References of "Delvigne, Frank"
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See detailFermentation profile of Saccharomyces cerevisiae and Candida tropicalis as starter cultures on barley malt medium
Alloue-Boraud, Mireille; N’Guessan, Kouadio; Djeni, Théodore et al

in Journal of Food Science & Technology (in press)

Saccharomyces cerevisiae C8-5 and Candida tropicalis F0-5 isolated from traditional sorghum beer were tested for kinetic parameters on barley malt extract, YPD (863 medium) and for alcohol production. The ... [more ▼]

Saccharomyces cerevisiae C8-5 and Candida tropicalis F0-5 isolated from traditional sorghum beer were tested for kinetic parameters on barley malt extract, YPD (863 medium) and for alcohol production. The results showed that C. tropicalis has the highest maximum growth rate and the lowest doubling time. Values were 0.22 h-1 and 0.32 h-1 for maximum growth rate, 3 h 09 min and 2 h 09 min for doubling time respectively on barley malt extract and YPD. On contrary, glucose consumption was the fastest with S. cerevisiae (-0.36 g/l/h and -0.722 g/l/h respectively on barley malt extract and YPD). When these two yeasts were used as starters in pure culture and co-culture at proportion of 1:1 and 2:1 (cell/cell) for barley malt extract fermentation, we noticed that maltose content increased first from 12.12 g/l to 13.62-16.46 g/l and then decreased. The highest increase was obtained with starter C. tropicalis + S. cerevisiae 2:1. On contrary, glucose content decreased throughout all the fermentation process. For all the starters used, the major part of the ethanol was produced at 16 h of fermentation. Values obtained in the final beers were 11.4, 11.6, 10.4 and 10.9 g/l for fermentation conducted with S. cerevisiae, C. tropicalis, C. tropicalis + S. cerevisiae 1:1 and C. tropicalis + S. cerevisiae 2:1. Cell viability measurement during the fermentation by using flow cytometry revealed that the lowest mean channel fluorescence for FL3 (yeast rate of death) was obtained with C. tropicalis + S. cerevisiae 2:1 after 48 h of fermentation. [less ▲]

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See detailDynamic single-cell analysis of Saccharomyces cerevisiae under process perturbation: comparison of different methods for monitoring the intensity of population heterogeneity
Delvigne, Frank ULg; Baert, Jonathan ULg; Gofflot, Sébastien et al

in Journal of Chemical Technology & Biotechnology (in press)

BACKGROUND: Single cell biology has attracted a lot of attention these past few years and has led to numerous fundamental results pointing out the heterogeneity of clonal cell populations. In this context ... [more ▼]

BACKGROUND: Single cell biology has attracted a lot of attention these past few years and has led to numerous fundamental results pointing out the heterogeneity of clonal cell populations. In this context, microbial phenotypic heterogeneity under bioprocessing conditions needs to be further investigated. In this study, yeast based processes have been investigated by using on-line flow cytometry in combination with a fluorescent transcriptional reporter (GFP) and viability fluorescence tags (propidium iodide, PI). Methods aiming at expressing the dispersion of these fluorescence tags among the yeast populations have been investigated for different bioreactor operating conditions. RESULTS: Yeast viability was determined on the basis of PI uptake. Segregation between PI negative and positive subpopulations could be efficiently quantified on the basis of the mean-to-median ratio or the amplitude of the interquartile range. On the other hand, the same quantification could not be made for the segregation occurring at the level of GFP synthesis. Indeed, when cells were exposed to sub-lethal or mild stresses (such as in scale-down reactors) two GFP subpopulations could be visualized by real-time FC, but quantification by one of the above-mentioned methods was not possible. CONCLUSIONS: Yeast population heterogeneity was observed in representative bioreactor operating conditions. Difficulties for the determination of segregation at the level of GFP synthesis point out the fact that one needs to understand the segregation mechanisms for the applied fluorescent reporters, to judge whether simple mathematical tools may be applied or if more sophisticated computational tools are needed for the quantification of the microbial population segregation. [less ▲]

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See detailFungal biofilm reactor improves the productivity of hydrophobin HFBII
Khalesi, Mohammadreza; Zune, Quentin ULg; Telek, Samuel ULg et al

in Biochemical Engineering Journal (in press)

Production and purification of hydrophobin HFBII has recently been the subject of intensive research, but the yield of production needs to be further improved for a generic use of this molecule at ... [more ▼]

Production and purification of hydrophobin HFBII has recently been the subject of intensive research, but the yield of production needs to be further improved for a generic use of this molecule at industrial scale. In a first step, the influence of different carbon sources on the growth of Trichoderma reesei and the production of HFBII was investigated. The optimum productivity was obtained by using 40 g/L lactose. Carbon starvation and excretion of extracellular enzyme were determined as two main conditions for the production of HFBII. In the second phase, and according to the physiological mechanisms observed during the screening phase, a bioreactor set up has been designed and two modes of cultures have been investigated, i.e. the classical submerged fermentation and a fungal biofilm reactor. In this last set-up, the broth is continuously recirculated on a metal packing exhibiting a high specific surface. In this case, the fungal biomass was mainly attached to the metal packing, leading to a simplification of downstream processing scheme. More importantly, the HFBII concentration increased up to 48.6 ± 6.2 mg/L which was 1.8 times higher in this reactor configuration and faster than the submerged culture. X-ray tomography analysis shows that the biofilm overgrowth occurs when successive cultures are performed on the same packing. However, this phenomenon has no significant influence on the yield of HFBII, suggesting that this process could be operated in continuous mode. Protein hydrolysis during stationary phase was observed by MALDI-TOF analysis according to the removal of the last amino acid from the structure of HFBII after 48 h from the beginning of fermentation in biofilm reactor. Hopefully this modification does not lead to alternation of the main physicochemical properties of HFBII. [less ▲]

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See detailBioRefine Project: Detection of bioavailability of Metallic Trace Elements in soils by the use of microbial biosensors
Tarayre, Cédric ULg; Hurdebise, Quentin ULg; Fischer, Christophe ULg et al

Poster (2014, September 09)

Zinc, lead and cadmium are the main Metallic Trace Elements (MTEs) found in soils contaminated by the mining industry in Europe. MTEs are spread in the environment because of the disruption of ... [more ▼]

Zinc, lead and cadmium are the main Metallic Trace Elements (MTEs) found in soils contaminated by the mining industry in Europe. MTEs are spread in the environment because of the disruption of biogeochemical cycles caused by human activities. Due to their low mobility and biodegradability, they accumulate in soils where they are strongly bound to particles. It has become necessary to understand interactions between MTEs and the environment and to implement remediation actions. This work is focused on remediation monitoring techniques by using whole cell microbial biosensors able to detect zinc, lead and cadmium. Biosensors provide a signal in response to the bio-available concentration in MTEs, which are valuable for the design of efficient techniques involving bioremediation. Whole cell biosensors used in this work are based on Escherichia coli strains carrying a fluorescent reporter system. The reporter element contains a promoter sensitive to MTEs and a gene coding for the Green Fluorescent Protein (GFP). MTEs activate the synthesis of GFP, which is a very stable protein, causing the accumulation of GFP inside the cells. Then, fluorescence can be measured by flow cytometry. In this study, two biosensors were investigated: E. coli pPzraPgfp and E. coli pPzntAgfp. The last strain provided a linear response to zinc up to 20 mg/l and a curvilinear response to cadmium up to 0.15 mg/l. No detection was highlighted regarding lead. In practical cases, soils and wastes are contaminated by several types of MTEs. Consequently, combined contaminations were also tested. This work allowed highlighting that the strain E. coli pPzntAgfp can be used to assess the bioavailability of cadmium in soils, although the experimental procedure must be improved. This work is supported by the BioRefine Project, a European project in which various member states focus on recovery of inorganics from organic wastestreams. We gratefully acknowledge the INTERREG IVB NWE programme, which financed the BioRefine Project (ref. 320J-BIOREFINE). [less ▲]

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See detailDesign of a fungal biofilm reactor for recombinant protein production from Aspergillus oryzae
Zune, Quentin ULg; Delepierre, Anissa ULg; Bauwens, Julien ULg et al

Poster (2014, September 07)

Fungi are microorganisms exhibiting high secretive power of various metabolites and have the ability to perform post-translational modifications during protein synthesis. In the field of fermentation ... [more ▼]

Fungi are microorganisms exhibiting high secretive power of various metabolites and have the ability to perform post-translational modifications during protein synthesis. In the field of fermentation industry, they are ideal hosts for secondary metabolites and recombinant protein production. At the industrial-scale, equipments usually required for solid-state or submerged fermentation of filamentous fungi have demonstrated their limitations in terms of productivity, mass transfers or products recovery (1, 2). Recently, fungal biofilm reactors were designed to combine advantages from submerged and solid-state culture and reveal their usefulness for greater secondary metabolites production relative to submerged culture conditions (3). In our work, we propose the design of a fungal biofilm reactor for a recombinant protein production from an Aspergillus oryzae strain containing a GFP reporter gene system under the control of a promoter specifically induced in solid-state conditions. The fungal biofilm reactor is composed of a metal structured packing, having the function of inert support for biofilm growth, immerged or aspersed by a liquid medium. Whereas recombinant protein production is not significantly different at the flask-scale between submerged and biofilm conditions, productivity is higher in the submerged conditions at the bioreactor-scale. Presence of recombinant proteins entrapped in the biofilm matrix highlights a diffusion constraint and a lower mass transfer in our fungal biofilm reactor. However, persistence of a free liquid biomass of low viscosity and fungal biomass retention on the support are attractive for the implementation of a continuous process in our fungal biofilm reactor. Further studies will consider a 2-D proteomic comparison of the extracellular medium from fungal biofilm reactor and submerged culture conditions in order to better understand proteins secretion and identify over-expressed proteins in biofilm conditions. [less ▲]

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See detailImplications of microbial phenotypic heterogeneity in large-scale bioprocessing conditions
Delvigne, Frank ULg; Gorret, Nathalie; Molina-Jouve, Carole et al

Conference (2014, September)

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See detailPathogenicity of Aspergillus clavatus produced in a fungal biofilm bioreactor toward Culex quinquefasciatus (Diptera: Culicidae)
Seye, Fawrou; Bawin, Thomas ULg; Boukraa, Slimane ULg et al

in Journal of Pesticide Science (2014), 39(3), 127-132

Many entomopathogenic fungi have been demonstrated to be potential agents for efficiently controlling mosquito populations. In the present study, we investigated a bioreactor system to produce metabolites ... [more ▼]

Many entomopathogenic fungi have been demonstrated to be potential agents for efficiently controlling mosquito populations. In the present study, we investigated a bioreactor system to produce metabolites and conidia by combining technological advantages of submerged and solid-state fermentations. The efficiency of fungal products was tested toward mosquitoes. Aspergillus clavatus (Eurotiales: Trichocomaceae) was grown by semi-solid-state fermentation in a bioreactor for up to 7 days. Depending on conidial doses (2.5×10^7, 5×10^7, 7.5×10^7, 10×10^7 and 12.5×10^7 conidia/mL), mortality ranged from 37.2±15.0 to 86.3±5.0% toward larvae and from 35.8±2.0 to 85.2±1.5% toward adults. The metabolites (10, 20, 40, 60, 80 and 100% v/v) yielded mortality from 23.7±15.0 to 100.0±0.1% toward larvae, and two sprayed volumes (5 and 10 mL) reached 45.5±1.4 and 75.6±2.6% mortality, respectively, toward adults. [less ▲]

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See detailDevelopment of entomopathogenic fungi in mosquito control: which kind of production for which efficiency?
Bawin, Thomas ULg; Delvigne, Frank ULg; Francis, Frédéric ULg

Poster (2014, August 06)

Mosquitoes (Diptera: Culicidae) are zoonotic vectors responsible for numerous infectious diseases of medical and veterinary importance such as filariasis, malaria and encephalitis. As part of an ... [more ▼]

Mosquitoes (Diptera: Culicidae) are zoonotic vectors responsible for numerous infectious diseases of medical and veterinary importance such as filariasis, malaria and encephalitis. As part of an integrated vector control, entomopathogenic fungi could be developed as biopesticides in two ways: spores and metabolites recognized as effective virulence factors. Solid-state fermentation enhances spore production and induces the secretion of metabolites quantitatively and qualitatively different from submerged fermentation, which impairs fungal metabolic efficiency. In this context, we showed high spore productivity of solid-state media based on agro-industrial substrates as wheat bran. Spores remained pathogenic, as revealed by classical toxicity tests and electron microscopy. However, the absence of free water makes culture parameter variations difficult to control in large-scale. Recently, we performed a bioreactor design intended for simultaneous spore and metabolite production, combining the technological advantages of submerged and solid-state fermentations. Biofilm fermentation (i.e. growth of fungal biomass on an inert support immerged in a nutrient medium) is a tremendous production system favouring the secretion of insecticidal metabolites in the liquid medium as we showed recently. This is also an interesting tool to provide an overview of the complexity of the metabolic pathways involved in the regulation of extracellular metabolites secretion because corresponding genes are reported to be differentially expressed from classical fermentation systems. Researches in vector control are currently intensified. In this context, the identification of genes and metabolites specifically expressed during biofilm fermentation will help to develop new technologies related both to the design of bioreactor and the production of insecticidal proteins. [less ▲]

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See detailEffect of entomopathogenic Aspergillus strains against the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae)
Seye, Fawrou; Bawin, Thomas ULg; Boukraa, Slimane ULg et al

in Applied Entomology and Zoology (2014)

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Entomopathogenic fungi are valuable tools for potential aphid control. As part of a ... [more ▼]

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Entomopathogenic fungi are valuable tools for potential aphid control. As part of a selection process, laboratory bioassays were carried with five different concentrations of Aspergillus clavatus (Desmazières), Aspergillus flavus (Link) and Metarhizium anisopliae ((Metschnikoff) Sorokin) spores against the pea aphid, Acyrthosiphon pisum (Harris). Aspergillus isolates induced higher mortalities than M. anisopliae which is a well-known entomopathogen in the literature. Lethal concentrations (LC50 and LC90) were 1.23 x 10^3 and 1.34 x 10^7 spores/ml for A. flavus, 4.95 x 10^2 and 5.65 x 10^7 spores/ml for A. clavatus, and 3.67 x 10^3 and 9.71 x 10^7 spores/ml for M. anisopliae five days after treatment. Mycelia development and sporulation on adult cadavers was observed 48 hours after incubation. The intrinsic growth rate of A. pisum decreased with increased spore concentration for all fungal strains suggesting an increase in pathogen fitness related to a consumption of host resources. In conclusion, Aspergillus species could be useful in aphid control as pest control agents despite their saprophytic lifestyle. This is also in our knowledge the first report of A. clavatus and A. flavus strains pathogenic to aphids. [less ▲]

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See detailBiorefine: Recovery of nutrients and metallic trace elements from different wastes by chemical and biochemical processes
Tarayre, Cédric ULg; Fischer, Christophe ULg; De Clercq, Lies et al

Conference (2014, June 05)

At present, most waste processing operations are not oriented towards the valorization of valuable reusable components such as nitrogen, phosphorus, potassium and even Metallic Trace Elements (MTEs ... [more ▼]

At present, most waste processing operations are not oriented towards the valorization of valuable reusable components such as nitrogen, phosphorus, potassium and even Metallic Trace Elements (MTEs). Currently, sewage sludge, for example is usually used as a fertilizer in agriculture, in energy production or in the field of construction. Ashes originating from sludge incineration contain heavy metals and minerals in large quantities. Manure is mainly used in agriculture, although considerable amounts of nutrients are lost and cause pollution. Digestate is also used in agriculture, but other alternatives have been proposed, such as the energetic valorization. Better valorization of these wastes in agriculture (or other sectors) is however largely constrained by a multitude of legal requirements. An important problematic point is the concentration in MTEs that is found in those wastes. Consequently, recovery of nutrients and MTEsmay be a key solution for optimal valorization of wastes. Many unit operations used in the field of chemical and biochemical engineering (mechanical operations on fluids, solids, mass and heat transfers, chemical reactions, etc.) could be used in order to achieve an efficient recovery yield of nutrients and trace elements. The aim of the BioRefine Project is to make an inventory of all recovery techniques of nutrients and MTEs in five countries: Belgium, France, Germany, United Kingdom and The Netherlands. Pilot plants will also be tested to assess the efficiency of new treatment techniques after which the most efficient processes will be chosen to be applied on a larger scale. In addition, the collected data will be used to propose exploitation scenarios taking into account legal constraints and optimized logistics.This work is supported through an INTERREG IVB NWE programme(ref. 320J-BIOREFINE). [less ▲]

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See detailResearch of phosphate accumulating microorganisms from WWTPs for the recovery of phosphorus from organic wastes (3BV.3.47)
Tarayre, Cédric ULg; Michels, Evi; Buysse, Jeroen et al

Poster (2014, June)

Many wastes containing reusable components, such as nitrogen, phosphorus and potassium, are not exploited through ideal processes. As an example, in Wallonia (Belgium), the main treatment applied to ... [more ▼]

Many wastes containing reusable components, such as nitrogen, phosphorus and potassium, are not exploited through ideal processes. As an example, in Wallonia (Belgium), the main treatment applied to sewage sludge consists in incinerating the material. Such a process is chosen when the heavy metals are too concentrated in the sludge, preventing an agricultural use. However, sewage sludge, as well as manure, slurry and digestate, contain notable amounts of nutrients (nitrogen, phosphorus and potassium). Some Waste Water Treatment Plants (WWTPs) are actually designed in order to promote Phosphate Accumulating Organisms (PAOs), able to store or release phosphorus in accordance with the environmental conditions. The aim of this work is to isolate PAOs from WWTPs and evaluate their applicability to phosphorus recovery from organic wastes. Metagenomics and metabolic properties are also considered. This work is supported by the BioRefine Project, a European project in which various member states focus on recovery of inorganics from organic wastestreams. [less ▲]

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See detailStochastic exposure to sub-lethal high temperature enhances exopolysaccharides (EPS) excretion and improves Bifidobacterium bifidum cell survival to freeze-drying
Nguyen, Huu Thanh ULg; Razafindralambo, Hary; Blecker, Christophe ULg et al

in Biochemical Engineering Journal (2014), 88

Exposure of microbial cells to sub-lethal stresses is known to increase cell robustness. In this work, a two-compartment bioreactor in which microbial cells are stochastically exposed to sub-lethal ... [more ▼]

Exposure of microbial cells to sub-lethal stresses is known to increase cell robustness. In this work, a two-compartment bioreactor in which microbial cells are stochastically exposed to sub-lethal temperature stresses has been used in order to investigate the response of the stress sensitive Bifidobacterium bifidum THT 0101 to downstream processing operations. A stochastic model validated by residence time distribution experiments has shown that in the heat-shock configuration, a two-compartment bioreactor (TCB) allows the exposure of microbial cells to sub-lethal temperature of 42°C for a duration comprised between 100 and 300 seconds. This exposure resulted in a significant increase of cell resistance to freeze-drying by comparison with cells cultivated in conventional bioreactors or in the TCB in the cold shock mode (CS-TCB). The mechanism behind this robustness seems to be related with the coating of microbial cells with exopolysaccharide (EPS), as assessed by the change of the zeta potential and the presence of higher EPS concentration after heat shock. Conditioning of Bifidobacteria on the basis of the heat shock technique is interesting from the practical and economical point of view since this strategy can be directly implemented in the bioreactor during stationary phase preceding cell recovery and freeze-drying [less ▲]

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See detailAccumulation lipidique par Yarrowia lipolytica: un nouvel outil d'observation
Bouchedja, Doria Naïla; Delvigne, Frank ULg; Boudjellal, Abdelghani et al

Conference (2014, April 06)

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See detailUse of on-line flow cytometry for the characterization of microbial stress dynamics during the bioprocess
Brognaux, Alison ULg; Han, Shanshan; Sorensen, Soren et al

Conference (2014, April 03)

Microbial cell population heterogeneity is now recognized as a major source of issues for the development and optimization of bioprocesses. Flow cytometry is a very powerful tool for the follow up of ... [more ▼]

Microbial cell population heterogeneity is now recognized as a major source of issues for the development and optimization of bioprocesses. Flow cytometry is a very powerful tool for the follow up of physiological properties of microbial cells in process-related conditions at the single cell level, and can be used to study the dynamics of segregation directly in bioreactors. In this context, specific interfaces have been developed in order to connect flow cytometer (FC) directly on bioreactor for automated analyses. In this work, we propose a simplified version of such interface and demonstrated its usefulness for multiplexed experiments. This automated FC system has been tested for the follow up of the dynamics of an E. coli pfis::gfpAAV fluorescent bio-reporter and its PI uptake, correlated with membrane permeability. This bioreporter is composed of a fis promoter, a growth dependent promoter-indicator of the nutrient status of cells, fused to a gene expressing an unstable variant of GFP. The results obtained showed that the dynamics of the GFP synthesis is complex and can be attributed to a complex set of biological parameters. Segregation in the membrane permeability has been noticed. This work demonstrates that a simplified version of on-line FC can be used at the process level for the investigation of the dynamics of complex physiological mechanisms. [less ▲]

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See detailCFD-based Compartment model for description of mixing in bioreactors
Delafosse, Angélique ULg; Calvo, Sébastien ULg; Collignon, Marie-Laure ULg et al

in Chemical Engineering Science (2014), 106

In most bioprocesses, it is fundamental to accurately predict the hydrodynamics behavior of bioreactors of different size and its interaction with the biological reaction. Computational Fluid Dynamics can ... [more ▼]

In most bioprocesses, it is fundamental to accurately predict the hydrodynamics behavior of bioreactors of different size and its interaction with the biological reaction. Computational Fluid Dynamics can provide detailed modeling about hydrodynamics and mixing. However, it is computationally intensive, especially when reactions are taken into account. Another way to predict hydrodynamics is the use of “Compartment” or “Network-of-zones” model which are much less demanding in computation time than CFD. However, compartments and fluxes between them are often defined by considering global quantities not representative of the flow complexity. To overcome the limitations of these two methods, a solution is to combine compartment modeling and CFD simulations. The aim of this study is to propose a compartment model where the flow rates between two adjacent compartments are easily computed from the velocity fields obtained by CFD. The mixing evolution predicted by the CFD-based compartment model have been then compared with mixing experiment results. Unlike a CFD mixing simulation and a classical compartment model, the CFD-based compartment model proposed in this work reproduces with a good accuracy the spatial distribution of concentrations during the mixing process and this, without any adjustable parameters. [less ▲]

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See detailScale-down effect on the extracellular proteome of Escherichia coli: correlation with membrane permeability and modulation according substrate heterogeneities
Brognaux, Alison ULg; Francis, Frédéric ULg; Twizere, Jean-Claude ULg et al

in Bioprocess and Biosystems Engineering (2014)

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane ... [more ▼]

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane permeability and the synthesis of several outer-membrane components allowing to cope with substrate limitation commonly found in high-cell density culture. A comparative analysis of protein leakage has thus been performed in well-mixed bioreactors and in scale-down devices. The extracellular proteome of E.coli has been investigated by 2D-gel electrophoresis and identified by subsequent MALDI-TOF analysis. On 110 picked spots, 67 proteins have been identified and the sub-localisation and the molecular function of these proteins have been determined. A majority of the extracellular proteome was composed of outer-membrane and periplasmic proteins (64%) confirming the fact that leakage is involved in high-cell density cultures. About 50% of this extracellular proteome was composed of transport and binding proteins. Furthermore, the more abundant spots on the gel corresponded to porin proteins and periplasmic transporters. In particular, the OmpC porin was found to be very abundant. Moreover, the scale-down effect on this extracellular proteome has been investigated by 2D-DIGE analysis (2-Dimensional Differential in-Gel Electrophoresis) and significant differences have been observed by comparison with culture carried out in well-mixed systems. Indeed, since substrate limitation signal is alleviated in this kind of apparatus, cell permeability was lowered as shown by flow cytometry. In scale-down conditions, protein leakage was thus less abundant. [less ▲]

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See detailScale-down effect on the extracellular proteome of Escherichia coli: correlation with membrane permeability and modulation according substrate heterogeneities
Brognaux, Alison ULg; Francis, Frédéric ULg; Twizere, Jean-Claude ULg et al

in Bioprocess and Biosystems Engineering (2014)

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane ... [more ▼]

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane permeability and the synthesis of several outer-membrane components allowing to cope with substrate limitation commonly found in high-cell density culture. A comparative analysis of protein leakage has thus been performed in well-mixed bioreactors and in scale-down devices. The extracellular proteome of E.coli has been investigated by 2D-gel electrophoresis and identified by subsequent MALDI-TOF analysis. On 110 picked spots, 67 proteins have been identified and the sub-localisation and the molecular function of these proteins have been determined. A majority of the extracellular proteome was composed of outer-membrane and periplasmic proteins (64%) confirming the fact that leakage is involved in high-cell density cultures. About 50% of this extracellular proteome was composed of transport and binding proteins. Furthermore, the more abundant spots on the gel corresponded to porin proteins and periplasmic transporters. In particular, the OmpC porin was found to be very abundant. Moreover, the scale-down effect on this extracellular proteome has been investigated by 2D-DIGE analysis (2-Dimensional Differential in-Gel Electrophoresis) and significant differences have been observed by comparison with culture carried out in well-mixed systems. Indeed, since substrate limitation signal is alleviated in this kind of apparatus, cell permeability was lowered as shown by flow cytometry. In scale-down conditions, protein leakage was thus less abundant. [less ▲]

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