References of "Brognaux, Alison"
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See detailCharacterisation of Phosphate Accumulating Organisms and Techniques for Polyphosphate Detection: A Review
Tarayre, Cédric ULg; Nguyen, Huu-Thanh; Brognaux, Alison ULg et al

in Sensors (2016), 16(797), 1-14

Phosphate minerals have long been used for the production of phosphorus-based chemicals used in many economic sectors. However, these resources are not renewable and the natural phosphate stocks are ... [more ▼]

Phosphate minerals have long been used for the production of phosphorus-based chemicals used in many economic sectors. However, these resources are not renewable and the natural phosphate stocks are decreasing. In this context, the research of new phosphate sources has become necessary. Many types of wastes contain non-negligible phosphate concentrations, such as wastewater. In wastewater treatment plants, phosphorus is eliminated by physicochemical and/or biological techniques. In this latter case, a specific microbiota, phosphate accumulating organisms (PAOs), accumulates phosphate as polyphosphate. This molecule can be considered as an alternative phosphate source, and is directly extracted from wastewater generated by human activities. This review focuses on the techniques which can be applied to enrich and try to isolate these PAOs, and to detect the presence of polyphosphate in microbial cells. [less ▲]

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See detailSingle cell analysis of Escherichia coli outer membrane porin composition in response to nutrient depletion
Delepierre, Anissa ULg; Brognaux, Alison ULg; Bauwens, Julien ULg et al

Poster (2016, May 12)

Characterization of outer membrane integrity into isoclonal population of Escherichia coli using fluorescent probe specific to cell viability: propidium iodide (PI) staining; combined to FACS cytometry ... [more ▼]

Characterization of outer membrane integrity into isoclonal population of Escherichia coli using fluorescent probe specific to cell viability: propidium iodide (PI) staining; combined to FACS cytometry and proteomic studies of sorted subpopulations. The results tend to reveal two distincts cellular strategies: Cells positively probed by PI offer a high outer membrane protein (OMP) content, indicating nutrient competence in response to substrate limitative conditions. Unprobed cells, characterized by a low OMPs rate, could suffer from growth arrest and develop stress responses (Crp, Cra, RpoS,RpoN, RpoH-dependent). [less ▲]

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See detailImpact of phenotypic heterogeneity and metabolic specialisation on metabolic engineering strategies: case of study of E.coli as a representative microbial cell factory
Brognaux, Alison ULg; Delepierre, Anissa ULg; Pecheux, Hélène ULg et al

Conference (2015, July 21)

The goal of this research is to directly highlight the simultaneous occurrence of several phenotypes with distinct metabolic function among clonal population of E. coli., often used for recombinant ... [more ▼]

The goal of this research is to directly highlight the simultaneous occurrence of several phenotypes with distinct metabolic function among clonal population of E. coli., often used for recombinant protein and pDNA production in bioprocesses. This phenotypic heterogeneity, first due to the noise, is reinforced by environmental heterogeneities occurring at large scale during fed-batch processes. This phenotypic heterogeneity has been tracked according to GFP reporter strains (biosensors) that circulate in the bioreactor and encounter environmental heterogeneities. First, we have highlighted the simultaneous occurrence of several phenotypes with distinct metabolic functions. Indeed, a diversity of glucose uptake strategies has recently been noticed with the PtsG and MglABC transporters. Moreover, when E.coli encounters zones of glucose excess, acetate is produced through the overflow metabolism. Only the sub-population with high acs expression (acetate transporter) could consume this acetate. GFP reporter strains have thus been constructed for PtsG, MgIABC and acs genes. In addition of the stable GFP, two destabilized GFP variants for each gene have been used to obtain more instantaneous responses. The response of these biosensors have been followed by on-line flow cytometry. In the end, this experimental strategy for direct phenotyping at the single cell level will also be used to investigate the impact of metabolic engineering strategies on phenotypic heterogeneity, robustness and fitness of microbial population in industrial conditions. [less ▲]

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See detailPhenotypic variability in bioprocessing conditions can be tracked on the basis of on-line flow cytometry and fits to a scaling law
Baert, Jonathan ULg; Kinet, Romain ULg; Brognaux, Alison ULg et al

in Biotechnology Journal (2015)

Noise in gene and protein expression is a major cause for bioprocess deviation. However, this phenomenon has been only scarcely considered in real bioprocessing conditions. In this work, a scaling-law ... [more ▼]

Noise in gene and protein expression is a major cause for bioprocess deviation. However, this phenomenon has been only scarcely considered in real bioprocessing conditions. In this work, a scaling-law derived from a genome-scale studies based on GFP reporter systems has been calibrated to an on-line flow cytometry device, allowing thus to get an insight at the level of promoter activity and associated noise during a whole microbial culture carried out in bioreactor. We show that most of the GFP reporter systems investigated and thus corresponding genes could be included inside the area covered by the scaling-law. The experimental results suggest that this scaling-law could be used to predict the dynamics of promoter activity, as well as the associated noise, in bioprocessing conditions. The knowledge acquired throughout this work could be used for the design of more robust expression systems. [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 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 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 ▲]

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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 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)

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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 ▲]

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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)

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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 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 ▲]

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