Applied Microbiology and Biotechnology

, Volume 101, Issue 14, pp 5889–5901 | Cite as

Characterization of an autotrophic bioreactor microbial consortium degrading thiocyanate

  • Mathew Paul Watts
  • Liam Patrick Spurr
  • Han Ming Gan
  • John William Moreau
Environmental biotechnology


Thiocyanate (SCN) forms as a by-product of cyanidation during gold ore processing and can be degraded by a variety of microorganisms utilizing it as an energy, nitrogen, sulphur and/or carbon source. In complex consortia inhabiting bioreactor systems, a range of metabolisms are sustained by SCN degradation; however, despite the addition or presence of labile carbon sources in most bioreactor designs to date, autotrophic bacteria have been found to dominate key metabolic functions. In this study, we cultured an autotrophic SCN-degrading consortium directly from gold mine tailings. In a batch-mode bioreactor experiment, this consortium degraded 22 mM SCN, accumulating ammonium (NH4 +) and sulphate (SO4 2−) as the major end products. The consortium consisted of a diverse microbial community comprised of chemolithoautotrophic members, and despite the absence of an added organic carbon substrate, a significant population of heterotrophic bacteria. The role of eukaryotes in bioreactor systems is often poorly understood; however, we found their 18S rRNA genes to be most closely related to sequences from bacterivorous Amoebozoa. Through combined chemical and phylogenetic analyses, we were able to infer roles for key microbial consortium members during SCN biodegradation. This study provides a basis for understanding the behaviour of a SCN degrading bioreactor under autotrophic conditions, an anticipated approach to remediating SCN at contemporary gold mines.


Bioremediation Thiocyanate Autotrophic Bioreactor Environmental biotechnology 



Funding for this research was provided by Newmarket Gold Inc. We gratefully acknowledge David Coe, Will Wettenhall, Yan Lim and Megan Parnaby for access to the field site, assistance when collecting the samples and providing access to historic chemical data.

Compliance with ethical standards


This study was funded by Newmarket Gold Inc.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2017_8313_MOESM1_ESM.pdf (978 kb)
ESM 1 (PDF 977 kb)


  1. Akcil A (2003) Destruction of cyanide in gold mill effluents: biological versus chemical treatments. Biotechnol Adv 21(6):501–511. doi: 10.1016/S0734-9750(03)00099-5 CrossRefPubMedGoogle Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410CrossRefPubMedGoogle Scholar
  3. Amaral-Zettler LA, McCliment EA, Ducklow HW, Huse SM (2009) Correction: a method for studying Protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes. PLoS One 4(12)Google Scholar
  4. Amarger N, Macheret V, Laguerre G (1997) Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules. Int J Syst Evol Microbiol 47(4):996–1006Google Scholar
  5. Anderson PM (1980) Purification and properties of the inducible enzyme cyanase. Biochemistry 19(13):2882–2888CrossRefPubMedGoogle Scholar
  6. Anderson PM, Y-c S, Fuchs JA (1990) The cyanase operon and cyanate metabolism. FEMS Microbiol Rev 7(3–4):247–252CrossRefPubMedGoogle Scholar
  7. Berben T, Overmars L, Sorokin DY, Muyzer G (2017) Comparative genome analysis of three thiocyanate oxidizing Thioalkalivibrio species isolated from Soda Lakes. Front Microbiol 8:254. doi: 10.3389/fmicb.2017.00254
  8. Bhunia F, Saha N, Kaviraj A (2000) Toxicity of thiocyanate to fish, plankton, worm, and aquatic ecosystem. Bull Environ Contam Toxicol 64(2):197–204CrossRefPubMedGoogle Scholar
  9. Boden R, Cleland D, Green PN, Katayama Y, Uchino Y, Murrell JC, Kelly DP (2012) Phylogenetic assessment of culture collection strains of Thiobacillus thioparus, and definitive 16S rRNA gene sequences for T. thioparus, T. denitrificans, and Halothiobacillus neapolitanus. Arch Microbiol 194(3):187–195CrossRefPubMedGoogle Scholar
  10. Brenner K, You L, Arnold FH (2008) Engineering microbial consortia: a new frontier in synthetic biology. Trends Biotechnol 26(9):483–489CrossRefPubMedGoogle Scholar
  11. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7(5):335–336CrossRefPubMedPubMedCentralGoogle Scholar
  12. Chaudhari AU, Kodam KM (2010) Biodegradation of thiocyanate using co-culture of Klebsiella pneumoniae and Ralstonia sp. Appl Microbiol Biotechnol 85(4):1167–1174CrossRefPubMedGoogle Scholar
  13. Chen Y, Wu L, Boden R, Hillebrand A, Kumaresan D, Moussard H, Baciu M, Lu Y, Murrell JC (2009) Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave. ISME J 3(9):1093–1104CrossRefPubMedGoogle Scholar
  14. Chung YC, Huang C, Tseng CP (1996) Biodegradation of hydrogen sulfide by a laboratory-scale immobilized Pseudomonas putida CH11 biofilter. Biotechnol Prog 12(6):773–778CrossRefPubMedGoogle Scholar
  15. Dash RR, Gaur A, Balomajumder C (2009) Cyanide in industrial wastewaters and its removal: a review on biotreatment. J Hazard Mater 163(1):1–11CrossRefPubMedGoogle Scholar
  16. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72(7):5069–5072CrossRefPubMedPubMedCentralGoogle Scholar
  17. Diange EA, Lee S-S (2013) Rhizobium halotolerans sp. nov., isolated from chloroethylenes contaminated soil. Curr Microbiol 66(6):599–605CrossRefPubMedGoogle Scholar
  18. du Plessis C, Barnard P, Muhlbauer R, Naldrett K (2001) Empirical model for the autotrophic biodegradation of thiocyanate in an activated sludge reactor. Lett Appl Microbiol 32(2):103–107CrossRefPubMedGoogle Scholar
  19. Eaton AD, Franson MAH (2005) Standard methods 4500-CN M in standard methods for the examination of water and wastewater. American Public Health Association, American Water Works Association, Water Environment Federation, 21st ednGoogle Scholar
  20. Ebbs S (2004) Biological degradation of cyanide compounds. Curr Opin Biotechnol 15(3):231–236. doi: 10.1016/j.Copbio.2004.03.006 CrossRefPubMedGoogle Scholar
  21. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19):2460–2461CrossRefPubMedGoogle Scholar
  22. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27(16):2194–2200CrossRefPubMedPubMedCentralGoogle Scholar
  23. Felföldi T, Székely AJ, Gorál R, Barkács K, Scheirich G, András J, Rácz A, Márialigeti K (2010) Polyphasic bacterial community analysis of an aerobic activated sludge removing phenols and thiocyanate from coke plant effluent. Bioresour Technol 101(10):3406–3414CrossRefPubMedGoogle Scholar
  24. Francis CA, Beman JM, Kuypers MM (2007) New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation. ISME J 1(1):19–27CrossRefPubMedGoogle Scholar
  25. Friedrich CG, Bardischewsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8(3):253–259CrossRefPubMedGoogle Scholar
  26. Gould WD, King M, Mohapatra BR, Cameron RA, Kapoor A, Koren DW (2012) A critical review on destruction of thiocyanate in mining effluents. Miner Eng 34:38–47CrossRefGoogle Scholar
  27. Grigor’eva N, Kondrat’eva T, Krasil’nikova E, Karavaiko G (2006) Mechanism of cyanide and thiocyanate decomposition by an association of Pseudomonas putida and Pseudomonas stutzeri strains. Microbiology 75(3):266–273Google Scholar
  28. Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21(3):494–504CrossRefPubMedPubMedCentralGoogle Scholar
  29. Happold F, Jones G, Pratt D (1958) Utilization of thiocyanate by Thiobacillus thioparus and T. thiocyanoxidans. Nature 182:266-267Google Scholar
  30. Huddy RJ, van Zyl AW, van Hille RP, Harrison ST (2015) Characterisation of the complex microbial community associated with the ASTER™ thiocyanate biodegradation system. Miner Eng 76:65–71CrossRefGoogle Scholar
  31. Joshi DR, Zhang Y, Tian Z, Gao Y, Yang M (2016) Performance and microbial community composition in a long-term sequential anaerobic-aerobic bioreactor operation treating coking wastewater. Appl Microbiol Biotechnol:1–12Google Scholar
  32. Kantor RS, Zyl AW, Hille RP, Thomas BC, Harrison ST, Banfield JF (2015) Bioreactor microbial ecosystems for thiocyanate and cyanide degradation unravelled with genome-resolved metagenomics. Environ MicrobiolGoogle Scholar
  33. Karavaiko G, Kondrat’eva T, Savari E, Grigor’eva N, Avakyan Z (2000) Microbial degradation of cyanide and thiocyanate. Microbiology 69(2):167–173CrossRefGoogle Scholar
  34. Katayama Y, Narahara Y, Inoue Y, Amano F, Kanagawa T, Kuraishi H (1992) A thiocyanate hydrolase of Thiobacillus thioparus. A novel enzyme catalyzing the formation of carbonyl sulfide from thiocyanate. J Biol Chem 267(13):9170–9175PubMedGoogle Scholar
  35. Kelly DP (1999) Thermodynamic aspects of energy conservation by chemolithotrophic sulfur bacteria in relation to the sulfur oxidation pathways. Arch Microbiol 171(4):219–229CrossRefGoogle Scholar
  36. Kelly DP, Wood AP (2000) Confirmation of Thiobacillus denitrificans as a species of the genus Thiobacillus, in the beta-subclass of the Proteobacteria, with strain NCIMB 9548 as the type strain. Int J Syst Evol Microbiol 50(2):547–550CrossRefPubMedGoogle Scholar
  37. Kelly DP, Shergill JK, Lu W-P, Wood AP (1997) Oxidative metabolism of inorganic sulfur compounds by bacteria. Antonie Van Leeuwenhoek 71(1–2):95–107CrossRefPubMedGoogle Scholar
  38. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO (2012) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41(1):e1Google Scholar
  39. Kossoff D, Dubbin W, Alfredsson M, Edwards S, Macklin M, Hudson-Edwards KA (2014) Mine tailings dams: characteristics, failure, environmental impacts, and remediation. Appl Geochem 51:229–245CrossRefGoogle Scholar
  40. Kwon HK, Woo SH, Park JM (2002) Thiocyanate degradation by Acremonium strictum and inhibition by secondary toxicants. Biotechnol Lett 24(16):1347–1351CrossRefGoogle Scholar
  41. Lay-Son M, Drakides C (2008) New approach to optimize operational conditions for the biological treatment of a high-strength thiocyanate and ammonium waste: pH as key factor. Water Res 42(3):774–780CrossRefPubMedGoogle Scholar
  42. Lee C, Kim J, Do H, Hwang S (2008) Monitoring thiocyanate-degrading microbial community in relation to changes in process performance in mixed culture systems near washout. Water Res 42(4–5):1254–1262. doi: 10.1016/j.watres.2007.09.017 CrossRefPubMedGoogle Scholar
  43. Lindemann SR, Bernstein HC, Song H-S, Fredrickson JK, Fields MW, Shou W, Johnson DR, Beliaev AS (2016) Engineering microbial consortia for controllable outputs. ISME J 10:2077–2084Google Scholar
  44. Little B, Ray R, Pope R (2000) Relationship between corrosion and the biological sulfur cycle: a review. Corrosion 56(4):433–443CrossRefGoogle Scholar
  45. Luther GW, Findlay AJ, MacDonald DJ, Owings SM, Hanson TE, Beinart RA, Girguis PR (2011) Thermodynamics and kinetics of sulfide oxidation by oxygen: a look at inorganically controlled reactions and biologically mediated processes in the environment. Front Microbiol 2:62CrossRefPubMedPubMedCentralGoogle Scholar
  46. Luthy RG, Bruce SG Jr (1979) Kinetics of reaction of cyanide and reduced sulfur species in aqueous solution. Environ Sci Technol 13(12):1481–1487CrossRefGoogle Scholar
  47. Magasanik B (1982) Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet 16(1):135–168CrossRefPubMedGoogle Scholar
  48. Martens-Habbena W, Berube PM, Urakawa H, José R, Stahl DA (2009) Ammonia oxidation kinetics determine niche separation of nitrifying archaea and bacteria. Nature 461(7266):976–979CrossRefPubMedGoogle Scholar
  49. Matějů V, Čižinská S, Krejčí J, Janoch T (1992) Biological water denitrification—a review. Enzym Microb Technol 14(3):170–183CrossRefGoogle Scholar
  50. Millero FJ, Hubinger S, Fernandez M, Garnett S (1987) Oxidation of H2S in seawater as a function of temperature, pH, and ionic strength. Environ Sci Technol 21(5):439–443CrossRefPubMedGoogle Scholar
  51. Mori K, K-i S, Urabe T, Sugihara M, Tanaka K, Hamada M, Hanada S (2011) Thioprofundum hispidum sp. nov., an obligately chemolithoautotrophic sulfur-oxidizing gammaproteobacterium isolated from the hydrothermal field on Suiyo Seamount, and proposal of Thioalkalispiraceae fam. nov. in the order Chromatiales. Int J Syst Evol Microbiol 61(10):2412–2418CrossRefPubMedGoogle Scholar
  52. Moses CO, Nordstrom DK, Herman JS, Mills AL (1987) Aqueous pyrite oxidation by dissolved oxygen and by ferric iron. Geochim Cosmochim Acta 51(6):1561–1571CrossRefGoogle Scholar
  53. Mudder TI, Botz M, Smith A (2001) Chemistry and treatment of cyanidation wastes. Mining Journal Books, LondonGoogle Scholar
  54. Müller T, Walter B, Wirtz A, Burkovski A (2006) Ammonium toxicity in bacteria. Curr Microbiol 52(5):400–406CrossRefPubMedGoogle Scholar
  55. Nordstrom DK, Southam G (1997) Geomicrobiology of sulfide mineral oxidation. Rev Mineral 35:361–390Google Scholar
  56. O’Dell JW (1993) Determination of ammonia nitrogen by semi-automated colorimetry (method 350.1-1). Environmental monitoring systems laboratory office of research and development, EPA/600/R-93/100Google Scholar
  57. Ogawa T, Noguchi K, Saito M, Nagahata Y, Kato H, Ohtaki A, Nakayama H, Dohmae N, Matsushita Y, Odaka M (2013) Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is one of the β-carbonic anhydrase family enzymes. J Am Chem Soc 135(10):3818–3825Google Scholar
  58. Oh KJ, Kim D, Lee I-H (1998) Development of effective hydrogen sulphide removing equipment using Thiobacillus sp. IW Environ Pollut 99(1):87–92CrossRefPubMedGoogle Scholar
  59. Palatinszky M, Herbold C, Jehmlich N, Pogoda M, Han P, von Bergen M, Lagkouvardos I, Karst SM, Galushko A, Koch H (2015) Cyanate as an energy source for nitrifiers. Nature 524(7563):105–108CrossRefPubMedPubMedCentralGoogle Scholar
  60. Park D-H, Cha J-M, Ryu H-W, Lee G-W, Yu E-Y, Rhee J-I, Park J-J, Kim S-W, Lee I-W, Joe Y-I (2002) Hydrogen sulfide removal utilizing immobilized thiobacillus sp. IW with Ca-alginate bead. Biochem Eng J 11(2):167–173CrossRefGoogle Scholar
  61. Rogerson A, Hannah F, Gothe G (1996) The grazing potential of some unusual marine benthic amoebae feeding on bacteria. Eur J Protistol 32(2):271–279CrossRefGoogle Scholar
  62. Ryu B-G, Kim W, Nam K, Kim S, Lee B, Park MS, Yang J-W (2015) A comprehensive study on algal–bacterial communities shift during thiocyanate degradation in a microalga-mediated process. Bioresour Technol 191:496–504Google Scholar
  63. Sorokin D (2002) Oxidation of inorganic sulfur compounds by obligatory organotrophic bacteria. Mikrobiologiia 72(6):725–739Google Scholar
  64. Sorokin DY, Teske A, Robertson LA, Kuenen JG (1999) Anaerobic oxidation of thiosulfate to tetrathionate by obligately heterotrophic bacteria, belonging to the Pseudomonas stutzeri group. FEMS Microbiol Ecol 30(2):113–123CrossRefPubMedGoogle Scholar
  65. Sorokin DY, Tourova TP, Lysenko AM, Kuenen JG (2001) Microbial thiocyanate utilization under highly alkaline conditions. Appl Environ Microbiol 67(2):528–538CrossRefPubMedPubMedCentralGoogle Scholar
  66. Sorokin DY, Tourova TP, Lysenko AM, Mityushina LL, Kuenen JG (2002) Thioalkalivibrio thiocyanoxidans sp. nov. and Thioalkalivibrio paradoxus sp. nov., novel alkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria capable of growth on thiocyanate, from soda lakes. Int J Syst Evol Microbiol 52(2):657–664CrossRefPubMedGoogle Scholar
  67. Sorokin DY, Abbas B, van Zessen E, Muyzer G (2014) Isolation and characterization of an obligately chemolithoautotrophic Halothiobacillus strain capable of growth on thiocyanate as an energy source. FEMS Microbiol Lett 354(1):69–74CrossRefPubMedGoogle Scholar
  68. Stafford D, Callely A (1969) The utilization of thiocyanate by a heterotrophic bacterium. J Gen Microbiol 55(2):285–289CrossRefPubMedGoogle Scholar
  69. Stott M, Franzmann P, Zappia L, Watling H, Quan L, Clark B, Houchin M, Miller P, Williams T (2001) Thiocyanate removal from saline CIP process water by a rotating biological contactor, with reuse of the water for bioleaching. Hydrometallurgy 62(2):93–105CrossRefGoogle Scholar
  70. Stratford J, Dias AEO, Knowles CJ (1994) The utilization of thiocyanate as a nitrogen source by a heterotrophic bacterium: the degradative pathway involves formation of ammonia and tetrathionate. Microbiology 140(10):2657–2662CrossRefPubMedGoogle Scholar
  71. Toran L, Harris RF (1989) Interpretation of sulfur and oxygen isotopes in biological and abiological sulfide oxidation. Geochim Cosmochim Acta 53(9):2341–2348CrossRefGoogle Scholar
  72. Trudinger P (1967) Metabolism of thiosulfate and tetrathionate by heterotrophic bacteria from soil. J Bacteriol 93(2):550–559PubMedPubMedCentralGoogle Scholar
  73. van Buuren C, Makhotla N, Olivier JW (2011) The ASTER process: technology development through to piloting, demonstration and commercialization. In: Proceedings of the ALTA, p 23–28Google Scholar
  74. van Zyl AW, Huddy R, Harrison STL, van Hille RP (2014) Evaluation of the ASTER™ process in the presence of suspended solids. Miner Eng 76:72–80Google Scholar
  75. Vazquez G, Jz Z, Millero FJ (1989) Effect of metals on the rate of the oxidation of H2S in seawater. Geophys Res Lett 16(12):1363–1366CrossRefGoogle Scholar
  76. Villemur R, Juteau P, Bougie V, Ménard J, Déziel E (2015) Development of four-stage moving bed biofilm reactor train with a pre-denitrification configuration for the removal of thiocyanate and cyanate. Bioresour Technol 181:254–262Google Scholar
  77. Vishniac W, Santer M (1957) The Thiobacilli. Bacteriol Rev 21(3):195PubMedPubMedCentralGoogle Scholar
  78. Watts MP, Moreau JW (2016) New insights into the genetic and metabolic diversity of thiocyanate-degrading microbial consortia. Appl Microbiol Biotechnol 100(3):1101–1108CrossRefPubMedGoogle Scholar
  79. Weekers PH, Bodelier PL, Wijen JP, Vogels GD (1993) Effects of grazing by the free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis on various bacteria. Appl Environ Microbiol 59(7):2317–2319PubMedPubMedCentralGoogle Scholar
  80. Whitlock JL (1990) Biological detoxification of precious metal processing wastewaters. Geomicrobiol J 8(3–4):241–249CrossRefGoogle Scholar
  81. Xu X, Hui D, King AW, Song X, Thornton PE, Zhang L (2015) Convergence of microbial assimilations of soil carbon, nitrogen, phosphorus, and sulfur in terrestrial ecosystems. Scientific reports 5Google Scholar
  82. Youatt JB (1954) Studies on the metabolism of Thiobacillus thiocyanoxidans. J Gen Microbiol 11(2):139–149CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Mathew Paul Watts
    • 1
  • Liam Patrick Spurr
    • 1
  • Han Ming Gan
    • 2
    • 3
  • John William Moreau
    • 1
  1. 1.School of Earth SciencesUniversity of MelbourneParkvilleAustralia
  2. 2.School of ScienceMonash University MalaysiaPetaling JayaMalaysia
  3. 3.Genomics Facility, Tropical Medicine and Biology PlatformMonash University MalaysiaPetaling JayaMalaysia

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