【作者】 刘宏伟；

【导师】 刘学端；

【作者基本信息】 中南大学 ， 生物学， 2013， 博士

【摘要】 化能自养微生物嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)与嗜酸异养菌Acidiphilium acidophilum之间在生物浸出及酸性矿坑水(AMD)之类极端酸性环境中存在的协同作用已引起广泛关注,但仍缺乏从生理和基因水平对其进行的全面而深入的研究。为深入了解此两种微生物之间的协同作用及其在生物浸出体系和AMD等极端酸性环境中的生态功能,并为AMD环境的修复提供一定参考,本文对这两种微生物组成的共培养进行了一系列的实验研究。本文研究内容主要包括：(1)评估RT-qPCR定量At. ferrooxidans和Aph. acidophilum细胞的相对数量的准确度；(2)用此两种微生物进行长期驯化形成共培养体系,然后对此两物种微生物群落的生长和生理活性进行检测；(3)从转录水平研究了At. ferrooxidans与碳代谢和铁代谢相关基因在不同培养体系中的表达差异；(4)检验此共培养体系分别在Cd2+, Cu2+, Ni2+和Mg2+胁迫下的稳定性；(5)共培养体系受到葡萄糖抑制时的生长和生理活性；(6)将此共培养体系应用于黄铁矿和低品位黄铜矿的生物浸出实验。上述研究所得结果如下：(1)利用RT-qPCR定量At. ferrooxidans以及Aph. acidophilum这两种微生物的相对数量时,用于DNA提取的培养物中微生物细胞总量应在6.52×109cells至2.61×1010cells之间,能得到准确的定量结果,并且在两种微生物混合的条件下其各自的特异性引物能确保实验结果互不产生干扰；(2)成功的构建由At. ferrooxidans和Aph. acidophilum这两种微生物组成的共培养物,并基于其RT-qPCR定量的生长动态变化可知,共培养体系的At. ferrooxidans能更快的进入对数生长期并且对数生长期延长2天以上,数量较其在纯培养中高出5倍以上。(3)亚铁氧化及其与铁代谢相关的基因表达研究结果表明,共培养中的At. ferrooxidans对能源的获取能力明显高于其纯培养。另外,对At. ferrooxidans中与CO2固定相关基因表达的结果分析说明编码RuBisCO的第二套结构基因cbbLS-2和正向调控基因cbbR在共培养体系中的表达均上调。(4)在Cd2+, Cu2+, Ni2+和Mg2+这4种金属离子分别存在的条件下异养菌Aph. acidophilum均能促进At. ferrooxidans对亚铁的氧化,提高其对能源利用的效率。共培养体系中的异养菌Aph. acidophilum使At.ferrooxidans对Cu2+的最大耐受浓度(MTC)由2.0g/L提高到5.0g/L而且共培养在5.0g/L Cu2+条件下的细胞数量与2.0g/L Cu2+条件下生长的At. ferrooxidans纯培养相似。另外,共培养中的At. ferrooxidans对Mg2+的MTC也由12.0g/L提高到17.0g/L。(5)无论是否加入葡萄糖,共培养对Fe2+氧化的效率均较At.ferrooxidans纯培养高。当葡萄糖浓度为5g/L时,At. ferrooxidans纯培养失去对Fe2+的氧化能力,而共培养仍能在100h内将所有的Fe2+氧化完,且加入葡萄糖越多的培养体系氧化终点的pH值也越高。在不加入葡萄糖的条件下At. ferrooxidans与Aph. acidophilum数量比在100：1的数量级,表明以这两种菌为代表的自养菌和异养菌在自然条件下生物量的比例。无论纯培养还是共培养的At. ferrooxidans数量均随葡萄糖浓度的提高而减少,且延滞期则变长；而异养生长的Aph. acidophilum则相反。(6)生物浸出实验中嗜酸异养菌Aph. acidophilum促进了At. ferrooxidans对黄铁矿样品的浸出,浸出率较其纯培养提高了22.7%；但在含铁量较低的低品位黄铜矿浸出体系中共培养和At. ferrooxidans纯培养的浸出率均低于33%。在加入2.0g/L Fe2+的低品位黄铜矿浸出体系中,共培养和At. ferrooxidans纯培养的浸出率均得到提高,分别达到52.22%和41.27%。综合上述所有结果表明,经长期驯化成功构建了由At. ferrooxidans和Aph. acidophilum组成的共培养体系,且利用RT-qPCR能准确定量此共培养中两种微生物的相对数量变化。异养菌Aph. acidophilum能与At. ferrooxidans一起在共培养中进行异养生长并对At. ferrooxidans的生长有促进作用；At. ferrooxidans通过上调表达与亚铁氧化相关的基因和第二套cbbLS结构基因来提高对亚铁的氧化和CO2的固定。由于Aph. acidophilum能促进At. ferrooxidans对亚铁的氧化,并能缓解或消除葡萄糖对At. ferrooxidans的抑制,所以不能以加入类似于葡萄糖的有机物作为AMD环境生物修复的手段,且适合进行Fe2+氧化的At. ferrooxidans与Aph. acidophilum的数量比例范围应在100：1的数量级。Aph. acidophilum与At. ferrooxidans共培养在一定的环境胁迫下仍能保持其稳定性并完成各自的生态功能,并且嗜酸异养菌Aph. acidophilum适合在含铁量较高的浸出体系中与铁氧化细菌共同作用来提高生物浸出的效率。更多还原

【Abstract】 Although, the synergetic interactions between chemolithoautotroph At. ferrooxidans and heterotroph Aph. acidophilum in bioleaching and acid mine drainage (AMD) environment have drawn a share of attention, in-depth research regarding synergetic interactions are still unknown on physiological and transcriptional level. To gain a better understanding of the synergic interactions and ecological functions between these two species that commonly occurred in bioleaching system and AMD environment, a series of research regarding the co-culture of these two species have been conducted.The content of researches included:(1) evaluation of the accuracy of RT-qPCR quantified growth dynamics of At. ferrooxidans and Aph. acidophilum;(2) a co-culture composed of At. ferrooxidans and Aph. acidophilum were successfully acclimated in this study, the growth dynamics and physiological activity were monitored;(3) the expression difference of carbon and iron metabolism related genes between At. ferrooxidans pure culture and its co-culture with Aph. Acidophilum was studied;(4) the stability of co-culture which consists of Aph. acidophilum and At. ferrooxidans separately exposed to four metal ions (Cd2+, Cu2-, Ni2+and Mg2+) was tested;(5) the growth dynamics and physiological activity of At. ferrooxidans and its natural co-culture with Aph. acidophilum in media with or without glucose were measured respectively;(6) this co-culture was also applied to bioleaching of pyrite and low grade chalcopyrite.The results of these researches are listed as follow:(1) for accurate relative cell number quantification of the At. ferrooxidans and Aph. acidophilum, the total cell number in culture sample should be between6.52×109cells and2.61×1010cells; and the specific primers of these two species ensured the specificity of the results respectively;(2) A co-culture composed of At. ferrooxidans and Aph. acidophilum has been successfully acclimated in this study, and depending on the RT-qPCR quantified growth dynamics, the At. ferrooxidans in co-culture entered earlier and had2days longer exponential phase, obtained5times more cell number than that in pure culture.(3) the ferrous iron concentration in culture medium and the expression of iron oxidation related genes revealed that the energy acquisition of At. ferrooxidans in co-culture was more efficient than that in pure culture. Furthermore, the analysis of CO2fixation related genes in At. ferrooxidans indicated that the second copy of RuBisCO encoding genes cbbLS-2and the positive regulator encoding gene cbbR were up-regulated in co-culture system;(4) In the Cd2+, Cu2+, Ni2+and Mg2+metal resistance experiment, heterotrophic bacteria Aph. acidophilum facilitated the ferrous iron oxidation by At. ferrooxidans and improved its efficiency of energy utilization. The maximum tolerant concentration (MTC) of At. ferrooxidans to Cu2+was improved from2.0g/L to5.0g/L by Aph. acidophilum, and the cell density of co-culture in5.0g/L Cu2+was almost the same as purely cultured At. ferrooxidans in2.0g/L Cu2+. In addition, the MTC of co-cultured At. ferrooxidans to Mg2+was also improved from12.0g/L to17.0g/L by Aph. acidophilum.(5) whether glucose was added in culture media or not, the Fe2+oxidation efficiency of At. ferrooxidans is higher in co-culture than that in pure culture. When the concentration of glucose is5g/L, pure culture of At. ferrooxidans couldn’t oxidize Fe2+while the co-culture could finish the Fe2+oxidation in100h, and the pH is higher when more glucose was added in both cultures. Without glucose, the cell number ratio of At. ferrooxidans to Aph. acidophilum in co-culture was about100:1, which suggested the usual cell number ratio between autotrophic bacteria and heterotrophic bacteria in AMD environment. In both pure and co-culture condition, the cell number of At. ferrooxidans decreased and the lag phase prolonged with the increase of glucose concentration; while in the case of Aph. acidophilum in co-culture, the cell number and lag phase showed a reverse trend;(6) In bioleaching experiment, the pyrite bioleaching efficiency of co-culture increased by22.70%as compared with that of purely cultured At. ferrooxidans. While in the low grade chalcopyrite bioleaching system with few iron, the bioleaching efficiency of both At. ferrooxidans and its co-culture with Aph. acidophilum were lower than33%. In the low grade chalcopyrite bioleaching system with pre-added2g/L Fe2+, the bioleaching efficiency of At. ferrooxidans and its co-culture with Aph. acidophilum were raised to41.27%and52.22%, respectively.In conclusion, a co-culture composed of At. ferrooxidans and Aph. acidophilum were successfully acclimated in this study and the relative cell number can be quantified accurately. Aph. acidophilum could heterotrophically grow with At. ferrooxidans and promote the growth of it. By means of activating iron oxidation related genes and the2nd set of cbbLS genes in At. ferrooxidans, the Aph. acidophilum facilitated the iron oxidation and CO2fixation by At. ferrooxidans. Since Aph. acidophilum facilitated the Fe2+oxidation by At. ferrooxidans and reduced the inhibition by glucose, the addition of organic compounds such as glucose may not be a good AMD bio-remediation strategy. For efficient Fe2+oxidiation, the proper cell number of At. ferrooxidans should be100times higher than that of Aph. acidophilum. At. ferrooxidans and Aph. acidophilum in co-culture could maintain their physiological stability and sustain their ecological function under environmental stress. The bioleaching results suggested that acidophilic heterotrophic bacteria Aph. acidophilum should be applied to the bioleaching system with high iron concentration, in which it could collaborate with iron oxidation bacteria to improve the bioleaching efficiency.更多还原