Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

An effective method of DNA extraction for bioleaching bacteria from acid mine drainage


An effective and versatile method for microorganism lysis and direct extraction of DNA from bioleaching bacteria was developed using pure cultures and an acid mine drainage (AMD) sediment sample. In the described method, microorganisms are treated at three different incubation temperatures: boiling water incubation for 6–10min, followed by 60 ± 5°C for 30min, then 72°C for 30min. The extracted DNA is purified using a phenol/chloroform/alcohol mixture and precipitated in absolute alcohol. The 16S ribosomal RNA (rRNA) and gyrB genes of the pure cultures were amplified using the polymerase chain reaction (PCR) and differentiated using repetitive intergenic DNA sequences amplification (Rep-PCR). For the AMD sediment sample, the 16S rRNA and gyrB genes of the amplicons were digested with Hin6I and MspI, and the restriction fragment length polymorphism analysis patterns were used as a fingerprint to discern community diversity. The results indicated that this method is a versatile, reproducible, effective, and rapid technique for routine DNA extraction from bioleaching bacteria. The low cost of this method also makes it attractive for large-scale studies.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Baker BJ, Banfield JF (2003) Microbial communities in acid mine drainage. FEMS Microbiol Ecol 44:139–152

  2. Barman SP, Lunsford L, Chambers P, Hedley ML (2000) Two methods for quantifying DNA extracted from poly(lactide-co-glycolide) microspheres. J Control Release 69:337–344

  3. Berensmeier S (2006) Magnetic particles for the separation and purification of nucleic acids. Appl Microbiol Biotechnol 73:495–504

  4. Bloem J, Bolhuis PR, Veringa MR, Weiringa J (1995) Microscopic methods for counting bacteria and fungi in soil. In: Alef K, Nannipieri P (eds) Methods in applied soil microbiology and biochemistry. Academic, London, pp 162–173

  5. Bond PL, Druschel GK, Banfield JF (2000) Comparison of AMD microbial communities in physically and geochemically distinct ecosystems. Appl Environ Microbiol 66:4962–4971

  6. Chartier M, Mercier G, Blais JF (2001) Partitioning of trace metals before and after biological removal of metals from sediments. Water Res 35:1435–1444

  7. Guay R, Silver-Can M (1975) Thiobacillus acidophilus sp. nov.: isolation and some physiological characteristics. J Microbiol 21:281–288

  8. Guthrie JN, Moriarty DJW, Blackall LL (2000) DNA extraction from coral reef sediment bacteria for the polymerase chain reaction. J Microbiol Meth 43:73–80

  9. Jakubowski H (2005) An approach based on Chemical logic. Biochemistry Online: Update, 9/25

  10. Kemp PF (1994) A philosophy of methods development: the assimilation of new methods and information into aquatic microbial ecology. Microb Ecol 28:159–162

  11. Krsek M, Wellington EMH (1999) Comparison of different methods for the isolation and purification of total community DNA from soil. J Microbiol Methods 39:1–16

  12. Marchesi JR, Sato T, Weightman AJ, Martin TA, Fry JC, Hiom SJ, Wade WG (1998) Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl Environ Microbiol 64:795–799

  13. Mercier G, Chartier M (1999) Decontamination of fly ash and used lime from municipal waste incinerator using Thiobacillus ferrooxidans. Environ Manag 24:517–528

  14. Niemi MR, Heiskanen I, Wallenius K, Lindstrom K (2001) Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia. J Microbiol Methods 45:155–165

  15. Peng H (2006) Research on the phenotypic and genetic diversity of Acidithiobacillus ferrooxidans strains. Master thesis, Central South University, China

  16. Peng H, Yang Y, Li X, Qiu G, Liu XD, Huang JF, Hu YH (2006) Structure analysis of 16S rRNA sequences from strains of Acidithiobacillus ferrooxidans. J Biochem Mol Biol 39:178–182

  17. Prodělalová J, Rittich B, Španová A, Petrová K, Beneš MJ (2004) Isolation of genomic DNA using magnetic cobalt ferrite and silica particles. J Chromatogr A 1056:43–48

  18. Qiu G, Fu B, Zhou HB, Liu X, Gao J, Liu FF, Chen XH (2007) Isolation of a strain of Acidithiobacillus caldus and its role in bioleaching of chalcopyrite. World J Microbiol Biotechnol 23:1217–1225

  19. Roberts LS, Janovy J Jr (1996) Foundations of parasitology, 5th edn. Wm. C. Brown Publishers, Boston, MA, USA, p 118

  20. Rogstad SH (1993) Surveying plant genomes for variable number of tandem repeat loci. Methods Enzymol 224:278–294

  21. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, pp 361–1570

  22. Schippers A, Hallmann R, Wentzien S, Sand W (1995) Microbial diversity in uranium mine waste heaps. Appl Environ Microbiol 61:2930–2935

  23. Versalovic J, Koeuth T, Lupski R (1991) Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 19:6823–6831

  24. Yamamoto S, Harayama S (1995) PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol 61:1104–1109

  25. Yin HQ (2007) Development and Application of the microarray technology during the research on the structure and function of microbial communities for acid mine drainages from the copper mines. Doctoral thesis, Central South University, China

  26. Zeng LP (2006) Identification of a deep-sea heterotrophic strain and the study of the effect of its pigment on cultured retinal neurons. Master thesis, Central South University, China

  27. Zhang ZH (2002) Research on bacterial leaching mechanism and its screening and breeding. Doctoral thesis, Central South University, China

  28. Zhang YF (2006) Isolation and bioleaching characterization of autotrophic and heterotrophic bacterium. Master thesis, Central South University, China

  29. Zhang YF, Yang Y, Liu J, Qiu G (2007) Isolation, characterization and phylogenetic analysis of Acidiphilium-like bacterium from acid mine drainage. Adv Mater Res 20–21:473–476

  30. Zhou J, Bruns MA, Tiedje JM (1996) Recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322

  31. Zhou HB, Zeng XX, Liu FF, Qiu G, Hu YH (2006) Screening, identification and desilication of a silicate bacterium. J Cent South Univ Technol 13:337–341

  32. Zhou HB, Liu X, Fu B, Qiu G, Huo Q, Zeng WM, Liu JSH, Chen XH (2007) Isolation and characterization of Acidithiobacillus caldus from several typical environments in China. J Cent South Univ Technol 14:163–169

Download references


This work was supported by grants from the Chinese Science Foundation for Distinguished Group (no. 50321402), the National Nature Science Foundation of China (no. 40376036), and the China Ocean Mineral Resources R & D Association (no. DY105-02-04-05).

Author information

Correspondence to Jufang Huang.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zeng, L., Huang, J., Zhang, Y. et al. An effective method of DNA extraction for bioleaching bacteria from acid mine drainage. Appl Microbiol Biotechnol 79, 881 (2008).

Download citation


  • Bioleaching bacteria
  • Acid mine drainage
  • DNA extraction
  • Rep-PCR