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. 2023 Nov 4;11(11):2705.
doi: 10.3390/microorganisms11112705.

Effect of Lyoprotective Agents on the Preservation of Survival of a Bacillus cereus Strain PBG in the Freeze-Drying Process

Diana Galeska Farfan Pajuelo  1 , Milena Carpio Mamani  1 , Gisela July Maraza Choque  1 , Dina Mayumi Chachaque Callo  1 , César Julio Cáceda Quiroz  1
Affiliations

Effect of Lyoprotective Agents on the Preservation of Survival of a Bacillus cereus Strain PBG in the Freeze-Drying Process

Diana Galeska Farfan Pajuelo et al. Microorganisms. .

Abstract

Lyophilization is a widely employed long-term preservation method in which the bacterial survival rate largely depends on the cryoprotectant used. Bacillus cereus strain PBC was selected for its ability to thrive in environments contaminated with arsenic, lead, and cadmium, tolerate 500 ppm of free cyanide, and the presence of genes such as ars, cad, ppa, dap, among others, associated with the bioremediation of toxic compounds and enterotoxins (nheA, nheB, nheC). Following lyophilization, the survival rates for Mannitol 2.5%, Mannitol 10%, and Glucose 1% were 98.02%, 97.12%, and 96.30%, respectively, with the rates being lower than 95% for other sugars. However, during storage, for the same sugars, the survival rates were 78.71%, 97.12%, and 99.97%, respectively. In the cake morphology, it was found that the lyophilized morphology showed no relationship with bacterial survival rate. The best cryoprotectant for the PBC strain was 1% glucose since it maintained constant and elevated bacterial growth rates during storage, ensuring that the unique characteristics of the bacterium were preserved over time. These findings hold significant implications for research as they report a new Bacillus cereus strain with the potential to be utilized in bioremediation processes.

Keywords: bacterial survival; glucose; lactose and Bacillus; lyophilization; mannitol.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Tolerance of bacterial strains to free cyanide under alkaline conditions.
Figure 2
Figure 2
Growth kinetics. (a) Kinetics of the natural logarithm of absorbance (OD 600) vs. time (hours). (OD 600) vs. time (hours). (b) Kinetics of the natural logarithm of colony forming units (CFU/mL) vs. time (hours).
Figure 3
Figure 3
Calibration curve CFU/mL vs. absorbance (OD 600) for Bacillus cereus strain PBG.
Figure 4
Figure 4
Graph of survival after lyophilization. The bars labeled with an asterisk (*) do not significantly differ from the control bar according to Dunnett’s tests. Meanwhile, bars marked with ‘A’ exhibit the highest survival rates compared to other cryoprotectants, while ‘B’ indicates lower survival rates.
Figure 5
Figure 5
Graph of survival after lyophilization and during storage of processed sugars. Values with differing letters are considered to have significant differences.
Figure 6
Figure 6
Morphology of lyophilized bacterial suspension observed. (a) Intact, (b) porous, (c) partial collapse, and (d) collapse.
Figure 7
Figure 7
Distribution graph of cake morphology features.
See this image and copyright information in PMC

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