Journal of Microbiology

, Volume 54, Issue 6, pp 413–419 | Cite as

Calculibacillus koreensis gen. nov., sp. nov., an anaerobic Fe(III)-reducing bacterium isolated from sediment of mine tailings

  • Ui-Gi Min
  • So-Jeong Kim
  • Heeji Hong
  • Song-Gun Kim
  • Joo-Han Gwak
  • Man-Young Jung
  • Jong-Geol Kim
  • Jeong-Geol Na
  • Sung-Keun RheeEmail author


A strictly anaerobic bacterium, strain B5T, was isolated from sediment of an abandoned coal mine in Taebaek, Republic of Korea. Cells of strain B5T were non-spore-forming, straight, Gram-positive rods. The optimum pH and temperature for growth were pH 7.0 and 30°C, respectively, while the strain was able to grow within pH and temperature ranges of 5.5–7.5 and 25–45°C, respectively. Growth of strain B5T was observed at NaCl concentrations of 0 to 6.0% (w/v) with an optimum at 3.0–4.0% (w/v). The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, an unknown phospholipid and three unknown polar lipids. Strain B5T grew anaerobically by reducing nitrate, nitrite, ferric-citrate, ferric-nitrilotriacetate, elemental sulfur, thiosulfate, and anthraquinone-2-sulfonate in the presence of proteinaceous compounds, organic acids, and carbohydrates as electron donors. The isolate was not able to grow by fermentation. Strain B5T did not grow under aerobic or microaerobic conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B5T is most closely related to the genus Tepidibacillus (T. fermentans STGHT; 96.3%) and Vulcanibacillus (V. modesticaldus BRT; 94.6%). The genomic DNA G+C content (36.9 mol%) of strain B5T was higher than those of T. fermentans STGHT (34.8 mol%) and V. modesticaldus BRT (34.5 mol%). Based on its phenotypic, chemotaxonomic, and phylogenetic properties, we describe a new species of a novel genus Calculibacillus, represented by strain B5T (=KCTC 15397T =JCM 19989T), for which we propose the name Calculibacillus koreensis gen. nov., sp. nov.


Calculibacillus koreensis coal mine tailings iron 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

12275_2016_6086_MOESM1_ESM.pdf (367 kb)
Supplementary material, approximately 368 KB.


  1. Aklujkar, M., Coppi, M.V., Leang, C., Kim, B.C., Chavan, M.A., Perpetua, L.A., Giloteaux, L., Liu, A., and Holmes, D.E. 2013. Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens. Microbiology 159, 515–535.CrossRefPubMedGoogle Scholar
  2. Beller, H.R., Han, R., Karaoz, U., Lim, H., and Brodie, E.L. 2013. Genomic and physiological characterization of the chromatereducing, aquifer-derived Firmicute Pelosinus sp. strain HCF1. Appl. Environ. Microbiol. 79, 63–73.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Benson, H.J. 2002. Microbiological applications; a laboratory manual in general microbiology. 8th ed. McGraw Hill, New York, USA.Google Scholar
  4. Blodau, C., Hoffmann, S., Peine, A., and Peiffer, S. 1998. Iron and sulfate reduction in the sediments of acidic mine lake 116 (Brandenburg, Germany): Rates and geochemical evaluation. Water Air Soil Pollut. 108, 249–270.CrossRefGoogle Scholar
  5. Buck, J.D. 1982. Nonstaining (KOH) method for determination of gram reactions of marine bacteria. Appl. Environ. Microbiol. 44, 992–993.PubMedPubMedCentralGoogle Scholar
  6. Caccavo, F., Blakemore, R.P., and Lovley, D.R. 1992. A hydrogenoxidizing, Fe(III)-reducing microorganism from the Great Bay estuary, New Hampshire. Appl. Environ. Microbiol. 58, 3211–3216.PubMedPubMedCentralGoogle Scholar
  7. Caccavo, F. Jr., Lonergan, D.J., Lovley, D.R., Davis, M., Stolz, J.F., and McInerney, M.J. 1994. Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism. Appl. Environ. Microbiol. 60, 3752–3759.PubMedPubMedCentralGoogle Scholar
  8. Childers, S.E., Ciufo, S., and Lovley, D.R. 2002. Geobacter metallireducens accesses insoluble Fe(III) oxide by chemotaxis. Nature 416, 767–769.CrossRefPubMedGoogle Scholar
  9. Felsenstein, J. 1981. Evolutionary trees from DNA sequences: A maximum likelihood approach. J. Mol. Evol. 17, 368–376.CrossRefPubMedGoogle Scholar
  10. Friese, K., Wendt-Potthoff, K., Zachmann, D.W., Fauville, A., Mayer, B., and Veizer, J. 1998. Biogeochemistry of iron and sulfur in sediments of an acidic mining lake in Lusatia, Germany. Water Air Soil Pollut. 108, 231–247.CrossRefGoogle Scholar
  11. Gonzalez, J.M. and Saiz-Jimenez, C. 2002. A fluorimetric method for the estimation of G+C mol% content in microorganisms by thermal denaturation temperature. Environ. Microbiol. 4, 770–773.CrossRefPubMedGoogle Scholar
  12. Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95–98.Google Scholar
  13. Hong, Y., Wu, P., Li, W., Gu, J., and Duan, S. 2012. Humic analog AQDS and AQS as an electron mediator can enhance chromate reduction by Bacillus sp. strain 3C3. Appl. Microbiol. Biotechnol. 93, 2661–2668.CrossRefPubMedGoogle Scholar
  14. Islam, F.S., Gault, A.G., Boothman, C., Polya, D.A., Charnock, J.M., Chatterjee, D., and Lloyd, J.R. 2004. Role of metal-reducing bacteria in arsenic release from Bengal delta sediments. Nature 430, 68–71.CrossRefPubMedGoogle Scholar
  15. Johnson, D.B., Rolfe, S., Hallberg, K.B., and Iversen, E. 2001. Isolation and phylogenetic characterization of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine. Environ. Microbiol. 3, 630–637.CrossRefPubMedGoogle Scholar
  16. Kaneda, T. 1991. Iso-and anteiso-fatty acids in bacteria: Biosynthesis, function, and taxonomic significance. Microbiol. Rev. 55, 288–302.PubMedPubMedCentralGoogle Scholar
  17. Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y. S., Lee, J.H., Yi, H., et al. 2012. Introducing EzTaxone-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62, 716–721.CrossRefPubMedGoogle Scholar
  18. Kim, K.K., Lee, J.S., Lee, K.C., Oh, H.M., and Kim, S.G. 2010. Pontibaca methylaminivorans gen. nov., sp. nov., a member of the family Rhodobacteraceae. Int. J. Syst. Evol. Microbiol. 60, 2170–2175.CrossRefPubMedGoogle Scholar
  19. Kimura, M. 1983. The neutral theory of molecular evolution. Cambridge University Press, Cambridge.CrossRefGoogle Scholar
  20. L’Haridon, S., Miroshnichenko, M.L., Kostrikina, N.A., Tindall, B.J., Spring, S., Schumann, P., Stackebrandt, E., Bonch-Osmolovskaya, E.A., and Jeanthon, C. 2006. Vulcanibacillus modesticaldus gen. nov., sp. nov., a strictly anaerobic, nitrate-reducing bacterium from deep-sea hydrothermal vents. Int. J. Syst. Evol. Microbiol. 56, 1047–1053.CrossRefPubMedGoogle Scholar
  21. Lane, D. 1991. 16S/23S rRNA sequencing, pp. 115–175. In Stackebrandt, E. and Goodfellow, M. (eds.), Nucleic acid techniques in bacterial systematics, Wiley, Chichester, UK.Google Scholar
  22. Li, X., Park, J.H., Edraki, M., and Baumgartl, T. 2013. Understanding the salinity issue of coal mine spoils in the context of salt cycle. Environ. Geochem. Health 36, 453–465.CrossRefPubMedGoogle Scholar
  23. Li, H., Peng, J., Weber, K.A., and Zhu, Y. 2011. Phylogenetic diversity of Fe(III)-reducing microorganisms in rice paddy soil: Enrichment cultures with different short-chain fatty acids as electron donors. J. Soils Sediments 11, 1234–1242.CrossRefGoogle Scholar
  24. Lovley, D.R. 1991. Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol. Rev. 55, 259–287.PubMedPubMedCentralGoogle Scholar
  25. Lovley, D.R., Chapelle, F.H., and Phillips, E.J. 1990. Fe(III)-reducing bacteria in deeply buried sediments of the Atlantic Coastal Plain. Geology 18, 954–957.CrossRefGoogle Scholar
  26. Lovley, D.R. and Phillips, E.J. 1986. Organic matter mineralization with reduction of ferric iron in anaerobic sediments. Appl. Environ. Microbiol. 51, 683–689.PubMedPubMedCentralGoogle Scholar
  27. Lovley, D.R. and Phillips, E.J. 1988. Novel mode of microbial energy metabolism: Organic carbon oxidation coupled to dissimilatory reduction of iron or manganese. Appl. Environ. Microbiol. 54, 1472–1480.PubMedPubMedCentralGoogle Scholar
  28. Meslé, M., Dromart, G., and Oger, P. 2013. Microbial methanogenesis in subsurface oil and coal. Res. Microbiol. 164, 959–972.CrossRefPubMedGoogle Scholar
  29. Minnikin, D.E., O’Donnell, A.G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A., and Parlett, J.H. 1984. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J. Microbiol. Methods 2, 233–241.CrossRefGoogle Scholar
  30. Nei, M., Kumar, S., and Takahashi, K. 1998. The optimization principle in phylogenetic analysis tends to give incorrect topologies when the number of nucleotides or amino acids used is small. Proc. Natl. Acad. Sci. USA 95, 12390–12397.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Nevin, K.P., Holmes, D.E., Woodard, T.L., Hinlein, E.S., Ostendorf, D.W., and Lovley, D.R. 2005. Geobacter bemidjiensis sp. nov. and Geobacter psychrophilus sp. nov., two novel Fe(III)-reducing subsurface isolates. Int. J. Syst. Evol. Microbiol. 55, 1667–1674.CrossRefPubMedGoogle Scholar
  32. Ogg, C.D. and Patel, B.K. 2009. Thermotalea metallivorans gen. nov., sp. nov., a thermophilic, anaerobic bacterium from the Great Artesian Basin of Australia aquifer. Int. J. Syst. Evol. Microbiol. 59, 964–971.CrossRefPubMedGoogle Scholar
  33. Pruesse, E., Quast, C., Knittel, K., Fuchs, B.M., Ludwig, W., Peplies, J., and Glockner, F.O. 2007. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 35, 7188–7196.CrossRefPubMedPubMedCentralGoogle Scholar
  34. Saitou, N. and Nei, M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.PubMedGoogle Scholar
  35. Sasser, M. 1990. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101., MIDI Inc, Newark, DE,USA.Google Scholar
  36. Schaeffer, A.B. and Fulton, M.D. 1933. A simplified method of staining endospores. Science 77, 194.CrossRefPubMedGoogle Scholar
  37. Semple, K., Westlake, D., and Krouse, H. 1987. Sulfur isotope fractionation by strains of Alteromonas putrefaciens isolated from oil field fluids. Can. J. Microbiol. 33, 372–376.CrossRefGoogle Scholar
  38. Shirling, E.B. and Gottlieb, D. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Evol. Microbiol. 16, 313–340.Google Scholar
  39. Si, O.J., Kim, S.J., Jung, M.Y., Choi, S.B., Kim, J.G., Kim, S.G., Roh, S.W., Lee, S., and Rhee, S.K. 2015. Leeuwenhoekiella polynyae sp. nov., isolated from a polynya in western Antarctica. Int. J. Syst. Evol. Microbiol. 65, 1694–1699.CrossRefPubMedGoogle Scholar
  40. Siegert, M., Cichocka, D., Herrmann, S., Grundger, F., Feisthauer, S., Richnow, H.H., Springael, D., and Krüger, M. 2011. Accelerated methanogenesis from aliphatic and aromatic hydrocarbons under iron- and sulfate-reducing conditions. FEMS Microbiol. Lett. 315, 6–16.CrossRefPubMedGoogle Scholar
  41. Slobodkin, A., Reysenbach, A.L., Strutz, N., Dreier, M., and Wiegel, J. 1997. Thermoterrabacterium ferrireducens gen. nov., sp. nov., a thermophilic anaerobic dissimilatory Fe(III)-reducing bacterium from a continental hot spring. Int. J. Syst. Bacteriol. 47, 541–547.CrossRefPubMedGoogle Scholar
  42. Slobodkina, G.B., Panteleeva, A.N., Kostrikina, N.A., Kopitsyn, D.S., Bonch-Osmolovskaya, E.A., and Slobodkin, A.I. 2013. Tepidibacillus fermentans gen. nov., sp. nov.: A moderately thermophilic anaerobic and microaerophilic bacterium from an underground gas storage. Extremophiles 17, 833–839.CrossRefPubMedGoogle Scholar
  43. Sororzano, L. 1969. Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol. Oceanogr. 14, 799–801.CrossRefGoogle Scholar
  44. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.CrossRefPubMedPubMedCentralGoogle Scholar
  45. Tan, B., Charchuk, R., Li, C., Nesbo, C., Abu Laban, N., and Foght, J. 2014. Draft genome sequence of uncultivated Firmicutes (Peptococcaceae SCADC) single cells sorted from methanogenic alkane-degrading cultures. Genome Announc. 2, e00909–14.PubMedPubMedCentralGoogle Scholar
  46. Trüper, H.G. and Schlegel, H.G. 1964. Sulphur metabolism in Thiorhodaceae. I. Quantitative measurements on growing cells of Chromatium okenii. Antonie van Leeuwenhoek 30, 225–238.CrossRefGoogle Scholar
  47. Tschech, A. and Pfennig, N. 1984. Growth yield increase linked to caffeate reduction in Acetobacterium woodii. Arch. Microbiol. 137, 163–167.CrossRefGoogle Scholar
  48. Weber, K.A., Achenbach, L.A., and Coates, J.D. 2006. Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction. Nat. Rev. Microbiol. 4, 752–764.CrossRefPubMedGoogle Scholar
  49. Weisburg, W.G., Barns, S.M., Pelletier, D.A., and Lane, D.J. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173, 697–703.PubMedPubMedCentralGoogle Scholar
  50. Widdel, F. and Bak, F. 1992. Gram-negative mesophilic sulfate-reducing bacteria, pp. 3352–3378. In Starr, M.P., Stolp, H., Trüper H.G., Balows, A., and Schlegal, H.G. (eds.), The Prokaryotes, 2nd ed vol. 1. Springer, Berlin, Germany.CrossRefGoogle Scholar
  51. Wrighton, K.C., Agbo, P., Warnecke, F., Weber, K.A., Brodie, E.L., DeSantis, T.Z., Hugenholtz, P., Andersen, G.L., and Coates, J.D. 2008. A novel ecological role of the Firmicutes identified in thermophilic microbial fuel cells. ISME J. 2, 1146–1156.CrossRefPubMedGoogle Scholar
  52. Yarza, P., Yilmaz, P., Pruesse, E., Glöckner, F.O., Ludwig, W., Schleifer, K.H., Whitman, W.B., Euzéby, J., Amann, R., and Rosselló-Móra, R. 2014. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat. Rev. Microbiol. 12, 635–645.CrossRefPubMedGoogle Scholar
  53. Zhang, Y.Z., Fang, M.X., Zhang, W.W., Li, T.T., Wu, M., and Zhu, X.F. 2013. Salimesophilobacter vulgaris gen. nov., sp. nov., an anaerobic bacterium isolated from paper-mill wastewater. Int. J. Syst. Evol. Microbiol. 63, 1317–1322.CrossRefPubMedGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Ui-Gi Min
    • 1
  • So-Jeong Kim
    • 1
  • Heeji Hong
    • 1
  • Song-Gun Kim
    • 2
  • Joo-Han Gwak
    • 1
  • Man-Young Jung
    • 1
  • Jong-Geol Kim
    • 1
  • Jeong-Geol Na
    • 3
  • Sung-Keun Rhee
    • 1
    Email author
  1. 1.Department of MicrobiologyChungbuk National UniversityCheongjuRepublic of Korea
  2. 2.Microbial Resources Center/KCTCKorea Research Institute of Bioscience and BiotechnologyJeongeupRepublic of Korea
  3. 3.Biomass and Waste Energy LaboratoryKorea Institute of Energy ResearchDaejeonRepublic of Korea

Personalised recommendations