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See detailSpatiotemporal monitoring of the antibiome secreted by Bacillus biofilms on plant roots using MALDI mass spectrometry imaging
Debois, Delphine ULg; Jourdan, Emmanuel; Smargiasso, Nicolas ULg et al

in Analytical Chemistry (2014), 86(9), 4431-4438

Some soil Bacilli living in association with plant roots can protect their host from infection by pathogenic microbes and are therefore being developed as biological agents to control plant diseases. The ... [more ▼]

Some soil Bacilli living in association with plant roots can protect their host from infection by pathogenic microbes and are therefore being developed as biological agents to control plant diseases. The plant protective activity of these bacteria has been correlated with the potential to secrete a wide array of antibiotic compounds upon growth as planktonic cells in isolated cultures under laboratory conditions. However, in situ expression of these antibiotics in the rhizosphere where bacterial cells naturally colonize root tissues is still poorly understood. In this work, we used Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) to examine spatio-temporal changes in the secreted antibiome of B. amyloliquefaciens developing as biofilms on roots. Non-ribosomal lipopeptides such as the plant immunity elicitor surfactin or the highly fungitoxic iturins and fengycins were readily produced albeit in different time-frames and quantities in the surrounding medium. Interestingly, MS/MS experiments performed directly from the gelified culture medium, also allowed to identify a new variant of surfactins released at later time points. However, no other bioactive compounds such as polyketides were detected at any time, strongly suggesting that the antibiome expressed in planta by B. amyloliquefaciens does not reflect the vast genetic arsenal devoted to the formation of such compounds. This first dynamic study reveals the power of MALDI MSI as tool to identify and map antibiotics synthesized by root-associated bacteria and more generally, to investigate plant-microbe interactions at the molecular level. [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 detailEnzymatic process for the fractionation of baker's yeast cell wall (saccharomyces cerevisiae)
Borchani, Chema; Fonteyn, Fabienne; Jamin, Guilhem et al

Conference (2014, April 07)

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See detailImaging MS: strategies for the identification of analytes
Debois, Delphine ULg; Smargiasso, Nicolas ULg; Jourdan, Emmanuel et al

Scientific conference (2014, April 04)

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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 detailRhodococcus erythropolis, a good candidate for an in-situ bioaugmentation starter
Masy, Thibaut ULg; Caterina, David; Tromme, Olivier et al

Conference (2014, March 05)

In-situ bioremediation is as a green and cheap process to clean soils from pollution compared to other techniques which often imply the excavation of soils. Amongst the bacteria used, Rhodococcus ... [more ▼]

In-situ bioremediation is as a green and cheap process to clean soils from pollution compared to other techniques which often imply the excavation of soils. Amongst the bacteria used, Rhodococcus erythropolis appears as one of the best candidates for bioaugmentation. In fact, this species forms biofilms and produces biosurfactants to solubilize hydrocarbons, which are consequently more available for this bacterium and the endogenous oil-degrading flora. Moreover, its large genome allows the degradation of various persistent pollutants, such as polyaromatic hydrocarbons or sulfur-containing hydrocarbons. In addition to these benefits, our strain Rhodococcus erythropolis T902.1, isolated from a dried polluted soil, resists to desiccation during industrial process or drought, and maintains its biodegradation capabilities. To test this strain in field conditions, a bioaugmentation experiment at a pilot scale was initiated in partnership with the Department ArGEnCo, Applied Geophysics of the University of Liège. The pilot contains 2 m3 of sand, in which a vertical lens of highly polluted clayey soil (7200 mg of hydrocarbons/g of dry weight) was inserted. During the first three months, 75% of the hydrocarbons content was degraded, whereas a previous biostimulation experiment with KNO3 and H2O2 did not lead to any depletion of the pollutant. This degradation was correlated with the increase of total and specific microorganisms (by a factor 13 and 10 respectively) and the almost complete NO3- consumption (from 50 to nearly 0 mg/L). Furthermore, electrical resistivity tomography images of the contaminated lens also depicted a switch in the bulk conductivity values that does not correspond to the trend followed by the aqueous conductivity. It could be explained by the implementation of the injected bacteria and their production of hydrophobic biosurfactants desorbing hydrocarbons from soil particles. This assumption is strengthened by the fact that low concentrations of hydrocarbons were detected in piezometers downstream of the contaminated area. Further experiments will be carried out at a smaller scale to validate this hypothesis. On the one hand, we are currently designing a protocol to follow the biofilm formation by Rhodococcus erythropolis T902.1 with spectral induced polarization (SIP) signature in sand columns of 1.5 L. On the other hand, the analysis of biosurfactants will be performed in liquid cultures containing diesel oil, to characterize the hydrophobicity developed by the strain in presence of a common but complex pollutant. To conclude, all these characteristics showed by Rhodococcus erythropolis T902.1 make it an ideal candidate for the production of a bioremediation starter to quickly treat hydrocarbons-polluted soils. . Furthermore, the better comprehension of geophysical signatures associated with such a process may lead in the future to use them as a low-cost monitoring tool for a better visualization of active remediation zones. [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 detailEffect of encapsulated nanoparticles on thermophillic anaerobic digestion
Al-Ahmad, Alaa Eddin ULg; Hiligsmann, Serge ULg; Lambert, Stéphanie ULg et al

Poster (2014, February 07)

Recently, enormous interest has been focused on biological applications of metal nanoparticles NPs due to their small size, high specified surface and their great potential in application to many science ... [more ▼]

Recently, enormous interest has been focused on biological applications of metal nanoparticles NPs due to their small size, high specified surface and their great potential in application to many science fields. The most studied process concerns zero valent palladium and iron NPs improving anaerobic biodegradation of chlorinated hydrocarbons (Windt et al., 2005). Moreover, investigation carried out in our lab showed that iron NPs encapsulated in silicate matrix may enhance hydrogen production by Clostridium butyricum (Beckers et al., 2013). Nevertheless the influences of metal NPs on methane producing anaerobic digestion have seldom been investigated. The present work investigates the enhancement effect of seven different metal NPs on methane production during the thermophilic anaerobic digestion. NPs of Cu, Pd, Pt, Ni, Co, Ag and Fe encapsulated in porous silica (SiO2) to prevent their coagulation and agglomeration, were added at concentration of 10-5mol/L in batch test (125ml serum bottles containing 70mL culture medium with 5g/L acetate monohydrate as the sole carbon substrate). Nickel, cobalt and iron NPs improved methane production from acetate. To confirm the previous results, the NPs were tested at different concentrations (10-4, 10-5, and 10-6 mol/L) with starch and glucose substrates. The results show that the impact increases with the increase of NPs concentrations up to 10-4 mol/L. The modified Gompertz equation was applied to describe the effect of NPs on anaerobic digestion. According to this model, the kinetic of methane production was particularly affected by nanoparticles addition. The values of the maximum methane production rate MPR (ml/day) was significantly higher 72.5% with nickel NPs at a concentration of 10-4 mol/L than the control without NPs. [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 detailNew alternatives to chemical pesticides: deciphering the action mechanisms of lipid based plant elicitors via complementary biophysical and biological approaches.
Nasir, Mehmet Nail ULg; Polo Lozano, Damien ULg; Luzuriaga Loaiza, Walter ULg et al

Poster (2014, February)

Nowadays, many health and environmental problems are caused by the use of chemical pesticides. In this context, an increasing demand for alternative products such as biopesticides has been observed. Among ... [more ▼]

Nowadays, many health and environmental problems are caused by the use of chemical pesticides. In this context, an increasing demand for alternative products such as biopesticides has been observed. Among biopesticides, elicitor molecules which are able to trigger immune defense responses in plants are one of the most promising options. Although numerous elicitors have been discovered, the mechanisms involved in the perception, by plants, of only a few molecules have been identified. These elicitors usually interact with proteic receptors but we have recently shown that they may also act on the lipid phase of the plasma membrane. This project first aims to improve our understanding of the molecular mechanisms involved in the recognition of specific lipid based elicitors (LBE). On that basis, the FIELD project will contribute to the design and the development of innovative compounds derived natural LBE. A multi-disciplinary approach, based on chemistry, bio-physics, bio-chemistry, and phytopathology will be followed by a consortium of different research groups from Gembloux Agro-Bio Tech in close collaboration with teams from foreign institutions. [less ▲]

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See detailThe role of protein modifications in senescence of freeze-dried Acetobacter senegalensis during storage
Shafiei, Rasoul ULg; Zarmehrkhorshid, Raziyeh ULg; Bentaib, Azeddine ULg et al

in Microbial Cell Factories (2014)

Background Loss of viability is one of the most important problems during starter culture production. Previous research has mostly focused on the production process of bacterial starters, but there are ... [more ▼]

Background Loss of viability is one of the most important problems during starter culture production. Previous research has mostly focused on the production process of bacterial starters, but there are few studies about cellular protein deterioration causing cell defectiveness during storage. In the present study, we investigated the influence of storage temperature (−21, 4, 35°C) on the cellular protein modifications which may contribute to the senescence of freeze-dried Acetobacter senegalensis. Results Heterogeneous populations composed of culturable cells, viable but non-culturable cells (VBNC) and dead cells were generated when freeze-dried cells were kept at −21 and 4°C for 12 months whereas higher storage temperature (35°C) mainly caused death of the cells. The analysis of stored cell proteome by 2D-DiGE demonstrated a modified pattern of protein profile for cell kept at 4 and 35°C due to the formation of protein spot trains and shift of Isoelectric point (pI). Quantification of carbonylated protein by ELISA showed that the cells stored at 4 and 35°C had higher carbonylated protein contents than fresh cells. 2D-DiGE followed by Western blotting also confirmed the carbonylation of cellular proteins involved in translation process and energy generation. The auto-fluorescent feature of cells kept at 35°C increased significantly which may be an indication of protein glycation during storage. In addition, the percentage of cellular unsaturated fatty acid and the solubility of cellular proteins decreased upon storage of cells at higher temperature suggesting that peroxidation of fatty acids and possibly protein lipidation and oxidation occurred. Conclusions High storage temperature induces some deteriorative reactions such as protein oxidation, lipidation and glycation which may cause further protein modifications like pI-shift, and protein insolubility. These modifications can partly account for the changes in cell viability. It can also be deduced that even moderate carbonylation of some critical cellular proteins (like ribosomal proteins) may lead to VBNC formation or death of freeze-dried bacteria. Moreover, it seems that other mechanisms of biomolecule deterioration preceding protein carbonylation lead to VBNC formation under very low storage temperature. [less ▲]

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See detailTechnologie de production de masse d’insectes - INSECTECH
Richard, Gaetan ULg; Hance, Thierry; Fauconnier, Marie-Laure ULg et al

Report (2014)

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See detailCharacterization of Lactobacillus strains isolated from Algerian children faeces for their probiotic properties
Bahri, F.; Lejeune, Annick ULg; Dubois-Dauphin, R. et al

in African Journal of Microbiology Research [=AJMR] (2014), 8(3), 297-303

Lactic acid bacteria termed probiotics have preventive as well as curative effects on several types of diarrhoea of different aetiologies. The main objective of this study was to screen lactobacilli ... [more ▼]

Lactic acid bacteria termed probiotics have preventive as well as curative effects on several types of diarrhoea of different aetiologies. The main objective of this study was to screen lactobacilli strains having probiotic traits, isolated from Algerian healthy children faeces on the purpose of using them further in children diarrheal illnesses. One hundred and twenty (120) lactic acid bacteria isolates were selected from faecal samples of healthy Algerian children aged between five and ten years. Gram positive rods and catalase negative bacteria (52 isolates) were screened, in vitro, for their probiotic potential properties including ability to survive in simulated gastro-intestinal conditions, adherence to Caco-2 cells and their antimicrobial activity. The results show that only five strains resisted in simulated gastric juice at pH 1.5 and pepsin. Four of them were resistant to simulated intestinal conditions at pH 8 and pancreatin and have a good adherence. In the end, three of them were retained as they display interesting probiotic profiles characterized by a strong antimicrobial effect against some intestinal pathogenic bacteria. They were identified by 16S rDNA sequencing as Lactobacillus plantarum F12, Lactobacillus brevis G6 and Lactobacillus paracasei B13. [less ▲]

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See detailIsolation of an amylolytic chrysophyte, Poterioochromonas sp. from the digestive tract of the termite R. santonensis
Tarayre, Cédric ULg; Bauwens, Julien ULg; Brasseur, Catherine ULg et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2014), 18(1),

The aim of this work was the isolation and cultivation of amylolytic protists living in the digestive tract of the termite Reticulitermes santonensis (Feytaud). A chrysophyte identified as ... [more ▼]

The aim of this work was the isolation and cultivation of amylolytic protists living in the digestive tract of the termite Reticulitermes santonensis (Feytaud). A chrysophyte identified as Poterioochromonas sp. was isolated in a special medium containing rice grains as a source of carbon and nitrogen. Then, the protist was grown in a medium containing starch as a carbon source, tryptone, and a phosphate buffer at different pH values (5, 6 and 7). Yeast extract was added or not. Ciprofloxacin was used to avoid the bacterial development. Other antibiotics were also tested but showed an inhibitive effect on the growth of Poterioochromonas sp. Yeast extract allowed reaching 1.9 (pH 5), 2.3 (pH 6) and 2.2 (pH 7) times higher final cell concentrations, and 2.8 (pH 5), 2.8 (pH 6) and 2.2 (pH 7) times higher biomass yields. The starch concentration did not decrease in the medium until 3 and 4 days of culture, with and without yeast extract, respectively. Eight days of culture were necessary for hydrolyzing the starch completely, with and without yeast extract. Maltose and maltotriose were detected in the culture media and were hydrolyzed progressively. Maximal maltose concentrations were 0.68, 0.66 and 0.51 g.l-1 in the medium containing yeast extract. Maltotriose concentrations were only 0.17, 0.14 and 0.12 g.l-1. Other glucose oligomers were also detected but in lower quantities. It was determined that the protist developed a weak amylase activity, particularly at a weakly acidic pH (5-6). Such a pH also allowed a better growth of the protist. A maximal amylase activity of 112 nkat.l-1 was measured with yeast extract at pH 5. No other enzymatic activity (protease, cellulase or xylanase) was detected except amylase. The degradation products of starch which were obtained by enzymatic hydrolysis allow the identification of α-amylase, amyloglucosidase and possibly β-amylase activities. [less ▲]

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See detailReprogramming of fatty acid and oxylipin synthesis in rhizobacteria-induced systemic resistance in tomato
Mariutto, Martin; Fauconnier, Marie-Laure ULg; Ongena, Marc ULg et al

in Plant Molecular Biology (2014), 84(4-5), 455-476

The rhizobacterium Pseudomonas putida BTP1 stimulates induced systemic resistance (ISR) in tomato. A previous work showed that the resistance is associated in leaves with the induction of the first enzyme ... [more ▼]

The rhizobacterium Pseudomonas putida BTP1 stimulates induced systemic resistance (ISR) in tomato. A previous work showed that the resistance is associated in leaves with the induction of the first enzyme of the oxylipin pathway, the lipoxygenase (LOX), leading to a faster accumulation of its product, the free 13-hydroperoxy octadecatrienoic acid (13-HPOT), 2 days after Botrytis cinerea inoculation. In the present study, we further investigated the stimulation of the oxylipin pathway: metabolites and enzymes of the pathway were analyzed to understand the fate of the 13-HPOT in ISR. Actually the stimulation began upstream the LOX: free linolenic acid accumulated faster in P. putida BTP1-treated plants than in control. Downstream, the LOX products 13-fatty acid hydroperoxides esterified to galactolipids and phospholipids were more abundant in bacterized plants than in control before infection. These metabolites could constitute a pool that will be used after pathogen attack to produce free fungitoxic metabolites through the action of phospholipase A2, which is enhanced in bacterized plants upon infection. Enzymatic branches which can use as substrate the fatty acid hydroperoxides were differentially regulated in bacterized plants in comparison to control plants, so as to lead to the accumulation of the most fungitoxic compounds against B. cinerea. Our study, which is the first to demonstrate the accumulation of an esterified defense metabolite during rhizobacteria-mediated induced systemic resistance, showed that the oxylipin pathway is differentially regulated. It suggests that this allows the plant to prepare to a future infection, and to respond faster and in a more effective way to B. cinerea invasion. [less ▲]

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See detailPlant defense stimulation by natural isolates of Bacillus depends on efficient surfactin production
Cawoy, Hélène ULg; Mariutto, Martin; Henry, Guillaume et al

in Molecular Plant-Microbe Interactions (2014), 27

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