SCREENIND AND DESIGN METHODS FOR OPTIMIZATION OF LIGNOCELLULOSIC BIOMASS SACCHARIFICATION BY NEW BIOCATALYSTS AND PROCESS STRATEGIES

Saccharification of lignocellulosic biomass (LB) is the core process of the sugar-based biorefinery and allowed the production of monosaccharides to be fermented in bio-ethanol. The improvement of the technologies enabling the fractionation and depolymerization of polysaccharides from LBs is necessary to support the decarbonization of the energy and chemical industry through the deployment of second-generation biorefineries. Indeed, more efficient biocatalytic processes and technologies will make feasible the use of multiple waste LBs through a proper seasonal and regional interchange of biorefinery feedstocks. In addition, multidisciplinary research efforts can lead to costs saving (e.g. biocatalyst consumption) through - the discovery and production of novel hydrolytic enzymes and the mechanism thereof in the depolymerization of cellulose and hemicellulose chains embedded in the LB structure [1] - the rational design of efficient and sustainable technologies and process layouts for LBs delignification and enzymatic hydrolysis [2, 3] Despite the finely tuned formulation of commercial hydrolytic enzyme cocktails, the industrial practice is based on the use of large excess cellulase enzymes to make possible satisfactory depolymerization of high solids loading in LBs slurries. The enzyme discovery of cellulases from (hyper)thermophilic environments by metagenomic approach ensures the identification of novel robust biocatalysts that are stable under the conditions required for LB treatment processes. Indeed, in extreme (hyper)thermophilic environments, such as hot springs or deep-sea hydrothermal vents, microorganisms have evolved to thrive under high-temperature conditions. These extremophiles possess enzymes, including cellulases, that are naturally adapted to function efficiently at elevated temperatures. Leveraging metagenomic techniques, we can tap into the genetic diversity of these microbial communities and explore the vast repertoire of cellulase genes present in their genomes. By using suitable bioinformatics pipelines it is possible to predict and identify genes associated with cellulose degradation in the metagenomic datasets. These putative cellulase genes will be then heterologously expressed and enzymatically characterized by using substrates analogous to the polysaccharides present in LB. Furthermore, the newly identified thermophilic cellulases may possess unique features and substrate specificities that make them promising in breaking down complex lignocellulosic structures. This could potentially lead to improved saccharification yields, reduced enzyme dosage, and enhanced overall process efficiency, contributing to the development of more cost-effective and sustainable biofuels production methods. In this framework, our recent efforts focused on the development of reliable and versatile protocols for (Fig.1) i) assessment of novel cellulases as industrial biocatalysts for sugar-based biorefinery against reference LB substrates; ii) LB substrates investigation as potential feedstock through standard delignification pretreatments and commercial cellulase cocktails; iii) optimization of novel delignification/EH process strategies with standard cellulase cocktails/EH conditions and widely used LB substrates. The first point was addressed thanks to a research collaboration funded by the Italian section of the ESFRI European Infrastructure IBISBA joint undertaking [4]. The developed methodology provides insight into the activity of novel endo- and exo-glucanases against reference LB substrates through a simple versatile procedure based on micro-litres scale tests of adsorption and hydrolysis of reference LB slurries. Along this path, the second and third steps have been addressed through a methodology applied to a pool of valuable LBs available in the Mediterranean area (cardoon and Arundo donax stalks, rice straw) as well as in the European food industry (coffee silverskin, apple pomace) [5]. The methodology goes through the adsorption of cellulolytic enzymes on LB substrates properly pretreated by well-established delignification processes (e.g. diluted alkali hydrolysis) and provides the optimal conditions of a two-step process enabling both the minimization of the enzyme loading and the maximization of sugars yields at high (8-12%w/v) solids content. Results show: - quantitative information about the heterogeneous EH of real LBs substrates for reactor design purposes (e.g. enzymes adsorption equilibria against LB slurries); - a repeatable feedstock agnostic protocol to minimize biocatalyst consumption; - a scalable process layout of the two-step adsorption/EH allowing the recycling of the excess biocatalyst.