References of "Delvigne, Frank"
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See detailFlow-cytometric assessment of damages to Acetobacter senegalensis during freeze-drying process and storage
Shafiei, Rasoul ULg; Delvigne, Frank ULg; Thonart, Philippe ULg

in Acetic Acid Bacteria (2013), 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. <br />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 <br />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 <br />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 ▲]

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See detailEvaluation of viability and growth of Acetobacter senegalensis under different stress conditions
Shafiei, Rasoul ULg; Thonart, Philippe ULg; Delvigne, Frank ULg et al

in International Journal of Food Microbiology (2013)

Acetic acid bacteria (AAB) are used in production of vinegars. During acetic acid fermentation, AAB encounter various aggressive conditions which may lead to a variety of cellular disorders. Previous ... [more ▼]

Acetic acid bacteria (AAB) are used in production of vinegars. During acetic acid fermentation, AAB encounter various aggressive conditions which may lead to a variety of cellular disorders. Previous researches mainly studied the influences of different carbon sources on tolerance of AAB to ethanol and acetic acid. In this study, different techniques were used comparatively to investigate the effects of preadaptation on the ability of A. senegalensis to tolerate ethanol and acetic acid. In general, the carbon sources used for preadaptation of A. senegalensis exhibited significant effects on the tolerance of cells to stressors. Flow-cytometric assessments of preadapted cells in ethanol showed that 87.3% of the cells perform respiration after exposure to a stress medium containing 5% (v/v) ethanol and 3% (w/v) acetic acid. However, 58.4% of these preadapted cells could keep their envelope integrity under the stress condition. They could also grow rapidly (μmax = 0.39/h) in the stress medium (E5A3) with a high yield (>80%). A. senegalensis grown in glucose exhibited a low tolerance to acetic acid. Analysis of their respiration capacity, membrane integrity and culturability revealed that almost all the population were dead after exposure to 5% (v/v) ethanol and 3% (w/v) acetic acid. In contrast, exposure of A. senegalensis preadapted in a mixture of glucose and acetic acid to a stress medium containing 5% (v/v) ethanol and 3% (w/v) acetic acid, exhibited an intact respiration system and cellular membrane integrity in 80.3% and 50.01% of cells, respectively. Moreover, just 24% of these cells could keep their culturability under that stress condition. [less ▲]

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See detailOn-line flow cytometry profiling of Escherichia coli stress response
Brognaux, Alison ULg; Han, Shanshan; Sorensen, Soren et al

Conference (2013, February 08)

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See detailOn-line profiling of Escherichia coli stress response
Brognaux, Alison ULg; Shanshan, Han; Sorensen, Soren et al

in Communications in Agricultural and Applied Biological Sciences (2013)

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See detailBiofilm formation on metal structured packing for the production of high added value biomolecules
Zune, Quentin ULg; Zune, Quentin ULg; Toye, Dominique ULg et al

in Récents Progrès en Génie des Procédés (2013)

Many white biotechnology bioprocesses apply techniques from chemical engineering based on bioreactors with mechanical stirring system commonly employed in pharmaceutical sector, food industry or energy ... [more ▼]

Many white biotechnology bioprocesses apply techniques from chemical engineering based on bioreactors with mechanical stirring system commonly employed in pharmaceutical sector, food industry or energy field (Dasilva, 2004). As in chemical engineering, scale-up of these bioprocesses induces physicochemical constraints that affect physiological pathways and decrease performances. In this context, it is essential to think new bioprocesses better suited to physiology of microorganisms, minimizing physicochemical constraints. The aim of this work consists to use stainless steel structured packing (SSP) with high specific area (500-750 m²/m³) as inert support for biomass immobilization in order to produce high added value biomolecules. These bioreactors are biocatalysts in which microbial system is immobilized biomass on the form of a biofilm performing bioconversion of a substrate into a specific product (Rosche, 2009). In this study, an experimental setting containing a SSP reproduces solid-state fermentation (SSF) like conditions. Two well known microorganisms for their ability to form biofilm and secrete metabolites are tested in the experimental setting : Bacillus subtilis for its lipopeptides and Aspergillus oryzae for its glucoamylase. Effectiveness of the bioprocess in term of dynamic of the excretion of the target biomolecule is compared with a classical submerged culture (SmF). For lipopeptides production from B. subtilis, SSP is located in a 20L bioreactor continuously aspersed by liquid medium required to the growth of the biofilm. In the case of A. oryzae, the SSP is partially immerged in a 250 mL shake flask. X-ray tomography of the SSP allows non-invasive visualization and quantification of biofilm repartition inside the support. Implementation of SSP permits almost total immobilization of biomass on the form of a mono-species biofilm to the detriment of the liquid phase. Processing of images obtained by X-ray tomography of the SSP provides relevant information for the optimization of the bioprocess. For both microorganism species, results indicate the influence of parameters such as hydrodynamics, aeration rate and microorganism specificity, on the biofilm morphology inside the support and the performances of the bioprocess. SSF-like conditions in the experimental setting lead to technologic progress, such as absence of foam formation, persistence of the microbial system, and improve the dynamic of metabolites excretion compared with conditions imposed by the submerged culture. Further experiment will consider hydrodynamics aspects and amount of carbon source on effectiveness of the bioprocess. [less ▲]

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See detailA low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors
Brognaux, Alison ULg; Han, Shanshan; Sorensen, Soren J et al

in Microbial Cell Factories (2013), 12(100), 1-14

Background Microbial cell population heterogeneity is now recognized as a major source of issues in the development and optimization of bioprocesses. Even if single cell technologies are available for the ... [more ▼]

Background Microbial cell population heterogeneity is now recognized as a major source of issues in the development and optimization of bioprocesses. Even if single cell technologies are available for the study of microbial population heterogeneity, only a few of these methods are available in order to study the dynamics of segregation directly in bioreactors. In this context, specific interfaces have been developed in order to connect a flow cytometer directly to a bioreactor for automated analyses. In this work, we propose a simplified version of such an interface and demonstrate its usefulness for multiplexed experiments.Results A low-cost automated flow cytometer has been used in order to monitor the synthesis of a destabilized Green Fluorescent Protein (GFP) under the regulation of the fis promoter and propidium iodide (PI) uptake. The results obtained showed that the dynamics of GFP synthesis are complex and can be attributed to a complex set of biological parameters, i.e. on the one hand the release of protein into the extracellular medium and its uptake modifying the activity of the fis promoter, and on the other hand the stability of the GFP molecule itself, which can be attributed to the protease content and energy status of the cells. In this respect, multiplexed experiments have shown a correlation between heat shock and ATP content and the stability of the reporter molecule. Conclusion This work demonstrates that a simplified version of on-line FC can be used at the process level or in a multiplexed version to investigate the dynamics of complex physiological mechanisms. In this respect, the determination of new on-line parameters derived from automated FC is of primary importance in order to fully integrate the power of FC in dedicated feedback control loops. [less ▲]

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See detailMicrobial characterization of probiotics-Advisory report of the Working Group "8651 Probiotics" of the Belgian Superior Health Council (SHC).
Huys, Geert; Botteldoorn, Nadine; Delvigne, Frank ULg et al

in Molecular Nutrition & Food Research (2013)

When ingested in sufficient numbers, probiotics are expected to confer one or more proven health benefits on the consumer. Theoretically, the effectiveness of a probiotic food product is the sum of its ... [more ▼]

When ingested in sufficient numbers, probiotics are expected to confer one or more proven health benefits on the consumer. Theoretically, the effectiveness of a probiotic food product is the sum of its microbial quality and its functional potential. Whereas the latter may vary much with the body (target) site, delivery mode, human target population, and health benefit envisaged microbial assessment of the probiotic product quality is more straightforward. The range of stakeholders that need to be informed on probiotic quality assessments is extremely broad, including academics, food and biotherapeutic industries, healthcare professionals, competent authorities, consumers, and professional press. In view of the rapidly expanding knowledge on this subject, the Belgian Superior Health Council installed Working Group "8651 Probiotics" to review the state of knowledge regarding the methodologies that make it possible to characterize strains and products with purported probiotic activity. This advisory report covers three main steps in the microbial quality assessment process, i.e. (i) correct species identification and strain-specific typing of bacterial and yeast strains used in probiotic applications, (ii) safety assessment of probiotic strains used for human consumption, and (iii) quality of the final probiotic product in terms of its microbial composition, concentration, stability, authenticity, and labeling. [less ▲]

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See detailDesign of growth-dependent biosensors based on destabilized GFP for the detection of physiological behavior of Escherichia coli in heterogeneous bioreactors
Han, Shanshan; Delvigne, Frank ULg; Brognaux, Alison ULg et al

in Biotechnology Progress (2013), 29(2), 553-563

In this work we present the design and characterization of GFP-based reporter systems designed in order to describe cellular activity in ‘complex’, heterogeneous bioreactors. The reporter systems consist ... [more ▼]

In this work we present the design and characterization of GFP-based reporter systems designed in order to describe cellular activity in ‘complex’, heterogeneous bioreactors. The reporter systems consist of Escherichia coli strains carrying growth dependent promoters fused to genes expressing stable and unstable variants of GFP, respectively. The response of Escherichia coli cells to transient exposure to glucose was studied in a two-compartment scale down bioreactor (SDR) consisting of a stirred tank reactor (STR) connected to plug-flow reactor (PFR). Such a SDR system is employed to mimic the situation that often encountered in large-scale, fed-batch bioreactors. The response of E. coli coli to oxygen-poor and glucose-rich regions was simulated by continuously pumping E. coli cells from STR to the PFR. A concentrated glucose pulse (400 g/L) was consecutively added at the entrance of the PFR and samples were taken from PFR. The GFP expressions were significantly marked after 10 hours of culture in STR (control reactor) and SDR, whereas, growth rates were rather similar. Additional experiments in chemostat (D=0.14 h-1) with programmed glucose perturbation (30 g/L, frequency: 100/900 s) suggested that the activities of the promoter are linked with the substrate limitation signal. Taken together with immunoblot analysis, we suppose protein leakage is responsible for the overexpression of fis and the related promoters, such as rrnB in this case study, but additional works are required in order to confirm this relationship. Our finding are of great interest for industrial application since the GFP signal can be detected very early during the culture and is related to relevant physiological changes. This investigation is useful for a better understanding of the fast dynamic phenomena occurring in heterogeneous large-scale bioreactors. [less ▲]

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See detailReal-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
Brognaux, Alison ULg; Bugge, Jörg; Schwartz, Friedel et al

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 ▲]

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See detailDirect and indirect use of GFP whole cell biosensors for the assessment of bioprocess performances: design of milliliter scale-down bioreactors
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

in Biotechnology Progress (2013), 29(1), 48-59

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 ... [more ▼]

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. [less ▲]

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See detailIntégration du stress microbien pour le scaling-up des bioréacteurs
Delvigne, Frank ULg

Scientific conference (2013)

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See detailUtilisation des termites comme source de microorganismes dans la filière de production du bioéthanol de seconde génération
Tarayre, Cédric ULg; Bauwens, Julien ULg; Brasseur, Catherine ULg et al

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 ▲]

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See detailScale-up - scale-down : vers une meilleure intégration industrielle des biotechnologies blanches
Delvigne, Frank ULg

Scientific conference (2012, November 14)

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See detailImplementation of structured metal packing for the design of biofilm reactor : analysis by high energy X-ray tomography and application to the production of lipopeptides by Bacillus subtilis
Zune, Quentin ULg; Soyeurt, Delphine; Ongena, Marc ULg et al

Poster (2012, October 08)

1. Whereas multi-species biofilm reactors are commonly used for treatments of water and gas effluents, new strategies are arising for the development of mono-species biofilm reactors in order to produce ... [more ▼]

1. Whereas multi-species biofilm reactors are commonly used for treatments of water and gas effluents, new strategies are arising for the development of mono-species biofilm reactors in order to produce high added value molecules. Thus, it is required to design new bioreactors able to promote the growth of the biomass on the form of a biofilm and to identify the key physico-chemical parameters involved in order to optimize the bioprocess. 2. Aim of this study was to investigate a pilot-scale biofilm reactor comprising a metal structured packing promoting growth of Bacillus subtilis as a biofilm for the production of lipopeptides, high added value compounds with high surface active properties. 3. In this work, the mechanical stirring system of a 20L stirred tank bioreactor has been removed and replaced by a metal structured packing positioned in the headspace of the vessel above a liquid phase. The culture medium is continuously recirculated on the packing thanks to a peristaltic pump and air supply is performed just above the liquid phase under the packing. High energy X-ray tomography was used to estimate non-invasively the biofilm distribution inside the packing and permitted to define parameters that affect scale-up. Performances of the biofilm reactor were compared with a submerged culture in a stirred tank reactor in terms of lipopeptides production. 4. After 72 hours of fermentation, 94 % of the total biomass adheres onto the metal packing on the form of a biofilm. The colonization of this latter has been visualized non-invasively by X-ray tomography directly inside the packing and shows a conical repartition of the biofilm mass (about 25% of the total volume of the packing) as well as the presence of clogging. However, unlike the submerged culture, no foam formation appeared during fermentation and surfactin yield reaches 345,4 ± 32,8 mg / L for the biofilm reactor against 277,3 ± 34,4 mg / L in the stirred tank reactor. 5. In conclusion, this experimental setting leads to a major technological progress avoiding foam formation and increasing surfactin production. Nevertheless, significant improvements are required at the level of the biofilm distribution in thin layers inside the packing in order to increase mass transfer and lipopeptides recoveries. Further investigations will be devoted to the optimization of the physico-chemical parameters involved in biofilm distribution. [less ▲]

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See detailImplementation of structured metal packing for the design of biofilm reactor : analysis by high energy X-ray tomography and application to the production of lipopeptides by Bacillus subtilis
Zune, Quentin; Soyeurt, Delphine; Ongena, Marc ULg et al

Poster (2012, October 08)

1. Whereas multi-species biofilm reactors are commonly used for treatments of water and gas effluents, new strategies are arising for the development of mono-species biofilm reactors in order to produce ... [more ▼]

1. Whereas multi-species biofilm reactors are commonly used for treatments of water and gas effluents, new strategies are arising for the development of mono-species biofilm reactors in order to produce high added value molecules. Thus, it is required to design new bioreactors able to promote the growth of the biomass on the form of a biofilm and to identify the key physico-chemical parameters involved in order to optimize the bioprocess. 2. Aim of this study was to investigate a pilot-scale biofilm reactor comprising a metal structured packing promoting growth of Bacillus subtilis as a biofilm for the production of lipopeptides, high added value compounds with high surface active properties. 3. In this work, the mechanical stirring system of a 20L stirred tank bioreactor has been removed and replaced by a metal structured packing positioned in the headspace of the vessel above a liquid phase. The culture medium is continuously recirculated on the packing thanks to a peristaltic pump and air supply is performed just above the liquid phase under the packing. High energy X-ray tomography was used to estimate non-invasively the biofilm distribution inside the packing and permitted to define parameters that affect scale-up. Performances of the biofilm reactor were compared with a submerged culture in a stirred tank reactor in terms of lipopeptides production. 4. After 72 hours of fermentation, 94 % of the total biomass adheres onto the metal packing on the form of a biofilm. The colonization of this latter has been visualized non-invasively by X-ray tomography directly inside the packing and shows a conical repartition of the biofilm mass (about 25% of the total volume of the packing) as well as the presence of clogging. However, unlike the submerged culture, no foam formation appeared during fermentation and surfactin yield reaches 345,4 ± 32,8 mg / L for the biofilm reactor against 277,3 ± 34,4 mg / L in the stirred tank reactor. 5. In conclusion, this experimental setting leads to a major technological progress avoiding foam formation and increasing surfactin production. Nevertheless, significant improvements are required at the level of the biofilm distribution in thin layers inside the packing in order to increase mass transfer and lipopeptides recoveries. Further investigations will be devoted to the optimization of the physico-chemical parameters involved in biofilm distribution. [less ▲]

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