typical autotrophic microorganism. The optimal growth conditions of strain YS-1 were: pH 2.0, shaking speed 150 rpm, inoculum 10%, temperature 35 鈩，
本文编号：2443136
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