On-line profiling of Escherichia coli stress response

in Communications in Agricultural and Applied Biological Sciences (in press)

On-line flow cytometry to detect cell growth rate and membrane damage during bioprocess: focus on the segregation of the microbial population

Conference (2013, April 24)

Flow-cytometric assessment of damages to Acetobacter senegalensis during freeze-drying process and storage

in Acetic acid bacteria journal (2013), volume 2(2(s1)), 10

Downstream processes have great influences on bacterial starter production. Different modifications occur to cellular compounds during freeze-drying process and storage of bacterial starters. Consequently ... [more ▼]

Downstream processes have great influences on bacterial starter production. Different modifications occur to cellular compounds during freeze-drying process and storage of bacterial starters. Consequently, viability and culturability (multiplication capacity) undergo some changes. In this study, the effects of freeze-drying process and storage conditions were examined on cell envelope integrity, respiration and culturability of Acetobacter senegalensis. Freezing of cells protected with mannitol (20% w/w) did not affect cell multiplication and respiration considerably; however, 19% of cells showed compromised cell envelope after freezing. After drying, 1.96×1011 CFU/g were enumerated, indicating that about 34% of the cells could survive and keep their culturability. Drying of the cells induced further leakage in cell envelope and finally 81% of cells appeared as injured ones; however, 87% of the dried cells maintained their respiration capacity. Storage temperature had significant effect on cell multiplication ability; higher storage temperature (35°C),caused 8.59-log reduction in cell culturability after nine-month period of storage. Collapse of cell envelop integrity and respiration wasobserved at 35°C. At lower storage temperature (4°C), the culturability decreased about one-log reduction after nine months. Cell envelope integrity was subjected to minor changes during a period of nine month-storage at 4°C whereas a heterogeneous population of cells with different respiration capacity emerged at 4°C. These results indicate that a major part of cells undergone drying process and storage entered into viable but non-culturable state. In addition, usage of different culture media didn’t improve resuscitation. Besides, it seems that sub-lethal damages to cell envelope caused uptake of propidium iodide, however these kinds of injuries could not impress cell multiplications and respiration. [less ▲]

On-line flow cytometry profiling of Escherichia coli stress response

Conference (2013, February 08)

Real-time monitoring of cell viability and cell density on the basis of a three dimensional optical reflectance method (3D-ORM): investigation of the effect of sub-lethal and lethal injuries

in Journal of Industrial Microbiology & Biotechnology (2013)

Cell density and cell viability have been followed on-line by using a three-dimensional optical reflectance method (3D-ORM) probe. This method has allowed to highlight the differences between a well-mixed ... [more ▼]

Cell density and cell viability have been followed on-line by using a three-dimensional optical reflectance method (3D-ORM) probe. This method has allowed to highlight the differences between a well-mixed and a scale-down bioreactor configured in order to reproduce mixing deficiencies during a fed-batch culture of E. coli. These differences have been observed both for the obscuration factor (OBF) and the coincidence probability (COP) delivered by the probe. These parameters are correlated to flow cytometry measurement based on the PI-uptake test and cell density based on optical density measurement. This first set of results has pointed out the fact that the 3D-ORM probe is sensitive to sub-lethal injuries encountered by microbial cells in process-related conditions. The effect of lethal injuries has been further investigated on the basis of additional experiments involving heat stress and a sharp increase of the OBF has been observed indicating that cells are effectively injured by the increase of temperature. However, further improvement of the probe are needed in order to give access to single-cell measurements. [less ▲]

Flow-cytometric assessment of damages to Acetobacter senegalensis during freeze-drying process and storage

in Acetic Acid Bacteria (2013)

Downstream processes have great influences on bacterial starter production. Different modifications occur to cellular compounds during freeze-drying process and storage of bacterial starters. Consequently ... [more ▼]

Downstream processes have great influences on bacterial starter production. Different modifications occur to cellular compounds during freeze-drying process and storage of bacterial starters. Consequently, viability and culturability (multiplication capacity) undergo some changes. In this study, the effects of freeze-drying process and storage conditions were examined on cell envelope integrity, respiration and culturability of Acetobacter senegalensis. Freezing of cells protected with mannitol (20% w/w) did not affect cell multiplication and respiration considerably; however, 19% of cells showed compromised cell envelope after freezing. After drying, 1.96×1011 CFU/g were enumerated, indicating that about 34% of the cells could survive and keep their culturability. Drying of the cells induced further leakage in cell envelope and finally 81% of cells appeared as injured ones; however, 87% of the dried cells maintained their respiration capacity. Storage temperature had significant effect on cell multiplication ability; higher storage temperature (35°C) caused 8.59-log reduction in cell culturability after nine-month period of storage. Collapse of cell envelop integrity and respiration was observed at 35°C. At lower storage temperature (4°C), the culturability decreased about one-log reduction after nine months. Cell envelope integrity was subjected to minor changes during a period of nine month-storage at 4°C whereas a heterogeneous population of cells with different respiration capacity emerged at 4°C. These results indicate that a major part of cells undergone drying process and storage entered into viable but non-culturable state. In addition, usage of different culture media didn’t improve resuscitation. Besides, it seems that sub-lethal damages to cell envelope caused uptake of propidium iodide, however these kinds of injuries could not impress cell multiplications and respiration. [less ▲]

Use of GFP whole cell microbial biosensors for the investigation of the scale-up and scale-down effect on biopharmaceutical processes

Book published by Momentum press (2013)

Utilisation des termites comme source de microorganismes dans la filière de production du bioéthanol de seconde génération

Poster (2012, November 14)

Les termites abritent une microflore symbiotique qui intervient dans la dégradation des fibres constitutives du bois, synthétisant des enzymes capables d’hydrolyser ses composants. Les sucres ... [more ▼]

Les termites abritent une microflore symbiotique qui intervient dans la dégradation des fibres constitutives du bois, synthétisant des enzymes capables d’hydrolyser ses composants. Les sucres fermentescibles libérés suite à cette hydrolyse sont utilisables dans le cadre de la production du bioéthanol de seconde génération. [less ▲]

Effects of glycerol on Pseudomonas fluorescens BTP1 freeze-dried

in International Journal of Biotechnology and Biochemistry. (2012), 8(2), 245-258

The storage stability of freeze-dried powders was studied by parameters such as loss of viability on the Plate Count Agar (PCA). Powder with glycerol (PG) contains 8.4x1010cfu/g before storage 1 ... [more ▼]

The storage stability of freeze-dried powders was studied by parameters such as loss of viability on the Plate Count Agar (PCA). Powder with glycerol (PG) contains 8.4x1010cfu/g before storage 1.1x1010cfug after 3 months at 4°C and 6.0x108cfu/g after 3 months at 20°C. The concentration of soluble proteins (mg/g) decrease during storage at 4°C from 3.77 to 0.80 after 90 days; and the ratios of unsaturated to saturated fatty acids (C18:3/C16:0 and C18:2/C16:0) decrease respectively from 0.05 to 0.04 and 0.007 to 0.004 after 3 months at 4°C. This ratio characterises the membrane fluidity. Powder without glycerol (PS) contains 1.1x1010 cfu/g before storage and 1.4 x 108 cfu/g after 3 months at 4°C and 1.4 x 107 cfu/g after 3 months at 20°C. The concentration of soluble proteins (mg/g) decrease during storage at 4°C from 4.08 to 0.42 after 90 days, the glutathione concentration decrease during storage at 4°C from 2.2 to 1.4. The beneficial effect of glycerol on fatty acid composition during freezedrying is shown and the ratios of unsaturated to saturated fatty acids (C18:2/C16:0 and C18:3/C16:0) decrease respectively from 0.019 to 0.004 and 0.054 to 0.036 after 90 days storage at 4°C. Analysis by flow cytometry was used to assess the physiological state in which cells are at the end of freeze-drying. We found 13.5% live cells, 36.1% dead cells and 50.4% cells in an intermediate state for powder with glycerol (PG) after freeze-drying. These results shows that glycerol play an important role in Pseudomonas fluorescens BTP1 desiccation during freeze-drying, by maintaining a degree of viability after freeze-drying and during storage. [less ▲]

Improvement of the cellulose hydrolysis yields and hydrolysate concentration by management of enzymes and substrate input

in Cerevisia : Belgian Journal of Brewing and Biotechnology (2012), 37

In order to improve the hydrolysis of cellulose fiber and to obtain highly concentrated hydrolysate, two methods based on successive addition of enzyme and substrate were assessed. The first method, which ... [more ▼]

In order to improve the hydrolysis of cellulose fiber and to obtain highly concentrated hydrolysate, two methods based on successive addition of enzyme and substrate were assessed. The first method, which required only substrate addition, allowed to increase by 50% the hydrolysate concentration and to decrease by 30% enzyme units needed. The second method highlighted the ability to reach very high concentrated hydrolysate (up to 170 g/l) by simultaneous addition of enzyme and substrate. In parallel, relationships between some limiting factors and the yields of hydrolysis were investigated. In conclusion, viscosity evolution of cellulose suspension during hydrolysis step was investigated with an aim to improve the management of enzyme and substrate addition. [less ▲]