Journal of Industrial Microbiology & Biotechnology

, Volume 40, Issue 12, pp 1403–1413 | Cite as

Viability of calcifying bacterial formulations in fly ash for applications in building materials

  • Navdeep Kaur Dhami
  • Abhijit Mukherjee
  • M. Sudhakara Reddy
Environmental Microbiology


Evidence of bacterial involvement in precipitation of calcium carbonates has brought a revolution in the field of applied microbiology, geotechnical sciences, environmental and civil engineering with its marked success in restoration of various building materials. For applications of these calcite binder-producing bacterial cultures, different expensive carrier materials have been used but their high costs have come in the way of their successful commercialization. In the present study, we have explored the potential of cheap industrial by-product fly ash as a carrier material for bacterial cells and investigated the viability of calcifying bacterial isolates: Bacillus megaterium, Bacillus cereus, and Lysinibacillusfusiformis in fly ash carrier at varying temperatures and moisture conditions along with biomineralization efficacy of these formulations. We used laser scanning confocal microscopy to analyze the viability of bacteria by florescent dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) along with the plate count method. Results revealed that fly ash successfully served as an effective carrier material and bacterial formulations stored at 4 °C provided longer shelf life than those stored at higher temperatures. Up to 106 cfu/g was found to sustain in all formulations at 4 °C compared to 104-105 cfu/g in case of higher temperatures up to 1 year. For 4 °C, higher moistures (50 %) were found to provide better survivability while for higher temperatures, lower moistures (30 %) favored higher viability. The biomineralization capability of fresh and formulated bacterial cells was compared on the basis of precipitation of carbonates and it was found that carbonate precipitation efficacy of formulated bacterial cells was comparable to fresh bacterial cells.


Bacillus Fly ash Inoculum formulations Temperature Moisture Cell viability 


  1. 1.
    De Jong JT, Soga K, Kavazanjian E, Burns S, Van Paassen LA, Al Qabany A, Aydilek A, Bang SS, Burbank M, Caslake LF, Chen CY, Cheng X, Chu J, Ciurli S, Esnault-Filet A, Fauriel S, Hamdan N, Hata T, Inagaki Y, Jefferis S, Kuo M, Laloui L, Larrahondo J, Manning DAC, Martinez B, Montoya BM, Nelson DC, Palomino A, Renforth P, Santamarina JC, Seagren EA, Tanyu B, Tsesarsky M, Weaver T (2013) Biogeochemical processes and geotechnical applications: progress, opportunities and challenges. Geotechnique 63:287–301CrossRefGoogle Scholar
  2. 2.
    Rodriguez-Navarro C, Jroundi F, Schiro M, Ruiz-Agudo E, González –Muñoz MT (2012) Influence of substrate mineralogy on bacterial mineralization of calcium carbonate: Implications in stone conservation. Appl Environ Microbiol 78:4017–4029PubMedCrossRefGoogle Scholar
  3. 3.
    Dhami NK, Mukherjee A, Reddy MS (2012) Biofilm and microbial applications in biomineralized concrete. In: Jong Seto (ed) Advanced topics in biomineralization, InTech, New York,  pp 137–164Google Scholar
  4. 4.
    Dick J, De Windt W, De Graef B, Saveyn H, Van der Meeren P, De Belie N, Verstraete W (2006) Bio-deposition of a calcium carbonate layer on degraded limestone by Bacillus species. Biodegradation 17:357–367PubMedCrossRefGoogle Scholar
  5. 5.
    Rodriguez-Navarro C, Rodriguez-Gallego M, Ben Chekroun K, Gonzalez-Muñoz MT (2003) Conservation of ornamental stone by Myxococcus xanthus induced carbonate biomineralization. Appl Environ Microbiol 69:2182–2193PubMedCrossRefGoogle Scholar
  6. 6.
    Stocks-Fischer S, Galinat JK, Bang SS (1999) Microbiological precipitation of CaCO3. Soil Biol Biochem 31:1563–1571CrossRefGoogle Scholar
  7. 7.
    De Muynck W, Belie N, Verstraete W (2010) Microbial carbonate precipitation in construction materials: a review. Ecol Eng 36:118–136CrossRefGoogle Scholar
  8. 8.
    Adams BL, Bates TC, Oliver JD (2003) Survival of Helicobacter pylori in a natural freshwater environment. Appl Environ Microbiol 69:7462–7466PubMedCrossRefGoogle Scholar
  9. 9.
    Créach V, Baudoux AC, Bertru G, Rouzic BL (2003) Direct estimate of active bacteria: CTC use and limitations. J Microbiol Methods 52:19–28PubMedCrossRefGoogle Scholar
  10. 10.
    Richardson RE, James CA, Bhupathiraju VK, Cohen LA (2002) Microbial activity in soils following steam treatment. Biodegradation 13:285–295PubMedCrossRefGoogle Scholar
  11. 11.
    Winding A, Binnerup SJ, Sorense J (1994) Viability of indigenous soil bacteria assayed by respiratory activity and growth. Appl Environ Microbiol 60:2869–2875PubMedGoogle Scholar
  12. 12.
    Montemor MF, Simoes AMP, Salta MM (2000) Effect of fly ash on concrete reinforcement corrosion studied by EIS. Cem Concr Comp 22:175–185CrossRefGoogle Scholar
  13. 13.
    Thomas MDA, Matthews JC (1992) The permeability of fly ash concrete. Mater Struct 25:388–396CrossRefGoogle Scholar
  14. 14.
    Khan MJ, Majid S, Mohidin FA, Khan N (2011) A new bioprocess to produce low cost powder formulations of biocontrol bacteria and fungi to control fusarial wilt and root-knot nematode of pulses. Biol Control 59:130–140CrossRefGoogle Scholar
  15. 15.
    Kumar V, Gupta P (2010) Studies on shelf-life of fly-ash based Azotobacter chroococcum formulation and its bio-efficacy in wheat. Res J Agric Biol Sci 6:280–282Google Scholar
  16. 16.
    Dhami NK, Mukherjee A, Reddy MS (2013) Biomineralization of calcium carbonate polymorphs by the bacterial strains isolated from calcareous sites. J Microbiol Biotechnol 23:707–714PubMedCrossRefGoogle Scholar
  17. 17.
    McGarity JW, Myers MG (1967) A survey of urease activity in soils of northern New South Wales. Plant Soil 27:217–238CrossRefGoogle Scholar
  18. 18.
    Achal V, Mukherjee A, Reddy MS (2009) Strain improvement of Sporosarcina pasteurii for enhanced urease and calcite production. J Ind Microbiol Biotechnol 36:981–988PubMedCrossRefGoogle Scholar
  19. 19.
    APHA (American Public Health Association) (1989) Standard methods for the examination of water and wastewater. In: 7th edn. American Public Health Association, Washington, DCGoogle Scholar
  20. 20.
    Reddy BVV, Gupta A (2005) Characteristics of soil-cement blocks using highly sandy soils. Material Struct 38:651–658Google Scholar
  21. 21.
    Achal V, Mukherjee A, Reddy MS (2011) Effect of calcifying bacteria on permeation properties of concrete structures. J Ind Microbiol Biotechnol 38:1229–1234PubMedCrossRefGoogle Scholar
  22. 22.
    Dhami NK, Mukherjee A, Reddy MS (2012) Improvement in strength properties of ash bricks by bacterial calcite. Ecol Eng 39:31–35CrossRefGoogle Scholar
  23. 23.
    Gaind S, Gaur AC (2004) Evaluation of fly ash as carrier material for diazotrophs and phosphobacteria. Bioresour Technol 95:187–190PubMedCrossRefGoogle Scholar
  24. 24.
    Gaind S, Gaur AC (1990) Influence of temperature on the efficiency of phosphate solubilizing microorganisms. Indian J Microbiol 30:305–310Google Scholar
  25. 25.
    Cigdem K, Merih K (2005) Effect of formulation on the viability of biocontrol agent, Trichoderma harzianum conidia. Afr J Biotechnol 85:483–486Google Scholar
  26. 26.
    Daza A, Santamaria C, Rodrigues-Navarro DN, Camach M, Orive R, Temprano F (2000) Perlite as a carrier for bacterial inoculants. Soil Biol Biochem 32:567–572CrossRefGoogle Scholar
  27. 27.
    Walker R, Rossall S, Asher MJC (2004) Comparison of application methods to prolong the survival of potential biocontrol bacteria on stored sugar-beet seed. J Appl Microbiol 97:293–305PubMedCrossRefGoogle Scholar
  28. 28.
    Sherr BF, Giorgio PD, Sherr EB (1999) Estimating abundance and single-cell characteristics of respiring bacteria via the redox dye CTC. Aquat Microb Ecol 18:117–131CrossRefGoogle Scholar
  29. 29.
    Nielsen JL, de Muro MA, Nielsen PH (2003) Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium chloride for activity studies by simultaneous use of microautoradiography and fluorescence in situ hybridization. Appl Environ Microbiol 69:641–643PubMedCrossRefGoogle Scholar
  30. 30.
    Cappitelli F, Toniolo L, Sansonetti A, Gulotta D, Ranalli G, Zanardini E, Sorlini C (2007) Advantages of using microbial technology over traditional chemical technology in removal of black crusts from stone surfaces of historical monuments. Appl Environ Microbiol 73:5671–5675PubMedCrossRefGoogle Scholar
  31. 31.
    Bachmeier KL, Williams AE, Warmington JR, Bang SS (2002) Urease activity in microbiologically induced calcite precipitation. J Biotechnol 93:171–181PubMedCrossRefGoogle Scholar
  32. 32.
    Qian C, Wang R, Cheng L, Wang J (2010) Theory of microbial carbonate precipitation and its application in restoration of cement-based materials defects. Chin J Chem 28:847–857CrossRefGoogle Scholar
  33. 33.
    De Muynck W, Leuridan S, Loo DV, Verbeken K, Cnudde V, De Belie N, Verstraete W (2011) Influence of pore structure on the effectiveness of a biogenic carbonate surface treatment for limestone conservation. Appl Environ Microbiol 77:6808–6820PubMedCrossRefGoogle Scholar
  34. 34.
    Achal V, Mukherjee A, Reddy MS (2010) Biocalcification by Sporosarcina pasteurii using corn steep liquor as nutrient source. J Ind Microbiol Biotechnol 6:170–174Google Scholar
  35. 35.
    Rivadeneyra MA, Parraga J, Delgado R, Ramos-Cormenzana A, Delgado G (1998) Biomineralisation of carbonates by Halomonas eurihalina in solid and liquid media with different salinities: crystal formation sequence. Res Microbiol 149:277–287PubMedCrossRefGoogle Scholar
  36. 36.
    De Muynck W, Cox K, De Belie N, Verstraete W (2008) Bacterial carbonate precipitation as an alternative surface treatment for concrete. Constr Build Mater 22:875–885CrossRefGoogle Scholar
  37. 37.
    Whiffin VS, van Paassen L, Harkes MP (2007) Microbial carbonate precipitation as a soil improvement technique. Geomicrobiol J 24:417–423CrossRefGoogle Scholar
  38. 38.
    Zamarreno DV, Inkpen R, May E (2009) Carbonate crystals precipitated by freshwater bacteria and their use as a limestone consolidant. Appl Env Microbiol 75:5981–5990CrossRefGoogle Scholar
  39. 39.
    Samonin VV, Elikova EE (2004) A study on the absorption of bacterial cells on porous materials. Microbiol 73:696–701CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2013

Authors and Affiliations

  • Navdeep Kaur Dhami
    • 1
  • Abhijit Mukherjee
    • 2
  • M. Sudhakara Reddy
    • 1
  1. 1.Department of BiotechnologyThapar UniversityPatialaIndia
  2. 2.Indian Institute of TechnologyAhmedabadIndia

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