Isolation, Identification Of Thermophilic Leaching Microorganisms And Basic Studies On Their Application And Bioleaching Potential

To obtain thermophilic sulfide mineral-oxidizing microorganisms for basic and applied studies,fourteen samples of acidic hydrothermal spring and acid mine drainage were collected from Guangdong,Yunnan, Jiangxi and Taiwan in China.From these samples more than thirty strains were isolated with a number of unique methods and identified by morphological,physiological and molecular analyses.The isolated strains can be classified into six species,with twenty-one strains in Acidithiobacillus caldus,one in Sulfobacillus thermosulfidooxidnas,one in Alicyclobacillus sendaiensis,two in Acidianus manzaensis,two in Sulfolobus metallicus and three in Metallosphaera sedula.However,only the strains belonging to the former four species are included in this dissertation.1)Isolation and identification of Acidithiobacillus caldusA total of twenty-one moderately thermo-acidophilic strains were isolated from fourteen samples of acidic hydrothermal spring and acid mine drainage,collected at several geographically insulated sites in China. These strains were rod-shaped and G^-,grew aerobically by oxidizing sulfur to sulfuric acid at optimum temperatures and pHs of 35-55℃and 2.0-3.0.Phylogenetic analyses based on 16S rRNA gene sequences showed that all of them belonged to Acidithiobacillus caldus,with 16S rDNA similarities over 99%between At.caldus and the strains.On the basis of these results,the 21 strains should be grouped into At.caldus.2)Random amplified polymorphic DNA analysis of Acidithiobacillus caldus strains recovered from different extreme environmentsGenomic DNA was extracted from each isolated strain,and PCR for each template DNA was carried out using fourteen 10-mer primers after the PCR conditions were carefully optimized.A RAPD(Randomly Amplification Polymorphic DNA)profile of 264 distinctive banding patterns was obtained,in which 1602 reproducible and distinguishable bands were included with an average of 114 bands per primer.The banding patterns were compared by scoring each DNA band as present or absent,and a genetic similarity matrix was constructed with Jaccard's coefficient.The matrix of generated similarities was analyzed by the unweighted pair-group method with arithmetic average(UPGMA cluster analysis)and a dendrogram was then built up.The Jaccard's coefficient demonstrated a similarity range from 0.438 to 0.884 between the strains and the twenty-two At.caldus strains studied were clustered into three groups at 0.6 similarity level by UPGMA clustering.The results of this study suggest that the strain variabilities in At.caldus are correlated with chemical variations in environments and RAPD analysis is a sensitive and reliable method for strain diversity assay in At.caldus if the RAPD-PCR conditions are carefully optimized.3)Heavy metal resistance of Acidithiobacillus caldus strainsTen moderately thermophilic sulfur-oxidizing strains(AN2,DB2,JX2,YN1,YN2,YN3,YN6,YN12,YN15 and TW26)in At.caldus and five heavy metal sulfates(CuSO₄·5H₂O,ZnSO₄·7H₂O,NiSO₄·6H₂O, 3CdSO₄·8H₂O and CoSO₄·7H₂O)were selected for heavy metal resistant tests.YN12 exhibited a very high degree of resistance to cadmium(â…¡), zinc(â…¡)and cobalt(â…¡),with original maximum tolerated concentrations (MTC)of 4.8,12.0 and 9.0 g·l^-1respectively.DB2 and YN2 were the most resistant to copper(â…¡)and Nickel(â…¡),with MTCs of 20 and 120 g·L^-1.YN12 could obtain an ultimate resistance of 31.5 g·l^-1Cd²⁺after adapted by a gradually increased concentration of 3CdSO₄·8H₂O. Continuously cultured in the medium containing 210 g·l^-13CdSO₄·8H₂O (equal to 31.5 g·l^-1Cd²⁺)for three generations,the strain was capable of restoring its growth and sulfur oxidation activity to an approximately normal level.The result of this study indicates that YN12 may play a potential role in cadmium-rich sulphide mineral bioleaching and cadmium pollution control.4)Isolation and identification of Sulfobacillus thermosulfidooxidans A moderate acidothermophilic bacterial strain,YN22,was isolated from an acid hot spring in Tengchong,Yunan,China.The gram-positive-staining cells of the strain were straight to slight curved rods,1.6-2.8μm long and 0.4-0.7μm in diameter.The temperature range for growth was 30 to 60℃with an optimum at 53℃.The pH range for growth at 53℃was from 1.0 to 5.0 with an optimum at 1.5.YN22,the chemoautotrophic strain,was capable of oxidizing Fe²⁺to Fe³⁺in media containing 0.02%(w/v)yeast extract to support fast growth,but did not effectively utilize sulfur or reduced sulphides as energy source.16s rRNA gene Analysis revealed this strain closely related to Sulfobacillus thermosulfidooxidans,with over 99%sequence similarity between each other.The G+C contents of DNA were also very similar between Sb. thermosulfidooxidans and strain YN22,which were 47.5 and 47.3 mol% respectively.Based on phenotypic,biochemical and genotypic analyses, YN22 is identified as a new strain of Sb.thermosulfidooxidans.It is the first time for this species to be isolated and identified in China.5)Jarosite-type precipitates mediated by YN22,Sulfobacillus thermosulfidooxidans,and their influences on the strainTo have a better understanding on the properties of the jarosite-type precipitate synthesized by Sb.thermosulfidooxidans,the evolution of the Sb.thermosulfidooxidans-mediated precipitation and the influence of the precipitate on this species,a newly isolated strain(YN22)of Sb. thermosulfidooxidans,was cultured in a medium containing Fe²⁺as energy source under optimal conditions(pH1.5,53℃,0.2g·L^-1yeast extract,30g·L^-1Fe₂SO₄·7H₂O and 170r·min^-1),added with or without glass beads.Remarkable differences were found in the oxidation rate of Fe²⁺,the precipitate yield of jarosite-type compounds and the population density between the two groups of cultures.The group with glass beads has a 6 h faster Fe²⁺oxidation,6 h earlier precipitation,78%higher precipitate yield and much lower population density than those without glass beads.XRD,EDS,FTIR and SEM analysis reveals that the precipitates originated from both groups are a mixture of potassium jarosite and ammoniojarosite,with morphological features similar to the latter.The results of the test referring to influence of the precipitates on YN22 show that the precipitate from the group without glass beads has no apparent influence on Fe²⁺oxidation rate of YN22 and only a limited influence on growth of the strain,whereas that from the group with glass beads remarkably inhibits the growth and Fe²⁺oxidation ability of YN22 when a precipitate content over 4 g·L^-1is used.6)Bioleaching of chalcopyrite and pyrite with Sulfobacillus thermosulfidooxidans strain YN22Bioleaching of chalcopyrite and pyrite by moderate thermophilic bacterium Sb.thermosulfidooxidans strain YN22 was studied.Copper dissolution reached 46.2%with the mixed culture of strain YN22 and strain YN2,a sulfur-oxidizing bacterium of Acidithiobacillus caldus,and 14.7%with the pure culture of Sb.thermosulfidooxidans strain YN22,at 53℃and 3%(w/v)pulp density,after 25 days.These results suggest that maximum metal recovery can be achieved by mixing strain YN22 with moderate thermophilic sulfur-oxidizer like A.caldus in leaching primary sulfide minerals such as chalcopyrite.However,rate data collected in a 25 d period of pyrite leaching with pure YN22 indicate that strain YN22 is an effective moderate thermophile for pyrite leaching.7)Isolation,characterization of heterotrophic acidothermophilic bacterial strainYNTC-1 and its influence on chalcopyrite and pyrite leaching by strain YN22A heterotrophic acidothermophilic bacterial strain,YNTC-1,was isolated from the same enriched sample of an acid spring from which Sb. thermosulfidooxidans strain YN22 was purified.Based on phenotypic and genotypic analyses,YNTC-1 was identified as a member of A. sendaiensis.Considering some important morphological and biochemical differences between the two bacteria,YNTC-1 may be proposed to be a novel subspecies of A.sendaiensis.However,this view has to be confirmed by further studies.Co-bioleaching of pyrite and chalcopyrite with Sb.thermosulfidooxidans shows that strain YNTC-1 has no evident influence on leaching rates of these two sulphide minerals.8)Isolation and identification of Acidianus manzaensis Two novel thermoacidophilic iron and sulfur-oxidizing archaeon strainsYN25 and YN26,were isolated from an in situ enriched acid hot spring sample collected in Yunnan,China,and identified as Acidianus manzaensis through morphological,physiological and molecular studies. Up to date,there are only three strains isolated and identified all over the world,Strain NA-1^T at NBRC and ATCC,Strain YN25 and YN26 at CSU.YN25 and YN26,the first found strains of A.manzaensis in China, will play an important role in basic and applied studies of thermophilic leaching microbes.9)Bioleaching of chalcopyrite and pyrite by A.manzaensis strain YN25Bioleaching ability of A.manzaensis strain YN25 for chalcopyrite and pyrite was tested.The results demonstrate that strain YN25 is one of the most effective sulfide oxidizers recorded,with a copper leaching rate of 79.16%for chalcopyrite after 24 d and Cu²⁺tolerated concentration increased by 80%through continuous leaching of chalcopyrite.Also YN25 appeared to dissolve more iron when bioleaching pyrite and the Fe_totalconcentration in YN25-bioleaching liquor was 2.4 times higher than that in YN22-bioleaching liquor after 28 d.It is the first time for this thermophilic archaea to be tested in sulfide mineral leaching.The parameters from these experiments may be used to evaluate the application potential of this species for sulfide ore bioleaching.