Thesis title: Un approccio green per il trattamento dei composti fenolici nei reflui oleari: Biocatalizzatore laccasi–trebbia di birra delignificata
Enzymes, thanks to their ability to catalyse specific reactions with high efficiency, are often used in biotechnological applications. Among these, laccase is an enzyme belonging to the multi-copper oxidoreductase family, capable of oxidising phenolic and aromatic compounds by coupling the reduction of molecular oxygen to water, and therefore often used for bioremediation and green chemistry applications. However, the industrial use of free-form laccases is limited by the difficulty of recovering the enzyme and therefore also by the cost of production. This research study investigated the catalytic capacity of a system based on laccase immobilised by physical adsorption on cellulose extracted from delignified spent grain (DSG). The ultimate goal was to evaluate the effectiveness of the system in degrading phenolic compounds present in olive mill waste water (OMWW), which still represent an open challenge in the disposal of this type of waste. The use of treated brewers' grains as a support for immobilisation allowed the synthesis of the system starting from a highly available material that is industrial waste and completely biodegradable. The cellulosic support was obtained by acid-base treatment of the spent grain, which, in addition to degrading the lignin, gave the surface structure a high porosity. These characteristics were confirmed by Scanning Electron Microscope analysis, which revealed a more irregular morphology dotted with microcavities, conditions particularly conducive to enzymatic adsorption. The immobilisation phase of laccase by adsorption was refined by varying the most relevant operating parameters: enzyme/support ratio, pH, temperature and incubation time. Degradation tests, conducted on both standardised polyphenol samples and real olive mill wastewater samples, showed that the immobilised enzyme had a slightly lower degradation capacity than free laccase, probably due to the reduced freedom of diffusion of the fixed enzyme. After 24 hours of treatment, immobilised laccase broke down an average of about 70% of the phenolic compounds, compared to values above 80% for free laccase. Although efficiency decreases, the advantage of being able to reuse the system for several consecutive cycles balances this out: in tests on real samples, the biocatalyst retained more than 40% of its initial degradation capacity even after six cycles of use, extending the overall duration to over 150 hours of operation. This performance, combined with the simplicity of the preparation process and the low cost of materials, makes the DSG-laccase biocatalyst a viable and sustainable alternative to traditional chemical or physicochemical methods for treating phenolic wastewater. Overall, the results demonstrate that laccase immobilised on delignified spent grain by adsorption is an efficient, stable and sustainable biocatalyst, capable of maintaining good catalytic performance even in complex matrices such as olive mill wastewater. This system is part of numerous studies seeking solutions to reduce the environmental impact of wastewater through the use of renewable materials from industrial waste, in line with the principles of green chemistry and the circular economy. The results obtained lay the foundations for further developments with a view to possible industrial scaling up and the integration of the biocatalyst into enzymatic purification processes.