诱变前后混合微生物对铜、锌硫化矿浸出能力比较及其纯种分离研究
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摘要
微生物浸矿是多种微生物共同作用的复杂生化过程。大部分浸矿微生物是化能自氧型,生长速度慢,浸矿效率低。如何提高它们对金属离子的浸出效率是工业应用的难题。目前,普遍的方法是从浸矿混合微生物中分离纯培养,对其诱变处理。但在工业生产中常常应用混合微生物,其中很大一部分的微生物得不到纯培养,因此单一菌种诱变方法来选育浸矿微生物有明显的局限性。
本研究采用几种理化诱变剂及其组合,对从江西、云南、广东和台湾等不同地点采集的浸矿混合微生物进行单一或复合诱变,以获得生长快和浸矿效率高的混合培养物。同时,对诱变后的混合微生物进行了菌株分离与纯化、16S rDNA鉴定和微生物群落的生态分析。结果如下:
通过混合微生物诱变和多次浸黄铜矿精矿实验,筛选得到了浸出Cu~(2+)能力较强的混合微生物A、B、C、D和E。
①在加入矿浆浓度为1%黄铜矿精矿的9K培养基中培养40 d,混合微生物A、B、C、D和E浸出的Cu~(2+)浓度比对照分别增加61.40%、125.57%、1.45%、58.98%和149.98%,浸出率分别提高17.56%、35.90%、0.41%、16.87%和42.88%。说明混合培养物诱变可提高微生物浸黄铜矿精矿的活性。
②在加入矿浆浓度为10%黄铜矿精矿的9K培养基中培养40 d,混合微生物A、B、C、D和E浸出的Cu~(2+)浓度比对照分别增加49.46%、71.68%、22.82%、44.85%和97.57%,浸出率分别提高3.65%、5.29%、1.69%、3.31%和7.20%。说明混合培养物诱变可提高微生物浸黄铜矿精矿的活性。
③在加入矿浆浓度为20%的黄铜矿精矿培养基中培养40 d,在9K和leathen培养基中,混合微生物E浸出Cu~(2+)浓度比对照分别增加87.22%和79.24%,浸出率分别提高3.60%和3.59%,表明混合微生物E的浸矿性能稳定。
④Cu~(2+)耐受实验表明,混合微生物E耐受Cu~(2+)能力明显提高,提示其具有工业应用的潜力。
⑤混合微生物A、B、C、D和E在9K培养基上生长到稳定期,它们的生长速度比未诱变的混合微生物快1-2d。混合微生物在含黄铜矿精矿的培养基中保存比在不加矿的培养基中保存,其生长到稳定期快1-2 d左右,表明加入选择性压力可以保持浸矿微生物的生长活性。
从不同地点采集的微生物,经过黄铜矿和闪锌矿的长期驯化后,通过混合微生物的复合诱变,得到浸出闪锌矿精矿能力强的混合微生物B、D和E。
①在加入矿浆浓度为1%闪锌矿精矿的9K培养基中培养40 d,混合微生物A、B、C、D和E浸出的Zn~(2+)浓度比对照分别增加8.06%、85.97%、-2.39%、77.61%和117.01%;浸出率分别提高1.54%、16.43%、-0.46%、14.83%和22.37%。
②在加入矿浆浓度为10%闪锌矿精矿的9K培养基中培养40 d,混合微生物B、D和E浸出Zn~(2+)浓度比对照分别增加49.14%、44.86%和54.77%;浸出率分别提高2.27%、2.07%和2.53%。
③在加入矿浆浓度为20%闪锌矿精矿的9K培养基中培养40 d,混合微生物B、D和E在9K培养基中培养,Zn~(2+)浸出率分别提高2.10%、1.59%和2.24%;在leathen培养基中,Zn~(2+)浸出率分别提高2.03%、1.89%和2.56%
④B混合微生物适合在矿浆浓度4%-16%下生长,E混合微生物适合在4%-32%矿浆浓度下生长,说明混合微生物B、E耐受高矿浆浓度,有良好的工业应用前景。
比较了诱变前后四个不同样品中微生物群落的多样性。结果表明,四个样品中微生物群落结构可以分为嗜铁钩端螺旋菌组、嗜酸氧化亚铁硫杆菌组和嗜酸氧化硫硫杆菌和喜温硫杆菌三大组。尽管随着培养条件的改变,样品中微生物的群落结构存在一定的变化,但在本实验条件下,微生物浸出体系中最主要的优势菌种仍然是嗜酸氧化亚铁硫杆菌和嗜酸氧化硫硫杆菌。
诱变处理使混合微生物中的嗜酸氧化硫硫杆菌(2~#菌株)由原来的次要菌株变成主要菌株,在含黄铜矿和闪锌矿培养基中分别占微生物总数的69%和57%,可能在微生物浸矿中起主要作用。
诱变前混合微生物中嗜酸氧化亚铁硫杆菌(1~#菌株)生长量最少,诱变处理显著提高了它在微生物群落中的数量。在含黄铜矿精矿和闪锌矿精矿培养基中培养时,它分别占微生物总数的20%和29%,可能在微生物浸矿中起重要作用。
在混合微生物样品中,进行微生物单菌株分离实验:
①分离出一株最适生长温度40℃,最适生长起始pH值为1.5的螺旋菌,16S rDNA序列分析表明,该菌为Leptospirillum ferriphilum。
②分离出一株最适生长温度45℃,最适生长起始pH值为1.8的杆菌,16S rDNA序列分析表明,该菌为Acidithiobacillus caldus。
③分离出一株最适生长温度53℃,最适生长起始pH值为2.0的杆菌,16S rDNA序列分析表明,该菌为Sulfobacillus thermosulfidooxidans。
④分离出K1和K2菌株,经初步鉴定分别为嗜酸氧化亚铁硫杆菌和嗜酸氧化硫硫杆菌。
Bioleaching is a complicated biochemical process in which a number of microbial genera are involved.Most of bioleaching microorganisms are chemoautotrophic.They grow slowly and have low efficiency in metal -leaching.How to improve the leaching efficiency is a problem of industrial applications.Up to date,most popular strategy is mutation of single microorganism isolated from mixed microorganisms of bioleaching.However,microorganisms for bioleaching industry are always in mixed form,and mostly,their pure culture may not be obtained. Therefore,there are limitations on mutation breed of single microorganism.
In present study,microbial samples were collected from acid mine drainage and spring of Jiangxi,Yunnan,Guangdong and Taiwan province, mixed and enriched.To obtain higher growth rate and higher leaching efficiency-mixed cultures,the mixed microorganisms were mutated using several physical and chemical mutagens and their combinations. Furthermore,single strain isolation,characterization of 16S rDNA,and ecological analysis were performed for the screened mixed cultures.The results are as follows:
The mixed cultures with higher concentrations of leaching Cu~(2+), designated A,B,C,D and E,were screened after mutation and leaching tests of chalcopyrite.
①The concentrations of leaching Cu~(2+)for mixed culture A,B,C, D and E were increased by 61.40%,125.57%,1.45%,58.98%and 149.98%and the leaching rates of Cu~(2+)in the cultures were correspondingly 17.56%,35.90%,0.41%,16.87%and 42.88%higher compared with the control,40 d after leaching in 9K medium containing 1%chalcopyrite pulp.The results indicate that Cu~(2+)leaching activities of mixed organisms can be enhanced by physical and chemical mutations.
②The amount of leaching Cu~(2+)for mixed culture A,B,C,D and E was increased by 49.46%,71.68%,22.82%,44.85%and 97.57%, respectively,with 3.65%,5.29%,1.69%,3.31%and 7.20%higher than the control in Cu~(2+)leaching rates,40 d after leaching in 9K medium each containing 10%Chalcopyrite pulp,again indicating that physical and chemical mutations may improve Cu~(2+)leaching activities of mixed organisms.
③When added with 20%chalcopyrite pulp and inoculated in 9K and Leathen mediums for 40 d,mixed culture E extracted 87.22%and 79.24%more Cu~(2+)from chalcopyrite and had 3.6%and 3.59 higher Cu~(2+) leaching rates than the unmutated control,suggesting the acquired higher characteristics of Cu~(2+)-leaching is stable.
④Mixed culture E was more resistant to Cu~(2+)than the unmutated control,suggesting that it possesses potentials for industrial applications.
⑤The growth of mixed culture A,B,C,D and E in 9k medium was 1-2 d higher than that of the unmutated control and the mixed cultures conserved on chalcopyrite grew 1-2 d faster than those not conserved on chalcopyrite.These results indicate that growth activity of leaching microorganisms can be kept by selective stress.
After three rounds of leaching tests on sphalerite,mixed cultures B, D and E with higher efficiency of Zn~(2+)-leaching were obtained from mixed microorganisms A,B,C,D and E mentioned above.
①Compared with the unmutated control,the concentrations of leaching Zn~(2+)in 9K medium added with 1%sphalerite pulp for mixed cultures A,B,C,D and E were increased by 8.06%、85.97%、-2.39%、77.61%and 117.01%,respectively,and the leaching rates of Zn~(2+)in the cultures were correspondingly 1.54%、16.43%、-0.46%、14.83%and 22.37%,indicating that mutation against mixed microorganisms may improve leaching effects.
②Compared with the unmutated control,the concentrations of leaching Zn~(2+)for mixed culture B,D and E inoculated in 9K medium with 10%sphalerite pulp were increased by 49.14%、44.86%and 54.77%, and the leaching rates of Zn~(2+)in the cultures were correspondingly 2.27%、2.07%and 2.53%,,respectively.
③When added with 20%sphalerite pulp and inoculated for 40 d, mixed culture B,D and E had 2.10%、1.59%and 2.24%higher Zn~(2+) leaching rates in 9K medium and 2.03%、1.89%and 2.56%higher Zn~(2+) leaching rates in Leathen medium than the unmutated control.
④Mixed culture B could adjust to 4%-16%pulp of sphalerite,and mixed culture E could adjust to 32%pulp of sphalerite.These results indicate that B and E are more tolerant to high concentrations of sphalerite pulp,suggesting that they have brighter prospects on industrial applications than D.
Diversity of microbial clones of CK,CKCu,Bcu and BZn were compared in this section.The results showed that the microbial populations of the four samples were categorized into three groups,i.e, Leptospirillum ferriphilum,Acidithiobacillus ferrooxidans,Thiobacillus thiooxidant and Acidithiobaeillus caldus.With the culture conditions varying,some changes may occur in the population structures of the mixed cultures.However,in the present study,the dominant microorganisms of the bioleaching systems were still Thiobacillus thiooxidant and Acidithiobacillus caldus.
The growth quantity of Acidithiobacillus ferrooxidan among mixed microoganisms is the smallest before mutation.After mutation,its quantity ratio in the culture of chalcopyrite and sphalerite is 20%and 29%respectively of all microorganisms and it propably plays an important role in bioleaching
The growth quantity of Thiobacillus thiooxidant among mixed microoganisms is small before mutation.After mutation,its quantity ratio in the culture of chalcopyrite and sphalerite is 69%and 57% respectively of all microorganisms and it propably plays an important role in bioleaching.
①A strain of Leptospirillum ferriphilum was isolated,and characterized by sequence analysis of 16S rDNA.Its optium growth temperature was 40℃,and its optium growth start pH was 1.5.
②A strain of Acidithiobacillus caldus was isolated,and characterized by sequence analysis of 16S rDNA.Its optium growth temperature was 45℃,and its optium growth start pH was 1.8.
③A strain of Sulfobacillus thermosulfidooxidans was isolated,and characterized by sequence analysis of 16S rDNA.Its optium growth temperature was 53℃,and its optium growth start pH was 2.0.
④No.1 strain of Acidithiobacillus ferrooxidans and No.2 strain of Thiobacillus thiooxidant were isolated.
本研究采用几种理化诱变剂及其组合,对从江西、云南、广东和台湾等不同地点采集的浸矿混合微生物进行单一或复合诱变,以获得生长快和浸矿效率高的混合培养物。同时,对诱变后的混合微生物进行了菌株分离与纯化、16S rDNA鉴定和微生物群落的生态分析。结果如下:
通过混合微生物诱变和多次浸黄铜矿精矿实验,筛选得到了浸出Cu~(2+)能力较强的混合微生物A、B、C、D和E。
①在加入矿浆浓度为1%黄铜矿精矿的9K培养基中培养40 d,混合微生物A、B、C、D和E浸出的Cu~(2+)浓度比对照分别增加61.40%、125.57%、1.45%、58.98%和149.98%,浸出率分别提高17.56%、35.90%、0.41%、16.87%和42.88%。说明混合培养物诱变可提高微生物浸黄铜矿精矿的活性。
②在加入矿浆浓度为10%黄铜矿精矿的9K培养基中培养40 d,混合微生物A、B、C、D和E浸出的Cu~(2+)浓度比对照分别增加49.46%、71.68%、22.82%、44.85%和97.57%,浸出率分别提高3.65%、5.29%、1.69%、3.31%和7.20%。说明混合培养物诱变可提高微生物浸黄铜矿精矿的活性。
③在加入矿浆浓度为20%的黄铜矿精矿培养基中培养40 d,在9K和leathen培养基中,混合微生物E浸出Cu~(2+)浓度比对照分别增加87.22%和79.24%,浸出率分别提高3.60%和3.59%,表明混合微生物E的浸矿性能稳定。
④Cu~(2+)耐受实验表明,混合微生物E耐受Cu~(2+)能力明显提高,提示其具有工业应用的潜力。
⑤混合微生物A、B、C、D和E在9K培养基上生长到稳定期,它们的生长速度比未诱变的混合微生物快1-2d。混合微生物在含黄铜矿精矿的培养基中保存比在不加矿的培养基中保存,其生长到稳定期快1-2 d左右,表明加入选择性压力可以保持浸矿微生物的生长活性。
从不同地点采集的微生物,经过黄铜矿和闪锌矿的长期驯化后,通过混合微生物的复合诱变,得到浸出闪锌矿精矿能力强的混合微生物B、D和E。
①在加入矿浆浓度为1%闪锌矿精矿的9K培养基中培养40 d,混合微生物A、B、C、D和E浸出的Zn~(2+)浓度比对照分别增加8.06%、85.97%、-2.39%、77.61%和117.01%;浸出率分别提高1.54%、16.43%、-0.46%、14.83%和22.37%。
②在加入矿浆浓度为10%闪锌矿精矿的9K培养基中培养40 d,混合微生物B、D和E浸出Zn~(2+)浓度比对照分别增加49.14%、44.86%和54.77%;浸出率分别提高2.27%、2.07%和2.53%。
③在加入矿浆浓度为20%闪锌矿精矿的9K培养基中培养40 d,混合微生物B、D和E在9K培养基中培养,Zn~(2+)浸出率分别提高2.10%、1.59%和2.24%;在leathen培养基中,Zn~(2+)浸出率分别提高2.03%、1.89%和2.56%
④B混合微生物适合在矿浆浓度4%-16%下生长,E混合微生物适合在4%-32%矿浆浓度下生长,说明混合微生物B、E耐受高矿浆浓度,有良好的工业应用前景。
比较了诱变前后四个不同样品中微生物群落的多样性。结果表明,四个样品中微生物群落结构可以分为嗜铁钩端螺旋菌组、嗜酸氧化亚铁硫杆菌组和嗜酸氧化硫硫杆菌和喜温硫杆菌三大组。尽管随着培养条件的改变,样品中微生物的群落结构存在一定的变化,但在本实验条件下,微生物浸出体系中最主要的优势菌种仍然是嗜酸氧化亚铁硫杆菌和嗜酸氧化硫硫杆菌。
诱变处理使混合微生物中的嗜酸氧化硫硫杆菌(2~#菌株)由原来的次要菌株变成主要菌株,在含黄铜矿和闪锌矿培养基中分别占微生物总数的69%和57%,可能在微生物浸矿中起主要作用。
诱变前混合微生物中嗜酸氧化亚铁硫杆菌(1~#菌株)生长量最少,诱变处理显著提高了它在微生物群落中的数量。在含黄铜矿精矿和闪锌矿精矿培养基中培养时,它分别占微生物总数的20%和29%,可能在微生物浸矿中起重要作用。
在混合微生物样品中,进行微生物单菌株分离实验:
①分离出一株最适生长温度40℃,最适生长起始pH值为1.5的螺旋菌,16S rDNA序列分析表明,该菌为Leptospirillum ferriphilum。
②分离出一株最适生长温度45℃,最适生长起始pH值为1.8的杆菌,16S rDNA序列分析表明,该菌为Acidithiobacillus caldus。
③分离出一株最适生长温度53℃,最适生长起始pH值为2.0的杆菌,16S rDNA序列分析表明,该菌为Sulfobacillus thermosulfidooxidans。
④分离出K1和K2菌株,经初步鉴定分别为嗜酸氧化亚铁硫杆菌和嗜酸氧化硫硫杆菌。
Bioleaching is a complicated biochemical process in which a number of microbial genera are involved.Most of bioleaching microorganisms are chemoautotrophic.They grow slowly and have low efficiency in metal -leaching.How to improve the leaching efficiency is a problem of industrial applications.Up to date,most popular strategy is mutation of single microorganism isolated from mixed microorganisms of bioleaching.However,microorganisms for bioleaching industry are always in mixed form,and mostly,their pure culture may not be obtained. Therefore,there are limitations on mutation breed of single microorganism.
In present study,microbial samples were collected from acid mine drainage and spring of Jiangxi,Yunnan,Guangdong and Taiwan province, mixed and enriched.To obtain higher growth rate and higher leaching efficiency-mixed cultures,the mixed microorganisms were mutated using several physical and chemical mutagens and their combinations. Furthermore,single strain isolation,characterization of 16S rDNA,and ecological analysis were performed for the screened mixed cultures.The results are as follows:
The mixed cultures with higher concentrations of leaching Cu~(2+), designated A,B,C,D and E,were screened after mutation and leaching tests of chalcopyrite.
①The concentrations of leaching Cu~(2+)for mixed culture A,B,C, D and E were increased by 61.40%,125.57%,1.45%,58.98%and 149.98%and the leaching rates of Cu~(2+)in the cultures were correspondingly 17.56%,35.90%,0.41%,16.87%and 42.88%higher compared with the control,40 d after leaching in 9K medium containing 1%chalcopyrite pulp.The results indicate that Cu~(2+)leaching activities of mixed organisms can be enhanced by physical and chemical mutations.
②The amount of leaching Cu~(2+)for mixed culture A,B,C,D and E was increased by 49.46%,71.68%,22.82%,44.85%and 97.57%, respectively,with 3.65%,5.29%,1.69%,3.31%and 7.20%higher than the control in Cu~(2+)leaching rates,40 d after leaching in 9K medium each containing 10%Chalcopyrite pulp,again indicating that physical and chemical mutations may improve Cu~(2+)leaching activities of mixed organisms.
③When added with 20%chalcopyrite pulp and inoculated in 9K and Leathen mediums for 40 d,mixed culture E extracted 87.22%and 79.24%more Cu~(2+)from chalcopyrite and had 3.6%and 3.59 higher Cu~(2+) leaching rates than the unmutated control,suggesting the acquired higher characteristics of Cu~(2+)-leaching is stable.
④Mixed culture E was more resistant to Cu~(2+)than the unmutated control,suggesting that it possesses potentials for industrial applications.
⑤The growth of mixed culture A,B,C,D and E in 9k medium was 1-2 d higher than that of the unmutated control and the mixed cultures conserved on chalcopyrite grew 1-2 d faster than those not conserved on chalcopyrite.These results indicate that growth activity of leaching microorganisms can be kept by selective stress.
After three rounds of leaching tests on sphalerite,mixed cultures B, D and E with higher efficiency of Zn~(2+)-leaching were obtained from mixed microorganisms A,B,C,D and E mentioned above.
①Compared with the unmutated control,the concentrations of leaching Zn~(2+)in 9K medium added with 1%sphalerite pulp for mixed cultures A,B,C,D and E were increased by 8.06%、85.97%、-2.39%、77.61%and 117.01%,respectively,and the leaching rates of Zn~(2+)in the cultures were correspondingly 1.54%、16.43%、-0.46%、14.83%and 22.37%,indicating that mutation against mixed microorganisms may improve leaching effects.
②Compared with the unmutated control,the concentrations of leaching Zn~(2+)for mixed culture B,D and E inoculated in 9K medium with 10%sphalerite pulp were increased by 49.14%、44.86%and 54.77%, and the leaching rates of Zn~(2+)in the cultures were correspondingly 2.27%、2.07%and 2.53%,,respectively.
③When added with 20%sphalerite pulp and inoculated for 40 d, mixed culture B,D and E had 2.10%、1.59%and 2.24%higher Zn~(2+) leaching rates in 9K medium and 2.03%、1.89%and 2.56%higher Zn~(2+) leaching rates in Leathen medium than the unmutated control.
④Mixed culture B could adjust to 4%-16%pulp of sphalerite,and mixed culture E could adjust to 32%pulp of sphalerite.These results indicate that B and E are more tolerant to high concentrations of sphalerite pulp,suggesting that they have brighter prospects on industrial applications than D.
Diversity of microbial clones of CK,CKCu,Bcu and BZn were compared in this section.The results showed that the microbial populations of the four samples were categorized into three groups,i.e, Leptospirillum ferriphilum,Acidithiobacillus ferrooxidans,Thiobacillus thiooxidant and Acidithiobaeillus caldus.With the culture conditions varying,some changes may occur in the population structures of the mixed cultures.However,in the present study,the dominant microorganisms of the bioleaching systems were still Thiobacillus thiooxidant and Acidithiobacillus caldus.
The growth quantity of Acidithiobacillus ferrooxidan among mixed microoganisms is the smallest before mutation.After mutation,its quantity ratio in the culture of chalcopyrite and sphalerite is 20%and 29%respectively of all microorganisms and it propably plays an important role in bioleaching
The growth quantity of Thiobacillus thiooxidant among mixed microoganisms is small before mutation.After mutation,its quantity ratio in the culture of chalcopyrite and sphalerite is 69%and 57% respectively of all microorganisms and it propably plays an important role in bioleaching.
①A strain of Leptospirillum ferriphilum was isolated,and characterized by sequence analysis of 16S rDNA.Its optium growth temperature was 40℃,and its optium growth start pH was 1.5.
②A strain of Acidithiobacillus caldus was isolated,and characterized by sequence analysis of 16S rDNA.Its optium growth temperature was 45℃,and its optium growth start pH was 1.8.
③A strain of Sulfobacillus thermosulfidooxidans was isolated,and characterized by sequence analysis of 16S rDNA.Its optium growth temperature was 53℃,and its optium growth start pH was 2.0.
④No.1 strain of Acidithiobacillus ferrooxidans and No.2 strain of Thiobacillus thiooxidant were isolated.
引文
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