Characterization of new bacterial glycoside hydrolases isolated from agricultural soils using a functional metagenomic approach

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 ▲]

Biomass hydrolyzing enzymes identified by functional screening of a metagenomic library from algal biofilms.

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 ▲]

Impact of the depth on bacterial diversity in an agricultural soil

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 ▲]

Characterization of three new carboxylic ester hydrolases isolated by functional screening of a forest-soil metagenomic library

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

Termites as a tool to improve lignocellulose biomass valorization : study of enzymatic complex in termites and its common symbionts by comprehensive metabolite profiling

Conference (2012, November 16)

Identification of new microbial enzymes from forest and marine ecosystems by functional metagenomics

Poster (2012, August 21)

IMPACT OF DEPTH AND SOIL COMPACTION ON BACTERIAL DIVERSITY IN SOIL

Poster (2012, August 19)

Bacteria are the most abundant and diverse microorganisms in soils. The amount of bacteria in soils can reach 10^10 cells per gram of soil. These organisms are involved in various processes in ... [more ▼]

Bacteria are the most abundant and diverse microorganisms in soils. The amount of bacteria in soils can reach 10^10 cells per gram of soil. These organisms are involved in various processes in agroecosystems such as nutrient cycling, contributing to plant nutrition, plant health and soil structure. The knowledge about this diversity is limited because only one percent of these organisms can be cultured by laboratory methods. During the last decades, many molecular-based techniques have been developed to assess the diversity of bacterial communities. The aim of this study was to determine the quantity and diversity of bacteria in two agricultural soils with differents soil management practices (tillage and no tillage) at different depths (10, 30 and 45 centimeters) and different compaction levels (high and low). Quantity was evaluated by real time PCR and diversity was analysed by the DGGE (Denaturing Gradient Gel Electrophoresis) technique. The results show that soil management has an impact on bacterial quantity at 45 centimeters and quantity is higher in till soil. Compaction level affects the bacterial quantity in till soil, quantity is higher in low compaction. And finally, depth influences the bacterial quantity in till and no till soil. In both soils, quantity decreases with the depth. The results will be presented and discussed on the poster. [less ▲]

Identification of bacteria community associated with earthworm gut

Poster (2012, July 26)

The role of earthworms in soil fertility and transformation of organic waste was regulary cited to be of first importance. Associated to these macro-invertebrates, a large diversity of micro-orgnisms are ... [more ▼]

The role of earthworms in soil fertility and transformation of organic waste was regulary cited to be of first importance. Associated to these macro-invertebrates, a large diversity of micro-orgnisms are found indirectly in their closed environment or directly in their gut. Functional aspects of these interactions and symbiosis in relation with soil characteristics and fertility rates are poorly developed. Here, the micro-organisms diversity and potential related functions of earthworm gut were investigated using a proteiomic approach for both protein and micro-organism identifications. Microbial community investigation was detected by proteomic approach based on bidimensional electrophoresis coupled with mass spectrometry using Matrix Assisted Laser Desorption Ionisation – time of flight (Maldi-Tof). Diversity of gut associated bacterial communities was discussed. Indeed, application of particular crop production practices such as crop residue management at the field level could regulate the gut bacterial communities in earthworm but also microbials in soils. Agricultural systems had to consider the microbial and associated organisms in the soil to enhance fertlility and crop production in sustainable ways. [less ▲]

Selection and cultivation of hydrolytic microorganisms extracted from the digestive tract of the termite Reticulitermes santonensis (3DV.1.55)

Poster (2012, June 21)

Biofuel production can be based on the use of fermentable substrates issued from the hydrolysis of lignocellulosic biomass stemming from agricultural residues and by-products. However, such substrates are ... [more ▼]

Biofuel production can be based on the use of fermentable substrates issued from the hydrolysis of lignocellulosic biomass stemming from agricultural residues and by-products. However, such substrates are not easy to degrade. Enzymes (cellulases, xylanases, etc.) can be used for this purpose and pre-treatments can increase their action by providing more available extremities. The digestive tract of the termite Reticulitermes santonensis contains various microorganisms (bacteria, molds, protists) able to degrade the wood components. These microorganisms act as consortia, leading to a better hydrolysis than in the cow rumen. Our purpose is the isolation of microorganisms from termite guts in order to evaluate their potential for hydrolysis of lignocellulosic materials. This approach led us to isolate and to study a bacteria (Bacillus sp.) displaying a xylanase activity, a mold (Aspergillus sp.) displaying a cellulase activity and a chrysophyte (protist) displaying an amylase activity. [less ▲]

Isolation of two antimicrobial activities using a functional metagenomic approach

Poster (2012, June)