References of "Brognaux, Alison"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailImplications of microbial phenotypic heterogeneity in large-scale bioprocessing conditions
Delvigne, Frank ULg; Gorret, Nathalie; Molina-Jouve, Carole et al

Conference (2014, September)

Detailed reference viewed: 8 (1 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 27 (9 ULg)
Full Text
See detailUtilisation de biocapteurs microbiens GFP pour la caractérisation des performances des bioréacteurs
Brognaux, Alison ULg

Doctoral thesis (2014)

The scale-up of bioprocesses is a critical step during bioproducts development. Actually, the mixing operation’s efficiency drops when the reactor volume increases: gradients of glucose and oxygen appear ... [more ▼]

The scale-up of bioprocesses is a critical step during bioproducts development. Actually, the mixing operation’s efficiency drops when the reactor volume increases: gradients of glucose and oxygen appear when operating in fed-batch mode, causing losses of production. The aim of this project is the scale-up and sizing of bioreactors based on the direct physiological parameters to consider this heterogeneity. Concretely, it consists in obtaining an on-line signal of the physiological status of micro-organisms. The coding sequence of the Green Fluorescent Protein (GFP) has been inserted after gene promoters of interest in Escherichia coli to built biosensors. A particular focus has been paid on promoters responding to general stress or specifically to lack of glucose and on those responding to cell growth rate. The GFP biosensors of interest have been tested in scale-down bioreactors, allowing to reproduce industrial hydrodynamic conditions at a laboratory scale. A mini-bioreactor platform has also been developed to enable a high throughput screening of biosensors. The intracellular accumulation of GFP has been measured by flow cytometry and GFP release has been monitored by western blot analyses. For biosensors sensitive to stress general response or glucose limitation, GFP has been induced during a glucose limitation and repressed by comparison when glucose heterogeneities appear. The use of a destabilized GFP has been considered in this project for ribosomal biosensors to approach more instantaneous physiological responses of microorganisms. For these ones, the response is proportional to growth rate during the batch phase, but more complex mechanisms take part during a prolonged glucose limitation. Membrane permeability has also been studied and has been noticed more important in homogeneous fed-batch bioreactors than in scale-down reactors. As GFP leakage has been noted in the extracellular medium, a study has also been carry out about proteins released in the extracellular medium (leakage), and correlated with the cell permeability. Finally, an on-line flow cytometer has been developed for the characterization of physiological status of micro-organisms during the bioprocess, and a 3D-ORM probe allowed to measure their viability on-line [less ▲]

Detailed reference viewed: 44 (22 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 31 (10 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 31 (10 ULg)
Full Text
Peer Reviewed
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

Poster (2014, February 07)

Detailed reference viewed: 13 (1 ULg)
Full Text
Peer Reviewed
See detailShort communication - Isolation of amylolytic, xylanolytic, and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere
Tarayre, Cédric ULg; Brognaux, Alison ULg; Bauwens, Julien ULg et al

in World Journal of Microbiology & Biotechnology (2013)

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO2 or CO2/H2 ... [more ▼]

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO2 or CO2/H2) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacills subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce β-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed β-glucosidase, amylase, cellulase, and xylanase activities. Finally, Bacillus subtilis strain ABGx produced xylanase and amylase. [less ▲]

Detailed reference viewed: 33 (11 ULg)
Full Text
Peer Reviewed
See detailUse of on-line flow cytometry for the characterization of physiological behavior in stress conditions during the bioprocess
Brognaux, Alison ULg; Han, Shanshan; Soren, Sorensen et al

Poster (2013, November 15)

Detailed reference viewed: 26 (8 ULg)
Peer Reviewed
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)

Detailed reference viewed: 28 (8 ULg)
Full Text
Peer Reviewed
See detailIsolation and Cultivation of a Xylanolytic Bacillus subtilis Extracted from the Gut of the Termite Reticulitermes santonensis
Tarayre, Cédric ULg; Brognaux, Alison ULg; Brasseur, Catherine ULg et al

in Applied Biochemistry and Biotechnology (2013)

The aim of this work was the isolation of xylanolytic microorganisms from the digestive tract of the termite Reticulitermes santonensis. The reducing sugars released after the hydrolysis of xylans can be ... [more ▼]

The aim of this work was the isolation of xylanolytic microorganisms from the digestive tract of the termite Reticulitermes santonensis. The reducing sugars released after the hydrolysis of xylans can be further fermented to provide bioethanol. A xylanolytic strain of Bacillus subtilis was isolated from the hindgut of the termite and displayed amylase and xylanase activities. The bacterium was grown on media containing agricultural residues: wheat bran, wheat distiller’s grains, and rapeseed oil cake. Wheat bran led to the highest induction of xylanase activity, although the development of the strain was less fast than in the other media. It was possible to reach maximal xylanase activities of 44.3, 33.5, and 29.1 I.U./ml in the media containing wheat bran, wheat distiller’s grains, and rapeseed oil cake, respectively. Mass spectrometry identified a wide range of xylose oligomers, highlighting an endoxylanase activity. The enzyme was stable up to 45 °C and displayed an optimal pH close to 8. [less ▲]

Detailed reference viewed: 48 (30 ULg)
Peer Reviewed
See detailOn-line flow cytometry profiling of Escherichia coli stress response
Brognaux, Alison ULg; Han, Shanshan; Sorensen, Soren et al

Conference (2013, February 08)

Detailed reference viewed: 37 (10 ULg)
Full Text
Peer Reviewed
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)

Detailed reference viewed: 57 (29 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 79 (28 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 28 (14 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 32 (8 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 51 (42 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 51 (26 ULg)