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See detailDevelopment of Hyphenated Methods for the Comprehensive Analysis of Complex Biological Samples
Brasseur, Catherine ULg

Doctoral thesis (2015)

The successful analysis of complex mixtures requires the combination of highly efficient separation techniques with advanced detection technologies to provide individual structural information. The ... [more ▼]

The successful analysis of complex mixtures requires the combination of highly efficient separation techniques with advanced detection technologies to provide individual structural information. The coupling of gas chromatography and mass spectrometry was one of the first steps in hyphenated method development. Nowadays, due to continuous hardware improvement, the term hyphenation has been extended to multidimensional separations and/or multiple detectors. The objective of the thesis is to develop hyphenated methods to extend the range of applications and propose solutions to improve the characterization of specifically selected complex samples. The main hyphenated technique that was used is comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS). Three thematic areas, presenting different levels and types of complexity were investigated: Forensic geotaphonomy with grave soil samples, lignocellulose biomass valorization with termite gut microbiota samples, and human monitoring for emerging organohalogen contaminants with blood samples. Data processing strategies were also developed in order to extract the relevant information from the large quantities of data produced. The different strategies included retention time alignment, comparative data processing, multivariate analysis using unsupervised learning algorithm such as principal component analysis (PCA), univariate analysis of variance (one-way ANOVA), and scripting for automated filtering of mass spectra. All developed methods allowed to successfully improve the characterization of samples originating from the different areas. For the first time, and because of the high level of specificity of GC×GC-TOFMS, the presence of methyl-branched alkane compounds was highlighted in grave soil samples, allowing to develop a screening method that has the capacity to differentiate between soils at proximity of buried decaying pig carcasses and control soils. This strategy could complement other approaches and contribute to aid forensic geotaphonomy investigators. In the biomass valorization area, more than 300 compounds were isolated and partly identified in the 1µL fluid volume available from the termite gut. A comparative study demonstrated that the adaptation of the termite system to non-optimal carbon sources is reflected in the metabolite profile. These results demonstrated the potential interest to investigate metabolite profiling with state-of-the-art separation science tools, to contribute to a better understanding of how termites efficiently degrades lignocellulose. In the human monitoring area, the analysis of serum samples using GC×GC-TOFMS and scripting allowed the efficient reduction of the data matrix from thousands of detected signals to a few important dozens, including the environmental contaminants of interest present in human blood plasma. Such an approach reduced data processing and reviewing time by several orders of magnitude while maintaining a high degree of identification power. This exploratory approach could result in the identification of emerging toxicants and help regulation bodies to gather better information before they rule on emerging issues. GC with high resolution mass spectrometry (HRMS) was also used, using a selective approach to evaluate human exposure to dechloranes, a family of emerging halogenated flame retardants recently reported as significantly present in the environment. A comprehensive approach was also developed using GC×GC coupled to high resolution TOFMS (HRTOFMS). The developed methodology will facilitate the monitoring of these emerging dechlorane contaminants and analogues in future exploratory studies. [less ▲]

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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 detailTrappist Beers: Exploring the Cloak of Secrecy by GC×GC-TOFMS
Allen, C; Loughnane, C; Stefanuto, Pierre-Hugues ULg et al

Poster (2014, May 21)

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See detailMeasurement of Trace levle Dechlorane Flame Retardants in Food and Feed by GC-MS/MS
L'Homme, Benjamin ULg; Calaprice, C; Brasseur, Catherine ULg et al

Poster (2014, May 21)

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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 detailEffect of land use on pollution status and risk of fish endocrine disruption in small farmland ponds
Mandiki; Gillardin; Martens et al

in Hydrobiologia (2014), 723

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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 detailDechloranes: The next Emerging POP?
Brasseur, Catherine ULg; L'Homme, Benjamin ULg; PIRARD, Catherine ULg et al

Conference (2013, November)

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See detailProjet Termitofuel: les termites et leurs symbiontes pour mieux valoriser la biomasse ligno-cellulosique
Bauwens, Julien ULg; Tarayre, Cédric ULg; Brasseur, Catherine ULg et al

Poster (2013, October 19)

L’exploitation actuelle de la biomasse ligno-cellulosique, extrêmement abondante sur Terre, est liée à la production de déchets relativement peu valorisables. C’est pourquoi le projet TERMITOFUEL étudie ... [more ▼]

L’exploitation actuelle de la biomasse ligno-cellulosique, extrêmement abondante sur Terre, est liée à la production de déchets relativement peu valorisables. C’est pourquoi le projet TERMITOFUEL étudie la digestion du bois chez les termites, grâce à une approche pluridisciplinaire, en vue d’améliorer la production de bioéthanol de seconde génération. Au sein de l’Unité d’Entomologie Fonctionnelle et Evolutive, l’approche protéomique est employée afin d’identifier les micro-organismes présents dans le tube digestif des termites et caractériser les activités enzymatiques. [less ▲]

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See detailInvestigation olfactive de la mort
Focant, Jean-François ULg; Stefanuto, Pierre-Hugues ULg; Brasseur, Catherine ULg et al

in Acta Clinica Belgica (2013, October), 68-6

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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 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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