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Isolation and Characterization of Ferroplasma Thermophilum Sp. Nov

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Isolation and Characterization of Ferroplasma Thermophilum Sp. Nov http://www.paper.edu.cn Journal of Applied Microbiology ISSN 1364-5072 ORIGINAL ARTICLE Isolation and characterization of Ferroplasma thermophilum sp. nov., a novel extremely acidophilic, moderately thermophilic archaeon and its role in bioleaching of chalcopyrite H. Zhou1,2, R. Zhang1,P.Hu1, W. Zeng1, Y. Xie1,C.Wu1,3 and G. Qiu1,2 1 School of Minerals Processing and Bioengineering, Central South University, Changsha, P.R. China 2 Key Laboratory of Biometallurgy, Ministry of Education, Central South University, Changsha, P.R. China 3 China Ocean Mineral Resources R&D Association, Beijing, P.R. China Keywords Abstract 16S rRNA gene, archaeon, chalcopyrite, T Ferroplasma sp., ferrous iron-oxidizing. Aims: To isolate Ferroplasma thermophilum L1 from a low pH environment and to understand its role in bioleaching of chalcopyrite. Correspondence Methods and Results: Using serial dilution method, a moderately thermophilic Guanzhou Qiu, School of Minerals Processing and acidophilic ferrous iron-oxidizing archaeon, named L1T, was isolated from and Bioengineering, Central South University, a chalcopyrite-leaching bioreactor. The morphological, biochemical and physio- Changsha, 410083, P.R. China. logical characteristics of strain L1T and its role in bioleaching of chalcopyrite E-mail: [email protected] were studied. Strain L1T was a nonmotile coccus that lacked cell wall. Strain T 2007 ⁄ 1566: received 26 September 2007, L1 had a temperature optimum of 45°C and the optimum pH for growth was T revised and accepted 24 January 2008 1Æ0. Strain L1 was capable of chemomixotrophic growth on ferrous iron and yeast extract. Results of fatty acid analysis, DNA–DNA hybridization, G+C con- doi:10.1111/j.1365-2672.2008.03807.x tent, and analysis based on 16S rRNA gene sequence indicated that strain L1T should be grouped in the genus Ferroplasma, and represented a new species, Ferroplasma thermophilum. Ferroplasma thermophilum in combination with Acidithiobacillus caldus and Leptospirillum ferriphilum could improve the copper dissolution in bioleaching of chalcopyrite. Conclusions: A novel extremely acidophilic, moderately thermophilic archaeon isolated from a bioleaching reactor has been identified as F. thermophilum that played an important role in bioleaching of chalcopyrite at low pH. Significance and Impact of the Study: This study contributes to understand the characteristics of F. thermophilum L1T and its role in bioleaching of sulfide ores. techniques have been applied to research the ecology of Introduction the mineral leaching environments, and the results sug- The use of micro-organisms to recover metals from gest that other species of moderately thermophilic bacte- low-grade ores has developed into a successful commer- ria, like Leptospirillum spp. and Acidithiobacillus caldus, cial biotechnology. For many years, it has been thought are dominant at certain mineral leaching environments that mesophilic Acidithiobacillus ferrooxidans (Temple (Goebel and Stackebrandt 1994; Norris et al. 2000). Okibe and Colmer 1951; Rawlings 2002), which grows opti- et al. (2003) found that Ferroplasma species were mally at 25–30°C is the most significant micro-organism dominant in the last bioleaching tank in a series of three. in the leaching of metal sulfides and a major contribu- This was probably the result of the low pH as a tor to acid mine drainage (Goebel et al. 2000; Rawlings result of sulfur compound oxidation, the high metal 2002). However, in recent years, molecular phylogenetic concentration and an increased organic carbon ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 1 转载 中国科技论文在线 http://www.paper.edu.cn Isolation of Ferroplasma thermophilum sp. nov. H. Zhou et al. concentration from dead cells from the previous leaching Materials and methods 1tanks in the series. Hawkes et al. (2006a,b) also found that Ferroplasma dominated in a chalcocite heap in Enrichment and isolation Myanmar. The microbial community inhabiting a low- grade copper sulfide run-of-mine test heap was analysed Leachate solution sample was taken from a column reac- 2by PCR-DGGE, and the result indicates that Ferroplasma tor processing chalcopyrite (Key Laboratory of Biometal- groups are quantitatively dominant at certain phases lurgy, Ministry of Education, China). The reactor was during the bioleaching process (Demergasso et al. 2005). originally inoculated with acidic mine drainage sample Ferroplasma groups are often flourishing at low pH, high collected from the Daye copper mine in Hubei province, amounts of total iron, ferrous iron and other heavy met- China. The operating temperature of the reactor was als (Johnson and Hallberg 2003). Reports suggest that 45°C and the pH was 1Æ3–1Æ9. Leachate solution was Ferroplasma spp. increase the sulfide’s leaching efficiency enriched with modified 9K medium (see later) containing at low pH and remove the dead biomass and cell 0Æ02% (w ⁄ v) yeast extract and trace elements (see later). secretions (Okibe and Johnson 2004). Therefore, this Strain L1T was isolated by serial dilution of the enrich- extreme micro-organism has potential commercial ment solution. Pure culture A. ferrooxidans type strain applications. ATCC23270 (AF465604) was purchased from ATCC. Pure culture of this type of organism was first Acidithiobacillus caldus strain s2 (DQ256484) and L. ferr- described in 2000 by Golyshina et al., designated Ferropl- iphilum strain YSK (DQ343299) used in the experiment asma acidiphilum strain YT. The strain is obligately auto- were isolated and conserved by our laboratory. Ferroplas- trophic and was isolated from a pyrite-leaching ma cupricumulans BH2T (AY907888) was kindly provided bioreactor in Kazakhstan (Golyshina et al. 2000). Second by Rebecca B. Hawkes (Hawkes et al. 2006a,b). species of the same genus, ‘Ferroplasma acidarmanus’ Fer1T, was subsequently isolated from a high concentra- Growth conditions tion of metals and extremely acidic mine drainage site at Iron Mountain and it is capable of chemo-organo- Unless otherwise stated, strain L1T was cultivated in basal trophic growth on yeast extract or sugars and chemo- salts of modified 9K medium (Silverman and Lundgren mixotrophic growth on ferrous iron and yeast extract or 1959), trace elements (Dopson and Lindstro¨m 1999), 30 g sugars (Edwards et al. 2000). In 2006, a new species of FeSO4Æ7H2O and 0Æ02% yeast extract. Modified 9K T the genus, Ferroplasma cupricumulans BH2 , was isolated medium contained (per litre): 3Æ0 g (NH4)2SO4,0Æ5g from an industrial mineral sulfide bioleach heap and KH2PO4,0Æ1 g KCl, 0Æ5 g MgSO4Æ7H2O, 0Æ01 g Ca(NO3)2. was only capable of chemomixotrophic growth on fer- Trace elements (per litre) comprised: 11Æ0 mg FeCl3Æ rous iron and yeast extract (Hawkes et al. 2006a,b). 6H2O, 0Æ5 mg CuSO4Æ5H2O, 50 mg Na2SO4,2Æ0mg Unlike mesophilic F. acidiphilum and ‘F. acidarmanus’, H3BO3,2Æ0 mg MnSO4,0Æ8mgNa2MoO4Æ2H2O, 0Æ6mg F. cupricumulans is a moderately thermophilic acido- CoCl2Æ6H2O, 0Æ9 mg ZnSO4Æ7H2O and 0Æ1mgNa2SeO4. phile. The pH of the medium was adjusted to 1Æ0 by adding Defined consortia of sulfur-oxidizing bacteria A. caldus 50% (v ⁄ v) H2SO4, was autoclaved, and then filter-steril- and iron-oxidizing bacteria, including Sulfobacillus ther- ized (0Æ2 lm filter paper) trace elements and FeSO4Æ7H2O 3mosulfidooxidans and A. ferrooxidans have been used to were added in the medium. Cultures were incubated in study the bioleaching of sulfide minerals (Dopson and rotary shakers at the indicated temperatures. Lindstro¨m 1999; Mcguire et al. 2001). The consortium The optimum temperature and pH for growth of the that comprises autotrophs Leptospirillum MT6 and A. cal- isolated strain L1T were determined by temperature- and dus KU, and the heterotroph Ferroplasma MT17 is the pH-controlled cultures in 250 ml shaking flasks with most efficient among all of those tested in a bioleaching 100 ml medium. Pure culture of strain L1T was inocu- of pyrite experiment (Okibe and Johnson 2004). lated to the liquid medium and the final cell density was In this study, a moderately thermophilic and acido- 1 · 106 cells per ml after inoculation. The incubation was philic ferrous iron-oxidizing archaeon Ferroplasma ther- performed at initial pH 1Æ0 and different temperatures or mophilum L1T was isolated from a chalcopyrite-leaching at 45°C and different initial pH values. After being incu- bioreactor and characterized. To further understand the bated for 4 days, the cell densities of cultures in the flasks role of genus Ferroplasma in mineral bioleaching, our were determined by a cell counting chamber. study was focussed on the copper dissolution from chal- The growth curve of strain L1T was determined by copyrite using pure culture A. ferrooxidans and mixed counting cell numbers in the culture after incubation at cultures A. caldus in combination with F. thermophilum an optimal growth temperature and growth pH value. L1T or Leptospirillum ferriphilum. Strain L1T was cultured on solid medium which contained ª 2008 The Authors 2 Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 中国科技论文在线 http://www.paper.edu.cn H. Zhou et al. Isolation of Ferroplasma thermophilum sp. nov. 1Æ0% agarose, and the plates were incubated at 45°C The growth of strain L1T on each substrate was moni- for 23 days. tored using a phase contrast microscope after three suc- To determine the optimum concentration of yeast cessive subcultures. extract, the basal salts medium with trace element and ) 30 g l 1 FeSO
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