Posilipo, Adriana (2024) Optimization of biochemical saccharification of lignocellulosic biomass. [Tesi di dottorato]

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Abstract

The aim of the PhD project was the optimization of the enzymatic saccharification of lignocellulosic biomasses. In particular, the activity addressed the study of pre- treatment and enzymatic hydrolysis process. The target biomasses were agro- industrial wastes, coffee silverskin, and energy crops present in the Mediterranean area (Cynara cardunculus and Arundo donax). The specific aims included the characterization of enzymes adsorption on the biomass as a key phenomenon affecting the perfomances of the biochemical delignification, catalyzed by laccases, and the enzymatic hydrolysis, catalyzed by cellulases. Results would address the optimization of the enzymatic hydrolysis step in terms of: i) minimal enzymes dosage providing satisfactory sugar yields; ii) the assessment of kinetic models for the overall glucose production rate. The biological pre-treatment was carried out with a fungal laccase, PoxA1b, from Pleurotus ostreatus. The study of enzymatic hydrolysis with the commercial cocktail Cellic ® CTec2 provided the characterization of the adsorption of the enzymes on the raw and pre-treated lignocellulosic biomasses as well as the optimization of the operating conditions to provide high yields of total fermentable sugars (larger than 70% in 24 h), high final sugar concentration, minimum specific amount of active adsorbed enzymes (about 4 mg/g). Results on the partially irreversible nature of the enzymes adsorption allowed to consider an enzymes recycling strategy aimed at process costs saving. The investigation of glucose production rate provided a simple semimechanistic kinetic model assessed and validated at lab scale for the system cardoon-Cellic® CTec2. This part of the study confirmed the importance of the type of mixing applied to the slurry during the enzymatic hydrolysis and revealed the occurrence of disgregating phenomena at the micro-scale of the biomass particles. These phenomena – very relevant at the early phase of the sugars production (1-4 h) - open perspectives for the future investigations aimed at optimization and control of the transport of enzyme molecules to the inner part of fibrous biomass for an efficient conversion of the polysaccharides and use of the biocatalyst. The research activity was carried out at the Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale of the Università di Napoli Federico II. During the six-month research secondment at the Institute of Biotechnology and Biochemical Engineering of University of Technology in Graz (Austria), the project was focused on the production of the multi-domain cellulase CelA from Cladicellulosiruptor bescii. The cellulase was tested as a promising alternative to commercial cocktail of fungal cellulases on the lignocellulosic cardoon biomass. Methods developed for the characterization of biocatalyst adsorption and its impact on the yield of hydrolysed sugars were used. The results showed a promising performance of the crude preparation from C. bescii culture with respect to both the purified protein and the commercial cocktail thus suggesting future perspectives on the use of this type of biocatalysts in the biorefinery of lignocellulosic feedstocks.

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