Ferroplasma thermophilum L1的分离、鉴定及应用于黄铜矿浸出的研究
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摘要
生物冶金技术是一种从矿物中提取金属的方法,特别适于处理贫矿和废矿,并具有成本低、投入小、能耗低、环境污染小等突出优点。在本文研究中,一株极端嗜酸中等嗜热的氧化亚铁古菌得到分离纯化,并鉴定为Ferroplasma属的一个新种。为了研究该古菌在硫化矿浸出中的作用,从古菌的生理生化特性出发,研究了喜温硫杆菌杆菌(Acidithiobacillus caldus)、嗜铁钩端螺旋菌(Leptospirillumferriphilum)、嗜酸硫化杆菌(Sulfobacillus acidophilus)与该古菌的不同组合对黄铜矿的浸出,同时考察了浸出过程中pH、亚铁离子浓度、三价铁离子浓度和铜离子浓度等参数的变化,并应用Real-timePCR技术考察了微生物种群的变化,讨论了以上各菌株浸出黄铜矿的机理。
从浸矿反应器的浸出液中分离得到一株氧化亚铁古菌L1。对菌株L1进行了形态、生理生化特性研究。球状,不可运动,没有细胞壁,菌体直径大小0.4～1.0μm。最适生长温度在45℃左右,最适初始生长pH为1.0,代时约为6.65 h。可在硫酸亚铁和酵母粉同时存在的条件下兼性营养型生长,但是在只有硫酸亚铁或酵母粉的情况下该菌株不能生长。该菌株不能利用蛋白胨、葡萄糖、乳糖和半乳糖等有机物以及硫磺、硫代硫酸钠等还原态的硫进行生长。菌株L1基因组DNA G+C含量为34.1mol%。基于16S rRNA序列同源性构建了系统发育树,结果表明此株菌是属于Ferroplasma属的古菌,与F.cupricumulans BH2~T具有99.0%的序列相似性。菌株L1与F.cupricumulans BH2~T的分子杂交相似度为46.3%。基于以上实验结果表明菌株L1应代表Ferroplasma属的一个新种,命名为F.thermophilum L1(CCTCC AB207143~T)。
利用喜温硫杆菌杆菌、嗜铁钩端螺旋菌、嗜酸硫化杆菌和菌株L1四种菌的不同组合进行黄铜矿摇瓶浸出实验。化能自养和化能兼性生长的中等嗜热菌同时存在能有效促进黄铜矿的浸出速率和浸出效率。具有不同生理特性(包括铁硫转换和有机物与无机物的利用)的菌株之间的相互作用有利于促进黄铜矿的浸出。在含有嗜铁钩端螺旋菌的浸出体系中,黄铜矿浸出速率在浸出15-20天后开始下降,这与浸出过程中在矿物表面形成的黄钾铁矾钝化膜有关。各浸矿体系矿渣的X-衍射分析表明Fe~(3+)浓度较低的浸矿液中没有黄钾铁矾的形成,相对于pH,Fe~(3+)的浓度可能是黄钾铁矾形成的更重要原因。
利用实时荧光定量PCR技术对浸矿微生物种群的分析表明,在浸矿初期喜温硫杆菌杆菌和嗜铁钩端螺旋菌占较大的比例,但是到了浸矿后期嗜铁钩端螺旋菌在种群中的比例明显降低。F.thermophilum在浸矿前期占据很少的数量,而到浸矿后期在种群中占据较大比例。嗜酸硫化杆菌在种群中的比例没有太大变化。实验结果也是与它们的生理生化特性相适应的。
Bioleaching is an economical method for the recovery of metals from minerals, especially from low grade ores, overburden and waste from current mining operations, which requires moderate capital investment with low operating cost. Furthermore, bioleaching are environmentally friendly. In the present study, an extremely acidophilic and moderately thermophilic iron-oxidizing archaeon was isolated and characterized, and the strain represents a new species of genus Ferroplasma. To understand the role iron-oxidizing archaeon plays in mineral bioleaching, according to its physiological characteristics we focused on the effects of variously defined combinations of At. caldus, L. ferriphilum, S. acidophilus and F. thermophilum on copper dissolution from chalcopyrite. The variation of pH, ferrous iron, ferric iron and copper was also investigated. The real-time quantitative PCR was applied to investigate the population dynamics of moderate thermophiles during bioleaching and the bioleaching mechanisms of chalcopyrite by the above microorganisms were discussed.
A ferrous iron-oxidizing archaeon, named L1, was isolated from a chalcopyrite-leaching bioreactor. Strain L1 is a non-motile coccus that lacks cell wall. The diameter of strain L1 ranges from 0.4 to 1.0μm. Strain L1 has a temperature optimum of45℃and the optimum initial pH for growth is 1.0. The generation time of strain L1 is 6.65 h. Strain L1 is capable of growth on ferrous iron and yeast extract. No growth occurred when ferrous iron or yeast extract presented as the sole energy source. The strain can not grow on following organic substrates: peptone, glucose, lactose and galactose. Strain L1 is also not capable of using elemental sulfur, sodium thiosulfate as energy sources. The morphological, biochemical and physiological characterization were investigated. The G+C content of strain L1 is 34.1%. Analysis based on 16S rRNA gene sequence indicated that the strain L1 should be grouped in the genus Ferroplasma, and stain L1 is most closely related to F. cupricumulans BH2~T with 99% similarity in gene sequence. The DNA-DNA similarity hybridization between F. cupricumulans BH2~T and strain L1 was 46.3%. Based on above results, strain L1 should represent a new species of genus Ferroplasma and is proposed the name F. thermophilum L1 (CCTCC AB207143~T).
The bioleaching of chalcopyrite by variously defined combinations of At. caldus, L. ferriphilum, S. acidophilus and F. thermophilum was studied in shake flask cultures. The complex consortia containing both chemoautotrophic and chemomixotrophic moderate thermophiles were found to be the most efficient in all of those tested. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, involving transformations of iron and sulfur and transfer of organic and inorganic compound, were considered to play a critical role in optimizing chalcopyrite dissolution. The decrease of chalcopyrite dissolution rate in leaching systems containing L. ferriphilum after 15-20 days coincided with the formation of jarosite precipitation on the mineral surface during the bioleaching as a passivation layer. The results of XRD indicated that low concentration of ferric iron reduced jarosite formation and the high concentration of ferric iron might be a more important parameter for forming jarosite precipitation than pH during bioleaching process.
The results of real-time PCR analysis indicate At. caldus and L. ferriphilum were the dominant organisms initial during bioleaching. The proportion of L. ferriphilum in total prokaryotes decreased in the latter stages. F. thermophilum was detected the numerically dominant organism in the latter stages, though the proportion of F. thermophilum in total prokaryotes was small initially. The proportion of S. acidophilus did not change obvious during bioleaching of chalcopyrite. The analysis results were consistent with physiological characteristics of these strains.
从浸矿反应器的浸出液中分离得到一株氧化亚铁古菌L1。对菌株L1进行了形态、生理生化特性研究。球状,不可运动,没有细胞壁,菌体直径大小0.4～1.0μm。最适生长温度在45℃左右,最适初始生长pH为1.0,代时约为6.65 h。可在硫酸亚铁和酵母粉同时存在的条件下兼性营养型生长,但是在只有硫酸亚铁或酵母粉的情况下该菌株不能生长。该菌株不能利用蛋白胨、葡萄糖、乳糖和半乳糖等有机物以及硫磺、硫代硫酸钠等还原态的硫进行生长。菌株L1基因组DNA G+C含量为34.1mol%。基于16S rRNA序列同源性构建了系统发育树,结果表明此株菌是属于Ferroplasma属的古菌,与F.cupricumulans BH2~T具有99.0%的序列相似性。菌株L1与F.cupricumulans BH2~T的分子杂交相似度为46.3%。基于以上实验结果表明菌株L1应代表Ferroplasma属的一个新种,命名为F.thermophilum L1(CCTCC AB207143~T)。
利用喜温硫杆菌杆菌、嗜铁钩端螺旋菌、嗜酸硫化杆菌和菌株L1四种菌的不同组合进行黄铜矿摇瓶浸出实验。化能自养和化能兼性生长的中等嗜热菌同时存在能有效促进黄铜矿的浸出速率和浸出效率。具有不同生理特性(包括铁硫转换和有机物与无机物的利用)的菌株之间的相互作用有利于促进黄铜矿的浸出。在含有嗜铁钩端螺旋菌的浸出体系中,黄铜矿浸出速率在浸出15-20天后开始下降,这与浸出过程中在矿物表面形成的黄钾铁矾钝化膜有关。各浸矿体系矿渣的X-衍射分析表明Fe~(3+)浓度较低的浸矿液中没有黄钾铁矾的形成,相对于pH,Fe~(3+)的浓度可能是黄钾铁矾形成的更重要原因。
利用实时荧光定量PCR技术对浸矿微生物种群的分析表明,在浸矿初期喜温硫杆菌杆菌和嗜铁钩端螺旋菌占较大的比例,但是到了浸矿后期嗜铁钩端螺旋菌在种群中的比例明显降低。F.thermophilum在浸矿前期占据很少的数量,而到浸矿后期在种群中占据较大比例。嗜酸硫化杆菌在种群中的比例没有太大变化。实验结果也是与它们的生理生化特性相适应的。
Bioleaching is an economical method for the recovery of metals from minerals, especially from low grade ores, overburden and waste from current mining operations, which requires moderate capital investment with low operating cost. Furthermore, bioleaching are environmentally friendly. In the present study, an extremely acidophilic and moderately thermophilic iron-oxidizing archaeon was isolated and characterized, and the strain represents a new species of genus Ferroplasma. To understand the role iron-oxidizing archaeon plays in mineral bioleaching, according to its physiological characteristics we focused on the effects of variously defined combinations of At. caldus, L. ferriphilum, S. acidophilus and F. thermophilum on copper dissolution from chalcopyrite. The variation of pH, ferrous iron, ferric iron and copper was also investigated. The real-time quantitative PCR was applied to investigate the population dynamics of moderate thermophiles during bioleaching and the bioleaching mechanisms of chalcopyrite by the above microorganisms were discussed.
A ferrous iron-oxidizing archaeon, named L1, was isolated from a chalcopyrite-leaching bioreactor. Strain L1 is a non-motile coccus that lacks cell wall. The diameter of strain L1 ranges from 0.4 to 1.0μm. Strain L1 has a temperature optimum of45℃and the optimum initial pH for growth is 1.0. The generation time of strain L1 is 6.65 h. Strain L1 is capable of growth on ferrous iron and yeast extract. No growth occurred when ferrous iron or yeast extract presented as the sole energy source. The strain can not grow on following organic substrates: peptone, glucose, lactose and galactose. Strain L1 is also not capable of using elemental sulfur, sodium thiosulfate as energy sources. The morphological, biochemical and physiological characterization were investigated. The G+C content of strain L1 is 34.1%. Analysis based on 16S rRNA gene sequence indicated that the strain L1 should be grouped in the genus Ferroplasma, and stain L1 is most closely related to F. cupricumulans BH2~T with 99% similarity in gene sequence. The DNA-DNA similarity hybridization between F. cupricumulans BH2~T and strain L1 was 46.3%. Based on above results, strain L1 should represent a new species of genus Ferroplasma and is proposed the name F. thermophilum L1 (CCTCC AB207143~T).
The bioleaching of chalcopyrite by variously defined combinations of At. caldus, L. ferriphilum, S. acidophilus and F. thermophilum was studied in shake flask cultures. The complex consortia containing both chemoautotrophic and chemomixotrophic moderate thermophiles were found to be the most efficient in all of those tested. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, involving transformations of iron and sulfur and transfer of organic and inorganic compound, were considered to play a critical role in optimizing chalcopyrite dissolution. The decrease of chalcopyrite dissolution rate in leaching systems containing L. ferriphilum after 15-20 days coincided with the formation of jarosite precipitation on the mineral surface during the bioleaching as a passivation layer. The results of XRD indicated that low concentration of ferric iron reduced jarosite formation and the high concentration of ferric iron might be a more important parameter for forming jarosite precipitation than pH during bioleaching process.
The results of real-time PCR analysis indicate At. caldus and L. ferriphilum were the dominant organisms initial during bioleaching. The proportion of L. ferriphilum in total prokaryotes decreased in the latter stages. F. thermophilum was detected the numerically dominant organism in the latter stages, though the proportion of F. thermophilum in total prokaryotes was small initially. The proportion of S. acidophilus did not change obvious during bioleaching of chalcopyrite. The analysis results were consistent with physiological characteristics of these strains.
引文
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