The necessity of using clean energy sources to meet the growing energy needs of the planet is a topic of strong current especially given the consequences, sometimes even tragic, that climate change brings. In this framework, emission due to the automotive sector is one of the crucial aspects to improve. Therefore the development of new low-emission energy sources becomes a must for research. Research efforts are needed to design and improve the biofuel synthesis process, which would produce sustainable and economically feasible transportation fuel. Second generation bio-ethanol is certainly one of the most promising biofuels in order to replace fossil fuels and to achieve a strong reduction of greenhouse gases. This PhD thesis has been focused on the optimization of two crucial steps in 2nd generation bio-ethanol synthesis: steam-explosion pretreatment and hydrolysis. The first chapter describes the massive problem of climate change and the consequent global warming and the crucial role that emissions of greenhouse gases have in this context. In order to reduce these emissions, a key role can be played by the replacement of traditional fossil fuels with biofuels. Among the latter, bioethanol from lignocellulosic biomass is certainly one of the most promising. The second chapter describes the bioethanol production process by focusing on second-generation bioethanol from lignocellulosic biomass. The chapter includes a thorough treatment of the biomass used and a detailed description of the several steps of the production process. The state of the art on enzymatic hydrolysis process is the subject of the third chapter. In this part of the thesis, it is described, in more detail, the process of enzymatic hydrolysis with particular attention to enzymes (cellulases) characteristics and functions. The fourth chapter describes the experimental work, conducted at the Department of Chemical Engineering, University of Lund (Sweden) addressed pretreatment step optimization. In this phase of research, several impregnating agents have been used in order to improve the steam explosion process. The fifth chapter describes the experimental activity, at the University of Lund, performed in order to test the process of hydrolysis within a bioreactor. This phase of the research was aimed to verify the benefits of the process by a more efficient mixing of reagents. The sixth chapter contains a description of the research work developed at the laboratories of the ENEA Research Center of Trisaia (Rotondella - MT). This phase of research was aimed to test the enzyme immobilization. The process of immobilization, in fact, is a technique that allows 10 to obtain more stable and more manageable enzymes and, above all, this methodology allows an easier recovery and reuse of the same enzymes.

This thesis work was done in collaboration with ENEA (Italian Agency for New Technologies, Energy and Sustainable Economic Development)- Research Center of Trisaia (Rotondella – MT). Research activity has developed in cooperation with the Department of Chemical Engineering of the University of Lund (Sweden).