References of "Vandenbol, Micheline"
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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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See detailStudy of bacterial diversity in the topsoil and below the hardpan in an agricultural soil by metagenomics following by two analysis pipelines
Stroobants, Aurore ULg; Lambert, Christrophe; Degrune, Florine ULg et al

Poster (2013, June 10)

On earth, Bacteria are ubiquitous and even present in extreme environments (pH, temperature,…). In soils in particular, bacteria are very abundant (up to 109 cells per gram of soil) but still poorly ... [more ▼]

On earth, Bacteria are ubiquitous and even present in extreme environments (pH, temperature,…). In soils in particular, bacteria are very abundant (up to 109 cells per gram of soil) but still poorly characterized. Thus, it is of paramount importance to use relevant study and analysis procedures to ensure that the results obtained closely reflect the real-life conditions. In the present work, we analyze the bacterial diversity in the topsoil and below the hardpan in an agricultural soil using the metagenomics approach, with the Ion Torrent PGM sequencer. The soil samples was collected at three depths : 10 cm (topsoil), 25 cm (topsoil above the hardpan) and 45 cm (below the hardpan), in a tilled and a no tilled plot. The taxonomic analysis of the reads obtained are carried out according to two different procedures with the RDP classifier program and with a confidence score threshold of 0 and 0.99. The 0 threshold is used to assign a species to all reads, each read being therefore assigned to its most closest known species. The threshold of 0.99 enables us to focus on reads being assigned to a species with a high degree of confidence. In this case, each read is assigned to the most specific rank having a confidence score higher than 0.99. The bacterial diversity was then compared between the different conditions. Results obtained demonstrate that the bacterial communities were not the same in the two horizons. For example, some classes of Acidobacteria were up to 11 fold more numerous in topsoil while others was until 12 fold more represented below the hardpan. The biomass and the bacterial diversity (Shannon index) were also greatly different between the two depths. [less ▲]

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See detailIsolation and cultivation of cellulolytic and xylanolytic bacteria and molds extracted from the gut of the termite Reticulitermes santonensis (3DV.1.14)
Tarayre, Cédric ULg; Bauwens, Julien ULg; Mattéotti, Christel et al

Poster (2013, June)

Biofuel production can be based on the use of agro-residues, consisting in a complex lignocellulosic structure which is not easily hydrolysable. The digestive tract of the termite Reticulitermes ... [more ▼]

Biofuel production can be based on the use of agro-residues, consisting in a complex lignocellulosic structure which is not easily hydrolysable. The digestive tract of the termite Reticulitermes santonensis contains a diversified microflora able to hydrolyze the wood components. Bacteria, molds and protists form efficient consortia, able to break the lignocellulosic complex by producing enzymes, such as xylanases and cellulases. Our purpose is the isolation of microbial strains from termite guts in order to evaluate their potential for hydrolysis of lignocellulosic materials. Termites were fed using different diets chosen to improve the xylanolytic and cellulolytic microflora: wood, microcristalline cellulose (added with lignin or not), α-cellulose (added with lignin or not) and birchwood xylan. Then, dissections were realized to isolate the potential xylanolytic and cellulolytic strains. This approach led us to isolate and to study several strains of bacteria (Bacillus sp. strain CTGx and Chryseobacterium sp. strain CTGx) and molds (Trichoderma virens strain CTGx and Sarocladium kiliense strain CTGx). These microorganisms were able to hydrolyze starch, xylan, cellulose, carboxymethylcellulose, esculin, β-glucan and Whatman® filter paper. They can produce glucose and xylose monomers and oligomers which can be further fermented to produce bioethanol. [less ▲]

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See detailResearch of New Enzyme Producing Strains in the Gut of the Termite Reticulitermes santonensis
Tarayre, Cédric ULg; Bauwens, Julien ULg; Mattéotti, Christel et al

Poster (2013, June)

Termites contain a complex microflora inside of their guts. Inferior termites contain bacteria, mycetes and protists that interact to degrade vegetable components. These strains act as consortia to break ... [more ▼]

Termites contain a complex microflora inside of their guts. Inferior termites contain bacteria, mycetes and protists that interact to degrade vegetable components. These strains act as consortia to break natural materials by secreting various enzymes. Our aim was the isolation and cultivation of microorganisms in order to produce new enzymes that can be further used in green chemistry. Termites were fed with different diets: pinewood, microcristalline cellulose (added with lignin or not), α-cellulose (added with lignin or not) and birchwood xylan. Then, dissections were realized to isolate interesting strains. All the microorganisms were subjected to enzyme assays. That technique allowed us to isolate and to cultivate various strains of bacteria, molds and protists. Three strains of bacteria, two strains of molds and one strain of protist were isolated and displayed different enzymatic activities. The bacteria Bacillus subtilis strain ABGx, Bacillus sp. strain CTGx and Chryseobacterium sp. strain CTGx displayed amylase, cellulase and xylanase activities. The molds Trichoderma virens strain CTGx and Sarocladium kiliense strain CTGx were also able to produce those enzymes. However, the protist Poterioochromonas sp. was found to produce only amylase. In conlusion, the termite gut is a complex culivation medium that provides a habitat for many microorganisms that show interesting enzymatic activities. [less ▲]

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See detailBiomass hydrolyzing enzymes identified by functional screening of a metagenomic library from algal biofilms.
Martin, Marjolaine ULg; Biver, Sophie ULg; Barbeyron, Tristan et al

Poster (2013, June)

Biomass hydrolyzing enzymes are increasingly searched for the production of biofuels and renewable chemical compounds using biomass. Microorganisms living on algaes are an interesting reservoir of biomass ... [more ▼]

Biomass hydrolyzing enzymes are increasingly searched for the production of biofuels and renewable chemical compounds using biomass. Microorganisms living on algaes are an interesting reservoir of biomass hydrolyzing enzymes, as they are in constant interaction with algal biomass. Therefore, we are interested in looking for some of those enzymes synthesized by the microflora living on the surface of the brown algae Ascophyllum nodosum. Algae samples were collected in the winter 2012 and a microbial DNA extraction method was developed. The whole extracted microbial genomes of the microorganisms living on the algae were restricted, inserted in a cloning vector and ligated products were used for transformation of cultivable Escherichia coli host cells. This metagenomic library was then screened for diverse enzymatic activities (lipolytic enzymes, cellulases, beta-glucosidases, alpha-amylases, arabinanases, xylanases and proteases) on agar plates with specific substrates. Five putative lipolytic enzymes, one cellulase and one beta-glucosidase were identified. Sequence analysis revealed low (<50%) sequence identities with known enzymes sequences, meaning new enzymes from unknown genomes have been discovered. To our knowledge this is the first functional screening that was realized with a metagenomic library from algal biofilms and this is the first cellulase identified by marine metagenomics. A second library has been constructed from algae sample from summer 2012 and is currently being screened. New enzymatic tests are being developed for the identification of enzymes degrading specific algal polysaccharides like agarases, carrageenases, alginate lyases, laminarinases,… Those very specific enzymes aren’t well known yet, and our metagenomic approach will probably help us to identify new families and structures of those algal biomass hydrolyzing enzymes. [less ▲]

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See detailMicroorganisms living on algae : An interesting reservoir of enzymes hydrolyzing algal biomass
Martin, Marjolaine ULg; Biver, Sophie ULg; Barbeyron, Tristan et al

Poster (2013, April 18)

Algal polysaccharides are increasingly used in food industry for their gelling properties and in pharmacology for their therapeutic properties. Furthermore, increasingly interest is taken on algae for ... [more ▼]

Algal polysaccharides are increasingly used in food industry for their gelling properties and in pharmacology for their therapeutic properties. Furthermore, increasingly interest is taken on algae for their use in the production of biofuels and bioenergies. To purify algal polysaccharides and degrade algal biomass, specific microbial enzymes are needed. Microorganisms living on algae are an interesting source of those enzymes, as they are in constant interaction with algal biomass. The aim of our study is to identify new enzymes degrading algae, produced by microorganisms living on the surface of algae. Therefore we developed a method for microbial DNA extraction from biofilms living on brown algae (Ascophyllum nodosum). Microbial DNA was extracted, restricted and inserted in cultivable host cells of Echerichia coli, for the construction of our metagenomic DNA library. This metagenomic library was first screened, on solid media with specific substrates, for enzymes generally used in the degradation of biomass (lipases, cellulases, proteases, beta-glucosidases, alpha-amylases, arabinanases and xylanases). Five lipolytic enzymes, one beta-glucosidase and one cellulase were identified. Those enzymes show very low percentages of sequences identities with known enzymes, meaning we identified new and unknown enzymes. Those enzymes and their activity are being characterized. Preliminary tests show interesting results, like a cellulase active at low temperature. Screening tests are now being developed to identify enzymes hydrolyzing algal polysaccharides like agarases, carrageenases, alginate lyases, laminarinases,… Those enzymes aren’t well known yet and we hope to identify new enzymes (families) with our rich DNA library by our approach. [less ▲]

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See detailFunctional screening of a metagenomic library from algal biofilms
Martin, Marjolaine ULg; Barbeyron, Tristan; Michel, Gurvan et al

in Smagghe, Guy; Boeckx, Pascal; Bossier, Peter (Eds.) et al Communications in Agricultural and Applied Biological Sciences (2013, February 08)

Macroalgae, and particularly their lignin-free polysaccharides, are increasingly used for their gelling and therapeutic properties and for the production of biofuels and renewable chemical compounds. To ... [more ▼]

Macroalgae, and particularly their lignin-free polysaccharides, are increasingly used for their gelling and therapeutic properties and for the production of biofuels and renewable chemical compounds. To extract, hydrolyze and purify this biomass, algae hydrolyzing enzymes are needed. Our work aims to identify and characterize algal biomass hydrolyzing enzymes expressed by microorganisms living on the surface of algae, by functional metagenomics. Therefore, a microbial DNA extraction method was developed to isolate the gDNA from the microorganisms of the brown algae Ascophyllum nodosum and a metagenomic library was constructed in Escherichia coli. The library was screened for diverse enzymatic activities (esterases, xylanases, cellulases, α-amylases, arabinanases, caseinases and β-glucosidases) on agar plates with specific enzymes substrates. Several new microbial enzymes (esterases, β-glucosidases, α-amylases and cellulases) were identified revealing the wealth of our library. Furthermore, those enzymes had less than 50% sequence identity with known protein sequences; meaning that our approach allows to identify new microbial enzymes expressed by uncultured microorganisms. Plate tests for medium-throughput screening of specific enzymes hydrolyzing algal polysaccharides (agarases, carrageenases and alginate lyases) are currently being developed. Our approach will probably allow us to identify new families of those ill-known enzymes, with particular enzymatic activities. [less ▲]

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See detailImpact of the depth on bacterial diversity in an agricultural soil
Stroobants, Aurore ULg; Degrune, Florine ULg; Lambert, Christophe et al

Poster (2013, February 08)

Bacteria are the most abundant and diverse microorganisms in soils. They play an important role in soil formation, contribute to plant nutrition and are involved in various processes in agroecosystems ... [more ▼]

Bacteria are the most abundant and diverse microorganisms in soils. They play an important role in soil formation, contribute to plant nutrition and are involved in various processes in agroecosystems such as nutrient cycling. The aim of this study was to evaluate the impact of the depth on bacterial diversity and quantity in an agricultural soil. Samples was collected on May 2011 and May 2012 at three different depths : 10, 25 and 45 centimeters. The quantity of total bacteria was measured by real time PCR and the analysis of the diversity was performed by the high throughput sequencing technology. Results obtained by these methods show that the biomass and the bacterial quantity and diversity (Shannon index) decrease with the depth, particularly at 45 centimeters. The biomass is, in average, 6.5 fold less important at 45 cm than at 10 cm and the quantity is 17 fold lower at 45 cm than at 10 cm. Our results also indicate that many taxa, such as Betaprotebacteria, Deltaproterobacteria, Gammaproteobacteria, Acidobacteria and Burkholderiales are influenced by the depth. The results will be presented in more details on the poster. [less ▲]

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See detailCharacterization of three new carboxylic ester hydrolases isolated by functional screening of a forest-soil metagenomic library
Biver, Sophie ULg; Vandenbol, Micheline ULg

in Journal of Industrial Microbiology & Biotechnology (2013), 40(2), 191-200

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See detailAn eYFP Reporter Gene for the Yeast Two-hybrid System
Damon, Coralie; Boxus, Mathieu; Twizere, Jean-Claude ULg et al

in Protein Journal (2013), 32(2), 126-130

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See detailSymbiont Diversity in Reticulitermes santonensis (Isoptera: Rhinotermitidae): Investigation Strategy Through Proteomics.
Bauwens, Julien ULg; Millet, Catherine ULg; Tarayre, Cédric ULg et al

in Environmental entomology (2013), 42(5), 882-7

The complex microbial community living in the hindgut of lower termites includes prokaryotes, flagellates, yeasts, and filamentous fungi. Many microorganisms are found in the termite gut, but only a few ... [more ▼]

The complex microbial community living in the hindgut of lower termites includes prokaryotes, flagellates, yeasts, and filamentous fungi. Many microorganisms are found in the termite gut, but only a few are thought to be involved in symbiotic association to participate in cellulose digestion. Proteomics provides analyses from both taxonomical and functional perspectives. We aimed to identify symbiont diversity in the gut of Reticulitermes santonensis (Feytaud), via complementary electrospray ionization associated to ion trap tandem mass spectrometry (LC-MS/MS) and two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry analysis. One specific challenge to the study of lower termites is the relatively few data available on abundant symbiotic flagellates. Analysis based on LC-MS/MS revealed few protein families showing assignments to eukaryotes and the taxonomic origin of highly represented actins could not be established. Tubulins proved to be the most suitable protein family with which to identify flagellate populations from hindgut samples using LC-MS/MS, compared with other protein families, although this method targeted few prokaryotes in our assay. Similarly, two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry did not succeed in identifying flagellate populations, but did permit the identification of most of the prokaryotic components of the symbiotic system. Finally, fungi and yeasts were identified by both methods. Owing to the lack of sequenced genes in flagellates, targeting tubulins for LC-MS/MS could allow fingerprints of flagellate populations to be established. Experimental and technical improvements might increase the efficiency of identification of prokaryotic populations in the near future, based on metaproteomic development. [less ▲]

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See detailInfluence of lignin in Reticulitermes santonensis: symbiotic interations investigated through proteomics
Bauwens, Julien ULg; Tarayre, Cédric ULg; Brasseur, Catherine ULg et al

in Symbiosis (2013)

The gut of lower termites is populated by numerous microbial species belonging to prokaryotes, fungi, yeasts and protists. These micro-organisms are organized in a complex symbiotic system, interacting ... [more ▼]

The gut of lower termites is populated by numerous microbial species belonging to prokaryotes, fungi, yeasts and protists. These micro-organisms are organized in a complex symbiotic system, interacting together and with the insect host. Their likely ability to degrade ligno-cellulosic compounds could lead to improvements in second generation biofuels production. Lignin elimination represents a critical point as this polymer significantly interferes with industrial process of cellulose. Although host produces its own lignin-degrading enzymes, some symbionts may participate in digestion of lignin and its degradation products in termite gut. Here, we compared gut proteomes from R. santonensis after rearing on artificial diets composed of cellulose with and without lignin. The effect of lignin in artificial diets on different parts of the digestive tract was compared through liquid chromatography associated with tandem mass spectrometry (LC-MS/MS) experiments. Enzymatic assays were performed to characterize activities present in R. santonensis digestive tract after feeding on artificial diets. Microscopic observations of microbial communities provided some information on population balances after feeding experiment. [less ▲]

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