Journal of Industrial Microbiology & Biotechnology

, Volume 44, Issue 12, pp 1613–1625 | Cite as

Evolution of copper arsenate resistance for enhanced enargite bioleaching using the extreme thermoacidophile Metallosphaera sedula

  • Chenbing Ai
  • Samuel McCarthy
  • Yuting Liang
  • Deepak Rudrappa
  • Guanzhou Qiu
  • Paul BlumEmail author
Genetics and Molecular Biology of Industrial Organisms - Original Paper


Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu2+ resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.


Extreme thermoacidophile Enargite bioleaching Metallosphaera sedula Arsenate resistance Mutation 



This work was supported by the Department of Energy Joint Genome Institute (DOE-JGI) under the Community Sequencing Program (CSP) (proposal ID 1515, project IDs 1036419, 1036422). And the University of Nebraska Cell Development Facility. This work was also financially supported by the China National Basic Research Program (No. 2010CB630901).

Supplementary material

10295_2017_1973_MOESM1_ESM.docx (297 kb)
Supplementary material 1 (DOCX 297 kb)


  1. 1.
    Ai C, McCarthy S, Eckrich V, Rudrappa D, Qiu G, Blum P (2016) Increased acid resistance of the archaeon, Metallosphaera sedula by adaptive laboratory evolution. J Ind Microbiol Biotechnol 43:1455–1465. doi: 10.1007/s10295-016-1812-0 CrossRefPubMedGoogle Scholar
  2. 2.
    Ai CB, McCarthy M, Schackwitz W, Martin J, Lipzen A, Blum P (2015) Complete genome sequences of evolved arsenate-resistant Metallosphaera sedula strains. Genome Announc 3:e01142-15. doi: 10.1128/genomeA.01142-15 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Allen MB (1959) Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch Mikrobiol 32:270–277CrossRefPubMedGoogle Scholar
  4. 4.
    Auernik KS, Maezato Y, Blum PH, Kelly RM (2007) The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism. Appl Environ Microbiol 74:682–692. doi: 10.1128/aem.02019-07 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Brierley CL, Brierley JA (2013) Progress in bioleaching: part B: applications of microbial processes by the minerals industries. Appl Microbiol Biotechnol 97:7543–7552. doi: 10.1007/s00253-013-5095-3 CrossRefPubMedGoogle Scholar
  6. 6.
    Bromfield L, Africa CJ, Harrison STL, van Hille RP (2011) The effect of temperature and culture history on the attachment of Metallosphaera hakonensis to mineral sulfides with application to heap bioleaching. Miner Eng 24:1157–1165. doi: 10.1016/j.mineng.2011.03.019 CrossRefGoogle Scholar
  7. 7.
    Deveci H (2002) Effect of solids on viability of acidophilic bacteria. Miner Eng 15:1181–1189CrossRefGoogle Scholar
  8. 8.
    Doerrler WT, Sikdar R, Kumar S, Boughner LA (2012) New functions for the ancient DedA membrane protein family. J Bacteriol 195:3–11. doi: 10.1128/jb.01006-12 CrossRefPubMedGoogle Scholar
  9. 9.
    Feng S, Yang H, Wang W (2015) Improved chalcopyrite bioleaching by Acidithiobacillus sp. via direct step-wise regulation of microbial community structure. Bioresour Technol 192:75–82. doi: 10.1016/j.biortech.2015.05.055 CrossRefPubMedGoogle Scholar
  10. 10.
    Feng S, Yang H, Wang W (2016) Insights to the effects of free cells on community structure of attached cells and chalcopyrite bioleaching during different stages. Bioresour Technol 200:186–193. doi: 10.1016/j.biortech.2015.09.054 CrossRefPubMedGoogle Scholar
  11. 11.
    Feng S, Yang H, Zhan X, Wang W (2014) Novel integration strategy for enhancing chalcopyrite bioleaching by Acidithiobacillus sp. in a 7-L fermenter. Bioresour Technol 161:371–378. doi: 10.1016/j.biortech.2014.03.027 CrossRefPubMedGoogle Scholar
  12. 12.
    Huber Gertrud, Spinnler Carola, Gambacorta Agata, Stetter KO (1989) Metallosphaera sedula gen. and sp. nov. represents a new genus of aerobic, metal-mobilizing, thermoacidophilic archaebacteria. Syst Appl Microbiol 12:38–47CrossRefGoogle Scholar
  13. 13.
    Hille RPV, Wyk NV, Harrison STL (eds) (2011) Review of the microbial ecology of BIOX® reactors illustrate the dominance of the genus Ferroplasma in many commercial reactors. Biohydrometallurgy: biotech key to unlock minerals resources value. Central South University Press, ChangshaGoogle Scholar
  14. 14.
    Kotze AA, Tuffin IM, Deane SM, Rawlings DE (2006) Cloning and characterization of the chromosomal arsenic resistance genes from Acidithiobacillus caldus and enhanced arsenic resistance on conjugal transfer of ars genes located on transposon TnAtcArs. Microbiology 152:3551–3560. doi: 10.1099/mic.0.29247-0 CrossRefPubMedGoogle Scholar
  15. 15.
    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, Maass A (2016) The bioleaching potential of a bacterial consortium. Bioresour Technol 218:659–666. doi: 10.1016/j.biortech.2016.07.012 CrossRefPubMedGoogle Scholar
  16. 16.
    Lattanzi P, Da Pelo S, Musu E, Atzei D, Elsener B, Fantauzzi M, Rossi A (2008) Enargite oxidation: a review. Earth Sci Rev 86:62–88. doi: 10.1016/j.earscirev.2007.07.006 CrossRefGoogle Scholar
  17. 17.
    Li B, Lin J, Mi S, Lin J (2010) Arsenic resistance operon structure in Leptospirillum ferriphilum and proteomic response to arsenic stress. Bioresour Technol 101:9811–9814. doi: 10.1016/j.biortech.2010.07.043 CrossRefPubMedGoogle Scholar
  18. 18.
    Maezato Y, Johnson T, McCarthy S, Dana K, Blum P (2012) Metal resistance and lithoautotrophy in the extreme thermoacidophile Metallosphaera sedula. J Bacteriol 194:6856–6863. doi: 10.1128/jb.01413-12 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    McCarthy S, Ai C, Wheaton G, Tevatia R, Eckrich V, Kelly R, Blum P (2014) Role of an archaeal PitA transporter in the copper and arsenic resistance of Metallosphaera sedula, an extreme thermoacidophile. J Bacteriol 196:3562–3570. doi: 10.1128/jb.01707-14 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    McCarthy S, Johnson T, Pavlik B, Payne S, Schackwitz W, Martin J, Lipzen A, Keffeler E, Blum P (2015) Expanding the limits of thermoacidophily in the archaeon Sulfolobus solfataricus by adaptive evolution. Appl Environ Microbiol 82(3):857–867. doi: 10.1128/aem.03225-15 CrossRefPubMedGoogle Scholar
  21. 21.
    Motamedi Ali Shafiee, Cai Sheng-Jian, Streicher Stanley L, Arison Byron H, Miller RR (1996) Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520. J Bacteriol 178:5243–5248CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Okibe N, Johnson DB (2004) Biooxidation of pyrite by defined mixed cultures of moderately thermophilic acidophiles in pH-controlled bioreactors: significance of microbial interactions. Biotechnol Bioeng 87:574–583. doi: 10.1002/bit.20138 CrossRefPubMedGoogle Scholar
  23. 23.
    Orell A, Navarro CA, Arancibia R, Mobarec JC, Jerez CA (2010) Life in blue: copper resistance mechanisms of bacteria and Archaea used in industrial biomining of minerals. Biotechnol Adv 28:839–848. doi: 10.1016/j.biotechadv.2010.07.003 CrossRefPubMedGoogle Scholar
  24. 24.
    Parada F, Jeffrey MI, Asselin E (2014) Leaching kinetics of enargite in alkaline sodium sulphide solutions. Hydrometallurgy 146:48–58. doi: 10.1016/j.hydromet.2014.03.003 CrossRefGoogle Scholar
  25. 25.
    Plackowski C, Hampton MA, Nguyen AV, Bruckard WJ (2013) Fundamental studies of electrochemically controlled surface oxidation and hydrophobicity of natural enargite. Langmuir 29:2371–2386. doi: 10.1021/la3043654 CrossRefPubMedGoogle Scholar
  26. 26.
    Rawlings DE, Johnson DB (2007) The microbiology of biomining: development and optimization of mineral-oxidizing microbial consortia. Microbiology 153:315–324. doi: 10.1099/mic.0.2006/001206-0 CrossRefPubMedGoogle Scholar
  27. 27.
    odrı́guez R, Ballester A, Blázquez ML, González F, Muñoz JA (2003) New information on the chalcopyrite bioleaching mechanism at low and high temperature. Hydrometallurgy 71:47–56. doi: 10.1016/s0304-386x(03)00173-7 CrossRefGoogle Scholar
  28. 28.
    Rudrappa D, White D, Yao AI, Singh R, Pavlik BJ, Blum P, Facciotti MT (2015) Identification of an archaeal mercury regulon by chromatin immunoprecipitation. Microbiology 161:2423–2433. doi: 10.1099/mic.0.000189 CrossRefPubMedGoogle Scholar
  29. 29.
    Ruiz MC, Vera MV, Padilla R (2011) Mechanism of enargite pressure leaching in the presence of pyrite. Hydrometallurgy 105:290–295. doi: 10.1016/j.hydromet.2010.11.002 CrossRefGoogle Scholar
  30. 30.
    Sasaki K, Takatsugi K, Ishikura K, Hirajima T (2010) Spectroscopic study on oxidative dissolution of chalcopyrite, enargite and tennantite at different pH values. Hydrometallurgy 100:144–151. doi: 10.1016/j.hydromet.2009.11.007 CrossRefGoogle Scholar
  31. 31.
    Sasaki K, Takatsugi K, Kaneko K, Kozai N, Ohnuki T, Tuovinen OH, Hirajima T (2010) Characterization of secondary arsenic-bearing precipitates formed in the bioleaching of enargite by Acidithiobacillus ferrooxidans. Hydrometallurgy 104:424–431. doi: 10.1016/j.hydromet.2009.12.012 CrossRefGoogle Scholar
  32. 32.
    Shiers DW, Ralph DE, Bryan CG, Watling HR (2013) Substrate utilisation by Acidianus brierleyi, Metallosphaera hakonensis and Sulfolobus metallicus in mixed ferrous ion and tetrathionate growth media. Miner Eng 48:86–93. doi: 10.1016/j.mineng.2012.10.006 CrossRefGoogle Scholar
  33. 33.
    Song J, Lin JQ, Gao L, Lin JQ, Qu YB (2008) Modeling and simulation of enargite bioleaching. Chin J Chem Eng 16:785–790CrossRefGoogle Scholar
  34. 34.
    Stookey LL (1970) Ferrozine-a new spectrophotometric reagent for iron. Anal Chem 42:779–781CrossRefGoogle Scholar
  35. 35.
    Struck A-W, Thompson ML, Wong LS, Micklefield J (2012) s-Adenosyl-methionine-dependent methyltransferases: highly versatile enzymes in biocatalysis, biosynthesis and other biotechnological applications. ChemBioChem 13:2642–2655CrossRefPubMedGoogle Scholar
  36. 36.
    Sun H, Chen M, Zou L, Shu R, Ruan R (2015) Study of the kinetics of pyrite oxidation under controlled redox potential. Hydrometallurgy 155:13–19. doi: 10.1016/j.hydromet.2015.04.003 CrossRefGoogle Scholar
  37. 37.
    Takatsugi K, Sasaki K, Hirajima T (2011) Mechanism of the enhancement of bioleaching of copper from enargite by thermophilic iron-oxidizing archaea with the concomitant precipitation of arsenic. Hydrometallurgy 109:90–96. doi: 10.1016/j.hydromet.2011.05.013 CrossRefGoogle Scholar
  38. 38.
    Uddin MN, Abdus Salam M, Hossain MA (2013) Spectrophotometric measurement of Cu(DDTC)2 for the simultaneous determination of zinc and copper. Chemosphere 90:366–373. doi: 10.1016/j.chemosphere.2012.07.029 CrossRefPubMedGoogle Scholar
  39. 39.
    Wang YG, Zeng WM, Qiu GZ, Chen XH, Zhou HB (2014) A moderately thermophilic mixed microbial culture for bioleaching of chalcopyrite concentrate at high pulp density. Appl Environ Microbiol 80:741–750CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Watling H, Shiers D, Collinson D (2015) Extremophiles in mineral sulphide heaps: some bacterial responses to variable temperature, acidity and solution composition. Microorganisms 3:364–390. doi: 10.3390/microorganisms3030364 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Watling HR, Watkin ELJ, Ralph DE (2010) The resilience and versatility of acidophiles that contribute to the bio-assisted extraction of metals from mineral sulphides. Environ Technol 31:915–933. doi: 10.1080/09593331003646646 CrossRefGoogle Scholar
  42. 42.
    R-B Zhang, M-M Wei, H-G Ji, X-H Chen, G-Z Qiu, H-B Zhou (2008) Application of real-time PCR to monitor population dynamics of defined mixed cultures of moderate thermophiles involved in bioleaching of chalcopyrite. Appl Microbiol Biotechnol 81:1161–1168. doi: 10.1007/s00253-008-1792-8 Google Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2017

Authors and Affiliations

  • Chenbing Ai
    • 1
    • 2
  • Samuel McCarthy
    • 2
  • Yuting Liang
    • 1
    • 2
  • Deepak Rudrappa
    • 2
  • Guanzhou Qiu
    • 1
  • Paul Blum
    • 2
    Email author
  1. 1.School of Minerals Processing and BioengineeringCentral South UniversityChangshaPeople’s Republic of China
  2. 2.School of Biological Sciences, Beadle Center for GeneticsUniversity of Nebraska-LincolnLincolnUSA

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