, Volume 22, Issue 6, pp 851–863 | Cite as

Bioleaching of copper- and zinc-bearing ore using consortia of indigenous iron-oxidizing bacteria

  • Wasim Sajjad
  • Guodong ZhengEmail author
  • Gaosen Zhang
  • Xiangxian Ma
  • Wang Xu
  • Suliman Khan
Original Paper


Indigenous iron-oxidizing bacteria were isolated on modified selective 9KFe2+ medium from Baiyin copper mine stope, China. Three distinct acidophilic bacteria were isolated and identified by analyzing the sequences of 16S rRNA gene. Based on published sequences of 16S rRNA gene in the GenBank, a phylogenetic tree was constructed. The sequence of isolate WG101 showed 99% homology with Acidithiobacillus ferrooxidans strain AS2. Isolate WG102 exhibited 98% similarity with Leptospirillum ferriphilum strain YSK. Similarly, isolate WG103 showed 98% similarity with Leptospirillum ferrooxidans strain L15. Furthermore, the biotechnological potential of these isolates in consortia form was evaluated to recover copper and zinc from their ore. Under optimized conditions, 77.68 ± 3.55% of copper and 70.58 ± 3.77% of zinc were dissolved. During the bioleaching process, analytical study of pH and oxidation–reduction potential fluctuations were monitored that reflected efficient activity of the bacterial consortia. The FTIR analysis confirmed the variation in bands after treatment with consortia. The impact of consortia on iron speciation within bioleached ore was analyzed using Mössbauer spectroscopy and clear changes in iron speciation was reported. The use of indigenous bacterial consortia is more efficient compared to pure inoculum. This study provided the basic essential conditions for further upscaling bioleaching application for metal extraction.


Bioleaching Iron-oxidizing bacteria Acidithiobacillus ferrooxidans Mössbauer spectroscopy 



This work was sponsored by CAS-TWAS President’s Fellowship for international Ph.D. students to WS, partially by the Natural Science Foundation of China (41572352) and Key Laboratory of Petroleum Resources, Gansu Province (SZDKFJJ20160601).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Acevedo F, Gentina JC, Valencia P (2004) Optimization of pulp density and particle size in the biooxidation of a pyritic gold concentrate by Sulfolobus metallicus [J]. World J Microbiol Biotechnol 20:865–869CrossRefGoogle Scholar
  2. Ahonen L, Tuovinen OH (1992) Bacterial oxidation of sulfide minerals in column leaching experiments at suboptimal temperatures. Appl Environ Microbiol 58:600–606PubMedPubMedCentralGoogle Scholar
  3. Anders BJ, Colin W (1995) Ferric sulphate oxidation using Thiobacillus ferrooxidans: a review. Process Biochem 30:225–236CrossRefGoogle Scholar
  4. Apel AW, Dugan RP (1978) Hydrogen ion utilization by iron grown Thiobacillus ferrooxidans [M]. In: Murr EL, Torma AE, Brierley AJ (eds) Metallurgical applications of bacterial leaching and related microbiological phenomena. Academic Press, New York, pp 45–58CrossRefGoogle Scholar
  5. Auld RR, Myre M, Mykytczuk NC, Leduc LG, Merritt TJ (2013) Characterization of the microbial acid mine drainage microbial community using culturing and direct sequencing techniques. J Microbiol Methods 93(2):108–115CrossRefPubMedGoogle Scholar
  6. Baba AA, Adekola FA, Atata RF, Ahmed RN, Panda S (2011) Bioleaching of Zn(II) and Pb(II) from Nigerian sphalerite and galena ores by a mixed culture of acidophilic bacteria. Trans Non-ferrous Met Soc China 21:2535–2541CrossRefGoogle Scholar
  7. Bhatti TM, Bigham JM, Carlson L, Tuovinen OH (1993) Mineral products of pyrrhotite oxidation by Thiobacillus ferrooxidans [J]. Appl Environ Microbiol 59:1984–1990PubMedPubMedCentralGoogle Scholar
  8. Cameron RA, Yeung CW, Greer CW, Gould WD, Mortazavi S, Bédard PL, Kennedy KJ (2010) The bacterial community structure during bioleaching of a low-grade nickel sulphide ore in stirred-tank reactors at different combinations of temperature and pH. Hydrometallurgy 104(2):207–215CrossRefGoogle Scholar
  9. Ciftci H, Akcil A (2010) Effect of biooxidation conditions on cyanide consumption and gold recovery from a refractory flotation gold concentrate. Hydrometallurgy 104:142–149CrossRefGoogle Scholar
  10. Das T, Ghosh MK, Chaudhury GR (2005) Assessment of the significant parameters influencing the bio-oxidation and bioprecipitation of iron from industrial leach liquor [J]. Miner Process Extr Metall 114:57–64CrossRefGoogle Scholar
  11. Deng Y, Liu XD, Liu HW, Jiang HD, Xu LF, Xiao YH, Liang YL (2017) Bioleaching of cadmium from contaminated paddy fields by consortium of autotrophic and indigenous cadmium-tolerant bacteria. Solid State Phenom 262:617–621CrossRefGoogle Scholar
  12. Deveci H, Akcil A, Alp I (2004) Bioleaching of complex zinc sulphides using mesophilic and thermophilic bacteria: comparative importance of pH and iron [J]. Hydrometallurgy 73:293–303CrossRefGoogle Scholar
  13. Ding JN, Jian GAO, Wu XL, Zhang CG, Wang DZ, Qiu GZ (2007) Jarosite-type precipitates mediated by YN22, Sulfobacillus thermosulfidooxidans, and their influences on strain. Trans Non-ferrous Met Soc China 17(5):1038–1044CrossRefGoogle Scholar
  14. Fu B, Zhou H, Zhang R, Qiu G (2008) Bioleaching of chalcopyrite by pure and mixed cultures of Acidithiobacillus spp. and Leptospirillum ferriphilum. Int Biodeterior Biodegrad 62:109–115CrossRefGoogle Scholar
  15. Ghassa S, Boruomand Z, Abdollahi H, Moradian M, Akcil A (2014) Bioleaching of high grade Zn–Pb bearing ore by mixed moderate thermophilic microorganisms. Sep Purif Technol 136:241–249CrossRefGoogle Scholar
  16. Guo Z, Zhang L, Cheng Y, Xiao X, Pan F, Jiang K (2010) Effects of pH, pulp density and particle size on solubilization of metals from a Pb/Zn smelting slag using indigenous moderate thermophilic bacteria. Hydrometallurgy 104(1):25–31CrossRefGoogle Scholar
  17. Haghshenas DF, Bonakdarpour B, Keshavarz E, Nasernejad B (2012) Optimization of physicochemical parameters for bioleaching of sphalerite by Acidithiobacillus ferrooxidans using shaking bioreactors. Hydrometallurgy 111–112:22–28CrossRefGoogle Scholar
  18. Hassanshahian M, Ghoebani S (2018) Isolation and characterization of iron and sulfur-oxidizing bacteria from Maiduk copper mine at Shahrbabk province in Iran. Geomicrobiology 35(4):261–265CrossRefGoogle Scholar
  19. Johnson DB (2013) Development and application of biotechnologies in the metal mining industry. Environ Sci Pollut Res 20(11):7768–7776CrossRefGoogle Scholar
  20. Johnson DB, Rolfe S, Hallberg KB, Iversen E (2001) Isolation and phylogenetic characterization of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine. Environ Microbiol 3(10):630–637CrossRefPubMedGoogle Scholar
  21. Johnson DB, Hallberg KB, Hedrich S (2014) Uncovering a microbial enigma: isolation and characterization of the streamer-generating, iron-oxidizing, acidophilic bacterium “Ferrovum myxofaciens”. Appl Environ Microbiol 80(2):672–680CrossRefPubMedPubMedCentralGoogle Scholar
  22. Keeling SE, Palmer ML, Caracatsanis FC, Johnson JA, Watling HR (2005) Leaching of chalcopyrite and sphalerite using bacteria enriched from a spent chalcocite heap [J]. Miner Eng 18:1289–1296CrossRefGoogle Scholar
  23. Khalid AM, Bhatti TM, Umar M (1993) An improved solid medium for isolation, enumeration and genetic investigations of autotrophic iron-and sulphur-oxidizing bacteria. Appl Microbiol Biotechnol 39(2):259–263CrossRefGoogle Scholar
  24. Latorre M, Cortés MP, Travisany D, Di Genova A, Budinich M, Reyes-Jara A, Hödar C, González M, Parada P, Bobadilla-Fazzini RA, Cambiazo V (2016) The bioleaching potential of a bacterial consortium. Bioresour Technol 218:659–666CrossRefPubMedGoogle Scholar
  25. Leahy MJ, Schwarz MP (2009) Modelling jarosite precipitation in isothermal chalcopyrite bioleaching columns. Hydrometallurgy 98:181–191CrossRefGoogle Scholar
  26. Libowitzky E, Rossman GR (1997) An IR adsorption for water in minerals. Am Mineral 82:1111–1115CrossRefGoogle Scholar
  27. Manafi Z, Abdollahi H, Tuovinen OH (2013) Shake flask and column bioleaching of pyritic porphyry copper sulphide ore. Int J Miner Process 119:16–20CrossRefGoogle Scholar
  28. Mishra S, Pradhan N, Panda S, Akcil A (2016) Biodegradation of dibenzothiophene and its application in the production of clean coal. Fuel Process Technol 152:325–342CrossRefGoogle Scholar
  29. Nowaczyk K, Juszczak A, Domka F, Siepak J (1998) The use of Thiobacillus ferrooxidans bacteria in the process of chalcopyrite leaching. Pol J Environ Stud 7:307–312Google Scholar
  30. Okibe N, Gericke M, Hallberg KB, Johnson DB (2003) Enumeration and characterization of acidophilic microorganisms isolated from a pilot plant stirred-tank bioleaching operation. Appl Environ Microbiol 69(4):1936–1943CrossRefPubMedPubMedCentralGoogle Scholar
  31. Olubambi PA, Ndlovu S, Potgieter JH, Borode JO (2008) Role of ore mineralogy in optimizing conditions for bioleaching low-grade complex sulphide ores. Trans Non-ferrous Met Soc China 18(5):1234–1246CrossRefGoogle Scholar
  32. Osorio H, Mangold S, Denis Y, Ñancucheo I, Esparza M, Johnson DB, Bonnefoy V, Dopson M, Holmes DS (2013) Anaerobic sulfur metabolism coupled to dissimilatory iron reduction in the extremophile Acidithiobacillus ferrooxidans. Appl Environ Microbiol 79:2172–2181CrossRefPubMedPubMedCentralGoogle Scholar
  33. Panda S, Sanjay K, Sukla LB, Pradhan N, Subbaiah T, Mishra BK, Prasad MSR, Ray SK (2012) Insights into heap bioleaching of lowgrade chalcopyrite ores: a pilot scale study. Hydrometallurgy 125–126:157–165CrossRefGoogle Scholar
  34. Panda S, Parhi PK, Nayak BD, Pradhan N, Mohapatra UB, Sukla LB (2013a) Two step meso-acidophilic bioleaching of chalcopyrite containing ball mill spillage and removal of the surface passivation layer. Bioresour Technol 130:332–338CrossRefPubMedGoogle Scholar
  35. Panda S, Pradhan N, Mohapatra UB, Panda SK, Rath SS, Nayak BD, Sukla LB, Mishra BK (2013b) Bioleaching of copper from pre and post thermally activated low grade chalcopyrite contained ball mill spillage. Front Environ Sci Eng 7:281–293CrossRefGoogle Scholar
  36. Panda S, Rout PC, Sarangi CK, Mishra S, Pradhan N, Mohapatra UB, Subbaiah T, Sukla LB, Mishra BK (2014) Recovery of copper from a surface altered chalcopyrite contained ball mill spillage through bio-hydrometallurgical route. Korean J Chem Eng 31:452–460CrossRefGoogle Scholar
  37. Panda S, Biswal A, Mishra S, Panda PK, Pradhan N, Mohapatra U, Akcil A (2015) Reductive dissolution by waste newspaper for enhanced meso-acidophilic bioleaching of copper from low grade chalcopyrite: a new concept of biohydrometallurgy. Hydrometallurgy 153:98–105CrossRefGoogle Scholar
  38. Panda S, Akcil A, Mishra S, Erust C (2017) Synergistic effect of biogenic Fe3+ coupled to S° oxidation on simultaneous bioleaching of Cu Co, Zn and As from hazardous pyrite ash waste. J Hazard Mater 325:59–70CrossRefPubMedGoogle Scholar
  39. Peng TJ, Shi LJ, Yu RL, Gu GH, Dan Z, Miao CN, Zeng WM (2016) Effects of processing pH stimulation on cooperative bioleaching of chalcopyrite concentrate by free and attached cells. Trans Non-ferrous Met Soc China 26(8):2220–2229CrossRefGoogle Scholar
  40. Petersen J, Dixon DG (2007) Modeling and optimization of heap bioleach processes. In: Rawlings DE, Johnson DB (eds) Biomining. Springer, Berlin, pp 153–176CrossRefGoogle Scholar
  41. Petrus HBTM, Wanta KC, Setiawan H, Perdana I, Astuti W (2018) Effect of pulp density and particle size on indirect bioleaching of Pomalaa nickel laterite using metabolic citric acid. IOP Conf Ser Mater Sci Eng 285(1):012004 (IOP Publishing) CrossRefGoogle Scholar
  42. Prasad PSR, Prasad KS, Chaitanya VK, Babu EVSSK, Sreedhar B, Murthy SR (2006) In situ FTIR study on the dehydration of natural goethite. J Asian Earth Sci 27(4):503–511CrossRefGoogle Scholar
  43. Rohwerder T, Gehrke T, Kinzler K, Sand W (2003) Bioleaching review part A: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. Appl Microbiol Biotechnol 63:239–248CrossRefPubMedGoogle Scholar
  44. Ruan HD, Frost RL, Kloprogge JT (2001) The behavior of hydroxyl units of synthetic goethite and its dehydroxylated product hematite. Spectrochim Acta Part A Mol Biomol Spectrosc 57(13):2575–2586CrossRefGoogle Scholar
  45. Russell JD, Fraser AR (1994) In: Wilson MJ (ed) Infrared methods in clay mineralogy: spectroscopic and chemical determinative methods. Chapman & Hall, London, pp 49–52Google Scholar
  46. Sajjad W, Zheng G, Zhang G, Ma X, Xu W, Ali B, Rafiq M (2018) Diversity of prokaryotic communities indigenous to acid mine drainage and related rocks from Baiyin open-pit copper mine stope, China. Geomicrobiology 35(7):580–600CrossRefGoogle Scholar
  47. Sand W, Gehrke T, Jozsa PG, Schippers A (2001) Biochemistry of bacterial leaching direct vs. indirect bioleaching. Hydrometallurgy 59:159–175CrossRefGoogle Scholar
  48. Sanket AS, Ghosh S, Sahoo R, Nayak S, Das AP (2017) Molecular identification of acidophilic manganese (Mn)-solubilizing bacteria from mining effluents and their application in mineral beneficiation. Geomicrobiology 34(1):71–80CrossRefGoogle Scholar
  49. Shiers DW, Collinson DM, Watling HR (2017) The impact of heap self-heating on microbial activity during the bioleaching of low-grade copper sulfide ores. Solid State Phenom 262:233–236 (Trans Tech Publications) CrossRefGoogle Scholar
  50. Silverman MP, Lundgren DG (1959) Studies on the chemoautotrophic iron bacterium Ferrobacillus ferrooxidans; an improved medium and a harvesting procedure for securing high cell yields. J Bacteriol 77(5):642PubMedPubMedCentralGoogle Scholar
  51. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526PubMedGoogle Scholar
  52. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  53. Tipre DR, Dave SR (2004) Bioleaching process for Cu–Pb–Zn bulk concentrate at high pulp density [J]. Hydrometallurgy 75:37–43CrossRefGoogle Scholar
  54. Tischler JS, Jwair RJ, Gelhaar N, Drechsel A, Skirl AM, Wiacek C, Schlömann M (2013) New cultivation medium for “Ferrovum” and Gallionella-related strains. J Microbiol Methods 95(2):138–144CrossRefPubMedGoogle Scholar
  55. Utimura SK, Rosario CGA, Botelho AB, Tenório JAS, Espinosa DCR (2017) Bioleaching process for metal recovery from waste materials. Energy Technol. (Springer, Cham) CrossRefGoogle Scholar
  56. Van der Grift CJG, Geus JW, Kappers MJ, Van der Maas JH (1989) Characterization of copper–silica catalysts by means of in situ diffuse reflectance infrared Fourier transform spectroscopy. Catal Lett 3(2):159–168CrossRefGoogle Scholar
  57. Wang Y, Su L, Zeng W, Wan L, Chen Z, Zhang L, Zhou H (2014) Effect of pulp density on planktonic and attached community dynamics during bioleaching of chalcopyrite by a moderately thermophilic microbial culture under uncontrolled conditions. Miner Eng 61:66–72CrossRefGoogle Scholar
  58. Watling HR (2006) The bioleaching of sulphide minerals with emphasis on copper sulphides—a review. Hydrometallurgy 84:81–108CrossRefGoogle Scholar
  59. Yang Y, Diao M, Liu K, Qian L, Nguyen AV, Qiu G (2013) Column bioleaching of low-grade copper ore by Acidithiobacillus ferrooxidans in pure and mixed cultures with a heterotrophic acidophile Acidiphilium sp. Hydrometallurgy 131:93–98CrossRefGoogle Scholar
  60. Yang Z, Zhang Z, Chai L, Wang Y, Liu Y, Xiao R (2016) Bioleaching remediation of heavy metal-contaminated soils using Burkholderia sp. Z-90. J Hazard Mater 301:145–152CrossRefPubMedGoogle Scholar
  61. Zeng X, Twardowska I, Wei S, Sun L, Wang J, Zhu J, Cai J (2015) Removal of trace metals and improvement of dredged sediment dewaterability by bioleaching combined with Fenton-like reaction. J Hazard Mater 288:51–59CrossRefPubMedGoogle Scholar
  62. Zheng GD, Fu BH, Takahashi Y, Kuno A, Matsuo M, Zhang JD (2010) Chemical speciation of redox sensitive elements during hydrocarbon leaching in the Junggar Basin, Northwest China. J Asian Earth Sci 39(6):713–723CrossRefGoogle Scholar
  63. Zhou HB, Zeng WM, Yang ZF, Xie YJ, Qiu GZ (2009) Bioleaching of chalcopyrite concentrate by a moderately thermophilic culture in a stirred tank reactor. Bioresour Technol 100:515–520CrossRefPubMedGoogle Scholar
  64. Zhu W, Xia J, Yang Y, Nie ZY, Zheng L, Ma CY, Zhang RY, Peng A, Tang L, Qiu G (2011) Sulfur oxidation activities of pure and mixed thermophiles and sulfur speciation in bioleaching of chalcopyrite. Bioresour Technol 102:3877–3882CrossRefPubMedGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Wasim Sajjad
    • 1
    • 2
  • Guodong Zheng
    • 1
    Email author
  • Gaosen Zhang
    • 3
  • Xiangxian Ma
    • 1
  • Wang Xu
    • 1
    • 2
  • Suliman Khan
    • 2
    • 4
  1. 1.Key Laboratory of Petroleum Resources, Gansu Province/Key Laboratory of Petroleum Resources Research, Institute of Geology and GeophysicsChinese Academy of SciencesLanzhouPeople’s Republic of China
  2. 2.University of Chinese Academy of SciencesBeijingPeople’s Republic of China
  3. 3.Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province/Key Laboratory of Desert and Desertification, Northwest Institute of Eco-Environment and ResourcesChinese Academy of SciencesLanzhouPeople’s Republic of China
  4. 4.The Key Laboratory of Aquatic Biodiversity and Conservation, Sciences, Institute of HydrologyChinese Academy of Sciences WuhanHubeiPeople’s Republic of China

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