Study of viability of Pseudomonas fluorescens BTP1 freeze-dried during storage at 4 and 20°C

Study of viability of Pseudomonas fluorescens BTP1 freeze-dried during storage at 4 and 20°C.

Wallon Center of Industrial Biology (CWBI), Gembloux Agro-Bio Tech, University of Liege, Belgium.

Abstract

The drying of bacteria remains a major alternative in order to keep them long term. After centrifugation, the bacterial pellet of Pseudomonas

fluorescens BTP1 was divided in two fractions one with and one without cryoprotectants (2% glycerol and 5% maltodextrine) and freeze-dried. After

freeze drying, powders were sealed in aluminium bag under vacuum and storage at 4 or 20°C. The storage stability of freeze-dried powders was studied by parameters such as loss of viability on the Plate Count Agar (PCA) (e.g. Concentration of Cells with glycerol (PG) at CFU/g before storage 109 _{and after 7 month, 10}8 _{at 4°C and 10}7 _{at 20°C) and evolution in membrane composition by measuring the ratio of unsaturated/saturated}

fatty acid. These ratios decrease in function of time (e.g. at 4°C the ratios of C18:3 and C18:2 by C16:0 decreases respectively of 0,013 to 0,001 and 0,05 to 0,03 after 60 days of storage). Viability (%) and concentration (CFU/g) of bacterial during storage at 4 or 20°C with aw = 0,32 was determined using a procedure published by (Kurtmann et al., 2009). In the present study, flow cytometric analysis was applied to evaluate the state in which are the cells at the end of storage time. Furthermore, we compared result the survival of bacteria as obtained by plate count with the flow cytometric analysis results.

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Key words: Viability, freeze-drying, flow cytometric, cryoprotectants, Cellular fatty acids

Conclusion

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Results

References:

Kurtmann, L., Carlsen, C. U., Risbo, J. and Skibsted, L. H. (2009). Cryobiology 58, 175-180. Yao, A. A., Coulibaly, I., Lognay, G., Fauconnier, M. L. and Thonart, P. (2008). Appl Microbiol Biotechnol 79, 1045-1052. Boyetchko, S., Pedersen, E., Punja, Z. and Reddy, M. (1999). In "Methods in Biotechnology" (F. R. Hall and J. J. Menn, eds), vol. 5, pp. 487-508. Hummana Press, Totawa N.J.

Material and methods

The strain used in our study is Pseudomonas fluorescens BTP1 of Wallon Center of Industrial Biology laboratory (CWBI). It was grown in bioreactor using 863 medium, for 20 hours and then concentrated by centrifugation. The pellet was divided in two parts, one with and one without cryoprotectants (2% w/w glycerol and 5% w/w maltodextrine) and freeze-dried in a low freeze-drier. The powders were sealed in aluminium bag under vacuum and storage at 4 or 20°C. The loss of viability by Plate Count Agar (PCA) compared by flow cytometer result and by fatty acid by gas chromatography (Yao et al., 2008 and Boyetchko et al., 1999).

It is now established that Pseudomonas fluorescens BTP1 can be storage in powder formulation in longue time. The figure 1 shows that storage at 4°C is best as storage at 20°C and glycerol is the best cryoprotectant during freeze drying and storage. The figure 2 shows that major cells after 210 days storage are in intermediate state (viable but no cultivable).This hypothesis is confirmed as shown in table 1. Lipids oxydation is one of factor responsable of death cell by study of unsaturated by saturated fatty acid ratio (fig. 3).

Fig.1 : Survival of freeze-drier Pseudomonas fluorescens BTP1 during

storage at 4°C (▲ cells with glycerol (PG), ♦ cells without cryoprotectant (PS), ■ cells with maltodextrine (PM)) or 20°C (∆ cells with glycerol (PG), ◊ cells without cryoprotectant (PS) and □ cells with maltodextrine.

a

b

c : cellules intermédiaires

Fig.2. Pseudomonas fluorescens BTP1 (PG) after PI/cFDA double staining.

(a) death cell 4,01%, (b) live cell and 5,54%(c) intermediate cells 90,45%. Legend. PG: Cells with Glycerol (2%).

Table1. Comparison of the viability result between plate counts and flow cytometric

A

B

Fig.3. A: C18:2/C16:0 and B: C18:3 / C16:0 ratio of Pseudomonas fluorescens