Skip to main content

Isolation of Leptospirillum ferriphilum by single-layered solid medium


According to physiological and biochemical characteristics of Leptospirillum ferriphilum, a strain of object bacteria was isolated successfully. Bacteria were enriched by selective liquid medium and plated on designed single-layered agar solid medium. Colony was cultured and bacteria were collected. The morphologies of the object bacteria were observed using crystal violet staining, scanning electron microscope(SEM) and transmission electron microscope (TEM). The result of 16S rDNA identification shows that this bacterium belongs to Leptospirillum ferriphilum and it is named as Leptospirillum ferriphilum strain D1. These results indicate that this new single-layered agar solid medium is efficient and simple for isolation of Leptospirillum ferriphilum. Additionally, physiological-biochemical characteristics show that the optimum initial pH value and its growth temperature are 1.68 and 40 °C, respectively. The culture of it is used to leach a complex concentrate chalocpyrite, the leaching efficiencies of copper and iron are 1.93% and 13.74%, respectively, and it is more effective than the A. ferrooxidans culture in the leaching of the complex concentrate chalcopyrite.

This is a preview of subscription content, access via your institution.


  1. BAKER B J, BANFIELD J F. Microbial communities in acid mine drainage[J]. FEMS Microbiology Ecology, 2003, 44(2): 139–152.

    Article  Google Scholar 

  2. PIVOVAROVA T A, MARKOSYAN G E, KARAVAIKO G I. The auxotrophic growth of Leptospirillum ferrooxidans[J]. Microbiology, 1981, 50(1): 339–344.

    Google Scholar 

  3. HIPPE H. Leptospirillum gen. nov. (ex Markosyan 1972), nom. rev., including Leptospirillum ferrooxidans sp. nov. (ex Markosyan 1972), nom. rev. and Leptospirillum thermoferrooxidans sp. nov. (Golovacheva et al. 1992) [J]. Int J Syst Evol Microbiol, 2000, 50(5): 501–503.

    Article  Google Scholar 

  4. DRUSCHEL G K, BAKER B J, GIHRING T, et al. Acid mine drainage biogeochemistry at Iron Mountain California [J]. Eochem Trans, 2004, 5(2): 13–17.

    Google Scholar 

  5. RAWLINGS D E, SILVER S. Mining with microbes[J]. Bio/Technology, 1995, 13(2): 773–778.

    Article  Google Scholar 

  6. NICOLETTE J C, RAWLINGS D E. Molecular relationship between two groups of the genus Leptospirillum and the finding that Leptospirillum ferriphilum sp. nov. dominates south african commercial biooxidation tanks that operate at 40 °C [J]. Appl Environ Microbiol, 2002, 68(1): 838–845.

    Google Scholar 

  7. RAWLINGS D E. Restriction enzyme analysis of 16S rRNA genes for the rapid identification of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Leptospirillum ferrooxidans strains in leaching environments[C]// JEREZ C A, VARGAS T, TOLEDO H, et al. Biohydrometallurgical Processing, vol. II. Santiago: University of Chile Press, 1995: 9–17

    Google Scholar 

  8. RAWLINGS D E, CORAM N J, GARDNER M N, et al. Thiobacillus caldus and Leptospirillum ferrooxidans are widely distributed in continuous flow biooxidation tanks used to treat a variety of metal containing ores and concentrates[C]// AMILS R, BALLESTER A. Biohydrometallurgy and the Environment: toward the Mining of the 21st Century, Part A. Amsterdam: Elsevier, 1999: 777–786.

    Google Scholar 

  9. RAWLINGS D E, TRIBUTSCH H, HANSFORD G S. Reasons why ‘Leptospirillum’-like species rather than Thiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial processes for biooxidation of pyrite and related ores[J]. Microbiology, 1999, 145(3): 5–13.

    Article  Google Scholar 

  10. JOHNSON D B. Selective solid media for isolating and enumerating acidophilic bacteria[J]. Journal of Microbiological Methods, 1995, 23(2): 205–218.

    Article  Google Scholar 

  11. TUOVINEN O H, KELLY D P. Studies on the growth of Thiobacillus ferrooxidans use of membrane filters and ferrous iron agar to determine viable numbers and comparison with 14CO2-fixation and iron-oxidation as measures of growth[J]. Arch Mikroboil, 1973, 88(10): 285–298.

    Article  Google Scholar 

  12. JOHNSON D B, MCGINNESS S. A highly efficient and universal solid medium for growing mesophilic and moderately thermophilic, iron-oxidizing, acidophilic bacteria[J]. Microbiol Mehods, 1991, 13(7): 113–122.

    Google Scholar 

  13. HARRISON J. A P. The acidophilic thiobacilli and other acidophilic bacteria that share their habitat[J]. Ann Rev Microbial, 1984, 38(2): 265–292.

    Article  Google Scholar 

  14. LIU Ying, LIU Xiang-mei, TIAN Ke-li. Growth and morphology of Leptospirillum ferrooxidans on solid medium[J]. Microbiology, 2003, 30(6): 70–72. (in Chinese)

    Google Scholar 

  15. LIU Ying, QI Fang-jun, LIU Xiang-mei. Phylogenetic analysis for 16S rDNA sequence of the vibrio shaped chemoautolithotrophic iron-oxidizing bacterium ML-04[J]. Journal of Shandon University, 2004, 39(5): 112–115. (in Chinese)

    Google Scholar 

  16. ZHOU J, BRUNS M A, TIEDJE J M. DNA recovery from soils of diverse composition[J]. Appl Environ Microbiol, 1996, 62(2): 316–322.

    Google Scholar 

  17. LANE D J. 16S/23S rRNA sequencing[C]// STACKEBRANDT E, GOODFELLOW M. Nucleic Acid Techniques in Bacterial Systematics. Chichester: John Wiley & Sons, 1991: 115–175.

    Google Scholar 

  18. ZHANG Zai-hai, QIU Guan-zhou, HU Yue-hua, et al. The investigation of the colony isolation of thiobacillus fenooxidans[J]. Multipurpose Utilization of Mineral Resources, 2001, (1): 19–23. (in Chinese)

  19. SAND W, ROHDE K, SOBOTKE B, et al. Evaluation of Leptospirillum ferrooxidans for leaching[J]. Appl Environ Microbiol, 1992, 58(1): 85–92.

    Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Liu Jian-she.

Additional information

Foundation item: Projects (50374075, 50321402) supported by the National Natural Science Foundation of China; Project(2004CB619204) supported by the National Basic Research and Development Program of China; Project(200549) supported by the Specialized Research Fund of the National; Excellent PhD Thesis

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Liu, Js., Xie, Xh., Xiao, Sm. et al. Isolation of Leptospirillum ferriphilum by single-layered solid medium. J Cent. South Univ. Technol. 14, 467–473 (2007).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Key words

  • Leptospirillum ferriphilum
  • isolation
  • purification
  • solid medium
  • bioleaching
  • concentrate chalcopyrite