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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 detailÉTUDE DU COMPLEXE ENZYMATIQUE DES SYMBIONTES DU TERMITE RETICULITERMES FLAVIPES (EX. SANTONENSIS)
Tarayre, Cédric ULg

Doctoral thesis (2014)

In the light of the economic, environmental and social context in which we live today, it has become obvious that the use of fossil fuels is not conceivable over the long term. Some alternatives have ... [more ▼]

In the light of the economic, environmental and social context in which we live today, it has become obvious that the use of fossil fuels is not conceivable over the long term. Some alternatives have therefore emerged in recent years. Second-generation biofuels are one of those alternatives and are based on the exploitation of vegetal biomass, also called lignocellulosic biomass. These materials require a hydrolysis step which can notably be achieved by enzymes. Some insects, such as termites, harbor complex microbial communities inside their digestive tracts. Those communities are able to produce enzymes which can be used in the field of the hydrolysis of vegetal biomass. This is what this thesis deals with. The main part of the work done focused on the research of enzyme-producing microorganisms (bacteria, mycetes and protists) responsible for the degradation of hemicelluloses and cellulose, the hydrolysis of which releases fermentable sugars. The insect which was used in this work was Reticulitermes flavipes (ex. santonensis), a lower termite, harboring a highly diversified internal microflora. This thesis describes the characterization of the microbial strains which were isolated and the enzymes they secrete. A complementary part of this research focussed on termites grown on artificial diets. The objective of this part was multiple : isolating enzyme-producing strains, not extractable according to the standard technique, and characterizing the microflora resulting from the applied artificial diets. This multidisciplinary approach was based on microscopy, proteomics, metagenomics and the assessment of metabolism applied to the different microbial consortia. [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 detailBioRefine Project: Detection of bioavailability of Metallic Trace Elements in soils by the use of microbial biosensors
Tarayre, Cédric ULg; Hurdebise, Quentin ULg; Fischer, Christophe ULg et al

Poster (2014, September 09)

Zinc, lead and cadmium are the main Metallic Trace Elements (MTEs) found in soils contaminated by the mining industry in Europe. MTEs are spread in the environment because of the disruption of ... [more ▼]

Zinc, lead and cadmium are the main Metallic Trace Elements (MTEs) found in soils contaminated by the mining industry in Europe. MTEs are spread in the environment because of the disruption of biogeochemical cycles caused by human activities. Due to their low mobility and biodegradability, they accumulate in soils where they are strongly bound to particles. It has become necessary to understand interactions between MTEs and the environment and to implement remediation actions. This work is focused on remediation monitoring techniques by using whole cell microbial biosensors able to detect zinc, lead and cadmium. Biosensors provide a signal in response to the bio-available concentration in MTEs, which are valuable for the design of efficient techniques involving bioremediation. Whole cell biosensors used in this work are based on Escherichia coli strains carrying a fluorescent reporter system. The reporter element contains a promoter sensitive to MTEs and a gene coding for the Green Fluorescent Protein (GFP). MTEs activate the synthesis of GFP, which is a very stable protein, causing the accumulation of GFP inside the cells. Then, fluorescence can be measured by flow cytometry. In this study, two biosensors were investigated: E. coli pPzraPgfp and E. coli pPzntAgfp. The last strain provided a linear response to zinc up to 20 mg/l and a curvilinear response to cadmium up to 0.15 mg/l. No detection was highlighted regarding lead. In practical cases, soils and wastes are contaminated by several types of MTEs. Consequently, combined contaminations were also tested. This work allowed highlighting that the strain E. coli pPzntAgfp can be used to assess the bioavailability of cadmium in soils, although the experimental procedure must be improved. This work is supported by the BioRefine Project, a European project in which various member states focus on recovery of inorganics from organic wastestreams. We gratefully acknowledge the INTERREG IVB NWE programme, which financed the BioRefine Project (ref. 320J-BIOREFINE). [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 detailBiorefine: Recovery of nutrients and metallic trace elements from different wastes by chemical and biochemical processes
Tarayre, Cédric ULg; Fischer, Christophe ULg; De Clercq, Lies et al

Conference (2014, June 05)

At present, most waste processing operations are not oriented towards the valorization of valuable reusable components such as nitrogen, phosphorus, potassium and even Metallic Trace Elements (MTEs ... [more ▼]

At present, most waste processing operations are not oriented towards the valorization of valuable reusable components such as nitrogen, phosphorus, potassium and even Metallic Trace Elements (MTEs). Currently, sewage sludge, for example is usually used as a fertilizer in agriculture, in energy production or in the field of construction. Ashes originating from sludge incineration contain heavy metals and minerals in large quantities. Manure is mainly used in agriculture, although considerable amounts of nutrients are lost and cause pollution. Digestate is also used in agriculture, but other alternatives have been proposed, such as the energetic valorization. Better valorization of these wastes in agriculture (or other sectors) is however largely constrained by a multitude of legal requirements. An important problematic point is the concentration in MTEs that is found in those wastes. Consequently, recovery of nutrients and MTEsmay be a key solution for optimal valorization of wastes. Many unit operations used in the field of chemical and biochemical engineering (mechanical operations on fluids, solids, mass and heat transfers, chemical reactions, etc.) could be used in order to achieve an efficient recovery yield of nutrients and trace elements. The aim of the BioRefine Project is to make an inventory of all recovery techniques of nutrients and MTEs in five countries: Belgium, France, Germany, United Kingdom and The Netherlands. Pilot plants will also be tested to assess the efficiency of new treatment techniques after which the most efficient processes will be chosen to be applied on a larger scale. In addition, the collected data will be used to propose exploitation scenarios taking into account legal constraints and optimized logistics.This work is supported through an INTERREG IVB NWE programme(ref. 320J-BIOREFINE). [less ▲]

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See detailResearch of phosphate accumulating microorganisms from WWTPs for the recovery of phosphorus from organic wastes (3BV.3.47)
Tarayre, Cédric ULg; Michels, Evi; Buysse, Jeroen et al

Poster (2014, June)

Many wastes containing reusable components, such as nitrogen, phosphorus and potassium, are not exploited through ideal processes. As an example, in Wallonia (Belgium), the main treatment applied to ... [more ▼]

Many wastes containing reusable components, such as nitrogen, phosphorus and potassium, are not exploited through ideal processes. As an example, in Wallonia (Belgium), the main treatment applied to sewage sludge consists in incinerating the material. Such a process is chosen when the heavy metals are too concentrated in the sludge, preventing an agricultural use. However, sewage sludge, as well as manure, slurry and digestate, contain notable amounts of nutrients (nitrogen, phosphorus and potassium). Some Waste Water Treatment Plants (WWTPs) are actually designed in order to promote Phosphate Accumulating Organisms (PAOs), able to store or release phosphorus in accordance with the environmental conditions. The aim of this work is to isolate PAOs from WWTPs and evaluate their applicability to phosphorus recovery from organic wastes. Metagenomics and metabolic properties are also considered. This work is supported by the BioRefine Project, a European project in which various member states focus on recovery of inorganics from organic wastestreams. [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 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 detailBioRefine: Recovery of Nutrients from Sewage Sludge, Manure and Digestate by a Combination of Chemical and Biochemical Unit Operations
Tarayre, Cédric ULg; Delvigne, Frank ULg; Michels, Evi et al

Poster (2013, December)

At this time, many wastes are exploited through processes that do not really consider applications potentially more profitable. Such wastes contain reusable components, such as nitrogen, phosphorus and ... [more ▼]

At this time, many wastes are exploited through processes that do not really consider applications potentially more profitable. Such wastes contain reusable components, such as nitrogen, phosphorus and potassium, whereas heavy metals may also be considered. Their composition depends on input materials, and considerable heterogeneities must be highlighted. Sewage sludge is usually exploited as a fertilizer in agriculture, in energy production or in the field of construction. The main application of manure is agriculture, although considerable amounts of nutrients are lost and cause pollution. Digestate is also used in agriculture, but other alternatives have been proposed, such as combustion. The use of waste in agriculture must respect many legal constraints. Another problematic point is the concentration of heavy metals that is found in those wastes. Consequently, recovery of nutrients and trace elements may be a key solution. Chemical and biochemical engineering propose many unit operations (mechanical operations on fluids, solids, mass and heat transfers, chemical reactions, etc.) that may be used to reach an efficient recovery yield of capital nutrients and trace elements. Here, we propose a methodology which consists in considering the unit operations separately with their own input and output flows, energy and heat consumption, investment, etc. and combining them to simulate industrial processes. The element concentrations and their forms will also be considered using a classification matrix. Combinations of unit operations will lead to reliable processes that should be applied on an industrial scale. This work is supported by the BioRefine Project, a European project in which various member states focus on recovery of inorganics from organic waste streams. We gratefully acknowledge the INTERREG IVB NWE programme, which financed the BioRefine Project (ref. 320J-BIOREFINE). [less ▲]

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See detailPrésentation des projets Interreg IVB BIOREFINE et RENEW
Tarayre, Cédric ULg; Delvigne, Frank ULg; Destain, Jacqueline ULg et al

Conference (2013, October 22)

Les projets BioRefine et Renew sont financés par le programme de coopération européen Interreg IVB. Leur but est de promouvoir les transferts de compétences, d'informations et de matières entre les ... [more ▼]

Les projets BioRefine et Renew sont financés par le programme de coopération européen Interreg IVB. Leur but est de promouvoir les transferts de compétences, d'informations et de matières entre les différents pays de la zone européenne du Nord-Ouest. Le projet BioRefine a pour but de récupérer les nutriments (N, P et K) ainsi que les éléments traces métalliques à partir de déchets bien spécifiques: le fumier, le lisier, les digestats de méthanisation et les boues de stations d'épuration. Cette récupération requiert l'élaboration de procédés industriels mis en place après une étude en laboratoire. Le projet Renew, lui, est beaucoup plus général. En ce qui concerne le rôle de Gembloux Agro-Bio Tech dans le projet, le travail sera essentiellement focalisé sur la production d'acide succinique à partir de déchets. Cette production sera assurée par la souche bactérienne Actinobacillus succinogenes. [less ▲]

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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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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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