几种高温浸矿菌的分离鉴定及其应用基础与浸矿潜力研究
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摘要
本论文根据不同高温环境样品的理化性质和目标菌株的生理生化特性,对不同温度级的高温浸矿菌进行了系统的富集、分离和鉴定,并对所分离的高温浸矿菌株进行了应用基础和浸矿潜力研究,以期为我国高温浸矿菌种资源库的建立提供菌种资源,为高温浸矿菌的基础和应用研究提供优良出发菌株。
作者从我国不同地域采集到各种环境样品14份,共从中分离鉴定出各类高温菌6属6种30余株,包括喜温嗜酸硫杆菌(Acidithiobacilluscaldus)21株(最适生长温度35-55℃)、嗜热硫氧化硫化杆菌(Sulfobacillus thermosulfidooxidnas)1株(最适生长温度53℃)、仙台脂环酸芽孢杆菌(Alicyclobacillus sendaiensis)1株(最适生长温度55℃)、万座酸菌(Acidianus manzaensis)2株(最适生长温度65℃)、金属硫叶菌(Sulfolobus metallicus)2株(最适生长温度67℃)和勤奋金属球菌(Metallosphaera sedula)3株(最适生长温度66.5℃)。在所有分离鉴定的高温菌中,除仙台脂环酸芽孢杆菌外,其它菌株均具有浸矿功能。由于篇幅所限,本论文只涉及到其中的前四属四种,即喜温嗜酸硫杆菌、嗜热硫氧化硫化杆菌、仙台脂环酸芽孢杆菌和万座酸菌。
本论文对上述四种高温菌的研究主要包括如下内容。
1)喜温嗜酸硫杆菌的分离鉴定
使用多种培养基,采用琼脂平板固体分离法从样品中分离到喜温嗜酸菌21株。经形态特征、生理生化特性研究及16S rRNA基因序列分析,鉴定这21个菌株为喜温嗜酸硫杆菌。根据样品来源,将这些菌株分为5个系列,即“YN”系列(来自云南腾冲、新华、瑞甸酸性热泉),“AN”系列(来自云南安宁酸性温泉),“TW”系列(来自台湾阳明山酸性热泉),“JX”和“DB”系列(分别来自江西德兴、广东大宝山酸性矿山废水)。
2)喜温嗜酸硫杆菌菌株随机扩增DNA多态性分析
21株喜温嗜酸硫杆菌来自多个地理位置相互隔离,物理化学性质各异的环境样品,虽然同属一种,但有些菌株在生理生化特性上表现出较大的差异。为揭示这些菌株之间的亲缘关系以及菌株差异性和环境条件之间的关联,从而为高效浸矿菌株的选育提供理论依据,作者在国内外首次运用随机扩增DNA多态性(RAPD)技术对这些菌株进行了多态性分析。结果表明这21个菌株可以分成三组,菌株分异主要与栖息环境的化学特性有关。硫、铁、铜、铝、钨、锌、镁、锰是影响菌群分异的主要因素,其中硫是关键元素。
3)喜温嗜酸硫杆菌菌株的重金属抗性研究
国内外有关喜温嗜酸硫杆菌对有毒离子的抗性研究多集中于砷。有关喜温嗜酸硫杆菌对其它有毒离子,特别是重金属离子抗性的研究报道较少。研究喜温嗜酸硫杆菌对重金属离子的抗性,对于提高其浸矿效率具有十分重要的意义。作者根据喜温嗜酸硫杆菌菌株的来源,选择了10个菌株和5种重金属硫酸盐,对所选菌株进行了重金属抗性研究。结果显示,YN12菌株对Zn~(2+)、Cd~(2+)和Co~(2+)的初始耐受浓度最高,对YN12的抗Cd~(2+)能力进行进一步的驯化增强后,其最终Cd~(2+)耐受浓度达到罕见的210 g·L~(-1)3CdSO_4·8H_2O(相当于31.5g·L~(-1)Cd~(2+)),是目前所报道的抗Cd~(2+)能力最强的菌株;DB2菌株对Cu~(2+)的初始耐受浓度最高;而YN2对Ni~(2+)的初始耐受能力最强。
4)嗜热硫氧化硫化杆菌的分离鉴定
根据嗜热硫氧化硫化杆菌能产生内生孢子,以抵抗不利环境的特点,本文作者采用前期以该菌的最适生长条件促进其营养生长,后期以该菌的临界生长条件促进其孢子生成的方式对该菌进行富集,最后采用促进营养生长的方式对该菌进行稀释分离,成功地从取自云南,pH4.5、温度约60℃的酸性热泉水样中分离到一株最适生长温度53℃,最适生长pH1.5(Fe~(2+)培养基)或2.5(S~0培养基)的杆状细菌YN22。经形态学、生理生化、分子生物学等方面的详细研究,确认YN22属于嗜热硫氧化硫化杆菌。这是国内首次分离,并经多种方法鉴定、确认的嗜热硫氧化硫化杆菌。
5)嗜热硫氧化硫化杆菌YN22菌株作用下形成的铁矾沉淀及其对该菌的影响
为了解嗜热硫氧化硫化杆菌作用下铁矾沉淀形成的规律以及铁矾沉淀对该菌生长的影响,从而为该菌浸矿参数的优化提供理论依据,作者在该菌最适生长条件下,在加或不加玻璃珠的培养基中,得到铁矾沉淀。加玻璃珠和不加玻璃珠两组间在Fe~(2+)氧化速率、沉淀产量和菌群密度上有显著差异,加玻璃珠一组Fe~(2+)氧化速率快6h,沉淀出现时间早6h,沉淀产量高78%。X衍射图谱、红外吸收光谱、能谱和扫描电镜图像揭示这两种沉淀都是黄钾铁矾和黄铵铁矾的混合物,形态上与后者相似。在未接种的加玻璃珠和不加玻璃珠对照组中,实验期间均无沉淀生成。产自不加玻璃珠组的沉淀对YN22菌株的生长和Fe~(2+)氧化能力没有明显影响,而产自加玻璃珠组的沉淀在浓度达到4g·L~(-1)时就会对YN22菌株的生长和Fe~(2+)氧化能力造成明显的抑制作用。实验结果提示,YN22用于实际浸矿,特别是用于搅拌式槽浸时,为防止铁矾沉淀的过早出现和过度累积,在其它参数较难调控的情况下,应将浸矿体系的pH控制在较低的合适范围内。
6)嗜热硫氧化硫化杆菌YN22菌株对黄铜矿和黄铁矿的浸出
到目前为止,国内尚未见嗜热硫氧化硫化杆菌浸出黄铜矿的报道。为评估其浸矿潜力,作者研究了YN22菌株纯菌以及和喜温嗜酸硫杆菌混合对黄铜矿和黄铁矿的浸出。结果显示,YN22菌株单独浸出黄铜矿的效率较低,25d铜的浸出率为14.7%。当YN22菌株与适度嗜热硫氧化菌—喜温嗜酸硫杆菌YN2菌株混合,浸矿效率大大改善,同期铜的浸出率提高到46.2%。YN22菌株对黄铁矿的浸出效果较好,经过25d的浸出,纯菌浸出液中总铁浓度达到5292 mg·L~(-1),是无菌浸出液的4.73倍。研究结果提示,用YN22菌株浸出黄铜矿一类的原生硫化矿时,最好与适度嗜热硫氧化菌混合。
7)异养菌YNTC-1的分离鉴定及其对YN22菌株浸矿的影响
为了解共栖异养菌对嗜热硫氧化硫化杆菌浸矿的影响,从分离YN22菌株的富集物中分离鉴定了一株与YN22共栖的专性异养菌YNTC-1,并利用该菌与YN22混合,进行了黄铜矿和黄铁矿的浸矿试验。基于表型、基因型和系统发育分析结果,YNTC-1菌株被鉴定为仙台脂环酸芽孢杆菌一新菌株。用YN22纯菌以及YN22与YNTC-1混合菌对黄铜矿和黄铁矿进行的浸矿实验表明,YNTC-1菌株对这两种硫化矿的浸出没有明显的促进作用。
8)万座酸菌的分离鉴定
运用原位富集法对云南腾冲的酸性热泉水样进行富集后,在实验室成功分离纯化了2株嗜热喜酸菌—YN25、YN26。经多种方法详细鉴定,确认两个菌株属于嗜热古菌—万座酸菌。万座酸菌是新近发现的酸菌属新种,目前全世界仅有3个菌株,即模式菌株NA-1~T和我们自云南酸性热泉中分离的YN25和YN26两个菌株。YN25和YN26是国内首次分离鉴定的万座酸菌菌株,该研究成果对于推动我国浸矿菌种,特别是高温浸矿菌种的基础和应用研究具有重要意义。
9)万座酸菌YN25菌株对黄铜矿和黄铁矿的浸出
万座酸菌YN25菌株对黄铜矿和黄铁矿的浸出实验表明,该菌株是目前已报道的浸矿菌中浸矿能力最强的菌株之一。黄铜矿的摇瓶实验结果显示,24d铜的浸出率达到79.16%。对黄铜矿的继代浸出实验进一步表明,通过Cu~(2+)定向驯化,YN25菌株对Cu~(2+)的耐受浓度可以在首次浸矿的基础上提高80%以上。YN25菌株对黄铁矿的浸出能力也远远高于YN22菌株,经过28d,YN25浸出液中Fe_(tot)浓度是YN22的3.4倍。这是目前国内外首次进行的万座酸菌浸矿实验,是该菌的重要应用基础研究,对于评价其应用前景具有重要的指导意义。
To obtain thermophilic sulfide mineral-oxidizing microorganisms for basic and applied studies,fourteen samples of acidic hydrothermal spring and acid mine drainage were collected from Guangdong,Yunnan, Jiangxi and Taiwan in China.From these samples more than thirty strains were isolated with a number of unique methods and identified by morphological,physiological and molecular analyses.The isolated strains can be classified into six species,with twenty-one strains in Acidithiobacillus caldus,one in Sulfobacillus thermosulfidooxidnas,one in Alicyclobacillus sendaiensis,two in Acidianus manzaensis,two in Sulfolobus metallicus and three in Metallosphaera sedula.However,only the strains belonging to the former four species are included in this dissertation.
1)Isolation and identification of Acidithiobacillus caldus
A total of twenty-one moderately thermo-acidophilic strains were isolated from fourteen samples of acidic hydrothermal spring and acid mine drainage,collected at several geographically insulated sites in China. These strains were rod-shaped and G~-,grew aerobically by oxidizing sulfur to sulfuric acid at optimum temperatures and pHs of 35-55℃and 2.0-3.0.Phylogenetic analyses based on 16S rRNA gene sequences showed that all of them belonged to Acidithiobacillus caldus,with 16S rDNA similarities over 99%between At.caldus and the strains.On the basis of these results,the 21 strains should be grouped into At.caldus.
2)Random amplified polymorphic DNA analysis of Acidithiobacillus caldus strains recovered from different extreme environments
Genomic DNA was extracted from each isolated strain,and PCR for each template DNA was carried out using fourteen 10-mer primers after the PCR conditions were carefully optimized.A RAPD(Randomly Amplification Polymorphic DNA)profile of 264 distinctive banding patterns was obtained,in which 1602 reproducible and distinguishable bands were included with an average of 114 bands per primer.The banding patterns were compared by scoring each DNA band as present or absent,and a genetic similarity matrix was constructed with Jaccard's coefficient.The matrix of generated similarities was analyzed by the unweighted pair-group method with arithmetic average(UPGMA cluster analysis)and a dendrogram was then built up.
The Jaccard's coefficient demonstrated a similarity range from 0.438 to 0.884 between the strains and the twenty-two At.caldus strains studied were clustered into three groups at 0.6 similarity level by UPGMA clustering.The results of this study suggest that the strain variabilities in At.caldus are correlated with chemical variations in environments and RAPD analysis is a sensitive and reliable method for strain diversity assay in At.caldus if the RAPD-PCR conditions are carefully optimized.
3)Heavy metal resistance of Acidithiobacillus caldus strains
Ten moderately thermophilic sulfur-oxidizing strains(AN2、DB2、JX2、YN1、YN2、YN3、YN6、YN12、YN15 and TW26)in At.caldus and five heavy metal sulfates(CuSO_4·5H_2O,ZnSO_4·7H_2O,NiSO_4·6H_2O, 3CdSO_4·8H_2O and CoSO_4·7H_2O)were selected for heavy metal resistant tests.
YN12 exhibited a very high degree of resistance to cadmium(Ⅱ), zinc(Ⅱ)and cobalt(Ⅱ),with original maximum tolerated concentrations (MTC)of 4.8,12.0 and 9.0 g·l~(-1)respectively.DB2 and YN2 were the most resistant to copper(Ⅱ)and Nickel(Ⅱ),with MTCs of 20 and 120 g·L~(-1).YN12 could obtain an ultimate resistance of 31.5 g·l~(-1)Cd~(2+)after adapted by a gradually increased concentration of 3CdSO_4·8H_2O. Continuously cultured in the medium containing 210 g·l~(-1)3CdSO_4·8H_2O (equal to 31.5 g·l~(-1)Cd~(2+))for three generations,the strain was capable of restoring its growth and sulfur oxidation activity to an approximately normal level.The result of this study indicates that YN12 may play a potential role in cadmium-rich sulphide mineral bioleaching and cadmium pollution control.
4)Isolation and identification of Sulfobacillus thermosulfidooxidans
A moderate acidothermophilic bacterial strain,YN22,was isolated from an acid hot spring in Tengchong,Yunan,China.The gram-positive-staining cells of the strain were straight to slight curved rods,1.6-2.8μm long and 0.4-0.7μm in diameter.The temperature range for growth was 30 to 60℃with an optimum at 53℃.The pH range for growth at 53℃was from 1.0 to 5.0 with an optimum at 1.5.YN22,the chemoautotrophic strain,was capable of oxidizing Fe~(2+)to Fe~(3+)in media containing 0.02%(w/v)yeast extract to support fast growth,but did not effectively utilize sulfur or reduced sulphides as energy source.16s rRNA gene Analysis revealed this strain closely related to Sulfobacillus thermosulfidooxidans,with over 99%sequence similarity between each other.The G+C contents of DNA were also very similar between Sb. thermosulfidooxidans and strain YN22,which were 47.5 and 47.3 mol% respectively.Based on phenotypic,biochemical and genotypic analyses, YN22 is identified as a new strain of Sb.thermosulfidooxidans.It is the first time for this species to be isolated and identified in China.
5)Jarosite-type precipitates mediated by YN22,Sulfobacillus thermosulfidooxidans,and their influences on the strain
To have a better understanding on the properties of the jarosite-type precipitate synthesized by Sb.thermosulfidooxidans,the evolution of the Sb.thermosulfidooxidans-mediated precipitation and the influence of the precipitate on this species,a newly isolated strain(YN22)of Sb. thermosulfidooxidans,was cultured in a medium containing Fe~(2+)as energy source under optimal conditions(pH1.5,53℃,0.2g·L~(-1)yeast extract,30g·L~(-1)Fe_2SO_4·7H_2O and 170r·min~(-1)),added with or without glass beads.Remarkable differences were found in the oxidation rate of Fe~(2+),the precipitate yield of jarosite-type compounds and the population density between the two groups of cultures.The group with glass beads has a 6 h faster Fe~(2+)oxidation,6 h earlier precipitation,78%higher precipitate yield and much lower population density than those without glass beads.XRD,EDS,FTIR and SEM analysis reveals that the precipitates originated from both groups are a mixture of potassium jarosite and ammoniojarosite,with morphological features similar to the latter.The results of the test referring to influence of the precipitates on YN22 show that the precipitate from the group without glass beads has no apparent influence on Fe~(2+)oxidation rate of YN22 and only a limited influence on growth of the strain,whereas that from the group with glass beads remarkably inhibits the growth and Fe~(2+)oxidation ability of YN22 when a precipitate content over 4 g·L~(-1)is used.
6)Bioleaching of chalcopyrite and pyrite with Sulfobacillus thermosulfidooxidans strain YN22
Bioleaching of chalcopyrite and pyrite by moderate thermophilic bacterium Sb.thermosulfidooxidans strain YN22 was studied.Copper dissolution reached 46.2%with the mixed culture of strain YN22 and strain YN2,a sulfur-oxidizing bacterium of Acidithiobacillus caldus,and 14.7%with the pure culture of Sb.thermosulfidooxidans strain YN22,at 53℃and 3%(w/v)pulp density,after 25 days.These results suggest that maximum metal recovery can be achieved by mixing strain YN22 with moderate thermophilic sulfur-oxidizer like A.caldus in leaching primary sulfide minerals such as chalcopyrite.However,rate data collected in a 25 d period of pyrite leaching with pure YN22 indicate that strain YN22 is an effective moderate thermophile for pyrite leaching.
7)Isolation,characterization of heterotrophic acidothermophilic bacterial strainYNTC-1 and its influence on chalcopyrite and pyrite leaching by strain YN22
A heterotrophic acidothermophilic bacterial strain,YNTC-1,was isolated from the same enriched sample of an acid spring from which Sb. thermosulfidooxidans strain YN22 was purified.Based on phenotypic and genotypic analyses,YNTC-1 was identified as a member of A. sendaiensis.Considering some important morphological and biochemical differences between the two bacteria,YNTC-1 may be proposed to be a novel subspecies of A.sendaiensis.However,this view has to be confirmed by further studies.Co-bioleaching of pyrite and chalcopyrite with Sb.thermosulfidooxidans shows that strain YNTC-1 has no evident influence on leaching rates of these two sulphide minerals.
8)Isolation and identification of Acidianus manzaensis
Two novel thermoacidophilic iron and sulfur-oxidizing archaeon strainsYN25 and YN26,were isolated from an in situ enriched acid hot spring sample collected in Yunnan,China,and identified as Acidianus manzaensis through morphological,physiological and molecular studies. Up to date,there are only three strains isolated and identified all over the world,Strain NA-1~T at NBRC and ATCC,Strain YN25 and YN26 at CSU.YN25 and YN26,the first found strains of A.manzaensis in China, will play an important role in basic and applied studies of thermophilic leaching microbes.
9)Bioleaching of chalcopyrite and pyrite by A.manzaensis strain YN25
Bioleaching ability of A.manzaensis strain YN25 for chalcopyrite and pyrite was tested.The results demonstrate that strain YN25 is one of the most effective sulfide oxidizers recorded,with a copper leaching rate of 79.16%for chalcopyrite after 24 d and Cu~(2+)tolerated concentration increased by 80%through continuous leaching of chalcopyrite.Also YN25 appeared to dissolve more iron when bioleaching pyrite and the Fe_(total)concentration in YN25-bioleaching liquor was 2.4 times higher than that in YN22-bioleaching liquor after 28 d.It is the first time for this thermophilic archaea to be tested in sulfide mineral leaching.The parameters from these experiments may be used to evaluate the application potential of this species for sulfide ore bioleaching.
作者从我国不同地域采集到各种环境样品14份,共从中分离鉴定出各类高温菌6属6种30余株,包括喜温嗜酸硫杆菌(Acidithiobacilluscaldus)21株(最适生长温度35-55℃)、嗜热硫氧化硫化杆菌(Sulfobacillus thermosulfidooxidnas)1株(最适生长温度53℃)、仙台脂环酸芽孢杆菌(Alicyclobacillus sendaiensis)1株(最适生长温度55℃)、万座酸菌(Acidianus manzaensis)2株(最适生长温度65℃)、金属硫叶菌(Sulfolobus metallicus)2株(最适生长温度67℃)和勤奋金属球菌(Metallosphaera sedula)3株(最适生长温度66.5℃)。在所有分离鉴定的高温菌中,除仙台脂环酸芽孢杆菌外,其它菌株均具有浸矿功能。由于篇幅所限,本论文只涉及到其中的前四属四种,即喜温嗜酸硫杆菌、嗜热硫氧化硫化杆菌、仙台脂环酸芽孢杆菌和万座酸菌。
本论文对上述四种高温菌的研究主要包括如下内容。
1)喜温嗜酸硫杆菌的分离鉴定
使用多种培养基,采用琼脂平板固体分离法从样品中分离到喜温嗜酸菌21株。经形态特征、生理生化特性研究及16S rRNA基因序列分析,鉴定这21个菌株为喜温嗜酸硫杆菌。根据样品来源,将这些菌株分为5个系列,即“YN”系列(来自云南腾冲、新华、瑞甸酸性热泉),“AN”系列(来自云南安宁酸性温泉),“TW”系列(来自台湾阳明山酸性热泉),“JX”和“DB”系列(分别来自江西德兴、广东大宝山酸性矿山废水)。
2)喜温嗜酸硫杆菌菌株随机扩增DNA多态性分析
21株喜温嗜酸硫杆菌来自多个地理位置相互隔离,物理化学性质各异的环境样品,虽然同属一种,但有些菌株在生理生化特性上表现出较大的差异。为揭示这些菌株之间的亲缘关系以及菌株差异性和环境条件之间的关联,从而为高效浸矿菌株的选育提供理论依据,作者在国内外首次运用随机扩增DNA多态性(RAPD)技术对这些菌株进行了多态性分析。结果表明这21个菌株可以分成三组,菌株分异主要与栖息环境的化学特性有关。硫、铁、铜、铝、钨、锌、镁、锰是影响菌群分异的主要因素,其中硫是关键元素。
3)喜温嗜酸硫杆菌菌株的重金属抗性研究
国内外有关喜温嗜酸硫杆菌对有毒离子的抗性研究多集中于砷。有关喜温嗜酸硫杆菌对其它有毒离子,特别是重金属离子抗性的研究报道较少。研究喜温嗜酸硫杆菌对重金属离子的抗性,对于提高其浸矿效率具有十分重要的意义。作者根据喜温嗜酸硫杆菌菌株的来源,选择了10个菌株和5种重金属硫酸盐,对所选菌株进行了重金属抗性研究。结果显示,YN12菌株对Zn~(2+)、Cd~(2+)和Co~(2+)的初始耐受浓度最高,对YN12的抗Cd~(2+)能力进行进一步的驯化增强后,其最终Cd~(2+)耐受浓度达到罕见的210 g·L~(-1)3CdSO_4·8H_2O(相当于31.5g·L~(-1)Cd~(2+)),是目前所报道的抗Cd~(2+)能力最强的菌株;DB2菌株对Cu~(2+)的初始耐受浓度最高;而YN2对Ni~(2+)的初始耐受能力最强。
4)嗜热硫氧化硫化杆菌的分离鉴定
根据嗜热硫氧化硫化杆菌能产生内生孢子,以抵抗不利环境的特点,本文作者采用前期以该菌的最适生长条件促进其营养生长,后期以该菌的临界生长条件促进其孢子生成的方式对该菌进行富集,最后采用促进营养生长的方式对该菌进行稀释分离,成功地从取自云南,pH4.5、温度约60℃的酸性热泉水样中分离到一株最适生长温度53℃,最适生长pH1.5(Fe~(2+)培养基)或2.5(S~0培养基)的杆状细菌YN22。经形态学、生理生化、分子生物学等方面的详细研究,确认YN22属于嗜热硫氧化硫化杆菌。这是国内首次分离,并经多种方法鉴定、确认的嗜热硫氧化硫化杆菌。
5)嗜热硫氧化硫化杆菌YN22菌株作用下形成的铁矾沉淀及其对该菌的影响
为了解嗜热硫氧化硫化杆菌作用下铁矾沉淀形成的规律以及铁矾沉淀对该菌生长的影响,从而为该菌浸矿参数的优化提供理论依据,作者在该菌最适生长条件下,在加或不加玻璃珠的培养基中,得到铁矾沉淀。加玻璃珠和不加玻璃珠两组间在Fe~(2+)氧化速率、沉淀产量和菌群密度上有显著差异,加玻璃珠一组Fe~(2+)氧化速率快6h,沉淀出现时间早6h,沉淀产量高78%。X衍射图谱、红外吸收光谱、能谱和扫描电镜图像揭示这两种沉淀都是黄钾铁矾和黄铵铁矾的混合物,形态上与后者相似。在未接种的加玻璃珠和不加玻璃珠对照组中,实验期间均无沉淀生成。产自不加玻璃珠组的沉淀对YN22菌株的生长和Fe~(2+)氧化能力没有明显影响,而产自加玻璃珠组的沉淀在浓度达到4g·L~(-1)时就会对YN22菌株的生长和Fe~(2+)氧化能力造成明显的抑制作用。实验结果提示,YN22用于实际浸矿,特别是用于搅拌式槽浸时,为防止铁矾沉淀的过早出现和过度累积,在其它参数较难调控的情况下,应将浸矿体系的pH控制在较低的合适范围内。
6)嗜热硫氧化硫化杆菌YN22菌株对黄铜矿和黄铁矿的浸出
到目前为止,国内尚未见嗜热硫氧化硫化杆菌浸出黄铜矿的报道。为评估其浸矿潜力,作者研究了YN22菌株纯菌以及和喜温嗜酸硫杆菌混合对黄铜矿和黄铁矿的浸出。结果显示,YN22菌株单独浸出黄铜矿的效率较低,25d铜的浸出率为14.7%。当YN22菌株与适度嗜热硫氧化菌—喜温嗜酸硫杆菌YN2菌株混合,浸矿效率大大改善,同期铜的浸出率提高到46.2%。YN22菌株对黄铁矿的浸出效果较好,经过25d的浸出,纯菌浸出液中总铁浓度达到5292 mg·L~(-1),是无菌浸出液的4.73倍。研究结果提示,用YN22菌株浸出黄铜矿一类的原生硫化矿时,最好与适度嗜热硫氧化菌混合。
7)异养菌YNTC-1的分离鉴定及其对YN22菌株浸矿的影响
为了解共栖异养菌对嗜热硫氧化硫化杆菌浸矿的影响,从分离YN22菌株的富集物中分离鉴定了一株与YN22共栖的专性异养菌YNTC-1,并利用该菌与YN22混合,进行了黄铜矿和黄铁矿的浸矿试验。基于表型、基因型和系统发育分析结果,YNTC-1菌株被鉴定为仙台脂环酸芽孢杆菌一新菌株。用YN22纯菌以及YN22与YNTC-1混合菌对黄铜矿和黄铁矿进行的浸矿实验表明,YNTC-1菌株对这两种硫化矿的浸出没有明显的促进作用。
8)万座酸菌的分离鉴定
运用原位富集法对云南腾冲的酸性热泉水样进行富集后,在实验室成功分离纯化了2株嗜热喜酸菌—YN25、YN26。经多种方法详细鉴定,确认两个菌株属于嗜热古菌—万座酸菌。万座酸菌是新近发现的酸菌属新种,目前全世界仅有3个菌株,即模式菌株NA-1~T和我们自云南酸性热泉中分离的YN25和YN26两个菌株。YN25和YN26是国内首次分离鉴定的万座酸菌菌株,该研究成果对于推动我国浸矿菌种,特别是高温浸矿菌种的基础和应用研究具有重要意义。
9)万座酸菌YN25菌株对黄铜矿和黄铁矿的浸出
万座酸菌YN25菌株对黄铜矿和黄铁矿的浸出实验表明,该菌株是目前已报道的浸矿菌中浸矿能力最强的菌株之一。黄铜矿的摇瓶实验结果显示,24d铜的浸出率达到79.16%。对黄铜矿的继代浸出实验进一步表明,通过Cu~(2+)定向驯化,YN25菌株对Cu~(2+)的耐受浓度可以在首次浸矿的基础上提高80%以上。YN25菌株对黄铁矿的浸出能力也远远高于YN22菌株,经过28d,YN25浸出液中Fe_(tot)浓度是YN22的3.4倍。这是目前国内外首次进行的万座酸菌浸矿实验,是该菌的重要应用基础研究,对于评价其应用前景具有重要的指导意义。
To obtain thermophilic sulfide mineral-oxidizing microorganisms for basic and applied studies,fourteen samples of acidic hydrothermal spring and acid mine drainage were collected from Guangdong,Yunnan, Jiangxi and Taiwan in China.From these samples more than thirty strains were isolated with a number of unique methods and identified by morphological,physiological and molecular analyses.The isolated strains can be classified into six species,with twenty-one strains in Acidithiobacillus caldus,one in Sulfobacillus thermosulfidooxidnas,one in Alicyclobacillus sendaiensis,two in Acidianus manzaensis,two in Sulfolobus metallicus and three in Metallosphaera sedula.However,only the strains belonging to the former four species are included in this dissertation.
1)Isolation and identification of Acidithiobacillus caldus
A total of twenty-one moderately thermo-acidophilic strains were isolated from fourteen samples of acidic hydrothermal spring and acid mine drainage,collected at several geographically insulated sites in China. These strains were rod-shaped and G~-,grew aerobically by oxidizing sulfur to sulfuric acid at optimum temperatures and pHs of 35-55℃and 2.0-3.0.Phylogenetic analyses based on 16S rRNA gene sequences showed that all of them belonged to Acidithiobacillus caldus,with 16S rDNA similarities over 99%between At.caldus and the strains.On the basis of these results,the 21 strains should be grouped into At.caldus.
2)Random amplified polymorphic DNA analysis of Acidithiobacillus caldus strains recovered from different extreme environments
Genomic DNA was extracted from each isolated strain,and PCR for each template DNA was carried out using fourteen 10-mer primers after the PCR conditions were carefully optimized.A RAPD(Randomly Amplification Polymorphic DNA)profile of 264 distinctive banding patterns was obtained,in which 1602 reproducible and distinguishable bands were included with an average of 114 bands per primer.The banding patterns were compared by scoring each DNA band as present or absent,and a genetic similarity matrix was constructed with Jaccard's coefficient.The matrix of generated similarities was analyzed by the unweighted pair-group method with arithmetic average(UPGMA cluster analysis)and a dendrogram was then built up.
The Jaccard's coefficient demonstrated a similarity range from 0.438 to 0.884 between the strains and the twenty-two At.caldus strains studied were clustered into three groups at 0.6 similarity level by UPGMA clustering.The results of this study suggest that the strain variabilities in At.caldus are correlated with chemical variations in environments and RAPD analysis is a sensitive and reliable method for strain diversity assay in At.caldus if the RAPD-PCR conditions are carefully optimized.
3)Heavy metal resistance of Acidithiobacillus caldus strains
Ten moderately thermophilic sulfur-oxidizing strains(AN2、DB2、JX2、YN1、YN2、YN3、YN6、YN12、YN15 and TW26)in At.caldus and five heavy metal sulfates(CuSO_4·5H_2O,ZnSO_4·7H_2O,NiSO_4·6H_2O, 3CdSO_4·8H_2O and CoSO_4·7H_2O)were selected for heavy metal resistant tests.
YN12 exhibited a very high degree of resistance to cadmium(Ⅱ), zinc(Ⅱ)and cobalt(Ⅱ),with original maximum tolerated concentrations (MTC)of 4.8,12.0 and 9.0 g·l~(-1)respectively.DB2 and YN2 were the most resistant to copper(Ⅱ)and Nickel(Ⅱ),with MTCs of 20 and 120 g·L~(-1).YN12 could obtain an ultimate resistance of 31.5 g·l~(-1)Cd~(2+)after adapted by a gradually increased concentration of 3CdSO_4·8H_2O. Continuously cultured in the medium containing 210 g·l~(-1)3CdSO_4·8H_2O (equal to 31.5 g·l~(-1)Cd~(2+))for three generations,the strain was capable of restoring its growth and sulfur oxidation activity to an approximately normal level.The result of this study indicates that YN12 may play a potential role in cadmium-rich sulphide mineral bioleaching and cadmium pollution control.
4)Isolation and identification of Sulfobacillus thermosulfidooxidans
A moderate acidothermophilic bacterial strain,YN22,was isolated from an acid hot spring in Tengchong,Yunan,China.The gram-positive-staining cells of the strain were straight to slight curved rods,1.6-2.8μm long and 0.4-0.7μm in diameter.The temperature range for growth was 30 to 60℃with an optimum at 53℃.The pH range for growth at 53℃was from 1.0 to 5.0 with an optimum at 1.5.YN22,the chemoautotrophic strain,was capable of oxidizing Fe~(2+)to Fe~(3+)in media containing 0.02%(w/v)yeast extract to support fast growth,but did not effectively utilize sulfur or reduced sulphides as energy source.16s rRNA gene Analysis revealed this strain closely related to Sulfobacillus thermosulfidooxidans,with over 99%sequence similarity between each other.The G+C contents of DNA were also very similar between Sb. thermosulfidooxidans and strain YN22,which were 47.5 and 47.3 mol% respectively.Based on phenotypic,biochemical and genotypic analyses, YN22 is identified as a new strain of Sb.thermosulfidooxidans.It is the first time for this species to be isolated and identified in China.
5)Jarosite-type precipitates mediated by YN22,Sulfobacillus thermosulfidooxidans,and their influences on the strain
To have a better understanding on the properties of the jarosite-type precipitate synthesized by Sb.thermosulfidooxidans,the evolution of the Sb.thermosulfidooxidans-mediated precipitation and the influence of the precipitate on this species,a newly isolated strain(YN22)of Sb. thermosulfidooxidans,was cultured in a medium containing Fe~(2+)as energy source under optimal conditions(pH1.5,53℃,0.2g·L~(-1)yeast extract,30g·L~(-1)Fe_2SO_4·7H_2O and 170r·min~(-1)),added with or without glass beads.Remarkable differences were found in the oxidation rate of Fe~(2+),the precipitate yield of jarosite-type compounds and the population density between the two groups of cultures.The group with glass beads has a 6 h faster Fe~(2+)oxidation,6 h earlier precipitation,78%higher precipitate yield and much lower population density than those without glass beads.XRD,EDS,FTIR and SEM analysis reveals that the precipitates originated from both groups are a mixture of potassium jarosite and ammoniojarosite,with morphological features similar to the latter.The results of the test referring to influence of the precipitates on YN22 show that the precipitate from the group without glass beads has no apparent influence on Fe~(2+)oxidation rate of YN22 and only a limited influence on growth of the strain,whereas that from the group with glass beads remarkably inhibits the growth and Fe~(2+)oxidation ability of YN22 when a precipitate content over 4 g·L~(-1)is used.
6)Bioleaching of chalcopyrite and pyrite with Sulfobacillus thermosulfidooxidans strain YN22
Bioleaching of chalcopyrite and pyrite by moderate thermophilic bacterium Sb.thermosulfidooxidans strain YN22 was studied.Copper dissolution reached 46.2%with the mixed culture of strain YN22 and strain YN2,a sulfur-oxidizing bacterium of Acidithiobacillus caldus,and 14.7%with the pure culture of Sb.thermosulfidooxidans strain YN22,at 53℃and 3%(w/v)pulp density,after 25 days.These results suggest that maximum metal recovery can be achieved by mixing strain YN22 with moderate thermophilic sulfur-oxidizer like A.caldus in leaching primary sulfide minerals such as chalcopyrite.However,rate data collected in a 25 d period of pyrite leaching with pure YN22 indicate that strain YN22 is an effective moderate thermophile for pyrite leaching.
7)Isolation,characterization of heterotrophic acidothermophilic bacterial strainYNTC-1 and its influence on chalcopyrite and pyrite leaching by strain YN22
A heterotrophic acidothermophilic bacterial strain,YNTC-1,was isolated from the same enriched sample of an acid spring from which Sb. thermosulfidooxidans strain YN22 was purified.Based on phenotypic and genotypic analyses,YNTC-1 was identified as a member of A. sendaiensis.Considering some important morphological and biochemical differences between the two bacteria,YNTC-1 may be proposed to be a novel subspecies of A.sendaiensis.However,this view has to be confirmed by further studies.Co-bioleaching of pyrite and chalcopyrite with Sb.thermosulfidooxidans shows that strain YNTC-1 has no evident influence on leaching rates of these two sulphide minerals.
8)Isolation and identification of Acidianus manzaensis
Two novel thermoacidophilic iron and sulfur-oxidizing archaeon strainsYN25 and YN26,were isolated from an in situ enriched acid hot spring sample collected in Yunnan,China,and identified as Acidianus manzaensis through morphological,physiological and molecular studies. Up to date,there are only three strains isolated and identified all over the world,Strain NA-1~T at NBRC and ATCC,Strain YN25 and YN26 at CSU.YN25 and YN26,the first found strains of A.manzaensis in China, will play an important role in basic and applied studies of thermophilic leaching microbes.
9)Bioleaching of chalcopyrite and pyrite by A.manzaensis strain YN25
Bioleaching ability of A.manzaensis strain YN25 for chalcopyrite and pyrite was tested.The results demonstrate that strain YN25 is one of the most effective sulfide oxidizers recorded,with a copper leaching rate of 79.16%for chalcopyrite after 24 d and Cu~(2+)tolerated concentration increased by 80%through continuous leaching of chalcopyrite.Also YN25 appeared to dissolve more iron when bioleaching pyrite and the Fe_(total)concentration in YN25-bioleaching liquor was 2.4 times higher than that in YN22-bioleaching liquor after 28 d.It is the first time for this thermophilic archaea to be tested in sulfide mineral leaching.The parameters from these experiments may be used to evaluate the application potential of this species for sulfide ore bioleaching.
引文
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