极端嗜酸硫杆菌在含砷难处理金矿的氧化预处理过程中的抗砷机制研究

Arsenic has obvious toxic effect to organisms. Many organisms can only tolerate very low dosage of arsenic. Arsenic in the environment is always found to be associated with several sulfide minerals like gold ore. However, high concentration of arsenic compounds existed in the arsenic-bearing refractory gold ores inhibits growth of a large number of bioleaching microorganisms and impedes the oxidation pretreatments of ores. Until now, some novel arsenic resistance genes and mechanisms have been proved to play key roles in certain microorganism strains which could resist high concentration of arsenic, besides those typical arsenic resistance operons such as arsRDABC and arsRBC found in Escherichia coli and other common strains. On the other hand, there have been only a few reports about arsenic resistance behaviors and mechanisms of Acidithiobacillus spp. in the bioleaching, and no paper on arsenic resistance mechanisms of Acidithiobacillus thiooxidans has been reported. Therefore, arsenic resistance mechanisms of microorganisms have not been illuminated completely. In our previous work, several strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans were successfully isolated and applicated in the oxidation pretreatments of arsenic-bearing refractory gold ores, and it was demonstrated that the arsenic resistance concentrations were higher than those of all the bioleaching microorganisms reported. In this study, differences between arsenic resistance mechanisms from different sources will be compared among arsenic resistance behaviors and mechanisms of several resistant strains of Acidithiobacillus spp. Furthermore, the hereditary conservations of ars operon and transposons, and whether there are some unknown arsenic resistance genes or mechanisms will be examined.

砷对生物体具有较明显的毒害作用，许多生物仅能耐受较低浓度的砷。自然环境中的砷多数与金等矿石伴生，含砷难处理金矿中含有的大量砷阻碍了多数浸矿微生物的正常生长和对矿石的氧化预处理。目前，除了在大肠杆菌等微生物中证实了arsRDABC和arsRBC等典型的砷抗性操纵子以外，还陆续在能耐受较高浓度砷的菌株中发现了一些新的抗砷基因和抗砷机制。关于极端嗜酸硫杆菌在微生物浸矿中的抗砷行为和机制的报道为数不多，尚未见关于嗜酸氧化硫硫杆菌抗砷机制的报道，微生物的抗砷机制至今仍未被完全解释。在我们的前期工作中，分离得到了若干株嗜酸氧化亚铁硫杆菌和嗜酸氧化硫硫杆菌，研究发现它们的抗砷浓度超过了目前已有报道的所有浸矿微生物。在本项目中，拟通过对比不同来源的抗性菌株之间抗砷机理的差异，考证传统的砷抗性操纵子、转座子模型的保守性以及是否存在新的抗砷基因及抗砷机制。

氧化亚铁硫杆菌等极端嗜酸硫杆菌，正在广泛应用于金、铜等贵金属和稀有金属的生物浸出。.在本项目中，分离、鉴定到了11株氧化亚铁硫杆菌，4株氧化硫硫杆菌，2株氧化亚铁钩端螺旋菌，2株隐藏嗜酸菌，1株疑似新菌（属）。微生物多样性分析结果发现煤矿GNM物种种类最丰富，LNJ1与LNZJK样品中细菌组成最相似。所有样品微生物归为Actinobacteria，Bacteriodetes，Firmicutes和Proteobacteria四大门，其中Proteobacteria分布最广。以氧化亚铁硫杆菌所代表的Acidithiobacillus属在GNM样品中丰度最高。.分别考察了初始pH值、初始Fe2+浓度、接种量、底物对极端嗜酸硫杆菌生长的影响；分别研究了氧化亚铁硫杆菌的菌落形态、细胞显微结构、生长曲线、硫氧化速率、铁氧化速率等生物学特征。结果发现，多株氧化亚铁硫杆菌的生长具有明显优势。.选择了五种重金属离子对氧化亚铁硫杆菌进行耐受性实验。结果表明：氧化亚铁硫杆菌具有较强的Cu2+和Cd2+抗性，Cu2+和Cd2+胁迫后，菌体细胞明显变小，近似于球状，表面粗糙，但基因组仍能保持完整。不同浓度、不同种类的重金属胁迫前后，铁氧还蛋白的表达存在显著差异，且Cd2+对菌体生长的影响比Cu2+明显。.对铁氧还蛋白基因fd-1、fd-2进行克隆、转化，并在E. coli BL21 (DE3)中进行了表达。Fd-1和Fd-2分别在25℃下加入0.5 mM IPTG诱导6 h、25 ℃下加入0.3 mM IPTG诱导6 h，可获得最大表达量。运用生物信息学分析软件对基因编码的氨基酸序列的理化性质、结构特征、功能结构域、可能参与的代谢通路等进行了预测。推测Fd-1参与胞内核酸转移和代谢，Fd-2作为光合作用和铁氧还蛋白的电子载体，在电子传递过程和各种酶促反应中起着重要作用。.采用RT-qPCR研究了氧化亚铁硫杆菌在Cu2+、Cd2+胁迫下基因afe-1948、afe-1862、afe-RS11495的差异表达，结果显示：这三个基因对Cu2+、Cd2+非常敏感，表达量随Cu2+、Cd2+浓度的增加而增大。在Cu2+为0.04 mol/L或Cd2+为0.06 mol/L时，表达量达到最大。生物信息学分析推测AFE-1948、AFE-1862、AFE-RS11495均参与金属离子的吸附转运等机制。