References of "Portetelle, Daniel"
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See detailWood digestion in lower termites: multidisciplinary approaches based on differential feeding
Bauwens, Julien ULg; Brasseur, Catherine ULg; Tarayre, Cédric ULg et al

Poster (2014, December)

Termites digestive tract and hindgut especially still holds many secrets despites hundreds of years of research. The complexity of the symbiotic microbial community and the contrast of physio-chemical ... [more ▼]

Termites digestive tract and hindgut especially still holds many secrets despites hundreds of years of research. The complexity of the symbiotic microbial community and the contrast of physio-chemical environments found in lower termites paunch are potentially the key point to explain the efficiency of ligno-cellulose digestion. Contribution of advancing technologies accelerates the progress of our knowledge in this field. Here, we present multiple approaches combining old and recent techniques used to highlight the effect of ligno-cellulosic compounds on termite gut and the role of populations from the symbiotic microbial community. Termites Reticulitermes flavipes (Kollar) submitted to various artificial diets showed variations in flagellates populations profile and enzymatic activities. Differential protein expression was investigated using 2D-DIGE MALDI-TOF-TOF and 2D-LC-MS/MS using high resolution orbitrap analyzer. Results from both proteomic experiments tend to support each-other and bring complementary points of view. The gel-free analysis resulted in highly contrasted identification of enzymes involved in ligno-cellulose digestion and metabolism. Finally, differential feeding experiments leaded to in vivo selection of different symbiotic communities. These communities were characterized following some metabolism assays and allowed the cultivation of diverse microbial consortia using media closely related to the respective artificial diets. This work provides relevant data on termite and associated microbial community response to alimentary diets. [less ▲]

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See detailIsolation and cultivation of xylanolytic and cellulolytic Sarocladium kiliense and Trichoderma virens from the gut of the termite Reticulitermes santonensis
Tarayre, Cédric ULg; Bauwens, Julien ULg; Brasseur, Catherine ULg et al

in Environmental Science & Pollution Research (2014)

The purpose of this work was the isolation and cultivation of cellulolytic and xylanolytic microorganisms extracted from the gut of the lower termite Reticulitermes santonensis. Microcrystalline cellulose ... [more ▼]

The purpose of this work was the isolation and cultivation of cellulolytic and xylanolytic microorganisms extracted from the gut of the lower termite Reticulitermes santonensis. Microcrystalline cellulose (with and without lignin) and beech wood xylan were used as diets instead of poplar wood in order to select cellulose and hemicellulose-degrading fungi. The strain Sarocladium kiliense (Acremonium kiliense) CTGxxyl was isolated from the termites fed on xylan, while the strain Trichoderma virens CTGxAviL was isolated from the termites fed on cellulose (with and without lignin). Both molds were cultivated in liquid media containing different substrates: agro-residues or purified polymers. S. kiliense produced maximal β-glucosidase, endo-1,4-β-D-glucanase, exo-1,4-β-D-glucanase and endo-1,4-β-D-xylanase activities of 0.103, 3.99, 0.53, and 40.8 IU/ml, respectively. T. virens produced maximal β-xylosidase, endo-1,4-β-D-glucanase, exo-1,4-β-D-glucanase, and endo-1,4-β-D-xylanase activities of 0.38, 1.48, 0.69, and 426 IU/ml. The cellulase and the xylanase of S. kiliense, less common than T. virens, were further investigated. The optimal activity of the xylanase was observed at pH 9–10 at 60 °C. The cellulase showed its maximal activity at pH 10, 70 °C. Zymography identified different xylanases produced by both molds, and some fragment sizes were highlighted: 35, 100, and 170 kDa for S. kiliense and 20, 40, 80, and 170 kDa for T. virens. In both cases, endo-1,4-β-D-xylanase activitieswere confirmed through mass spectrometry. [less ▲]

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See detailMultidisciplinary approaches and fractionations to study lower termite symbiotic system and ligno-cellulose digestion
Bauwens, Julien ULg; Brasseur, Catherine ULg; Tarayre, Cédric ULg et al

Poster (2014, August)

Wood-feeding termites are a considerable source of enzymes active on ligno-cellulosic compounds. These enzymes are produced by the termite host and some representatives of its symbiotic microbial ... [more ▼]

Wood-feeding termites are a considerable source of enzymes active on ligno-cellulosic compounds. These enzymes are produced by the termite host and some representatives of its symbiotic microbial community, and are of particular interest in regard second generation biofuel. However, the complexity of microbial interactions renders micro-organisms isolation very difficult. Culture-independent methods permitted to gather a large amount of data and to understand a little more the role of each microbial population, particularly the prokaryotes. Proteomics allows working on the final product of gene expression, and corresponds more to the real operation of the digestive system. In order to investigate such a complex system, it is necessary to use multidisciplinary approaches and to fractionate this system. Zymography or affinity chromatography are used in parallel of routine proteomics techniques such as two-dimensional gel electrophoresis associated to MALDI-TOF mass spectrometry and nano-LC ESI-MS/MS. We used an artificial-diet based rearing to induce changes in microbial population balance. We performed preliminary assay to investigate the glycosylated proteome in the hindgut of a lower termite, using Multi-Lectin Affinity Chromatography (M-LAC) and enzymatic activity of harvested fractions was assessed on cellulosic substrates. [less ▲]

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See detailIdentification and Characterization of a Halotolerant, Cold-Active Marine Endo-β-1,4-Glucanase by Using Functional Metagenomics of Seaweed-Associated Microbiota
Martin, Marjolaine ULg; Biver, Sophie ULg; Steels, Sébastien ULg et al

in Applied and Environmental Microbiology (2014), 80(16), 4958-4967

A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two ... [more ▼]

A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-β-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions. [less ▲]

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See detailNew carbohydrate-active enzymes identified by screening two metagenomic libraries derived from the soil of a winter wheat field
Stroobants, Aurore ULg; Portetelle, Daniel ULg; Vandenbol, Micheline ULg

in Journal of Applied Microbiology (2014)

Aims Soils are rich, diversified environments where β-glucosidases abound because of their importance in organic matter degradation. The aim of this work was to discover new β-glucosidases by constructing ... [more ▼]

Aims Soils are rich, diversified environments where β-glucosidases abound because of their importance in organic matter degradation. The aim of this work was to discover new β-glucosidases by constructing two metagenomic DNA libraries from soil samples collected in winter and spring from a field of winter wheat. Methods and Results Both libraries were screened on esculin-supplemented medium so as to isolate candidates showing β-glucosidase activity. Candidate analysis revealed seven putative β-glycosidases and two putative glycosyltransferases, displaying 25 to 82% identity to known enzymes. The putative β-glycosidases belong to families GH1, GH3 and GH20 and the two putative glycosyltransferases, probably, to new families. In characterization tests performed on bacteria in suspension or spread on agar plates, some candidates appeared to hydrolyse several natural and synthetic substrates. These tests also highlighted interesting industrial characteristics, such as the activity of four β-glycosidases under alkaline conditions and the esculin-hydrolysing activity of a β-glucosidase candidate in the presence of glucose. Conclusions Seven putative β-glycosidases and two putative glycosyltransferases were found by functional screening of two metagenomic DNA libraries derived from agricultural soil. Significance and Impact of the Study This study has identified β-glycosidases and putative glycosyltransferases that have or may have interesting industrial characteristics. [less ▲]

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See detailDiversity of Bacterial Communities in a Profile of a Winter Wheat Field: Known and Unknown Members
Stroobants, Aurore ULg; Degrune, Florine ULg; Olivier, Claire et al

in Microbial Ecology (2014)

In soils, bacteria are very abundant and diverse. They are involved in various agro-ecosystem processes such as the nitrogen cycle, organic matter degradation, and soil formation. Yet, little is known ... [more ▼]

In soils, bacteria are very abundant and diverse. They are involved in various agro-ecosystem processes such as the nitrogen cycle, organic matter degradation, and soil formation. Yet, little is known about the distribution and composition of bacterial communities through the soil profile, particularly in agricultural soils, as most studies have focused only on topsoils or forest and grassland soils. In the present work, we have used bar-coded pyrosequencing analysis of the V3 region of the 16S rRNA gene to analyze bacterial diversity in a profile (depths 10, 25, and 45 cm) of a well-characterized field of winter wheat. Taxonomic assignment was carried out with the Ribosomal Database Project (RDP) Classifier program with three bootstrap scores: a main run at 0.80, a confirmation run at 0.99, and a run at 0 to gain information on the unknown bacteria. Our results show that biomass and bacterial quantity and diversity decreased greatly with depth. Depth also had an impact, in terms of relative sequence abundance, on 81 % of the most represented taxonomic ranks, notably the ranks Proteobacteria, Bacteroidetes, Actinobacteridae, and Acidobacteria. Bacterial community composition differed more strongly between the topsoil (10 and 25 cm) and subsoil (45 cm) than between levels in the topsoil, mainly because of shifts in the carbon, nitrogen, and potassium contents. The subsoil also contained more unknown bacteria, 53.96 % on the average, than did the topsoil, with 42.06 % at 10 cm and 45.59 % at 25 cm. Most of these unknown bacteria seem to belong to Deltaproteobacteria, Actinobacteria, Rhizobiales, and Acidobacteria. [less ▲]

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See detailScreening of two agricultural genomic DNA libraries to seek new glycoside hydrolases
Stroobants, Aurore ULg; Portetelle, Daniel ULg; Vandenbol, Micheline ULg

Poster (2014, February 07)

Soils are very rich environments where the diversity of microorganisms is very high. These microorganisms play an important role in the degradation of organic matter with enzymes able to degrade it. This ... [more ▼]

Soils are very rich environments where the diversity of microorganisms is very high. These microorganisms play an important role in the degradation of organic matter with enzymes able to degrade it. This work aims to discover, by functional screening, new microbial glycoside hydrolases from soils collected in winter and spring in a winter wheat crop. The genomic DNA was extracted from both soils to construct two libraries in Escherichia coli. These libraries were then screened for beta-glucosidase activities on 2YT agar media containing 0.5% esculin and 0.1% ammonium iron (III) citrate. At this time, about 250.000 clones from each library have been screened. Two beta-glucosidases have already been found in the winter library while five beta-glucosidases and two glycosyltransferases were identified in the spring library. Sequence analyses with the BLASTX program revealed putative enzymes showing between 25% and 72% sequence identity with known enzymes and belonging to three glycoside hydrolase families (GH1, GH3 and GH20) and to two probably new glycosyltransferase families. Biochemical characterisation of the candidates at several pH values and temperatures, and with four substrates, is in progress. [less ▲]

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See detailMicroorganisms living on macroalgae: diversity, interactions, and biotechnological applications
Martin, Marjolaine ULg; Portetelle, Daniel ULg; Michel, Gurvan et al

in Applied Microbiology & Biotechnology (2014)

Marine microorganisms play key roles in every marine ecological process, hence the growing interest in studying their populations and functions. Microbial communities on algae remain underexplored ... [more ▼]

Marine microorganisms play key roles in every marine ecological process, hence the growing interest in studying their populations and functions. Microbial communities on algae remain underexplored, however, despite their huge biodiversity and the fact that they differ markedly from those living freely in seawater. The study of this microbiota and of its relationships with algal hosts should provide crucial information for ecological investigations on algae and aquatic ecosystems. Furthermore, because these microorganisms interact with algae in multiple, complex ways, they constitute an interesting source of novel bioactive compounds with biotechnological potential, such as dehalogenases, antimicrobials, and alga-specific polysaccharidases (e.g., agarases, carrageenases, and alginate lyases). Here, to demonstrate the huge potential of alga-associated organisms and their metabolites in developing future biotechnological applications, we first describe the immense diversity and density of these microbial biofilms. We further describe their complex interactions with algae, leading to the production of specific bioactive compounds and hydrolytic enzymes of biotechnological interest. We end with a glance at their potential use in medical and industrial applications. [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 detailRapport scientifique et technique final du projet HYDRASANTE
Boudry, Christelle ULg; François, Emmanuelle ULg; Nollevaux, Géraldine et al

Report (2013)

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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 detailBacillus subtilis as a tool for screening soil metagenomic libraries for antimicrobial activities
Biver, Sophie ULg; Steels, Sébastien ULg; Portetelle, Daniel ULg et al

in Journal of Microbiology and Biotechnology (2013), 23(6), 850-855

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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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See detailFunctional screening of a winter and a spring genomic DNA libraries obtained from soils in a winter wheat crop
Stroobants, Aurore ULg; Portetelle, Daniel ULg; Vandenbol, Micheline ULg

Poster (2013, June 10)

Soils are very rich environments where the diversity of microorganisms is very high. These microorganisms play important role in the degradation of organic matter with enzymes able to degrade it. The aim ... [more ▼]

Soils are very rich environments where the diversity of microorganisms is very high. These microorganisms play important role in the degradation of organic matter with enzymes able to degrade it. The aim of this work is to discover by functional screening new enzymatic activities of microorganisms from soils collected in winter and spring in a winter wheat crop. The genomic DNA was extracted from both soils to construct two libraries in Escherichia coli. These libraries were then screened for several enzymes such as lipase, beta-glucosidase, cellulase, α-amylase,… At this time, 2 beta-glucosidases and 3 lipases have already been found in the winter library and 3 beta-glucosidases and 1 lipase in the spring library. Sequence analyses with the BLASTX program revealed that two beta-glucosidases have less than 65% of sequence identity with known beta-glucosidases, one have 64% of identity with a known beta-galactosidase and one have 59% of identity with a glycoside hydrolase. The fifth seems to be a phosphorylase kinase (54% identity) which have a glucoamylase domain responsible for the activity. This ORF is interrupted by a transposase. Three of the four lipases have less than 60% of sequence identity with known lipases/esterases. The fourth show 55% of identity with a known beta-lactamase. [less ▲]

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See detailCharacterization of new bacterial glycoside hydrolases isolated from agricultural soils using a functional metagenomic approach
Biver, Sophie ULg; Dubois, Benjamin; Stroobants, Aurore ULg et al

Poster (2013, June 10)

Microorganisms play key roles in soil ecosystem functioning, notably through their ability to degrade plant cell wall polymers. For this, bacteria and fungi produce various enzymes such as cellulases ... [more ▼]

Microorganisms play key roles in soil ecosystem functioning, notably through their ability to degrade plant cell wall polymers. For this, bacteria and fungi produce various enzymes such as cellulases, xylanases, glucosidases, esterases or laccases. Finding new enzymes hydrolyzing cellulose, hemicellulose or lignin is not only interesting for a better understanding of the roles of the soil microflora still largely unknown but these enzymes are also useful for various biotechnological applications such as the production of renewable energy from lignocellulosic material. So here, we used a functional metagenomic approach to isolate new bacterial β-glucosidases, which were then biochemically characterized. The new enzymes were identified by functional analysis of agricultural-soil metagenomic libraries hosted in Escherichia coli and screened on medium containing esculin. After sequence analysis and preliminary estimation of the activity of the new β-glucosidases using p-nitrophenol derivatives on intact bacterial cells, the coding sequences of three of them were cloned into a bacterial expression vector so as to overproduce and purify them by affinity chromatography. The chosen enzymes show only 52-64% sequence identity to known family 3 (GH3) or 1 (GH1) glycoside hydrolases of different phyla (Actinobacteria, Acidobacteria and Proteobacteria). Analysis of the E. coli cells expressing each of them revealed that both GH1 proteins (ASEsc9 and ASEsc10) are thermophilic enzymes more active at mildly acidic to neutral pH while the GH3 enzyme (ASEsc6) is an alkaline, mesophilic, β-glucosidase also displaying xylosidase activity. Their coding sequences have been cloned in fusion with a carboxy-terminal His-tag and placed under the control of the IPTG-inducible promoter of the pET-30b vector. The proteins will be overproduced and purified for further characterization. [less ▲]

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