Quantitative Characterization of the Growth of Deinococcus geothermalis DSM-11302: Effect of Inoculum Size, Growth Medium and Culture Conditions
Abstract
:1. Introduction
2. Materials and Methods
2.1. Microorganisms, Media and Growth Conditions
2.1.1. Bacterial Strains
2.1.2. Media and Growth Conditions
2.1.3. Erlenmeyer Flask Experiments
2.1.3.1. Culture of the Three Strains of Deinococcus geothermalis
2.1.3.2. Effect of Yeast Extract Concentration
2.1.3.3. Effect of Inoculation Size and Incubation Duration of the Preculture
2.1.3.4. Effect of One- to Three-Stage Inoculum Cultures in Complex Medium
2.1.3.5. Effect of Dilution of the Residual Yeast Extract in the Medium with Cell Washing Before Inoculation
2.1.3.6. Growth of Deinococcus geothermalis DSM-11302 in Defined Media on Carbon Sources and Complex Nutrients
Vitamins | Concentration (mg·L−1) |
---|---|
Thiamine | 0.105 |
Riboflavine | 0.625 |
Pyridoxine | 0.12 |
Nicotinic acid | 3 |
Panthotenic acid | 0.525 |
Folic acid | 0.03 |
Choline | 7.5 |
Biotine | 0.02 |
2.1.4. Determination of the Respiratory Type of Deinococcus geothermalis DSM-11302
2.1.5. Culture in Bioreactor
2.1.6. Chemical Products
2.2. Analytical Methods
2.2.1. Biomass Analysis
2.2.2. Supernatant Analysis
3. Results and Discussion
3.1. Comparison of the Growth of Deinococcus geothermalis Strains DSM-11300, DSM-11301 and DSM-11302
Strains | Deinococcus geothermalis DSM 11300 | Deinococcus geothermalis DSM 11301 | Deinococcus geothermalis DSM 11302 | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Media | DMG | CMG | TH162 | Holland | DMG | CMG | TH162 | Holland | DMG | CMG | TH162 | Holland | |
24 h | Number of generations | 1.3 | 3.7 | 1.4 | 0.1 | 1.6 | 3.7 | 1.6 | 0.3 | 2.8 | 3.7 | 1.6 | 0.4 |
Glucose uptake (%) | 16 | 75 | / | / | 41 | 86 | / | / | 55 | 94 | / | / | |
48 h | Number of generations | 1.6 | 3.7 | 1.4 | 0.1 | 1.6 | 3.7 | 1.6 | 0.3 | 3.2 | 4.1 | 1.6 | 0.4 |
Glucose uptake (%) | 24 | 100 | / | / | 81 | 100 | / | / | 89 | 100 | / | / |
3.2. Effect of the Temperature on the Growth of Deinococcus geothermalis DSM-11302 and Respiratory Type
3.3. Effect of Yeast Extract Concentration
3.4. Effect of Method of Strain Preservation, Frozen Storage Period, Type of Strain Revivification and Variability between CFU on Petri Dishes
3.5. Influence of Inoculation Size and Duration of Incubation Phase of Pre-Culture
Medium | CMG | ||||||
---|---|---|---|---|---|---|---|
Preservation method | Freeze dried cells | Glycerol stocks (−80 °C) | |||||
Storage period (months) | – | 20 | 12 | ||||
Revivification | Petri dishes culture | Direct tube inoculation | Petri dishes culture | Petri dishes culture | Petri dishes culture | Petri dishes culture | Direct tube inoculation |
Initial OD600 nm | 0.282 | 0.334 | 0.286 | 0.270 | 0.270 | 0.338 | 0.324 |
Final OD600 nm | 1.58 | 1.77 | 1.63 | 1.72 | 1.72 | 1.62 | 1.72 |
Number of generations | 2.5 | 2.4 | 2.5 | 2.7 | 2.7 | 2.3 | 2.4 |
µ (h−1) | 0.76 | 0.75 | 0.75 | 0.76 | 0.76 | 0.76 | 0.71 |
Glucose uptake (%) | 44 | 42 | 41 | 50 | 60 | 40 | 42 |
Medium | DMG | ||||||
---|---|---|---|---|---|---|---|
Preservation method | Freeze dried cells | Glycerol stocks (–80 °C) | |||||
Storage period (months) | – | 20 | 12 | ||||
Revivification | Petri dishes culture | Direct tube inoculation | Petri dishes culture | Petri dishes culture | Petri dishes culture | Petri dishes culture | Direct tube inoculation |
Initial OD600 nm | 0.232 | 0.224 | 0.216 | 0.233 | 0.246 | 0.22 | 0.218 |
Final OD600 nm | 1.37 | 1.37 | 1.34 | 1.35 | 1.36 | 1.36 | 1.52 |
Number of generations | 2.6 | 2.6 | 2.6 | 2.5 | 2.5 | 2.6 | 2.8 |
µ (h−1) | 0.17 | 0.17 | 0.16 | 0.15 | 0.16 | 0.15 | 0.15 |
Glucose uptake (%) | 38 | 36 | 42 | 50 | 26 | 40 | 51 |
YS,X (gx·gGlc−1) | 0.40 | 0.44 | 0.36 | 0.31 | 0.59 | 0.41 | 0.35 |
Age of Pre-Culture (h) | 4 | 8 | 12 | 16 | 20 | 24 | |
---|---|---|---|---|---|---|---|
Inoculation size (%) | 10 | 10 | 10 | 20 | 10 | 10 | 10 |
Initial OD600 nm | 0.201 | 0.270 | 0.213 | 0.439 | 0.203 | 0.231 | 0.260 |
Final OD600 nm | 1.72 | 1.79 | 1.61 | 2.00 | 1.41 | 1.51 | 1.57 |
Number of generations | 3.0 | 2.7 | 2.9 | 2.2 | 2.7 | 2.7 | 2.6 |
µ (h−1) | 0.17 | 0.11 | 0.14 | 0.14 | 0.09 | 0.09 | 0.08 |
Glucose uptake (%) | 45 | 42 | 40 | 55 | 37 | 28 | 40 |
YS,X (gx·gGlc−1) | 0.26 | 0.31 | 0.19 | 0.21 | 0.22 | 0.29 | 0.23 |
3.6. Influence of Successive Subcultures, Dilution of The Residual Yeast Extract in the Medium and Cell Washing before Inoculation on the Growth of Deinococcus geothermalis DSM-11302 in DMG
3.7. Growth of Deinococcus geothermalis DSM-11302 in Defined Media on Carbon Sources and Complex Nutrients
Precultures nb | 1 | 2 | 3 | 1 | 1 | 1 |
---|---|---|---|---|---|---|
Cell washing nb | 1 | 1 | 1 | 1 | 2 | 3 |
Dilution of residual yeast extract | 2.10−4 | 2.10−4 | 2.10−4 | 2.10−4 | 4.10−7 | 8.10−10 |
Initial OD600 nm | 0.192 | 0.245 | 0.233 | 0.292 | 0.285 | 0.239 |
Final OD600 nm | 1.76 | 1.84 | 1.90 | 1.70 | 1.66 | 1.62 |
Number of generations | 3.2 | 2.9 | 3.0 | 2.5 | 2.5 | 2.8 |
µ (h−1) | 0.13 | 0.10 | 0.06 | 0.13 | 0.13 | 0.13 |
Glucose uptake (%) | 63 | 67 | 45 | 50 | 53 | 44 |
YS,X (gx·gGlc−1) | 0.14 | 0.15 | 0.25 | 0.20 | 0.18 | 0.25 |
Carbon Sources | CMC (Carboxy Methyl Cellulose) | Mannose | Trehalose | Galactose | Fructose | Maltose | Lactose | Sucrose | Glucose |
---|---|---|---|---|---|---|---|---|---|
Initial OD600 nm | 0.269 | 0.257 | 0.245 | 0.296 | 0.272 | 0.261 | 0.248 | 0.240 | 0.242 |
Final OD600 nm | 0.222 | 1.880 | 2.120 | 0.690 | 1.540 | 1.990 | 0.218 | 1.990 | 2.000 |
Number of generations | 0 | 2.9 | 3.1 | 1.2 | 2.5 | 2.9 | 0 | 3.1 | 3.1 |
Complex Sources | YE (Yeast Extract) Vitamins | Casamino Acids | D. geothermalis Extract | S. cerevisiae Extract | E. coli extract | None |
---|---|---|---|---|---|---|
Initial OD600 nm | 0.242 | 0.248 | 0.288 | 0.301 | 0.230 | 0.246 |
Final OD600 nm | 1.840 | 1.650 | 1.850 | 2.000 | 1.760 | 1.860 |
Number of generations | 2.9 | 2.7 | 2.7 | 2.7 | 2.9 | 2.9 |
Glucose uptake (%) | 100 | 90 | 100 | 100 | 100 | 94 |
Y S,X (gx·gGlc−1) | 0.23 | 0.21 | 0.23 | 0.23 | 0.23 | 0.23 |
3.8. Quantification of Deinococcus geothermalis DSM-11302 Growth on a Mineral Medium
Physiological Parameters | Phase I | Phase II | Phase III |
---|---|---|---|
Growth rate (h−1) | 0.28 | 0.16 | 0.04 |
Number of generations | 1.2 | 1.5 | – |
qO2 (mmolO2·CmolX−1·h−1) | 216 | 186 105 | 67 |
qCO2 (mmolCO2·CmolX−1·h−1) | 226 | 206 121 | 73 |
qGlc (CmolGlc·CmolX−1·h−1) | 2.2 | 0.22 | 0.22 |
QR | 1.7 | 1.12 | 1.12 |
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Appendix
References
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Bornot, J.; Molina-Jouve, C.; Uribelarrea, J.-L.; Gorret, N. Quantitative Characterization of the Growth of Deinococcus geothermalis DSM-11302: Effect of Inoculum Size, Growth Medium and Culture Conditions. Microorganisms 2015, 3, 441-463. https://doi.org/10.3390/microorganisms3030441
Bornot J, Molina-Jouve C, Uribelarrea J-L, Gorret N. Quantitative Characterization of the Growth of Deinococcus geothermalis DSM-11302: Effect of Inoculum Size, Growth Medium and Culture Conditions. Microorganisms. 2015; 3(3):441-463. https://doi.org/10.3390/microorganisms3030441
Chicago/Turabian StyleBornot, Julie, Carole Molina-Jouve, Jean-Louis Uribelarrea, and Nathalie Gorret. 2015. "Quantitative Characterization of the Growth of Deinococcus geothermalis DSM-11302: Effect of Inoculum Size, Growth Medium and Culture Conditions" Microorganisms 3, no. 3: 441-463. https://doi.org/10.3390/microorganisms3030441