Characterization of the response of GFP microbial biosensors sensitive to substrate limitation in scale-down bioreactors

Delvigne, Frank; Brognaux, Alison; Gorret, Nathalie; Neubauer, Peter; Delafosse, Angélique; Collignon, Marie-Laure; Toye, Dominique; Crine, Michel; Boxus, Mathieu; Thonart, Philippe

doi:10.1016/j.bej.2011.03.016

Cited by 21 publications

(24 citation statements)

References 34 publications

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“…Basically, the scale-up procedure was performed by keeping the volumetric power constant at a value of 500 W/m 3 . Mixing time values reporter in Table 1 were extracted from previous studies [5,6].…”

Section: Bioreactor Operating Scalesmentioning

confidence: 99%

Scalable temperature induced stress for the large-scale production of functionalized Bifidobacteria

Nguyen

Razafindralambo

Richel³

et al. 2015

Journal of Industrial Microbiology and Biotechnology

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The application of sub-lethal stresses is known to be an efficient strategy to enhance survival of probiotic bacteria during drying processes. In this context, we previously showed that the application of heat stress upon the entry into stationary phase increased significantly the viability of Bifidobacterium bifidum. However, this heat shock has been considered only in small-scale bioreactor and no information is available for a possible scaling-up strategy. Five different operating scales (0.2, 2, 20, 200 and 2000 L) have thus been tested and the results showed that the viability of B. bifidum increases from 3.15 to 6.57 folds, depending on the scale considered. Our observations pointed out the fact that the heat stress procedure is scalable according to the main outcome, i.e., increases in cell viability, but other factors have to be taken into account. Among these factors, dissolved carbon dioxide seems to play a significant role, since it explains the differences observed between the test performed at laboratory scale and in industrial conditions.

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Section: Bioreactor Operating Scalesmentioning

confidence: 99%

Scalable temperature induced stress for the large-scale production of functionalized Bifidobacteria

Nguyen

Razafindralambo

Richel³

et al. 2015

Journal of Industrial Microbiology and Biotechnology

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“…Whole cell microbial biosensors responsive to carbon limitation are very useful for the detection of spatial substrate heterogeneities, as demonstrated in our previous works:15, 16 the behavior of a csiE biosensor has already been studied in conventional stirred reactors. In the case of these specific reporter systems, GFP is actively synthesized under homogeneous conditions (environmental conditions met in the case of a lab‐scale reactor) and is deactivated when the biosensor is exposed to glucose heterogeneities in a scale‐down reactor able to reproduce industrial conditions 15. Another aspect that will be investigated in this work involves indirect use of the biosensors by analyzing the amount of extracellular GFP coming either from permeabilized membranes (protein leakage) or from lysed cells.…”

Section: Introductionmentioning

confidence: 94%

“…The combination and parallelization of these two feeding modes can be the basis of a high‐throughput scale‐down system. These investigations were carried out by using microbial GFP biosensors as physiological tracers for the environmental fluctuations met in industrial heterogeneous bioreactors 14, 15. Whole cell microbial biosensors responsive to carbon limitation are very useful for the detection of spatial substrate heterogeneities, as demonstrated in our previous works:15, 16 the behavior of a csiE biosensor has already been studied in conventional stirred reactors.…”

Section: Introductionmentioning

confidence: 99%

“…These investigations were carried out by using microbial GFP biosensors as physiological tracers for the environmental fluctuations met in industrial heterogeneous bioreactors 14, 15. Whole cell microbial biosensors responsive to carbon limitation are very useful for the detection of spatial substrate heterogeneities, as demonstrated in our previous works:15, 16 the behavior of a csiE biosensor has already been studied in conventional stirred reactors. In the case of these specific reporter systems, GFP is actively synthesized under homogeneous conditions (environmental conditions met in the case of a lab‐scale reactor) and is deactivated when the biosensor is exposed to glucose heterogeneities in a scale‐down reactor able to reproduce industrial conditions 15.…”

Section: Introductionmentioning

confidence: 99%

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Direct and indirect use of GFP whole cell biosensors for the assessment of bioprocess performances: Design of milliliter scale‐down bioreactors

Brognaux

Thonart

Delvigne

et al. 2012

Biotechnology Progress

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Substrate limitation responsive biosensors have been used for the development of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli, i.e., uspA::gfp, csiE::gfp, and yciG::gfp. Our previous studies have shown that these kinds of promoters are induced in response to substrate limitation and are significantly repressed when cultures are carried out in heterogeneous bioreactors. This sensitivity to substrate limitation has been confirmed in the case of the csiE and yciG biosensors. A mini-scale-down platform is proposed as a high throughput tool to rapidly investigate the usefulness of a given microbial biosensor. This platform is composed of shake flasks able to operate in fed-batch mode either using the slow release or the intermittent feeding principle. Local heterogeneities were reproduced at the level of these mini-bioreactors (operating under the intermittent feeding principle) and caused a decrease in GFP expression as in conventional scale-down reactors. The presence of GFP in supernatants was also noted and seems to be correlated with the substrate limitation signal for the three cultivation systems considered in this work (i.e., chemostat, conventional and mini-bioreactors) and with membrane permeability.

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“…Detection of nutrient limitation in Escherichia coli was demonstrated using the csiE [19,20], yciG [21 ] and fis promoters [22] to control GFP expression. The constructs were used as tools to evaluate how cellular physiology changes in response to different bioreactor conditions including in scaled down bioreactors [20] and in shake flasks operated under different conditions [21 ]. In eukaryotes, glucose deprivation and amino acid starvation [23,24] are also linked to the unfolded protein response (see below).…”

Section: Extrinsic Stressmentioning

confidence: 99%

Genetically-encoded biosensors for monitoring cellular stress in bioprocessing

Polizzi

Kontoravdi

2015

Current Opinion in Biotechnology

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With the current wealth of transcriptomic data, it is possible to design genetically-encoded biosensors for the detection of stress responses and apply these to high-throughput bioprocess development and monitoring of cellular health. Such biosensors can sense extrinsic factors such as nutrient or oxygen deprivation and shear stress, as well as intrinsic stress factors like oxidative damage and unfolded protein accumulation. Alongside, there have been developments in biosensing hardware and software applicable to the field of genetically-encoded biosensors in the near future. This review discusses the current state-of-the-art in biosensors for monitoring cultures during biological manufacturing and the future challenges for the field. Connecting the individual achievements into a coherent whole will enable the application of genetically-encoded biosensors in industry.

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Characterization of the response of GFP microbial biosensors sensitive to substrate limitation in scale-down bioreactors

Cited by 21 publications

References 34 publications

Scalable temperature induced stress for the large-scale production of functionalized Bifidobacteria

Scalable temperature induced stress for the large-scale production of functionalized Bifidobacteria

Direct and indirect use of GFP whole cell biosensors for the assessment of bioprocess performances: Design of milliliter scale‐down bioreactors

Genetically-encoded biosensors for monitoring cellular stress in bioprocessing

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