Archives of Microbiology

, Volume 180, Issue 1, pp 60–68 | Cite as

Novel thermo-acidophilic bacteria isolated from geothermal sites in Yellowstone National Park: physiological and phylogenetic characteristics

  • D. Barrie JohnsonEmail author
  • Naoko Okibe
  • Francisco F. Roberto
Original Paper


Moderately thermophilic acidophilic bacteria were isolated from geothermal (30–83 °C) acidic (pH 2.7–3.7) sites in Yellowstone National Park. The temperature maxima and pH minima of the isolates ranged from 50 to 65 °C, and pH 1.0–1.9. Eight of the bacteria were able to catalyze the dissimilatory oxidation of ferrous iron, and eleven could reduce ferric iron to ferrous iron in anaerobic cultures. Several of the isolates could also oxidize tetrathionate. Six of the iron-oxidizing isolates, and one obligate heterotroph, were low G+C gram-positive bacteria (Firmicutes). The former included three Sulfobacillus-like isolates (two closely related to a previously isolated Yellowstone strain, and the third to a mesophilic bacterium isolated from Montserrat), while the other three appeared to belong to a different genus. The other two iron-oxidizers were an Actinobacterium (related to Acidimicrobium ferrooxidans) and a Methylobacterium-like isolate (a genus within the α-Proteobacteria that has not previously been found to contain either iron-oxidizers or acidophiles). The other three (heterotrophic) isolates were also α-Proteobacteria and appeared be a novel thermophilic Acidisphaera sp. An ARDREA protocol was developed to discriminate between the iron-oxidizing isolates. Digestion of amplified rRNA genes with two restriction enzymes (SnaBI and BsaAI) separated these bacteria into five distinct groups; this result was confirmed by analysis of sequenced rRNA genes.


Acidophiles Biodiversity Iron oxidation Iron reduction Moderate thermophiles Yellowstone National Park 



Amplified ribosomal DNA restriction enzyme analysis


Ferrous sulfate overlay medium


Ferrous sulfate/potassium tetrathionate overlay medium


Tryptone soya broth



Naoko Okibe is grateful for financial assistance provided by The Institution of Mining and Metallurgy (UK), the Gen Foundation and Glaxo Ltd. We also appreciate the help of Zhe-Xue Quan (Korean Advanced Institute of Science and Technology, Taejon) with the bacterial growth experiments, and thank Lynn Petzke for her assistance in re-analyzing the phlyogenetic tree.


  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedGoogle Scholar
  2. Atkinson T, Cairns S, Cowan DA, Danson MJ, Hough DW, Johnson DB, Norris PR, Raven N, Robson R, Robinson C, Sharp RJ (2000) A microbiological survey of Montserrat island hydrothermal biotopes. Extremophiles 4:305–313PubMedGoogle Scholar
  3. Bridge TAM, Johnson DB (1998) Reduction of soluble iron and reductive dissolution of ferric iron-containing minerals by moderately thermophilic iron-oxidizing bacteria. Appl Environ Microbiol 64:2181–2186PubMedGoogle Scholar
  4. Brock TD (1978) Thermophilic Microorganisms and Life at High Temperatures. Springer- Verlag, New YorkGoogle Scholar
  5. Brock TD (2001) The origins of research on thermophiles. In: Reysenbach AL, Voytek A (eds) Thermophiles: biodiversity, ecology and evolution. Kluwer Academic/Plenum, New York, pp 1–9Google Scholar
  6. Clark DA, Norris PR (1996) Acidimicrobium ferrooxidans gen. nov., sp. nov.: mixed culture ferrous iron oxidation with Sulfobacillus species. Microbiology 141:785–90Google Scholar
  7. Coram NJ, Rawlings DE (2002) Molecular relationship between two groups of the genus Leptospirillum and the finding that Leptospirillum ferriphilum sp. nov. dominates South African commercial bioleaching tanks that operate at 40°C. Appl Environl Microbiol 68:838–845CrossRefGoogle Scholar
  8. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991) Touchdown PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19:4008–4008PubMedGoogle Scholar
  9. Felsenstein J (1989) PHYLIP-phylogeny inference package (version 3.5c). Caldistics 5:164–166Google Scholar
  10. Golyshina OV, Pivovarova TA, Karavaiko GI, Kondrat'eva TF, Moore ERB, Abraham WR, Lunsdorf H, Timmis KN, Yakimov MM, Golyshin PN (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 50:997–1006Google Scholar
  11. Green PN, Bousfield IJ, Hood D (1988). "A new Methylobacterium species - Methylobacterium rhodesianum sp. nov., Methylobacterium zatmanii sp. nov., and Methylobacterium fujisawaense sp. nov. Int J Syst Bacteriol. 38:124–127Google Scholar
  12. Hallberg KB, Johnson DB (2001) Biodiversity of acidophilic prokaryotes. Adv Appl Microbiol 49:37–84PubMedGoogle Scholar
  13. Hiraishi A, Matsuzawa Y, Kanbe T, Wakao N (2000) Acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments. Int J Syst Evol Microbiol 50:1539–1546PubMedGoogle Scholar
  14. Johnson DB (1995) Selective solid media for isolating and enumerating acidophilic bacteria. J Microbiol Meth 23:205–218CrossRefGoogle Scholar
  15. Johnson DB (2001) Importance of microbial ecology in the development of new mineral technologies. Hydrometallurgy 59:147–158CrossRefGoogle Scholar
  16. Johnson DB, Body DA, BridgeTAM, Bruhn DF, Roberto FF (2001a) Biodiversity of acidophilic moderate thermophiles isolated from two sites in Yellowstone National Park, and their roles in the dissimilatory oxidoreduction of iron. In: Reysenbach AL, Voytek A (eds) Thermophiles: biodiversity, ecology and evolution. Kluwer Academic/Plenum Publishers, New York, pp 23–39Google Scholar
  17. Johnson DB, Bacelar-Nicolau P, Okibe N, Yahya A, Hallberg KB (2001b) Role of pure and mixed cultures of gram-positive eubacteria in mineral leaching. In: Ciminelli VST, Garcia Jr O (eds) Biohydrometallurgy: fundamentals, technology and sustainable development: Process Metallurgy 11A. Elsevier, Amsterdam, pp 461–470Google Scholar
  18. Johnson DB, Rolfe S, Hallberg KB, Iversen E (2001c) Isolation and phylogenetic characterisation of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine. Environ Microbiol 3:630–637PubMedGoogle Scholar
  19. Jukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism. Academic Press, New York, pp 21–132Google Scholar
  20. Karavaiko GI, Tourova TP, Tsaplina IA, Bogdanova TI (2000) Investigation of the phylogenetic position of aerobic, moderately thermophilic bacteria oxidizing Fe2+, S0, and sulfide minerals and affiliated to the genus Sulfobacillus. Microbiologiya (English traslation) 69:857–860Google Scholar
  21. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, editors. Nucleic Acid Techniques in Bacterial Systematics. New York: Wiley, p. 115–175Google Scholar
  22. Lovley DR, Phillips EJP (1987) Rapid assay for microbially reducible ferric iron in aquatic sediments. Appl Environ Microbiol 53:1536–1540Google Scholar
  23. Page RDM (1996) TREEVIEW: An application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358PubMedGoogle Scholar
  24. Rawlings DE (1995) Restriction enzyme analysis of 16S rDNA genes for the rapid identification of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Leptospirillum ferrooxidans strains in leaching environments. In: Vargas T, Jerez CA, Wiertz JV, Toledo H (eds) Biohydrometallurgical Processing II. University of Chile, Santiago, pp. 9–18Google Scholar
  25. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  26. Selenska-Pobell S, Otto A, Kutschke S (1998) Identification and discrimination of thiobacill using ARDREA, RAPD and rep-APD. J Appl Microbiol 84:1085–1091CrossRefGoogle Scholar
  27. Shima, S, Suzuki, KI (1993) Hydrogenobacter acidophilus sp. Nov., a thermoacidophilic, aerobic, hydrogen-oxidizing bacterium requiring elemental sulfur for growth. Int J Syst Bacteriol 43:703–708Google Scholar
  28. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedGoogle Scholar
  29. Yahya A, Johnson DB (2002) Bioleaching of pyrite at low pH and low redox potentials by novel mesophilic gram-positive bacteria. Hydrometallurgy 63:181–188CrossRefGoogle Scholar
  30. Yahya A, Roberto FF, Johnson DB (1999) Novel mineral-oxidising bacteria from Montserrat (W.I.): physiological and phylogenetic characteristics. In: Amils R, Ballester A (eds) Biohydrometallurgy and the environment toward the mining of the 21st century: Process Metallurgy 9A. Elsevier, Amsterdam, pp 729–740Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • D. Barrie Johnson
    • 1
    Email author
  • Naoko Okibe
    • 1
  • Francisco F. Roberto
    • 2
  1. 1.School of Biological SciencesUniversity of WalesBangorUK
  2. 2.Biotechnology DepartmentIdaho National Engineering and Environmental LaboratoryIdaho FallsUSA

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