Biotechnology Letters

, Volume 32, Issue 10, pp 1405–1412 | Cite as

An investigation into the preservation of microbial cell banks for α-amylase production during 5 l fed-batch Bacillus licheniformis fermentations

  • Nichola H. Hancocks
  • Colin R. Thomas
  • Stuart M. Stocks
  • Christopher J. Hewitt
Original Research Paper

Abstract

Fluorescent staining techniques were used for a systematic examination of methods used to cryopreserve microbial cell banks. The aim of cryopreservation here is to ensure subsequent reproducible fermentation performance rather than just post thaw viability. Bacillus licheniformis cell physiology post-thaw is dependent on the cryopreservant (either Tween 80, glycerol or dimethyl sulphoxide) and whilst this had a profound effect on the length of the lag phase, during subsequent 5 l fed-batch fermentations, it had little effect on maximum specific growth rate, final biomass concentration or α-amylase activity. Tween 80 not only protected the cells during freezing but also helped them recover post-thaw resulting in shorter process times.

Keywords

Bacteria Flow cytometry Membrane potential Membrane integrity Cryopreservation Fermentation Bacillus licheniformis 

List of symbols

F

Feed rate (l h−1)

S

Substrate concentration in the feed solution (g l−1)

μ

Desired specific growth rate (h−1)

Yxs

Maximum biomass yield with a limiting substrate (g/g)

X0

Initial amount of cells at the start of feeding (g)

m

Maintenance coefficient (gg−1 h−1)

t

Time after feeding commences (h)

References

  1. Beal C, Fonseca F, Corrieu G (2001) Resistance to freezing and frozen storage of Streptococcus thermophilus is related to membrane fatty acid composition. J Dairy Sci 84:2347–2356CrossRefPubMedGoogle Scholar
  2. Endo Y, Kamisada S, Fujimoto K, Saito T (2006) Trans fatty acids promote the growth of some Lactobacillus strains. J Gen Appl Microbiol 52:29–35CrossRefPubMedGoogle Scholar
  3. Fuller BJ (2004) Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo Lett 25:375–388Google Scholar
  4. Gnoth S, Jenzsch M, Simitis R, Lübbert A (2007) Process Analytical Technology (PAT): batch-to-batch reproducibility of fermentation process by robust process operational design and control. J Biotechnol 132:180–186CrossRefPubMedGoogle Scholar
  5. Gurtovenko AA, Anwar J (2007) Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulphoxide. J Phys Chem B 111:10453–10460CrossRefPubMedGoogle Scholar
  6. Hewitt CJ, Nebe-Von-Caron G (2001) An industrial application of multi-parameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations. Cytometry 44:179–187CrossRefPubMedGoogle Scholar
  7. Hewitt CJ, Nebe-Von-Caron G (2004) The application of multi-parameter flow cytometry to monitor individual microbial cell physiological state. Adv Biochem Eng Biotechnol 89:197–223PubMedGoogle Scholar
  8. Hewitt CJ, Solomons G (1996) The production of alpha-amylase by Bacillus amyloliquefaciens B20 in a complex and totally defined synthetic culture medium. J Ind Microbiol 17:96–99CrossRefGoogle Scholar
  9. Hornbæk T, Nielsen AK, Dynesen J, Jakobsen M (2002) Use of fluorescence ratio imaging microscopy and flow cytometry for estimation of cell vitality for Bacillus licheniformis. FEMS Microbiol Lett 215:261–265CrossRefPubMedGoogle Scholar
  10. Hornbæk T, Nielsen AK, Dynesen J, Jakobsen M (2004) The effect of inoculum age and solid versus liquid propagation on inoculum quality of an industrial Bacillus licheniformis strain. FEMS Microbiol Lett 236:145–151PubMedGoogle Scholar
  11. Hubálek Z (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology 46:205–229CrossRefPubMedGoogle Scholar
  12. Johannsen E (1972) Malt extract as protective medium for lactic acid bacteria in cryopreservation. J Appl Microbiol 35:423–429CrossRefGoogle Scholar
  13. Kirsop BE, Snell JJS (1984) Maintenance of microorganisms: a manual of laboratory methods. Academic Press, LondonGoogle Scholar
  14. Lorentz K (1998) Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 9. IFCC method for α-amylase (1,4-α-D-glucan4-glucanohydrolase, EC 3.2.1.1). Clin Chem Lab Med 36:185–203CrossRefPubMedGoogle Scholar
  15. Lovelock EJ, Bishop MWH (1959) Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 183:1394–1395CrossRefPubMedGoogle Scholar
  16. Morgan CA, Herman N, White PA, Vesey G (2006) Preservation of micro-organisms by drying: a review. J Microbiol Methods 66:183–193CrossRefPubMedGoogle Scholar
  17. Nebe-Von-Caron G, Stephens PJ, Hewitt CJ, Powell JR, Badley RA (2000) Analysis of bacterial function by multi-colour and single cell sorting. J Microbiol Methods 42:97–114CrossRefPubMedGoogle Scholar
  18. Polge C, Smith AU, Parkes AS (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666CrossRefPubMedGoogle Scholar
  19. Simione FP (1998) Cryopreservation manual. Nalge Nunc International Corp., New York, pp 1–8Google Scholar
  20. Smittle RB, Gilliland SE, Speck ML, Walter WM (1974) Relationship of cellular fatty acid composition to survival of Lactobacillus bulgaricus in liquid nitrogen. Appl Microbiol 27:738–743PubMedGoogle Scholar
  21. Strandberg L, Andersson L, Enfors S (1994) The use of fed-batch cultivation for achieving high cell densities in the production of recombinant protein in Escherichia coli. FEMS Microbiol Rev 14:53–56CrossRefPubMedGoogle Scholar
  22. Webb C, Kamat SP (1993) Improving fermentation consistency through better inoculum preparation. World J Microbiol Biotechnol 9:308–312CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Nichola H. Hancocks
    • 1
  • Colin R. Thomas
    • 1
  • Stuart M. Stocks
    • 2
  • Christopher J. Hewitt
    • 3
  1. 1.School of Chemical EngineeringUniversity of BirminghamBirminghamUK
  2. 2.Novozymes A/SBagsværdDenmark
  3. 3.Department of Chemical Engineering, Centre for Biological EngineeringLoughborough UniversityLoughboroughUK

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