Annals of Microbiology

, Volume 57, Issue 2, pp 171–176 | Cite as

Isolation and identification of a strain ofLeptospirillum ferriphilum from an extreme acid mine drainage site

  • Jian Gao
  • Cheng-Gui Zhang
  • Xue-Ling Wu
  • Hai-Hua Wang
  • Guan-Zhou Qiu
Ecological and Environmental Microbiology Original Articles


Based on a new selective isolation strategy that mimicked physiological characteristics of leptospirilla, such as pH, temperature and its less sensitivity to the high ferric-ferrous iron ratio, a bacterial strain, called strain YSK, was isolated from an extreme acid mine drainage (AMD) site. Cells were Gram-negative, small curved rods measuring 0.27–0.52 by 0.81–3.17 μm. The cell shape suggested that strain YSK was likely a strain ofLeptospirillum ferrooxidans. However, based on the 16S rDNA sequence analysis, strain YSK possessed 100% sequence similarity with that of the typicalLeptospirillum ferriphilum strain Fairview; furthermore, the G+C content, the size of the 16S–23S rRNA gene spacer regions and the ability to grow at 45°C further indicated that strain YSK belonged to the speciesLeptospirillum ferriphilum.

Key words

Leptospirillum ferriphilum strain YSK selective isolation strategy 16S rDNA sequence G+C content 16S–23S rRNA gene spacer regions 


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  1. Asish K.D., Avery B.A., Wyandt C.M. (2006). Development and validation of a stability-indicating reversed-phase high performance liquid chromatography method for NPC 1161C, a novel 8-aminoquinoline anti-malarial drug. J. Chromatogr. A, 1110: 35–45.CrossRefGoogle Scholar
  2. Bond P.L., Smriga S.P., Banfield J.F. (2000). Phylogeny of microorganisms populating a thick, subaerial predominantly lithotrophic biofilm at an extreme acid mine drainage site. Appl. Environ. Microbiol., 66: 3842–3849.CrossRefPubMedGoogle Scholar
  3. Coram N.J., Rawlings D.E. (2002). Molecular relationship between two groups of the genusLeptospirillum and finding thatLeptospirillum ferriphilum sp. nov. dominates South Africa commercial biooxidation tanks that operate at 40 °C. Appl. Environ. Microbiol., 68 (2): 838–845.CrossRefPubMedGoogle Scholar
  4. Dong X.Z., Cai M.Y. (1999). Manual of ordinary bacteriology. Science Press, Beijing, pp. 410–412.Google Scholar
  5. Dopson M., Baker-Austin C., Hind A.,et al. (2004). Characterization ofFerroplasma isolates andFerroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments. Appl. Environ. Microbiol., 70 (4): 2079–2088.CrossRefPubMedGoogle Scholar
  6. Falco L., Pogliani C., Curutchet G.,et al. (2003). A comparison of bioleaching of covellite using pure cultures ofAcidithiobacillus ferrooxidans andAcidithiobacillus thiooxidans or a mixed culture ofLeptospirillum ferrooxidans andAcidithiobacillus thiooxidans. Hydrometallurgy, 71: 31–36.CrossRefGoogle Scholar
  7. Goebel B.M., Stackebrandt E. (1994). Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments. Appl. Environ. Microbiol., 60: 1614–1621.PubMedGoogle Scholar
  8. Goebel B.M., Stackebrandt E. (1995). Molecular analysis of microbial diversity in a natural acidic environment, In: Jerez C.A., Vargas T., Toledo H., Weirtz J.V., Eds, Biohydrometallurgical Processing, vol. II. University of Chile Press, Santiago Chile, pp. 43–52.Google Scholar
  9. Golyshina O.V., Pivovarova T.A., Karavaiko G.I.,et al. (2000).Ferroplasma acidiphilum gen. nov., sp. nov., an acidophilic, autotrophic, ferrous-iron-oxidizing, cell-wall-lacking, mesophilic member of the Ferroplasmaceae fam. nov., comprising a distinct lineage of the Archaea. Int. J. Syst. Evol. Microbiol., 50: 997–1006.PubMedGoogle Scholar
  10. Gürtler V., Stanisich V.A. (1996). New approaches to typing and identification of bacteria using the 16S–23S rDNA spacer region. Microbiology, 142: 3–16.CrossRefPubMedGoogle Scholar
  11. Harrison A.P. Jr., Norris P.R. (1985).Leptospirillum ferrooxidans and similar bacteria: some characteristics and genomic diversity. FEMS Microbiol. Lett., 30: 99–102.CrossRefGoogle Scholar
  12. Hawkes R.B., Franzmann P.D., O’hara G.,et al. (2006).Ferroplasma cupricumulans sp. nov., a novel moderately thermophilic, acidophilic archaeon isolated from an industrial-scale chalcocite bioleach heap. Extremophiles, 10 (6): 525–530.CrossRefPubMedGoogle Scholar
  13. Hippe H. (2000).Leptospirillum gen. nov. (ex Markosyan 1972), nom. rev., includingLeptospirillum ferrooxidans sp. nov. (ex Markosyan 1972) nom. rev. andLeptospirillum thermoferrooxidans sp. nov. (Golovachevaet al. 1992). Int. J. Syst. Evol. Microbiol., 50: 501–503.PubMedGoogle Scholar
  14. Jensen M.A., Webster J.A., Straus N. (1993). Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl. Environ. Microbiol., 59: 945–952.PubMedGoogle Scholar
  15. Johnson D.B. (1995). Selective solid media for isolating and enumerating acidophilic bacteria. J. Microbiol. Meth., 23 (2): 205–218.CrossRefGoogle Scholar
  16. Johnson D.B., Okibe N., Hallberg K.B. (2005). Differentiation and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction enzyme analysis. J. Microbiol. Meth., 60: 299–313.CrossRefGoogle Scholar
  17. Kelly D.P., Wood A.P. (2000). Reclassification of some species ofThiobacillus to the newly designated generaAcidithiobacillus gen. nov.,Halothiobacillus gen. nov. andThermithiobacillus gen. nov. Int. J. Syst. Evol. Microbiol., 50: 511–516.PubMedGoogle Scholar
  18. Leblond-Bourget N., Philippe H., Mangin I.,et al. (1996). 16S rRNA and 16S to 23S internal transcribed spacer analysis reveal inter- and intraspecificBifidobacterium phylogeny. Int. J. Syst. Bacteriol., 4691: 102–111.CrossRefGoogle Scholar
  19. Norris P.R., Barr D.W., Hinson D. (1988). Iron and mineral oxidation by acidophilic bacteria: affinities for iron and attackment to pyrite. In: Norris P.R., Kelly D.P., Eds, Biohydrometallurgy. Proceedings of the International Symposium. Science and Technology Letters, Kew, United King, pp. 43–59.Google Scholar
  20. Peng H., Yang Y., Li X.,et al. (2006). Structure analysis of 16S rDNA sequences from strains ofAcidithiobacillus ferrooxidans. J. Biochem. Mol. Biol., 39 (2): 178–182.PubMedGoogle Scholar
  21. Poulin R., Lawrence R.W. (1996). Economic and environmental niches of biohydrometallurgy. Miner. Eng., 9 (8): 799–810.CrossRefGoogle Scholar
  22. Rawlings D.E. (1995). Restriction enzyme analysis of 16S rRNA genes for therapid identification ofThiobacillus ferrooxidans, Thiobacillus thiooxidans andLeptospirillus ferrooxidans strains in leaching environments, In: Jerez C.A., Vargas T., Toledo H., Wiertz J.V., Eds, Biohydrometallurgical Processing, vol. II. University of Chile Press, Santiago, Chile, pp. 9–17.Google Scholar
  23. Rawlings D.E., Tributsch H., Hansford G.S. (1999). Reasons why ‘Leptospirillum’-like species rather thanThiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial processes for the biooxidation of pyrite and related ores. Microbiology, 145: 5–13.CrossRefPubMedGoogle Scholar
  24. Rawlings D.E. (2002). Heavy metal mining using microbes. Annu. Rev. Microbiol., 56: 65–91.CrossRefPubMedGoogle Scholar
  25. Sand W., Rohde K., Sobotke B.,et al. (1992). Evaluation ofLeptospirillum ferrooxidans for leaching. Appl. Environ. Microbiol., 58: 85–92.PubMedGoogle Scholar
  26. Schrenk M.O., Edward K.J., Goodman R.M.,et al. (1998). Distribution ofThiobacillus ferrooxidans andLeptospirillum ferrooxidans: implications for generation of acid mine drainage. Science, 279: 1519–1522.CrossRefPubMedGoogle Scholar
  27. Silverman M.P., Lundgren D.C. (1959). Study on the chemoautotrophic iron bacteriumthiobacillus ferrooxidans: I. an improved medium and harvesting procedure for securing high cell yield. J. Bacteriol., 77: 642–647.CrossRefPubMedGoogle Scholar
  28. Stanley J.T., Bryant M.P., Pfennig N.,et al. (1989). Bergey’s Manual of Systematic Bacteriology, Bergey D.H. (1860–1937), Volume 3, Williams and Wilkins, Baltimore, MD.Google Scholar
  29. Sugita T., Takashima M., Hamamoto M.,et al. (1997).Bensingtonia sakaguchii sp. nov. isolated from a leaf ofBischofia javanica in the Ogasawara Islands. J. Gen. Appl. Microbiol., 43: 231–235.CrossRefPubMedGoogle Scholar
  30. Thompson J.D., Gibson T.J., Plewniak F.,et al. (1997). The Clustal X windows interface: fexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res., 24: 4876–4882.CrossRefGoogle Scholar
  31. Tuffin I.M., Hector S.B., Deane S.M.,et al. (2006). Resistance determinants of a highly arsenic-resistant strain ofLeptospirillum ferriphilum isolated from a commercial biooxidation tank. Appl. Environ. Microbiol., 72 (3): 2247–2253.CrossRefPubMedGoogle Scholar
  32. Tyson G.W., Lo I., Baker B.J.,et al. (2005) Genome-directed isolation of the key nitrogen fixerLeptospirillum ferrodiazotrophum sp. nov. from an acidophilic microbial community. Appl. Environ. Microbiol., 71 (10): 6319–6324.CrossRefPubMedGoogle Scholar
  33. Van Aswegen P.C. (1993). Commissioning and operation of biooxidation plants for the treatment of refractory gold ores. In: Hiskey J.B., Warren G.W., Eds, Hydrometallurgy; Fundamentals, Technology and Innovations. Soc. Min. Metall. Explor. AIME, pp. 709–810.Google Scholar
  34. Wu Y., Li H.Z., Ma Y.P.,et al. (2005). Determination of the G+C mol% of DNA in a novel strain ofBifidobacterium bifidum by reversed-phase high performance liquid chromatography. Journal of Southwest China Normal University (Natural Science), 30 (1): 96–100.Google Scholar

Copyright information

© University of Milan and Springer 2007

Authors and Affiliations

  • Jian Gao
    • 1
    • 2
  • Cheng-Gui Zhang
    • 1
  • Xue-Ling Wu
    • 1
  • Hai-Hua Wang
    • 2
  • Guan-Zhou Qiu
    • 1
  1. 1.School of Resources Processing and BioengineeringCentral South UniversityChangshaChina
  2. 2.School of Life ScienceHunan University of Science and TechnologyXiangtanChina

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