Sustainable production of fuels and chemicals from biomass has become essential considering the societal and political demands for alternatives to petrochemical commodities. Efficient enzymatic conversion of plant biomass (i.e. lignocellulose) into monomeric sugars is one of the key challenges that need to be overcome. Lignocellulolytic micro-organisms, bacteria and fungi, are a reservoir for identifying new enzymes of high potential for carbohydrate modification. ZyBiom is an ambitious project that aims at mining bacterial and fungal biodiversity through the exploration of (i) metagenomic libraries generated from Canadian mammals intestinal microbiotes and (ii) secretomes of fungi from the Polyporales order isolated from natural biodiversity. In order to assess the enzymatic modification of carbohydrates from biomass, a state-of-the-art methodological approach will be developed at University of Toronto. After treatment of biomass by microbial enzymes, (i) the solid residue will be analysed in a high-throughput mode by Tof SIMS and (ii) the soluble fraction will be analysed by the quantification of sugars using high-throughput assays. This approach will lead to the selection of novel carbohydrate modifying enzymes. Further in-depth characterization of selected enzymes will provide new information to understand the mechanisms by which microorganisms deconstruct lignocellulose, as well as new candidates for efficient industrial processes.