Journal of Microbiology

, Volume 55, Issue 6, pp 440–447 | Cite as

Non-ureolytic calcium carbonate precipitation by Lysinibacillus sp. YS11 isolated from the rhizosphere of Miscanthus sacchariflorus

  • Yun Suk Lee
  • Hyun Jung Kim
  • Woojun ParkEmail author
Microbial Ecology and Environmental Microbiology


Although microbially induced calcium carbonate precipitation (MICP) through ureolysis has been widely studied in environmental engineering fields, urea utilization might cause environmental problems as a result of ammonia and nitrate production. In this study, many non-ureolytic calcium carbonate-precipitating bacteria that induced an alkaline environment were isolated from the rhizosphere of Miscanthus sacchariflorus near an artificial stream and their ability to precipitate calcium carbonate minerals with the absence of urea was investigated. MICP was observed using a phase-contrast microscope and ion-selective electrode. Only Lysinibacillus sp. YS11 showed MICP in aerobic conditions. Energy dispersive X-ray spectrometry and X-ray diffraction confirmed the presence of calcium carbonate. Field emission scanning electron microscopy analysis indicated the formation of morphologically distinct minerals around cells under these conditions. Monitoring of bacterial growth, pH changes, and Ca2+ concentrations under aerobic, hypoxia, and anaerobic conditions suggested that strain YS11 could induce alkaline conditions up to a pH of 8.9 and utilize 95% of free Ca2+ only under aerobic conditions. Unusual Ca2+ binding and its release from cells were observed under hypoxia conditions. Biofilm and extracellular polymeric substances (EPS) formation were enhanced during MICP. Strain YS11 has resistance at high pH and in high salt concentrations, as well as its spore-forming ability, which supports its potential application for self-healing concrete.


Lysinibacillus sp. YS11 MICP aeration urea aerobic MICP X-ray diffraction 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ahmed, I., Yokota, A., Yamazoe, A., and Fujiwara, T. 2007. Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. Int. J. Syst. Evol. Microbiol. 57, 1117–1125.CrossRefPubMedGoogle Scholar
  2. Atlas, R.M. 2005. Mineral salts agar, In Handbook of Media for Environmental Microbiology. 2nd ed. CRC Press, Taylor and Francis, New York, USA.Google Scholar
  3. Barabesi, C., Galizzi, A., Mastromei, G., Rossi, M., Tamburini, E., and Perito, B. 2007. Bacillus subtilis gene cluster involved in calcium carbonate biomineralization. J. Bacteriol. 189, 228–235.CrossRefPubMedGoogle Scholar
  4. Boquet, E., Boronat, A., and Ramos-Cormenzana, A. 1973. Production of calcite (calcium carbonate) crystals by soil bacteria is a general phenomenon. Nature 246, 527–529.CrossRefGoogle Scholar
  5. Braissant, O., Cailleau, G., Dupraz, C., and Verrecchia, E.P. 2003. Bacterially induced mineralization of calcium carbonate in terrestrial environments: the role of exopolysaccharides and amino acids. J. Sedimentary Res. 73, 485–490.CrossRefGoogle Scholar
  6. Braissant, O., Decho, A.W., Dupraz, C., Glunk, C., Przekop, K.M., and Visscher, P.T. 2007. Exopolymeric substances of sulfate-reducing bacteria: interactions with calcium at alkaline pH and implication for formation of carbonate minerals. Geobiology 5, 401–411.CrossRefGoogle Scholar
  7. Dhami, N.K., Reddy, M.S., and Mukherjee, A. 2013. Biomineralization of calcium carbonates and their engineered applications: a review. Front. Microbiol. 4, 314.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Dupraz, C., Reid, R.P., Braissant, O., Decho, A.W., Norman, R.S., and Visscher, P.T. 2009. Processes of carbonate precipitation in modern microbial mats. Earth Sci. Rev. 96, 141–162.CrossRefGoogle Scholar
  9. Dupraz, C., Visscher, P.T., Baumgartner, L.K., and Reid, R.P. 2004. Microbe-mineral interactions: early carbonate precipitation in a hypersaline lake (Eleuthera Island, Bahamas). Sedimentology 51, 745–765.CrossRefGoogle Scholar
  10. Ehrlich, H.L. 2002. Geomicrobiology. 4th ed. Taylor and Francis, New York, USA.Google Scholar
  11. Ercole, C., Cacchio, P., Botta, A.L., Centi, V., and Lepidi, A. 2007. Bacterially induced mineralization of calcium carbonate: the role of exopolysaccharides and capsular polysaccharides. Microsc. Microanal. 13, 42–50.CrossRefPubMedGoogle Scholar
  12. Falkowski, P.G., Fenchel, T., and Delong, E.F. 2008. The microbial engines that drive earth’s biogeochemical cycles. Science 320, 1034–1039.CrossRefPubMedGoogle Scholar
  13. Farhangi, M.B., Safari Sinegani, A.A., Mosaddeghi, M.R., Unc, A., and Khodakaramian, G. 2013. Impact of calcium carbonate and temperature on survival of Escherichia coli in soil. J. Environ. Manage. 119, 13–19.CrossRefPubMedGoogle Scholar
  14. Ferris, F.G., Stehmeier, L.G., Kantzas, A., and Mourits, F.M. 1997. Bacteriogenic mineral plugging. J. Can. Pet. Technol. 36, 56–61.CrossRefGoogle Scholar
  15. Fujita, Y., Ferris, F.G., Lawson, R.D., Colwell, F.S., and Smith, R.W. 2000. Calcium carbonate precipitation by ureolytic subsurface bacteria. Geomicrobiol. J. 17, 305–318.CrossRefGoogle Scholar
  16. Gadd, G.M. 2010. Metals, minerals and microbes: geomicrobiology and bioremediation. Microbiology 156, 609–643.CrossRefPubMedGoogle Scholar
  17. Hamdan, N., Edward K.J., Rittmann, B.E., and Karatas, I. 2011. Carbonate mineral precipitation for soil improvement through microbial denitrification. Geo-Frontiers Congress 2011, 3925–3934.Google Scholar
  18. Hammes, F., Boon, N., De Villiers, J., Verstraete, W., and Siciliano, S.D. 2003. Strain-specific ureolytic microbial calcium carbonate precipitation. Appl. Environ. Microbiol. 69, 4901–4909.CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hammes, F. and Verstraete, W. 2002. Key roles of pH and calcium metabolism in microbial carbonate precipitation. Rev. Environ. Sci. Biotechnol. 1, 3–7.CrossRefGoogle Scholar
  20. Hazen, R.M., Papineau, D., Bleeker, W., Downs R.T., Ferry, J.M., McCoy, T.J, Sverjensky, D.A., and Yang, H. 2008. Mineral evolution. Am. Mineral. 93, 1693–1720.CrossRefGoogle Scholar
  21. Kim, H.J., Eom, H.J., Park, C., Jung, J., Shin, B., Kim, W., Chung, N., Choi, I.G., and Park, W. 2016a. Calcium carbonate precipitation by Bacillus and Sporosarcina strains isolated from concrete and analysis of the bacterial community of concrete. J. Microbiol. Biotechnol. 26, 540–548.CrossRefPubMedGoogle Scholar
  22. Kim, J.G., Park, S.J., Damstéc, J.S.S., Schoutenc, S., Rijpstra, W.I.C., Jung, M.Y., Kima, S.J., Gwaka, J.H., Honga, H., Sia, O.J., et al. 2016b. Hydrogen peroxide detoxification is a key mechanism for growth of ammonia-oxidizing archaea. Proc. Natl. Acad. Sci. USA 113, 7888–7893.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Mishra, V. 2015. Modelling of the batch biosorption system: study on exchange of protons with cell wall-bound mineral ions. Environ. Technol. 36, 3194–3200.CrossRefPubMedGoogle Scholar
  24. Nolan, R.A. 1971. Amino acids and growth factors in vitamin-free casamino acids. Mycologia 63, 1231–234.CrossRefPubMedGoogle Scholar
  25. Park, S.J., Park, Y.M., Chun, W.Y., Kim, W.J., and Ghim, S.Y. 2010. Calcite-forming bacteria for compressive strength improvement in mortar. J. Microbiol. Biotechnol. 20, 782–788.PubMedGoogle Scholar
  26. Phillips, A.J., Gerlach, R., Lauchnor, E., Mitchell, A.C., Cunningham, A.B., and Spangler, L. 2013. Engineered applications of ureolytic biomineralization: a review. Biofouling 29, 715–733.CrossRefPubMedGoogle Scholar
  27. Priest, F.G., Goodfellow, M., and Todd, C. 1988. A numerical classification of the genus Bacillus. J. Gen. Microbiol. 134, 1847–1882.PubMedGoogle Scholar
  28. Reeburgh, W.S. 2007. Oceanic methane biogeochemistry. Chem. Rev. 107, 486–513.CrossRefPubMedGoogle Scholar
  29. Riding, R. 2000. Microbial carbonates: the geological record of calcified bacterial-algal mats and biofilms. Sedimentology 47, 179–214.CrossRefGoogle Scholar
  30. Rodriguez-Navarro, C., Rodriguez-Gallego, M., Ben Chckroun, K., and Gonzalez-Munoz, M.T. 2003. Conservation of ornamental stone by Myxococcus xanthus-induced carbonate biomineralization. Appl. Environ. Microbiol. 69, 2182–2193.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Seifan, M., Samani, A.K., and Berenjian, A. 2017. New insights into the role of pH and aeration in the bacterial production of calcium carbonate (CaCO3). Appl. Microbiol. Biotechnol. 101, 3131–3142.CrossRefPubMedGoogle Scholar
  32. Shirakawa, M.A., Cincotto, M.A., Atencio, D., Gaylarde, C.C., and John, V.M. 2011. Effect of culture medium on biocalcification by Pseudomonas putida, Lysinibacillus sphaericus and Bacillus subtilis. Braz. J. Microbiol. 42, 499–507.CrossRefPubMedPubMedCentralGoogle Scholar
  33. Silva, F.B., Boon, N., De Belie, N., and Verstraete, W. 2015. Industrial application of biological self-healing concrete: challenges and economical feasibility. J. Commer. Biotechnol. 21, 31–38.CrossRefGoogle Scholar
  34. Thomas, K.J. and Rice, C.V. 2014. Revised model of calcium and magnesium binding to the bacterial cell wall. Biometals 27, 1361–1370.CrossRefPubMedPubMedCentralGoogle Scholar
  35. Van Paassen, L.A., Daza, C.M., Staal, M., Sorokin, D.Y., Van Der Zon, W., and Van Loosdrecht, M.C. 2010. Potential soil reinforcement by biological denitrification. Ecol. Eng. 36, 168–175.CrossRefGoogle Scholar
  36. Wan, S., Li, G., An, T., Guo, B., Sun, L., Zu, L., and Ren, A. 2010. Biodegradation of ethanethiol in aqueous medium by a new Lysinibacillus sphaericus strain RG-1 isolated from activated sludge. Biodegradation 21, 1057–1066.CrossRefPubMedGoogle Scholar
  37. Wang, J., Ersan, Y.C., Boon, N., and Belie N.D. 2016. Application of microorganisms in concrete: a promising sustainable strategy to improve concrete durability. Appl. Microbiol. Biotechnol. 100, 2993.CrossRefPubMedGoogle Scholar
  38. Warren, L.A. and Ferris, F.G. 1998. Continuum between sorption and precipitation of Fe (III) on microbial surfaces. Environ. Sci. Technol. Lett. 21, 2331–2337.CrossRefGoogle Scholar
  39. Warren, L.A., Maurice, P.A., Parmar, N., and Ferris, F.G. 2001. Microbially mediated calcium carbonate precipitation: implications for interpreting calcite precipitation and for solid-phase capture of inorganic contaminants. Geomicrobiol. J. 18, 93–115.CrossRefGoogle Scholar
  40. Zhu, T. and Dittrich, M. 2016. Carbonate precipitation through microbial activities in natural environment, and their potential in biotechnology: a review. Front. Bioeng. Biotechnol. 4, 4.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological EngineeringKorea UniversitySeoulRepublic of Korea

Personalised recommendations