Studies On Selective Isolation, Identification And Characteristics Of Leptospirillum Ferriphilum From The Extreme Environments

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.