Applied Microbiology and Biotechnology

, Volume 99, Issue 8, pp 3559–3571 | Cite as

Improving survival and storage stability of bacteria recalcitrant to freeze-drying: a coordinated study by European culture collections

  • Jindrich Peiren
  • Joke Buyse
  • Paul De Vos
  • Elke Lang
  • Dominique Clermont
  • Sylviane Hamon
  • Evelyne Bégaud
  • Chantal Bizet
  • Javier Pascual
  • María A. Ruvira
  • M. Carmen Macián
  • David R. Arahal
Methods and protocols

Abstract

The objective of this study is to improve the viability after freeze-drying and during storage of delicate or recalcitrant strains safeguarded at biological resource centers. To achieve this objective, a joint experimental strategy was established among the different involved partner collections of the EMbaRC project (www.embarc.eu). Five bacterial strains considered as recalcitrant to freeze-drying were subjected to a standardized freeze-drying protocol and to seven agreed protocol variants. Viability of these strains was determined before and after freeze-drying (within 1 week, after 6 and 12 months, and after accelerated storage) for each of the protocols. Furthermore, strains were exchanged between partners to perform experiments with different freeze-dryer-dependent parameters. Of all tested variables, choice of the lyoprotectant had the biggest impact on viability after freeze-drying and during storage. For nearly all tested strains, skim milk as lyoprotectant resulted in lowest viability after freeze-drying and storage. On the other hand, best freeze-drying and storage conditions were strain and device dependent. For Aeromonas salmonicida CECT 894T, best survival was obtained when horse serum supplemented with trehalose was used as lyoprotectant, while Aliivibrio fischeri LMG 4414T should be freeze-dried in skim milk supplemented with marine broth in a 1:1 ratio. Freeze-drying Campylobacter fetus CIP 53.96T using skim milk supplemented with trehalose as lyoprotectant resulted in best recovery. Xanthomonas fragariae DSM 3587T expressed high viability after freeze-drying and storage for all tested lyoprotectants and could not be considered as recalcitrant. In contrary, Flavobacterium columnare LMG 10406T did not survive the freeze-drying process under all tested conditions.

Keywords

Freeze-drying Bacteria Lyoprotectant Viability Biological resource centers Residual moisture content 

Supplementary material

253_2015_6476_MOESM1_ESM.pdf (215 kb)
ESM 1(PDF 214 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Jindrich Peiren
    • 1
  • Joke Buyse
    • 2
  • Paul De Vos
    • 1
    • 2
  • Elke Lang
    • 3
  • Dominique Clermont
    • 4
  • Sylviane Hamon
    • 4
  • Evelyne Bégaud
    • 4
  • Chantal Bizet
    • 4
  • Javier Pascual
    • 5
  • María A. Ruvira
    • 5
  • M. Carmen Macián
    • 5
  • David R. Arahal
    • 5
  1. 1.Belgian Coordinated Collections of Microorganisms/Bacteria Collection (BCCM/LMG)Ghent UniversityGhentBelgium
  2. 2.Laboratory of MicrobiologyGhent UniversityGhentBelgium
  3. 3.Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)BraunschweigGermany
  4. 4.Centre de Ressources Biologiques de l’Institut Pasteur (CRBIP)Institut PasteurParisFrance
  5. 5.Colección Española de Cultivos Tipo (CECT) & Dpto. Microbiología y EcologíaUniversitat de ValènciaValenciaSpain

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