References of "Delvigne, Frank"
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See detailUse of on-line flow cytometry to detect segregation in the microbial population
Brognaux, Alison ULg; Zune, Quentin ULg; Han, Shanshan et al

Poster (2013, October 08)

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See detailStress microbien lors du processus d’extrapolation: Approche physique et biologique
Lejeune, Annick ULg; Thonart, Philippe ULg; Delvigne, Frank ULg

in Cerevisia : Belgian Journal of Brewing and Biotechnology (2013), 38(3), 89-101

Les micro-organismes sont utilisés dans de nombreux domaines (agro-alimentaire, pharmaceutique, environnemental, énergétique,…) que ce soit pour la production de biomasse ou de métabolites particuliers ... [more ▼]

Les micro-organismes sont utilisés dans de nombreux domaines (agro-alimentaire, pharmaceutique, environnemental, énergétique,…) que ce soit pour la production de biomasse ou de métabolites particuliers. Le passage de l’échelle du laboratoire à l’échelle industrielle est souvent problématique car les micro-organismes sont sensibles aux conditions environnementales développées au sein du volume réactionnel. De plus, la perte de l’efficacité d’homogénéisation des bioréacteurs industriels entraîne des perturbations au niveau du métabolisme des cellules. C’est pourquoi il est important d’étudier les conditions hydrodynamiques développées au sein du réacteur. Cet article présente des méthodes de calcul et de modélisation de ces conditions. L’impact de l’hydrodynamique sur le métabolisme microbien peut être étudié par l’utilisation de réacteurs scale-down. Les techniques nouvelles permettant de réaliser un scale-up prennent en compte des caractères physiologiques des cellules, qui estiment l’impact des conditions environnementales sur le métabolisme microbien et donc la bonne réussite du procédé. Enfin, l’étude de Saccharomyces cerevisiae, micro-organisme de grand intérêt biotechnologique, est réalisée. [less ▲]

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See detailDéveloppement d'un dispositif de trajectographie optique nécessaire pour la caractérisation expérimentale par une approche Euler-Lagrange des écoulements dans des bioréacteurs à cuve agitée
Collignon, Marie-Laure ULg; Delafosse, Angélique ULg; Delvigne, Frank ULg et al

in Récents Progrès en Génie des Procédés (2013, October)

Les bioréacteurs à cuve agitée sont des dispositifs largement utilisés dans l’industrie pharmaceutique. Les performances globales des cultures menées dans ces bioréacteurs dépendent fortement de ... [more ▼]

Les bioréacteurs à cuve agitée sont des dispositifs largement utilisés dans l’industrie pharmaceutique. Les performances globales des cultures menées dans ces bioréacteurs dépendent fortement de l’environnement physico-chimique et hydrodynamique local rencontré par les microorganismes et ce au cours du temps. Pour parvenir à caractériser cet historique de conditions locales rencontrées, une approche Euler-Lagrange doit être adoptée. La difficulté dans ce type d’approche est d’être capable de mesurer de manière fiable la trajectoire suivie par le microorganisme au sein du bioréacteur. Pour ce faire, le Laboratoire de Génie Chimique de l’Université de Liège a conçu un dispositif de trajectographie optique. L’objectif de cet article est de présenter ce dispositif, les développements qui ont été nécessaire à sa mise en œuvre et les résultats obtenus. Le dispositif se compose de deux caméras observant selon deux axes de l’espace un bioréacteur rétro-éclairé par des panneaux leds. La trajectoire suivie par une particule noire en gel d’alginate de calcium de 491 µm de diamètre est construite à partir des images acquises par ces deux caméras préalablement modélisée par un modèle pinhole et traitées par une approche rationnée. La validité des trajectoires mesurées a été prouvée dans un bioréacteur de 20 L mélangé par une hélice TTP en comparant les champs de vitesse moyen et d’énergie cinétique turbulent extraits de la trajectoire avec ceux obtenus par la technique eulérienne Stéréo P.I.V. La convergence de ces deux champs avec le temps d’acquisition de la trajectoire a également été démontrée. [less ▲]

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See detailPhysiological response of yeast to process perturbations: A mini-bioreactor approach
Lejeune, Annick ULg; Delvigne, Frank ULg; Thonart, Philippe ULg

in Cerevisia : Belgian Journal of Brewing and Biotechnology (2013), 38(1), 15-19

Large-scale production of yeast (Saccharomyces cerevisiae) is difficult to control, considering the drop of mixing and mass transfer efficiency during scale-up. The drop of hydrodynamic efficiency in ... [more ▼]

Large-scale production of yeast (Saccharomyces cerevisiae) is difficult to control, considering the drop of mixing and mass transfer efficiency during scale-up. The drop of hydrodynamic efficiency in large-scale bioreactors induces the formation of heterogeneities, i.e. mainly substrate and dissolved oxygen in process conditions. These extracellular fluctuations have several impacts at the level of the physiology of microorganisms, from metabolic shift to specific gene expression (stress response). Microbial cell responses to extracellular fluctuations are actually not fully understood. In this work, we propose to reproduce the main extracellular fluctuations at the level of a mini bioreactor platform. These mini-reactors are shake flasks equipped with dissolved oxygen probes. The cultures are realized with different fed-batch control strategies. A scale-down approach has been developed by considering slow release techniques and intermittent feeding, in order to reproduce the glucose and dissolved oxygen fluctuations experienced in large-scale reactors. The mini-reactor has been used to screen the response of several green fluorescent protein (GFP) reporter strains (adh2, tps2, pdc6 and hxt2). The GFP content of cells has been determined by flow cytometry in order to take into account population heterogeneity. In front of these results, the methodology presented in this work can be proposed as a scale-down tool. [less ▲]

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See detailApplication of three entomopathogenic fungi for aphid control
Bawin, Thomas ULg; Seye, Fawrou; Boukraa, Slimane ULg et al

Poster (2013, May 21)

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Chemical pesticide application is the most commonly used method for aphid control. Due to ... [more ▼]

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Chemical pesticide application is the most commonly used method for aphid control. Due to rapid developments of resistance to pesticides and environmental pollution, integrated pest management, including biological control was promoted. In this context, entomopathogenic fungi are valuable tools for potential aphid control since various fungal strains are already commercially available. The present study aims to evaluate and compare the insecticidal activity of two Metarhizium and one Aspergillus strains against Acyrthosiphon pisum aphid. Fungi were cultivated on wheat bran media in flasks up to sporulation. The solid media were washed with aqueous solutions containing 0.05% Tween 80 before filtration. The content of conidia was determined by haemocytometer. Doses ranging from 10^3 to 10^7 conidia/ml were then applied on young plants with 20 adult parthenogenetic aphids. Batches were incubated at a 16L/8D photoperiod, 25±2°C and 75-80% relative humidity. Adult mortality was assessed in order to determine LD50, LD90 and LT50 values, and the number of nymphs produced was daily recorded. Five days after treatment, mortality rates ranged from 30 to 91% depending on the fungal strain and tested dose. Corresponding LD50 and LD90 values were 1.23 x 10^3 and 1.34 x 10^7, 3.67 x 10^3 and 9.71 x 10^7, 4.95 x 10^2 and 5.65 x 10^7 conidia/ml for Metarhizium sp., Metarhizium anisopliae and Aspergillus clavatus respectively. At the higher dose, the LT50 were reached within 2, 4 and 3 days respectively, whereas the LT50 were never reached in the controls. By contrast, the intrinsic growth rates were significantly different from uninfected aphids only in the case of A. clavatus with 10^6 and 10^7 conidia/ml doses five days after exposure. In conclusion, these fungal isolates induced A. pisum mortality with a similar impact and A. clavatus infection appeared to alter the adults’ fitness. This suggests that these fungi may be candidates for aphid control. Further investigations should be made in order to assess their host range specificity. Toxic metabolites leading to death have to be identified and their safety towards non-target organisms confirmed. Finally, their persistence in the environment as well as the compatibility with over means of aphid control must be verified in a view of a broad integrated pest management. [less ▲]

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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 detailHigh-energy X-ray tomography analysis of a metal packing biofilm reactor for the production of lipopeptides by Bacillus subtilis
Zune, Quentin ULg; Soyeurt, Delphine; Toye, Dominique ULg et al

in Journal of Chemical Technology & Biotechnology (2013)

BACKGROUND: Whereas multi-species biofilm reactors are commonly used for the treatment of liquid and solid wastes, new strategies are progressing for the development of single species biofilm for the ... [more ▼]

BACKGROUND: Whereas multi-species biofilm reactors are commonly used for the treatment of liquid and solid wastes, new strategies are progressing for the development of single species biofilm for the production of high-value metabolites. Technically, this new concept relies on the design of bioreactors able to promote biofilm formation and on the identification of the key physico-chemical parameters involved in biofilm formation. RESULTS: An experimental setting comprising a liquid continuously recirculated on a metal structured packing has been used to promote Bacillus subtilis GA1 biofilm formation. The colonization of the packing has been visualized non-invasively by X-ray tomography. This analysis revealed an uneven, conical, distribution of the biofilm inside the packing. Compared with a submerged culture carried out in a stirred tank reactor, significant modification of the lipopeptide profile has been observed in the biofilm reactorwith the disappearance of fengycin and iturin fractions and an increase of the surfactin fraction. In addition, considering the biofilm reactor design, no foam formation has been observed during the culture. CONCLUSIONS: The configuration of this biofilm reactor set-up allows for a higher surfactin production by comparison with a submerged culture while avoiding foam formation. Additionally, scale-up could easily be performed by increasing the number of packing elements. [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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