【作者】 高健；

【导师】 邱冠周；

【作者基本信息】 中南大学 ， 矿物加工工程， 2007， 博士

【摘要】 各种证据表明，在温度≥40℃、pH≤1.4时，钩端螺旋菌在工业连续流生物反应槽中属于主要的铁氧化细菌。目前，在钩端螺旋菌属现存的三个种当中，仅仅嗜铁钩端螺旋菌能够在45℃生长，此外，除了两个抗砷基因被鉴定外，有关这个种的其他生物学特性的报道非常有限。因此，它已成为未来生物学研究的热点。然而，由于纯培养的困难，有限的嗜铁钩端螺旋菌菌株被分离。在本研究中，以嗜铁钩端螺旋菌为主要研究对象，开展了其分离方法、生理生化特性、分子生物学鉴定和磁小体的提取等研究工作，其主要内容如下：1．建立了一种分离嗜铁钩端螺旋菌的选择性方法。目前，分离嗜铁钩端螺旋菌的最成功方法是双层平板法。其特点是采用了双层平板以及异养细菌-Acidiphilium SJH的加入。尽管这种方法在铁氧化细菌的分离方面非常有效，但双层平板的制备比较繁琐，加入的SJH菌株也并不容易获得。因此，我们根据钩端螺旋菌适宜的生长温度、pH和某些生理生化特性，建立了一种相对简单有效的选择分离性方法，其主要特点是给嗜铁钩端螺旋菌提供有利的生长条件，如低pH(≤1.4)、较高培养温度(≥40℃)和较高氧化还原电位等。基于这种方法，经过连续稀释，我们获得了12株嗜铁钩端螺旋菌纯培养物。此外，还用酵母HJM代替SJH菌株进行了双层平板分离嗜铁钩端螺旋的尝试。2．以YSK菌株为研究对象，从形态学、生理生化及分子水平等方面进行了鉴定。钩端螺旋菌属的各个成员之间在形态学和生理学上并没有明显的差异。形态学表明，YSK菌株可能属于一株氧化亚铁钩端螺旋菌。然而，基于以下几种证据，证明YSK菌株属于嗜铁钩端螺旋菌：①45℃时的生长能力。②与嗜铁钩端螺旋菌典型菌株Fairyiew的16S rDNA序列的100％的一致性；③基因组G+C的相对百分含量为58.7％；④与表3—2中其他菌株间16S-23S间隔区大小的差异；⑤对黄铁矿高效的浸出能力与对黄铜矿有限的浸出能力。3．嗜铁钩端螺旋菌gyrB基因的PCR扩增与克隆有关嗜铁钩端螺旋菌gyrB基因的研究还未见相关报道。在本研究中，采用本研究室设计的一对寡核苷酸简并引物，首次报道了嗜铁钩端螺旋菌gyrB基因的PCR扩增与克隆，该基因的克隆为嗜铁钩端螺旋菌分子系统学的研究提供了分子标记。4．嗜铁钩端螺旋菌的16S rDNA和gyrB基因的序列分析从功能基因gyrB和16S rRNA基因的角度探讨了嗜铁钩端螺旋菌不同菌株间的系统发育关系。结果表明：①探测到了14个信号位点，其中9个信号位点分布于470—490位点之间。这些信号位点很有可能被用作识别嗜铁钩端螺旋菌不同菌株间的一种分子标记；②15株嗜铁钩端螺旋菌可分成明显的三组。相似性比较发现，系统发育间的相似性与矿物学特征间很可能存在某种必然的相关性；5．磁小体的分离与纯化首次通过蔗糖密度梯度离心和SDS处理法，从YSK菌株中分离出了纳米磁性颗粒。透射电境表明，磁性颗粒近似球形，平均直径为44nm。颗粒的大小属于单磁畴结构；能谱分析表明，这种磁性纳米颗粒主要由铁和氧两种元素组成。由此初步推断，这种颗粒就是磁小体。6．不同铁氧化细菌作用下形成的沉淀差异性分析通过ICP-AES，SEM，XRD和FTIR等方法，在不同温度条件下，对不同铁氧化细菌作用下形成的赭黄色高铁沉淀进行了分析。结果表明，无论是嗜铁钩端螺旋菌DY菌株(L. ferriphium)，还是嗜酸氧化亚铁硫杆菌GF菌株(At. ferrooxidans)作用下形成的沉淀都是黄钾铁矾和黄铵铁矾的混合物。其中，GF作用下形成的沉淀混合物中其黄钾铁矾的含量比DY作用下形成的黄钾铁矾约高出5.53％，而DY作用下形成的沉淀中，其黄铵铁矾的含量比GF作用下形成的沉淀中的黄铵铁矾的含量约高出15.24％。7．酸性矿坑水中极端嗜酸又能耐受中强碱酵母的分离与鉴定采用平板划线分离法，从江西某铜矿酸性矿坑水中分离出一株极端嗜酸、又能耐受中度碱性条件的异养微生物，命名为HJM菌株。该菌株能在pH1.5-10.0的范围内生长。形态学以及18S rDNA和26S rDNA D1／D2区序列分析表明，HJM菌株属于P. guilliermondii。金属抗性试验表明，该菌株对重金属铜离子的抗性可高达45mmol／L，因此，它的分离为研究极端环境中微生物的抗铜机制提供了材料。更多还原

【Abstract】 All kinds of evidences indicate that Leptospirilla are the primaryiron-oxidizers in industrial continuous-flow biooxidation tanks. Presently,of the three species documented, the species L. ferriphilum, as a newlyisolated bioleaching bacterium, has been the focus of biology researchdue to its ability to grow at 45℃and limited reports about its biologicalcharacteristics except that two arsenic-resistant genes were identifiedfrom the L. ferriphilum strain Fairview.However, as a result of difficulty to cultivate on solid media, thelimited L. ferriphilum strains have been isolated. In this work, studies onmethod for the isolation, physio-biochemical characteristics, molecularidentification and magnetosomes of L. ferriphilum were carried on, withL. ferriphilum being developed objective of this research. The primarysubjects in this dissertation are as follows:1. A selective method for isolating L. ferriphilum was set upAt present, the most successful approach to using laboratory mediahas been the development of a double-layer plate technique whichinvolve an acidophilic heterotrophic bacteria designated as AcidiphiliumSJH. Although the technique is efficient in isolating the iron-oxidizingbacteria, its need for acidophilic heterotrophic bacteria makes the processfor isolation very complex. In this study, we set up a new selectiveisolation strategy that mimicked physiological characteristics ofLeptospirilla, such as pH, temperature and its less sensitivity to the highferric-ferrous iron ratio. Based on this method, 12 strains of L.ferriphilum were obtained by the serial dilution. Furthermore, an attemptwas made to isolate L. ferriphilum by a double-layer plate technique inwhich yeast HJM was substituted for Acidiphilium SJH.2. Strain YSK was selected as a typical research objectStudies of mesophilic leptospirilla indicate that there are not distinctdifferences between mesophilic leptospirilla in morphology andphysiology. The cell shape suggested that strain YSK was likely a strainof Leptospirillum ferrooxidans. However, strain YSK was identified as L. ferriphilum and the evidences obtained were as follows:1 the ability to grow at 45℃;2 sequence analysis of 16S rRNA gene showed that strain YSKhad 100% sequence similarity with a typical Leptospirillumferriphilum strain Fairview;3 the G+C moles percent rates of strain YSK was 58.7%;4 the differences in size of the IRs between strain YSK andother strains listed in Table3-2;5 efficient ability to leach iron from pyrite and limited ability toleach copper from chalcopyrite.3. PCR amplification and cloning of gyrB gene from L.ferriphilumIn this work, according to two conserved regions PGMYIG andQRY(F)KGLGEM of the amino acid sequences of the DNA gyrasesubunit B proteins of bacteria from Genbank database and ICB database,the gyrB gene was first amplified and cloned from L.ferriphilum strainYSK with a pair of degenerate primer designed by ourself in ourlaboratory. Thus, the gyrB gene will be a molecular maker for researchingmolecular systematics of L.ferriphilum.4. Sequences analysis of 16S rDNA and gyrB gene from L.ferriphilumFrom the aspects of functional gene (gyrB gene) and evolutionarilyconserved gene (16S rRNA gene), respectively, phylogeneticrelationships between different L.ferriphilum strains were studied. Thisresult showed that nucleotide variables were detected in 14 positions andmost of them were found in the first 600 bp from 5’ terminal exceptposition 1019, 1023 and 1275. Interestingly, 9 positions were found in the470 to 490 bp, and these signature sites could be used as molecular makerto discern the subspecies of L.ferriphilum. Moreover, comparativeanalysis revealed that there are some certain links between phylogenicsimilarity and the mineralogical characteristics.5. Extraction and purification of magnetosomesThe magnetic nanoparticles were first extracted from Leptospirillumferriphilum strain YSK, isolated from acid mine drainages by treatmentwith sodium dodecyl sulfate (SDS) and centrifugation through a sucrosedensity gradient. TEM indicated that the nanoparticles were approximately spherical with a mean diameter of 44nm, and magnetitecrystals in this size range are single magnetic domains; Energy-dispersiveX-ray analysis showed that the nanoparticles primarily contained twokinds of elements, iron and oxygen. Thus it can be concluded that themagnetic particles are magnetosomes.6. Discrepant analysis of the iron precipitates formed by differentiron-oxidizing bacteriaThe yellow ochre-colored iron precipitates formed by two strains ofdifferent iron-oxidizing bacteria at different temperature were analyzedby using ICP-AES, SEM, XRD and FTIR. The results indicated that theprecipitates produced by L. ferriphium strain DY and At. ferrooxidansstrain GF were composed of jarosite and ammonium jarosite. However,the content of jarosite in the precipitates formed by strain GF was 5.53%higher than that formed by strain DY, but, the content of Ammoniumjarosite in the precipitates formed by strain DY was 15.24% higher thanthat formed by strain GF.7. Characteristics and molecular identification of a strain of heterotrophicmicroorganism isolated from acid mine drainageBy streak plate method, a strain of heterotrophic microorganismwhich was extremely acidophilic, alkalitolerant, named by HJM, wasisolated from an acid mine drainage of a certain copper mineral in JiangxiProvince. This strain was able to grow under pH value of 1.5-10.0.Morphological characteristics and sequences analysis of 16S rDNA and26S rDNA D1/D2 region revealed that it belonged to the species P.guilliermondii. Resistant experiment to metals showed that it could resistcopper of 45mmol/L, so it was an important strain used to study theresistance mechanism of copper.更多还原