中等嗜热菌浸出黄铜矿的研究
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摘要
生物冶金技术由于成本低、无污染、操作简单而日益受到人们的重视,尤其适用于我国矿产资源品位低、成分复杂的现实情况。中等嗜热菌在矿物浸出的应用中,由于减少了工业反应器的冷却设备,提供了更多的优越性,具有极大的应用前景。尽管中等嗜热菌浸出铜硫化矿在工业应用上面取得了一定的进展,但其微生物学机理研究还不够深入,铜的浸出效率还有待提高。本文从中国的多个铜矿区筛选出能高效浸出黄铜矿的中等嗜热富集物和纯菌,分析富集物的群落组成,考察该富集物在浸矿反应器中浸出黄铜矿的浸矿参数变化,研究反应器中微生物的群落结构和种群动态规律。通过人为构建浸矿微生物群落,考察构建群落的浸矿性能和微生物群落的变化。研究工作加深了对中等嗜热微生物浸矿特性的了解,为处理不同特性矿物的菌种群落的构建提供了依据,并为中等嗜热菌处理黄铜矿的工业应用提供了可供借鉴的数据。
本文的具体研究内容及研究结果概括如下：
一、中等嗜热富集物的筛选及其群落解析
1.中高温酸性条件下,以黄铁矿、黄铜矿、硫酸亚铁和硫粉混合物为主要能源物质的培养体系在稳定浸出时期,Sulfobacillus thermotolerans, Acidithiobacillus caldes和Leptospirillum ferriphilum 3种细菌均占有较高丰度。
2.不同能源对浸矿微生物的群落组成有显著影响。以黄铁矿为主要能源物质的培养体系中L. ferriphilum所占比例最高。在以硫酸亚铁和硫粉混合物为能源的培养体系中,L. ferriphilum所占比例均在50%左右,前一种培养体系中S. thermotolerans和At. caldus比例接近1:1,后一种培养体系中S. thermotolerans和At. caldus的比例接近4:1。黄铜矿中等高温浸出体系中古菌群落中绝大多数属于Ferroplasma属古菌。
二、中等嗜热纯菌的分离及其鉴定
1.从江西德兴铜矿废弃的酸性矿坑水中分离到一株中等嗜热嗜酸铁氧化菌ZW-1菌株为革兰氏阳性菌,长杆状,两端钝圆,菌体大小(0.8-1.5)μm×0.4μm,最适生长温度为48℃,最适生长pH值为1.9。可利用亚铁、单质硫自养生长和酵母粉、蛋白胨异养生长,不能利用葡萄糖。以16S rRNA序列的同源性为基础构建的系统发育树分析表明ZW-1菌株与Sulfobacillus acidophilus处于同一进化分支上,相似性达99.0%以上。该菌对Fe2+具有较高的氧化能力,最高达到0.295g/L·h,且能耐受较高浓度的Fe3+和Cu2+,分别为25g/L和35 g/L。采用ZW-1菌单独浸出黄铜矿(12 g/L矿浆浓度),20天后铜的浸出率46.4%。
2.从中国的四个典型的酸性环境中分离到的六株中等嗜热的硫氧化菌具有相似的形态及生理生化特性,最适生长温度为45-50℃,最适合生长pH为2.5-3.5。可以利用元素硫,硫代硫酸钠和连四硫酸钾为能源进行自养生长,添加少量葡萄糖可以刺激它们的生长。系统发育分析的结果表明六株细菌菌与Genbank上登陆的Acidithiobacillus caldus菌株的同源性非常高,均在99%以上,可初步鉴定为Acidithiobacillus sp.。
三、中等嗜热富集物浸出黄铜矿的研究
1.从中国多个黄铜矿酸性矿坑水富集到的中等嗜热浸矿微生物具有较强的黄铜矿浸出能力。经驯化,混合菌耐受矿浆浓度从10 g/L提高到50 g/L。在浸矿液中加入0.4 g/L的酵母粉,转速为180 rpm的条件下,中等嗜热混合驯化菌可浸出黄铜矿精矿中74%的铜。通过反应器搅拌浸出实验,该混合菌浸出黄铜矿的效果有了进一步提升。50 g/L的矿浆浓度下,搅拌浸出20天后,铜的浸出率为81%。在小规模柱浸反应器中,该中等嗜热浸矿富集微生物对于低品位黄铜矿具有一定的浸出能力,60天的浸出时间黄铜矿浸出率为40.8%。
2.在60 d柱浸的前、中、后三个阶段中,存在相类似的细菌种群类型,主要包括：L.ferriphilum, S. thermotolerans以及At. caldus。在反应器运行初期(20 d), L.ferriphilum在黄铜矿浸出体系中占有很高丰度(81%),随着浸矿的进行,L.ferriphilum丰度逐渐降低,在浸矿60 d时,L.ferriphilum所占比例降低到13%；前期S. thermotolerans的丰度为16%,浸矿末期(60 d), S. thermotolerans在总的微生物群落中所占比例显著上升,达到79%。
四、人工构建中等嗜热微生物群落浸出黄铜矿的研究
1.通过将分离纯化的中等嗜热菌混合可以构建浸矿能力强的微生物群落。中等嗜热混合菌At. caldus S2、L. ferriphilum YSK、F. thermophilum L1和Sulfobacillus sp.ZW-1的组合对含有大量碱性脉石、难处理的黄铜矿具有较好浸出效果。
2.当浸矿体系中同时含有自养(At. caldus S2和L. ferriphilumYSK)和兼性营养(F. thermophilum L1和Sulfobacillus sp.ZW-1)微生物时,它们之间表现出协同作用。生物冶金环境中微生物之间的协同作用包括铁、硫的转化,有机物的消除等,对黄铜矿的溶解起着重要作用。这为构建高效浸矿微生物菌群或者调控浸矿过程微生物的种群动态提供了重要依据。
为了提高生物浸矿效率,降低提取成本,发展和推广中等高温生物浸出工艺,应该从菌种选育、群落特征与浸矿效率关系、浸矿体系与过程优化控制以及浸矿添加剂等方面开展广泛深入研究。
Bioleaching is paid the increasing attention due to low cost, no pollution and simple operation, especially for low-grade and complex composition of mineral resources in China. Moderate thermophiles have great application prospect in the leaching of minerals and provide more advantages owing to reduced cooling equipment for industrial reactor. Despite some progress has been made in the industrial application of copper sulfide ores leaching by moderate thermophiles, but mechanism of microbiologies is not enough and the leaching efficiency of copper need to be improved. In this paper, moderate thermophilic isolates and enrichment with a high capacity of leaching chalcopyrite were obtained from a number of copper areas in China; the community composition of the enrichment was analysed; chalcopyrite leaching parameters by the enrichment in the reactor were investigated; microbial community structure and population dynamics in the reactor were studied. By artificially building bioleaching microbial communities, the bioleaching performance and microbial community varieties were surveyed. The research work enhances the understanding of leaching characteristics of moderate thermophiles and provides reference data and experience for industrial applications.
The specific research contents and results are summarized as follows:
1 Moderate thermophilic enrichment and community analysis
1.1 In this paper, under the moderate high temperature and acidic conditions, with addition of the pyrite, chalcopyrite, a mixture of ferrous sulfate and elemental sulfur as the main energy sources, moderate thermophilic microbial community structure in the stable phase was investigated by PCR-RFLP method, and community compositions of three culture systems were compared. Results showed that Sulfobacillus thermotolerans, Acidithiobacillus caldus and Leptospirillum ferriphilum had a high abundance in the three culture systems.
1.2 Different energy sources had a significant effect on microbial community compositions. In the culture system with pyrite as the main energy source, L. ferriphilum accounted for the highest proportion. In both culture systems culture system with a mixture of ferrous sulfate and elemental sulfur as energy sources, L ferriphilum accounted for about 50%, and differences in the proportion of bacteria were mainly the varieties of S. thermotolerans and A. caldus. In the former culture system the ratio of S. thermotolerans and A. caldus was about 1:1. And in the latter, the ratio of S. thermotolerans, and A. caldus was nearly 4:1. Most members of archaeal community in moderate thermophilic chalcopyrite leaching system were related to the genena of Ferroplasma.
2 Isolation and identification of moderate thermophilic bacteria
2.1 Moderately thermo-acidophilic and iron-oxiding strains ZW-1 was isolated from acid mine drainage in Dexing Mine, Jiangxi Province and its morphological, physiological and biochemical characteristics,16S rRNA sequences and leaching properties were studied. Results revealed that the strain was G- and rod-shaped, size (0.8-1.5)μm×0.4μm and with optimum temperatures and pH of 48℃and 1.9. The strain grew autotrophically by using ferrous and elemental sulfur as energy sources and heterotrophically by using yeast powder and peptone, but it can not use glucose. Phylogenetic tree based on 16S rRNA gene sequences revealed that the strain ZW-1 and Sulfobacillus acidophilus were at the same evolutionary branch with a similarity of 99.0%. Leaching performance studies showed that the strain ZW-1 had a high oxidation capacity of Fe2+ up to 0.295 g/L-h, and could tolerate a high concentration of 25 g/L Fe3+ and 35 g/L Cu2+. When leaching chalcopyrite (12 g/L pulp density), percentage copper released by the strain ZW-1 increased to 46.4% after 20 days.
2.2 Six moderate-thermophilic sulfur-oxidizing strains with similar morphological, physiological and biochemical characteristics were isolated from the four typical acidic environments in China. Their optimum growth temperatures and pH were 45-50℃and 2.5-3.5, respectively. They can grow autotrophically by using elemental sulfur, sodium thiosulfate and potassium tetrathionate as energy sources and trace glucose can stimulate their growth. Phylogenetic analysis showed that all six bacteria had high homology with Acidithiobacillus caldus in Genbank (more than 99%), and were initially identified as Acidithiobacillus sp.。
3 Bioleaching of chalcopyrite with moderate thermophilic enrichment
3.1 Moderate thermophilic microorganisms with strong chalcopyrite leaching capability were enriched from several acid mine drainage in China. By domestication, tolerance of pulp concentration increased from 10 g/L to 50 g/L. Under the conditions of 0.4 g/L yeast powder and 180rpm rotational speed, moderate thermophilic enrichment can leach 74% copper of chalcopyrite. Through agitation leaching experiments in the reactor, leaching of chalcopyrite by the consortia was further improved. When leaching chalcopyrite, the leaching rate of chalcopyrite in 50 g/L pulp density was 81% after 8 days. Acid consumption experiments showed that it was necessary to the high leaching of chalcopyrite by adding a large number of acid during leaching. In the small-scale column leaching reactor, the moderate thermophilic enrichment has a certain ability to low-grade chalcopyrite leaching, and the leaching rate of chalcopyrite was 40.8% after 60 days.
3.2 In the pre-mid-late three stages of 60d leaching, there were similar bacterial populations including:L. ferriphilum, S. thermotolerans and At. caldus. In the initial stages (20 d), L. ferriphilum had a high proportion of 81% in the leaching system of chalcopyrite. With leaching, the proportion of L. ferriphilum decreased gradually. In the 60 d, L. ferriphilum decreased to 13%. In the initial stages, S. thermotoleran had the proportion of 16%. In the late stages of leaching (60 d), the proportion of S. thermotolerans increased to 79%.
4 Bioleaching of chalcopyrite by "artificial design" of consortia of moderate thermophiles
4.1 We utilized the isolated microorganisms to construct effective moderate thermophilic consortia. The consortia including At. caldus S2, L. ferriphilum YSK and Sulfobacillus sp.TPY had higher leaching efficiency when processing the recalcitrant chalcopyrite containing basic gangue.
4.2 It had also shown that consortia containing autotrophic and heterotrophic acidophiles tend to show cooperation between different groups. Moderately thermophilic F. thermophilum and Sufobacillus sp. may enhance mineral oxidation by removing soluble organic materials that might inhibit some chemolithotrophic bacteria. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, including transformations of iron and sulfur and transfer of organic compound, were considered to play a critical role in promoting chalcopyrite dissolution. The results provided the information to the design of effective moderate thermophilic acidophiles consortia.
In order to enhance the dissolution rate, reduce cost, develop and popularize the moderate thermophilic bioleaching technology, we should carry out these studies on strain screening, correlation on community structure and leaching efficiency, leaching systems and process optimation and additives.
本文的具体研究内容及研究结果概括如下：
一、中等嗜热富集物的筛选及其群落解析
1.中高温酸性条件下,以黄铁矿、黄铜矿、硫酸亚铁和硫粉混合物为主要能源物质的培养体系在稳定浸出时期,Sulfobacillus thermotolerans, Acidithiobacillus caldes和Leptospirillum ferriphilum 3种细菌均占有较高丰度。
2.不同能源对浸矿微生物的群落组成有显著影响。以黄铁矿为主要能源物质的培养体系中L. ferriphilum所占比例最高。在以硫酸亚铁和硫粉混合物为能源的培养体系中,L. ferriphilum所占比例均在50%左右,前一种培养体系中S. thermotolerans和At. caldus比例接近1:1,后一种培养体系中S. thermotolerans和At. caldus的比例接近4:1。黄铜矿中等高温浸出体系中古菌群落中绝大多数属于Ferroplasma属古菌。
二、中等嗜热纯菌的分离及其鉴定
1.从江西德兴铜矿废弃的酸性矿坑水中分离到一株中等嗜热嗜酸铁氧化菌ZW-1菌株为革兰氏阳性菌,长杆状,两端钝圆,菌体大小(0.8-1.5)μm×0.4μm,最适生长温度为48℃,最适生长pH值为1.9。可利用亚铁、单质硫自养生长和酵母粉、蛋白胨异养生长,不能利用葡萄糖。以16S rRNA序列的同源性为基础构建的系统发育树分析表明ZW-1菌株与Sulfobacillus acidophilus处于同一进化分支上,相似性达99.0%以上。该菌对Fe2+具有较高的氧化能力,最高达到0.295g/L·h,且能耐受较高浓度的Fe3+和Cu2+,分别为25g/L和35 g/L。采用ZW-1菌单独浸出黄铜矿(12 g/L矿浆浓度),20天后铜的浸出率46.4%。
2.从中国的四个典型的酸性环境中分离到的六株中等嗜热的硫氧化菌具有相似的形态及生理生化特性,最适生长温度为45-50℃,最适合生长pH为2.5-3.5。可以利用元素硫,硫代硫酸钠和连四硫酸钾为能源进行自养生长,添加少量葡萄糖可以刺激它们的生长。系统发育分析的结果表明六株细菌菌与Genbank上登陆的Acidithiobacillus caldus菌株的同源性非常高,均在99%以上,可初步鉴定为Acidithiobacillus sp.。
三、中等嗜热富集物浸出黄铜矿的研究
1.从中国多个黄铜矿酸性矿坑水富集到的中等嗜热浸矿微生物具有较强的黄铜矿浸出能力。经驯化,混合菌耐受矿浆浓度从10 g/L提高到50 g/L。在浸矿液中加入0.4 g/L的酵母粉,转速为180 rpm的条件下,中等嗜热混合驯化菌可浸出黄铜矿精矿中74%的铜。通过反应器搅拌浸出实验,该混合菌浸出黄铜矿的效果有了进一步提升。50 g/L的矿浆浓度下,搅拌浸出20天后,铜的浸出率为81%。在小规模柱浸反应器中,该中等嗜热浸矿富集微生物对于低品位黄铜矿具有一定的浸出能力,60天的浸出时间黄铜矿浸出率为40.8%。
2.在60 d柱浸的前、中、后三个阶段中,存在相类似的细菌种群类型,主要包括：L.ferriphilum, S. thermotolerans以及At. caldus。在反应器运行初期(20 d), L.ferriphilum在黄铜矿浸出体系中占有很高丰度(81%),随着浸矿的进行,L.ferriphilum丰度逐渐降低,在浸矿60 d时,L.ferriphilum所占比例降低到13%；前期S. thermotolerans的丰度为16%,浸矿末期(60 d), S. thermotolerans在总的微生物群落中所占比例显著上升,达到79%。
四、人工构建中等嗜热微生物群落浸出黄铜矿的研究
1.通过将分离纯化的中等嗜热菌混合可以构建浸矿能力强的微生物群落。中等嗜热混合菌At. caldus S2、L. ferriphilum YSK、F. thermophilum L1和Sulfobacillus sp.ZW-1的组合对含有大量碱性脉石、难处理的黄铜矿具有较好浸出效果。
2.当浸矿体系中同时含有自养(At. caldus S2和L. ferriphilumYSK)和兼性营养(F. thermophilum L1和Sulfobacillus sp.ZW-1)微生物时,它们之间表现出协同作用。生物冶金环境中微生物之间的协同作用包括铁、硫的转化,有机物的消除等,对黄铜矿的溶解起着重要作用。这为构建高效浸矿微生物菌群或者调控浸矿过程微生物的种群动态提供了重要依据。
为了提高生物浸矿效率,降低提取成本,发展和推广中等高温生物浸出工艺,应该从菌种选育、群落特征与浸矿效率关系、浸矿体系与过程优化控制以及浸矿添加剂等方面开展广泛深入研究。
Bioleaching is paid the increasing attention due to low cost, no pollution and simple operation, especially for low-grade and complex composition of mineral resources in China. Moderate thermophiles have great application prospect in the leaching of minerals and provide more advantages owing to reduced cooling equipment for industrial reactor. Despite some progress has been made in the industrial application of copper sulfide ores leaching by moderate thermophiles, but mechanism of microbiologies is not enough and the leaching efficiency of copper need to be improved. In this paper, moderate thermophilic isolates and enrichment with a high capacity of leaching chalcopyrite were obtained from a number of copper areas in China; the community composition of the enrichment was analysed; chalcopyrite leaching parameters by the enrichment in the reactor were investigated; microbial community structure and population dynamics in the reactor were studied. By artificially building bioleaching microbial communities, the bioleaching performance and microbial community varieties were surveyed. The research work enhances the understanding of leaching characteristics of moderate thermophiles and provides reference data and experience for industrial applications.
The specific research contents and results are summarized as follows:
1 Moderate thermophilic enrichment and community analysis
1.1 In this paper, under the moderate high temperature and acidic conditions, with addition of the pyrite, chalcopyrite, a mixture of ferrous sulfate and elemental sulfur as the main energy sources, moderate thermophilic microbial community structure in the stable phase was investigated by PCR-RFLP method, and community compositions of three culture systems were compared. Results showed that Sulfobacillus thermotolerans, Acidithiobacillus caldus and Leptospirillum ferriphilum had a high abundance in the three culture systems.
1.2 Different energy sources had a significant effect on microbial community compositions. In the culture system with pyrite as the main energy source, L. ferriphilum accounted for the highest proportion. In both culture systems culture system with a mixture of ferrous sulfate and elemental sulfur as energy sources, L ferriphilum accounted for about 50%, and differences in the proportion of bacteria were mainly the varieties of S. thermotolerans and A. caldus. In the former culture system the ratio of S. thermotolerans and A. caldus was about 1:1. And in the latter, the ratio of S. thermotolerans, and A. caldus was nearly 4:1. Most members of archaeal community in moderate thermophilic chalcopyrite leaching system were related to the genena of Ferroplasma.
2 Isolation and identification of moderate thermophilic bacteria
2.1 Moderately thermo-acidophilic and iron-oxiding strains ZW-1 was isolated from acid mine drainage in Dexing Mine, Jiangxi Province and its morphological, physiological and biochemical characteristics,16S rRNA sequences and leaching properties were studied. Results revealed that the strain was G- and rod-shaped, size (0.8-1.5)μm×0.4μm and with optimum temperatures and pH of 48℃and 1.9. The strain grew autotrophically by using ferrous and elemental sulfur as energy sources and heterotrophically by using yeast powder and peptone, but it can not use glucose. Phylogenetic tree based on 16S rRNA gene sequences revealed that the strain ZW-1 and Sulfobacillus acidophilus were at the same evolutionary branch with a similarity of 99.0%. Leaching performance studies showed that the strain ZW-1 had a high oxidation capacity of Fe2+ up to 0.295 g/L-h, and could tolerate a high concentration of 25 g/L Fe3+ and 35 g/L Cu2+. When leaching chalcopyrite (12 g/L pulp density), percentage copper released by the strain ZW-1 increased to 46.4% after 20 days.
2.2 Six moderate-thermophilic sulfur-oxidizing strains with similar morphological, physiological and biochemical characteristics were isolated from the four typical acidic environments in China. Their optimum growth temperatures and pH were 45-50℃and 2.5-3.5, respectively. They can grow autotrophically by using elemental sulfur, sodium thiosulfate and potassium tetrathionate as energy sources and trace glucose can stimulate their growth. Phylogenetic analysis showed that all six bacteria had high homology with Acidithiobacillus caldus in Genbank (more than 99%), and were initially identified as Acidithiobacillus sp.。
3 Bioleaching of chalcopyrite with moderate thermophilic enrichment
3.1 Moderate thermophilic microorganisms with strong chalcopyrite leaching capability were enriched from several acid mine drainage in China. By domestication, tolerance of pulp concentration increased from 10 g/L to 50 g/L. Under the conditions of 0.4 g/L yeast powder and 180rpm rotational speed, moderate thermophilic enrichment can leach 74% copper of chalcopyrite. Through agitation leaching experiments in the reactor, leaching of chalcopyrite by the consortia was further improved. When leaching chalcopyrite, the leaching rate of chalcopyrite in 50 g/L pulp density was 81% after 8 days. Acid consumption experiments showed that it was necessary to the high leaching of chalcopyrite by adding a large number of acid during leaching. In the small-scale column leaching reactor, the moderate thermophilic enrichment has a certain ability to low-grade chalcopyrite leaching, and the leaching rate of chalcopyrite was 40.8% after 60 days.
3.2 In the pre-mid-late three stages of 60d leaching, there were similar bacterial populations including:L. ferriphilum, S. thermotolerans and At. caldus. In the initial stages (20 d), L. ferriphilum had a high proportion of 81% in the leaching system of chalcopyrite. With leaching, the proportion of L. ferriphilum decreased gradually. In the 60 d, L. ferriphilum decreased to 13%. In the initial stages, S. thermotoleran had the proportion of 16%. In the late stages of leaching (60 d), the proportion of S. thermotolerans increased to 79%.
4 Bioleaching of chalcopyrite by "artificial design" of consortia of moderate thermophiles
4.1 We utilized the isolated microorganisms to construct effective moderate thermophilic consortia. The consortia including At. caldus S2, L. ferriphilum YSK and Sulfobacillus sp.TPY had higher leaching efficiency when processing the recalcitrant chalcopyrite containing basic gangue.
4.2 It had also shown that consortia containing autotrophic and heterotrophic acidophiles tend to show cooperation between different groups. Moderately thermophilic F. thermophilum and Sufobacillus sp. may enhance mineral oxidation by removing soluble organic materials that might inhibit some chemolithotrophic bacteria. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, including transformations of iron and sulfur and transfer of organic compound, were considered to play a critical role in promoting chalcopyrite dissolution. The results provided the information to the design of effective moderate thermophilic acidophiles consortia.
In order to enhance the dissolution rate, reduce cost, develop and popularize the moderate thermophilic bioleaching technology, we should carry out these studies on strain screening, correlation on community structure and leaching efficiency, leaching systems and process optimation and additives.
引文
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