Isolation and identification of native sulfuroxidizing bacterium capable of uranium extraction

Document Type: Research Paper


1 Nuclear Fuel Cycle Research School, Nuclear Science and Technology Research Institute, Tehran, I.R. Iran

2 Department of Chemical Engineering, Faculty of Engineering, University of Mazandaran, Babolsar, Iran


Bioleaching is the extraction of metals from their ores through the use of microorganisms. In
this process, the use of native bacteria leads to achieve more yields of metals. So, in the present
study, native sulfur-oxidizing bacterium in potentiality of uranium extraction was isolated from
Ghachin mine in Iran and identified by partial gene sequencing. For this purpose, the water
samples were collected from Ghachin mine and cultivated in Starkey medium. In following, the
isolate was inoculated into individual Starkey plates and incubated until the colonies indicating
the purified bacterium appeared. Then, the identification was carried out based on phenotypic
characteristics and 16s rDNA sequencing. After that, bioleaching of uranium experiments
carried out using uranium ore at 2.5 and 5% pulp densities. The result showed that after 15 days
of incubation, the bacteria in the fresh samples was grown. Following 5-7 days of the plate's
incubation, we obtained the single purified colonies of the bacteria. On the basis of 16s rDNA
nucleotide sequencing, the bacteria showed 99.71% similarity to A. thiooxidans ATCC 19377.
Besides, the bioleaching experiments indicated that the bacterium is capable of uranium
extraction in 2.5 and 5% pulp densities during 3 and 5 days. In conclusion, in this study, for the
first time, we isolated the native sulfur-oxidizing bacterium capable of uranium extraction, from
uranium mine of Gachin in Bandar Abbas, Iran.


Main Subjects

1. Borisovich, U.A., Mihaylovich, K.A. (2013) Bioleaching of low grade uranium ore containing
pyrite using A. ferrooxidans and A. thiooxidans. Journal of Radioanalytical and Nuclear
Chemistry., 295, 151-156.
2. Rawlings, E. (1997) Biomining: theory, microbes and industrial processes. Bioscience,
Georgetown, Tex.
3. Glazer, A.N., Nikaido, H. (1995) Application of biotechnology for mineral processing. Microbial
biotechnology., 268-287.
4. Steudel, R. (1989) On the nature of the "elemental sulfur" (S0) produced by sulfuroxidizing
bacteria. In: Schegel, H.G., Bowien, B. (Eds.), A model for S0 globules. Biology of Autotrophic
Bacteria. Science Tech Publication., 289-303.
5. Bond, P.L., Druschel, G.K., Banfield, J.F. (2000) Comparison of acid mine drainage microbial
communities in physically and geochemicaly distinct ecosystems. Applied and Environmental
Microbiology., 66, 4962-4971.
6. Dopson, M., Craig, B.A., Koppineedi, P., Philip, L. (2003) Growth in sulfidic mineral
environments, metal resistance mechanisms in acidophilic micro-organisms. Microbiology., 149,
7. Waksman, S.A., Joffe, J.S. (1922) The chemistry of the oxidation of sulfurby microorganisms to
sulfuric acid and transformation of insoluble phosphates into soluble forms. Jour. Biol. Chem., 50,
8. Rohwerder, T., Gehrke, T., Kinzler, K., Sand, W. (2003) Progress in bioleaching: fundamentals and
mechanisms of bacterial metal sulphide oxidation. Appl Microbiol Biotechnology., 63, 239-248.
9. Sand, W., Gehrke, T. (2006) Extracellular polymeric substances mediate bioleaching/biocorrosion
via interfacial processes involving iron(III) ions and acidophilic bacteria. Res Microbiol., 157, 49-56.
10. Bergamo, R.F., Novo, M., Verissimo, R., Paulino, L., Stoppe, N., Sato, M., Manfio, G., Prado, P.,
Garsia, O., Ottoboni, L. (2004) Differentiation of Acidithiobacillus ferrooxidans and
Acidithiobacillus thiooxidans strains based on 16s-23s rDNA spacer polyphormism analysis. Res.
Microbiol., 155, 559-567.
11. Rawlings, E. (2005) Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms
used for the recovery of metals from minerals and their concentrates. Microbial Cell Factories.,4-13.
12. Ageeva, S.N., Kondrat'eva, T.F., Karavaiko, G.I. (2001) Phenotypic characteristics of Thiobacillus
ferrooxidans strains. Mikrobiologiia., 70, 226-234.
13. Altschul, S.F., Gish, W,. Miller, W., Myers, E.W., Lipman, D.J. (1990) Basic local alignment
search tool. J Mol Biol., 215, 403-410.
14. DSMZ. List of media. (2002) Deutsche Sammlung zon Mikroorganismen und Zellkulturen Gmb
15. Starkey, R.L., Collins, V.G. (1923) Autotrophs. Methods in Microbiology., 38, 55-73.

16. Waksman, S.A. (1922) Microorganisms concerned in oxidation of sulur in the soil. J Bacteriol.,
7(6), 605-608.
17. Shahroz, K., Faizul, H., Fariha, H., Kausar, S., Rahat, U. (2012) Growth and Biochemical
Activities of Acidithiobacillus thiooxidans Collected from Black Shale. Microbiology Research.,
2, 78-83.
18. Gram, H.C. (1884) "Über die isolierte Färbung der Schizomyceten in Schnitt- und
Trockenpräparaten" (in German). Fortschritte Medizin., 185-189.
19. Escobar, B., Bustos, K., Morales, G., Salazar, O. (2008) Rapid and specific detection of
Acidithiobacillus ferrooxidans and leptospirillum ferrooxidans by PCR. Hydrometalurgy., 92,
20. Ai, O., Satoshi, W., Tadayoshi, K., Tsuyoshi, S., Kazuo, K. (2005) Diversity of 16s ribosomal
DNA-defined bacterial population in acid rock drainage from japanese pyrite mine. Bioscience
and Bioengineering., 100, 644–652.
21. Leloup, J., Loy, A., Knab, N.J., borowski ,C., wagner, M., Jorgensen, B.B. (2007) Diversity and
abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine
sediment, Black Sea. Environ. Microbiol., 9, 131-142.
22. Sambrook, J., Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. New York: Cold
Spring Harbor Laboratory Press.
23. Altschul, S.F., Madden, T.L., Schaeffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J.
(1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res., 25, 33-89.
24. Chun, J., Lee, J.H., Jung, Y., Kim, M., Kim, S., Kim, B.K., Lim, Y.W. (2007) EzTaxon: a webbased
tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J
Syst Evol Microbiol., 57, 2259-2261.
25. Ronald, M.A. (1997) Handbook of Microbiological Media. Second Ed. Robert Stern Publisher,
New York.
26. Underwood, A.L., Day, R.A. (1991) Quantitative Analysis. Prentice Hall, London., 645-646.
27. Ryu, H.W., Cho, K.S., Chang, Y.K., Kim, S.D., Mori, T. (1995) Refinement of Low-Grade Clay
by Microbial Removal of Sulfur and Iron Compounds Using Thiobacillus ferrooxidans. Journal of
Fermentation and Bioengineering., 80, 46.
28. Vidyalakshmi, R., Srida, R. (2006) Isolation and characterization of sulphur oxidizing bacteria.
Journal of Culture Collections., 5, 73-75.
29. Ward, G. (1916) Laboratory Manual In General Microbiology, 1st edn. New York.
30. Rojas-avelizapa, N.G., Gómez-ramírez, M., Hernández-gama, R., Aburto, J., García, D.E. (2013)
Isolation and Selection of Sulfur-oxidizing Bacteria for the Treatment of Sulfur-containing
HazardousWastes. Chem. Biochem. Eng. Q., 27(1), 109-117.
31. Qiang, L.I., Cong, W., Baobin, L., Cunmin, S., Fei, D., Cunjiang, S., Shufang, W. (2012) Isolation
of Thiobacillus spp. and its application in the removal of heavy metals from activated sludge.
African Journal of Biotechnology., 97, 16336-16341.

32. Sivaji, R., Leslie, R. (1971) Berger G. The requirement of low pH for growth of Thiobacillus
thiooxidans . Archiv für Mikrobiologie., 79, 338-344.
33. Carmen, M. (2010) The taxonomic and physiologic diversity of the acidophilic chemolithotrophic
bacteria of the genus thiobacillus used in ores solubilization processes. Trav. Inst. Spéol. «Émile
34. Selman, A., Waksman, S.A. (1922) Microorganisms concerned in the oxidation of sulfur in the
soil. New Jersey Agricultural Experiment Station, Department of Soil Chemistry and
Bacteriology., 84, 605-608.
35. Ryu, H.W., Moon, H.S., Lee, E.Y., Cho, K.S., Choi, H. (2003) Leaching Characteristics of Heavy
Metals from Sewage Sludge by Acidithiobacillus thiooxidans MET. J. Environ. Qual., 32, 751-759.
36. Vishniac, W.V. (1974) The genus Thiobacillus. Bergey's manual of determinative bacteriology,
8th edn.
37. Kempner, E. (1966) Acid Production by Thiobacillus thiooxidans. Journal of Bacteriology., 92,
38. Rao, T. (2005) Advances in Water and Wastewater Treatment. American Society of Civil
39. Swinnen, I.A.M., BernaertS, K., Dens, E.J.J., Geeraerd, A.H., Vanimpe, J.F. (2004) Predictive
modelling of the microbial lag phase: a review. Int. J. Food Microbiol., 94, 137-159.
40. Al-qadiri, H., Al-alami, N., Lin, M., Al-holy, M., Cavinato, A., Rasco, B. (2008) Studying of the
bacterial growth phases using fourier transform infrared spectroscopy and multivariate analysis.
Journal of Rapid Methods & Automation in Microbiology., 16, 73-89.
41. Tsukasa, I., Kenichi, S., Satoshi, O. (2004) Isolation, Characterization, and In Situ Detection of a
Novel Chemolithoautotrophic Sulfur-Oxidizing Bacterium in Wastewater Biofilms Growing under
Microaerophilic Conditions. Appl Environ Microbiol., 70(5), 3122-3129.
42. Paulino, L., Rog´erio, F., Maricilda, P., Oswaldo, G., Gilson, P., Laura, M. (2001) Molecular
characterization of Acidithiobacillus ferrooxidans and A. thiooxidans strains isolated from mine
wastes in Brazil. Antonie van Leeuwenhoek., 80, 65–75.
43. Xia, J., Peng, A., He, H., Yang, Y., Liu, X., Qiu, G. (2004) Acidithiobacillus albertensis BY-05, a
new strain for bioleaching of metal sulfides ores. Project (50321402) supported by Nature Science
Foundation of China for innovation research group; Project (2004CB619204) supported by
National Major Basic Research Item, China.
44. Yongqing, N., Dongshi, W., Kaiyu, H. (2008) 16s rDNA and 16s–23s internal transcribed spacer
sequence analyses reveal inter- and intraspecific Acidithiobacillus phylogeny. Microbiology., 154,
45. Scott, P. (2013) What is Biomining. Wise Geek.
46. Acevedo, F., Gentina, J. (1989) Process engineering aspects of the bioleaching of copper ores.
Bioprocess Engineering., 4, 223-229.
47. Newman, L.A., Doty, S.L., Gery, K.L., Heilman, P.E., Muiiznieks, I., Shang, T.Q., Siemieniec,
S.T., Strand, S.E., Wang, X., Wilson, A.M., Gordon, M.P. (1998) Phytoremediation of organic contaminants: a review of phytoremediation research at the University of Washington. J Soil Commun., 7, 531-542.

48. Ownby, D.R., Newman, M.C. (2003) Advances in quantitative ion character-activity relationships
(QICARs): Using metal-ligand binding characteristics to predict metal toxicity. QSAR &
Combinatorial Science., 22(2), 241-246.