Impact of glycerol and storage temperature on gluatathione concentration and physiological state of Pseudomonas fluorescens BTP1 freeze-dried.

Mputu Kanyinda J-N., Pierart C., Delvigne F., Destain J. and Thonart P.

  Wallon Center of Industrial Biology (CWBI), Gembloux Agro-Bio Tech, University of Liege, Passage des Déportés, 2-5030 Gembloux Belgium.

Impact of glycerol and storage temperature on gluatathione concentration and physiological state of Pseudomonas fluorescens BTP1 freeze-dried.

Mputu Kanyinda J-N., Pierart C., Delvigne F., Destain J. and Thonart P.

  Wallon Center of Industrial Biology (CWBI), Gembloux Agro-Bio Tech, University of Liege, Passage des Déportés, 2-5030 Gembloux Belgium.

Abstract Pseudomonas fluorescens is commonly used as bio-fungicides in agriculture. For this use it requires formulations as either liquid or powder. Formulations have two advantages, storage and transport. Freeze-drying is a commonly used method to preserve bacteria. However, freeze-drying damages the cells, which results in loss of viability. Protective compounds are used to reduce loss of viability during process (freeze-drying and storage). In our study we used flow cytometry analysis to assess the physiological state in which cells are at the end of freeze-drying and Glutathione (GSH) was measured before and during storage.

Results

Materials and methodsThe strain used in our study is Pseudomonas fluorescens BTP1 of Wallon Center of Industrial Biology laboratory (CWBI). The pellet was divided in two parts, one with and one without glycerol, freeze-dried and storage at 4 or 20°C. The glutathione was measured by spectrophotometer at 412 nm of cell by flow cytometry (Mputu et al., 2012).

ConclusionThe decrease of glutathione during storage indicates that we have oxidation phenomenon, and this phenomenon is more pronounced at 20°C, major cells after freeze-drying are in intermediate state (viable but no cultivable). The beneficial effect to use glycerol in the conservation of Pseudomonas fluorescens is demonstrated.

References: Mputu Kanyinda, J.N., Pierart, C., Weekers, F., et al., 2012. Impact of protecting compounds on the viability, physiological state and lipid degradation of freeze-dried Pseudomonas fluorescens BTP1 during storage. Inter. Journal Biotech. Biochem. 8: 17-26

Fig.2. Concentration of glutathione (GSH) in Pseudomonas fluorescens BTP1during storage at 4 and 20°C.

DiscussionGlutathione (GSH) is a protective compound naturally produced by the cell. The concentration of glutathione decrease during storage (fig.2) respectively at 4°C and 20°C for powder without glycerol; from 2.2 µmol/ g before storage to 1.4 µmol/ g and 0.6 µmol/ g after 90 days storage. And for powder with glycerol, we have a decrease from 2.2 µmol/ g before storage to 1.7 µmol/ g after 90 days storage at 4°C and a decrease from 2.2 µmol/ g before storage to 0.6 µmol/ g after 90 days storage at 20°C. Figure 1. Shows that for the cream before freeze-drying we have 1.4% of dead cells, 4% of intermediate cells and 94.5% live cells, for powder without glycerol we have 44.9% of dead cells, 47.2% of intermediate cells and 7.9% of live cells and for powder with glycerol we have 36.1% of dead cells, 50.4% cells and 13.5% of live cells.

Fig.1. Pseudomonas fluorescens BTP1 after PI/cFDA double staining.

Legends: A: Cream of cells before freeze-drying,B: Powder with 2% glycerol,C: Powder without glycerol

Live cells 94.5%

Intermediate cells 4.0%

A

Intermediate cells50,4%

Live cells 13.5% 

B

7.9%

Dead cells44.9%

C