【作者】 刘毅；

【导师】 刘学端；

【作者基本信息】 中南大学 ， 微生物学， 2011， 博士

【摘要】 在生物冶金领域,为了构建和优化浸矿微生物群落,常常需要引入具有一定特性的嗜酸微生物。关于土著浸矿微生物群落引种机制的研究还没有相关报道。本文利用构建的共培养体系或驯化后的自然嗜酸微生物群落作为土著群落,在其不同的生长时期,引入不同代谢类型的外来浸矿菌种。对土著浸矿微生物群落的引种机制进行了研究,为找出浸矿过程中对土著群落引种的最佳类型和时间提供理论依据。采用液体梯度稀释分离法从不同酸性矿坑水样品中分离到4株弧状或螺旋状菌株。对四株分离物的纯度、生理特征、16S rDNA和gyrB基因序列、形态学以及对黄铁矿浸出能力的分析后,最终确定四株分离物均为嗜铁钩端螺旋菌(L. ferriphilum)。构建了一个包括At. caldus、L. ferriphilum、Ferroplasma、At. ferrooxidans、Acidiphillum spp和S. thermosulfidooxidans的共培养体系,其在40℃、pH为1.5和矿浆浓度为0.5%(g/100m1)时对黄铁矿的浸出效率相对较好；在此条件下,At. caldus、L. ferriphilum和Ferroplasma为共培养体系中的优势种群,而At. ferrooxidans、 Acidiphillum spp.和S. thermosulfidooxidans为劣势种群；其各个生长时期可划分为：0-48小时为生长适应期；48-96小时为快速生长期；96-192小时为稳定生长期；192-240小时为衰亡期。以上述共培养体系作为土著群落,At. thiooxidans A01作为外来菌种分别在第66和138小时引入土著群落后,对黄铁矿浸出效率分别提高了10%和13%。实时定量PCR结果显示：At. thiooxidans A01的引入促进L. ferriphilum、Ferroplasma和Acidiphillum spp.的生长；在一定程度上抑制At. caldus的生长；而对At. ferrooxidans和S. thermosulfidooxidans的影响不大。功能基因芯片结果表明：来自L.ferriphilum的编码protoheme ferrolyase、formate hydrogenlyase、ADP heptose、phosphoheptose isomerase和glycosyltransferase的基因,以及来自Ferroplasma sp的编码ferredoxin oxidoreductase的基因和来自Acidiphillum spp编码acetyl-CoA carboxylase的基因,在引种后0-20小时表达水平上调；来自At. caldus的doxD基因和来自S.thermosulfidooxidans编码ribulose-1,5-bisphosphate carboxylase/oxygenase的基因,在引种后0-20小时表达水平都下调；来自At.ferrooxidans编码ADP-heptose Synthase的基因在引种后0-12小时表达水平上调,而在12-20小时下调。对上述共培养体系的组分进行微调(At. thiooxidans A01加入,而L. ferriphilum YSK移除,其他组分不变),在相同实验条件下,L.ferriphilum YSK作为外来菌种分别在第0、66和138小时引入后,对黄铁矿的浸出效率分别提高了9%、14%和19%。实时定量PCR结果显示：L. ferriphilum YSK引入后,促进At. caldus、At. thiooxidans和Acidiphillum spp.的生长；抑制Ferroplasma和S. thermosulfidooxidans的生长；而对At. ferrooxidans的影响不大。运用功能基因芯片对第138小时引种后功能基因表达水平的变化进行分析后发现：来自L.ferriphilum的编码ADPheptose、phosphoheptose isomerase glycosyltransferase、Biotin carboxylase和protoheme ferrolyase的基因,以及来自Acidiphillum spp编码acetyl-CoA carboxylase的基因和来自At. caldus的doxD基因,在引种后0-20小时表达水平上调；来自At.ferrooxidans编码lipid A disaccharide synthase LpxB、glycosyl transferase和ADP-heptose Synthase的基因,在引种后0-8小时表达水平上调,而在8-20小时下调；来自Ferroplasma sp的编码ferredoxin oxidoreductase的基因在引种后0-4小时表达水平上调,而4-16小时下调,16-20小时上调；来自At. ferrooxidans的CbbS基因,在引种后0-20小时表达水平下调。对来自不同酸性矿坑水中的嗜酸微生物混合,在低品位黄铜矿的浸矿体系中经过连续五次驯化后,其对黄铜矿的浸出效率提高了近45%。RFLP结果显示：驯化前的群落中共检测到八种常见浸矿微生物,包括At. ferrooxidans, At. caldus, Sulfobacillus sp., L. ferriphilum, S. thermosulfidooxidans, Acidiphilium sp., Alicyclobacillus sp.和Pseudomonas sp.;而在驯化后的群落中,只检测到了At. ferrooxidans, At. caldus, Sulfobacillus sp., L.ferriphilum和Acidiphilium的存在。功能基因芯片结果表明：At. caldus、L. ferriphilum、Sulfobacillus sp.、 Sulfolobus sp.和Acidiphilium sp.在群落中的丰度随着驯化而逐渐增多；而S. thermosulfidooxidans、Alicyclobacillus sp.和At. ferrooxidans的丰度逐渐减少。来自At. caldus和Sulfolobus sp.的与硫代谢相关的功能基因的丰度逐渐增多,而来自At. ferrooxidans的与硫代谢相关的功能基因的丰度逐渐减少；来自L. ferriphilum的与铁代谢相关的编码protoheme ferrolyase的基因的丰度逐渐增加；来自At. ferrooxidans, At. caldus, L. ferriphilum, Sulfolobus spp.和Acidiphilium sp.的与金属抗性相关的功能基因的丰度逐渐增加。以上述从酸性矿坑水中驯化所得群落作为土著群落,At.thiooxidans A01作为外来菌种分别在第0、24和36天引入(浸矿周期为48天)。结果表明：在没有引种,以及第0、24和36天引种的各个浸矿体系中,最终Cu2+浸出浓度依次为：204.3、215.1、230.8和251.5mg/l。运用RFLP方法对在第36天引种后的土著群落进行分析后发现：在引种前第36天和引种后第48天的群落中,虽然同时检测到L. ferriphilum, At. ferrooxidans, At. caldus, Sulfobacillus sp., At. albertensis和Acidiphilium的存在,但优势种群At. caldus、 L.ferriphilum和At. ferrooxidans在群落中所占比例分别由38.4%、30.4%和17.6%相应变化为29.6%、40%和12%。而在没有引种的第48天的对照组中,我们只检测到了除At. albertensis外的其他5个种群,且At. caldus、L. ferriphilum和At. ferrooxidans的比例为43.2%、36%和10.4%。实时定量PCR和功能基因芯片结果显示：At.thiooxidansA01在第36天引入后首先主要吸附在矿石表面,并在第42天吸附量达到最大值,随后逐渐下降；而浮游的At. thiooxidans A01开始时菌体浓度较低,直到第45天才达到最大值,随后逐渐下降。吸附和浮游的At. caldus和Sulfobacillus sp在引种后的群落中的丰度都相对下降；而L. ferriphilum、At. ferrooxidans和Acidiphilium sp都相对上升。来自L. ferriphilum的编码protoheme ferrolyase和来自At.ferrooxidans的编码sulfide-quinone reductase的基因的丰度不管是在吸附还是浮游样品中,在引种后的群落中都显著增加；相反,来自At. caldus和Sulfolobus sp.的与硫代谢相关的一些基因(如doxD基因)却表现为丰度的下降。而来自At. ferrooxidans, L. ferriphilum和Acidiphilium sp等与金属抗性相关的基因都表现为丰度的增加。更多还原

【Abstract】 Special acidophilic species are usually introduced into the bioleaching systems for constructing and optimizing microbial community during bioleaching process. However, little is known about the mechanism of species introduction into original bioleaching consortia. The built co-culture system or nature bioleaching microbial community, after adaptation, functioned as the original bacterial consortium. In this study, exogenous bacteria, belonging to different metabolic type, were introduced into original bioleaching systems at different growth periods. The introduction mechanism was studied in order to determine the most appropriate introduction type and time for the original bioleaching microbial community, as well as provide some theoretical basis.Four vibrio or spiral-shaped bacteria strains were isolated from acid mineral drainage (AMD) using liquor gradient dilution method. Based on the analysis of purity, physiological property,16S rDNA and gyrB gene sequence, morphology, and bioleaching capacity towards pyrite, the four strains were identified as L. ferriphilum.A co-culture system consisted of At. caldus、L. ferriphilum、 Ferroplasma、Acidiphillum spp.、S. thermosulfidooxidans and At. ferrooxidans was built. It was found that the co-culture system demonstrated superior pyrite bioleaching capacity under the condition (40℃, pH1.5and0.5%(g/100ml) pulp density). Besides, At. caldus、L. ferriphilum and Ferroplasma were the dominant groups compared with At. ferrooxidans、Acidiphillum spp. and S. thermosulfidooxidans. The growth period can be divided into four phases:adaptation phase from0to48hours, rapid-growth phase from48to96hours, stable growth phase from96to192hours, and decline phase from192to240hours.The above co-culture system served as the indigenous community. At. thiooxidans A01, the exogenous species, was introduced into the original consortium at66and138hours, and the pyrite bioleaching rate increased by10%and13%, respectively. Based on the real-time PCR analysis, the introduction of At. thiooxidans A01promoted the growth of L. ferriphilum, Ferroplasma and Acidiphillum spp., inhibited the growth of At. Caldus, and demonstrated inconspicuous effect on the growth of At. ferrooxidans and S. thermosulfidooxidans. Based on the functional gene arrays data, protoheme ferrolyase、 formate hydrogenlyase、ADP heptose、 phosphoheptose isomerase and glycosyltransferase genes of L. ferriphilum, ferredoxin oxidoreductase gene of Ferroplasma sp. and acetyl-CoA carboxylase gene of Acidiphillum spp. were up-regulated from0to20hours after the introduction. In contrast, the doxD gene of At. caldus and ribulose-1,5-bisphosphate carboxylase/oxygenase gene of S. thermosulfidooxidans were down-regulated from0to20hours after the introduction. The ADP-heptose Synthase gene of At. ferrooxidans was up-regulated from0to12hours after the introduction, and then down-regulated from12to20hours.The composition of the above co-culture was slightly adjusted in the following experiment. To be specific, along with the removal of L. ferriphilum, At. thiooxidans was added in the system. Under the same experimental condition, L. ferriphilum YSK serving as the exogenous species, was introduced into the indigenous consortium at0,66and138hours, and the pyrite bioleaching rate increased by9%,14%and19%, respectively. Based on the real-time PCR analysis, the introduction of L. ferriphilum YSK promoted the growth of At. caldus, At. thiooxidans and Acidiphillum spp., inhibited the growth of Ferroplasma and S. thermosulfidooxidans, and demonstrated inconspicuous effect on the growth of At. ferrooxidans. The shift of functional gene expression level from "138hours introduction" was analyzed using the functional gene arrays. It was found that ADP heptose、phosphoheptose isomerase、 glycosyltransferase、Biotin carboxylase and protoheme ferrolyase genes of L. ferriphilum, acetyl-CoA carboxylase gene of Acidiphillum spp. and doxD gene of At. caldus were up-regulated from0to20hours after the introduction. In addition, lipid A disaccharide synthase LpxB、glycosyl transferase and ADP-heptose Synthase genes of At. ferrooxidans showed up-regulated expression from0to8hours after the introduction, and then down-regulated expression from8to20hours. The situation was more complex with ferredoxin oxidoreductase gene of Ferroplasma sp. In detail, this gene was up-regulated from0to4hours, down-regulated from16to20hours, and then up-regulated again from16to20hours after the introduction. CbbS gene of At. ferrooxidans showed down-regulated expression from0to20hours after the introduction.The acidophilic microorganism mixture from different AMD environment was adapted for five generations in chalcopyrite bioleaching system, and then was used in chalcopyrite bioleaching. An increase by45%of the bioleaching rate was observed. According to the RFLP analysis, the microbial community consisted of eight common bioleaching microorganisms, including At. ferrooxidans, At. caldus, Sulfobacillus sp., L. ferriphilum, S. thermosulfidooxidans, Acidiphilium sp., Alicyclobacillus sp. and Pseudomonas sp., however, only the existence of At. ferrooxidans, At. caldus, Sulfobacillus sp., L ferriphilum and Acidiphilium was detected after adaptation. Based on the analysis of functional gene arrays, functional gene abundance of At. Caldus, L.ferriphilum, Sulfobacillus sp., Sulfolobus sp. and Acidiphilium sp. was increased along with the adaptation, however, the abundance of S. thermosulfidooxidans、Alicyclobacillus sp. and At. ferrooxidans was through a decline. The functional genes of At. caldus and Sulfolobus sp. involved in sulfur metabolism, demonstrated increasing gene abundance, while the functional genes of At. ferrooxidans involved in sulfur metabolism showed decreasing abundance. In addition, protoheme ferrolyase gene, functional genes involved in iron metabolism of L. ferriphilum showed increasing abundance. The functional genes involved in metal resistance of At. ferrooxidans, At. caldus, L. ferriphilum, Sulfolobus spp. and Acidiphilium sp. also demonstrated increasing abundance.At. thiooxidans A01, the exogenous species, was introduced into the indigenous consortium, the adapted microbial community from AMD environment, respectively at the0th,24th and36th day (bioleaching cycle spanned48days). The results showed that the copper ion concentration was respectively204.3,215.1,230.8and251.5mg/L in the bioleaching system without and with introduction of At. thiooxidans A01at0th,24th and36th day. Microbial community structure was analyzed using RFLP method. After the inroduciton of At. thiooxidans A01, it suggested that L. ferriphilum, At. ferrooxidans, At. caldus, Sulfobacillus sp., At. albertensis and Acidiphilium were together detected in bioleaching all the way. Moreover, the proportion of dominant groups, namely, At. caldus、L. ferriphilum and At. ferrooxidans, in these two systems, changed from38.4%,30.4%and17.6%at36th day to29.6%,40%and12%at48th day, respectively. However, only five microbial species except for At. albertensis were detected in the control group without introduction of At. thiooxidans A01. The real-time PCR and functional gene arrays data suggested that At. thiooxidans A01, introduced at the the36th day, primarily attached on the mineral surface, and the adsorbance reached the peak at the42th day, followed by the decreasing amount. For the supernatant At. thiooxidans A01, the cell number was initially low, reached the peak at the45th day, and then turned into a decreasing tendency. The abundance of both attached and free At. caldus and Sulfobacillus sp. was decreased after the introduction, while, that of L. ferriphilum, At. ferrooxidans and Acidiphilium sp. were all increased. The abundance of protoheme ferrolyase gene of L. ferriphilum and sulfide-quinone reductase gene of At. ferrooxidans were increased after introduction in both attached and free bacteria. However, the genes involved in sulfur metabolism, such as doxD gene, showed decreasing abundance. Moreover, the metal resistance genes of At. ferrooxidans, L. ferriphilum and Acidiphilium sp. showed an increasing abundance.更多还原