Nowadays, the production of bioethanol from lignocellulosic biomass has attracted significant attention worldwide. This is mainly due to the rise in petroleum oil prices and the depletion of oil reserves, which have led to a highly volatile energy market. Every year, all the petroleum used in Ethiopia is imported from abroad. Dependence on foreign petroleum is a real threat to the national economy, as the supply of most commodities is affected by petroleum prices. Hence, promoting bioethanol is one of the initiatives for the development of a sustainable economy. In Ethiopia, there are diverse and abundant agro-industrial lignocellulosic residues that are not yet utilized for bioethanol production. Enset fiber and sugarcane bagasse are among them with a high concentration of cellulose and hemicellulose, making them potential candidates for the sustainable production of bioethanol. The ability to convert these agro-industrial lignocellulosic feedstocks to bioethanol is a key to making it competitive with petroleum-based fuels. However, the recalcitrant nature of lignocellulosic biomass makes this conversion a challenging process. An effective pretreatment can reduce the recalcitrant of lignocellulosic biomass by removing lignin, decreasing cellulose crystallinity, and increasing the specific surface area for efficient and higher conversion to fermentable sugars during hydrolysis. Fermenting organisms then convert the sugars into bioethanol. Among the chemical pretreatments, alkaline pretreatment is highly efficient in selectively removing lignin and hemicellulose without degrading cellulose and it can also be conducted under mild process conditions. The disadvantages of mild alkaline pretreatment are the long pretreatment duration and the requirement of a high amount of corrosive chemicals. Therefore, the research study presented in this PhD thesis addressed some of the challenges associated with the conversion of lignocellulosic biomass into fermentable sugars. The study specifically aims to investigate potential agro-industrial residue pretreatment techniques to provide a more digestible substrate that is highly susceptible to enzymatic attack. The biomass feedstocks were first pretreated using novel technologies such as ball milling, ultrasonication and deep eutectic solvents to intensify or replace the conventional mild alkaline pretreatment process. Following pretreatment, samples were hydrolyzed using cellulase to determine the reducing sugar release potential of the pretreated samples. The delignification extent of the pretreatment methods was evaluated using National Renewable Energy Laboratory (NREL) protocols. Furthermore, the pretreated samples were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and laser diffraction particle size analyzers to understand the physicochemical changes induced by the pretreatments. This study was divided into three main goals. The first goal was to investigate the effects of a single, sequential and simultaneous (dry chemomechanical) coupling of ball milling and mild VIII alkaline pretreatment on the physicochemical characteristics and enzymatic saccharification of Enset fiber. It was found that the simultaneous (dry chemomechanical) pretreatment was superior to other pretreatment schemes. The simultaneous action of alkali and planetary ball milling in the dry chemomechanical pretreatment acted synergistically to intensify the pretreatment and enhance the production of glucose. The maximum glucose yield achieved from the dry chemomechanical method was 581 g/kg of pretreated Enset fiber, which is 86 and 22% higher than that achieved from single alkaline and single dry ball milling pretreatments, respectively. Compared to the sequential pretreatments, the dry chemomechanical pretreatment resulted in comparable glucose yield from hydrolysis while cutting the pretreatment time from 120 to 90 min. Moreover, the energy efficiency of dry chemomechanical pretreatment was 1.3, 5.3 and 7.8 times higher than the energy efficiency of dry ball milling, sequential and alkaline pretreatments, respectively. Therefore, the action of alkali and planetary ball milling in the dry chemomechanical pretreatment acted synergistically to intensify the pretreatment and enhance the production of glucose. In the second stage of this work, the effect of different processing parameters such as ultrasonic power density, ultrasonication time and alkaline concentration in the conventional and ultrasound-assisted alkaline pretreatment on the extent of delignification, physicochemical characteristics, and enzymatic saccharification of sugarcane bagasse and Enset fiber have been investigated. The obtained results show that sugarcane bagasse subjected to ultrasound-assisted alkaline pretreatment (3% NaOH, 1.88W/ml ultrasonic power density and 60 min duration) released significantly higher glucose from subsequent enzymatic saccharification, 487 g/kg biomass, compared to that obtained from the conventional approach, 373 g/kg biomass. On the other hand, ultrasound-assisted alkaline pretreatment of Enset fiber (1.5% NaOH, 1.88W/ml ultrasonic power density and 30 min duration) resulted in a maximum glucose yield of 540 g/kg biomass compared to 431 g/kg biomass for the conventional alkaline pretreatment. In addition, ultrasound-assisted alkaline pretreatment showed a 65% reduction in NaOH consumption and a 30 min reduction in pretreatment time, compared to the conventional approach. Overall, the synergy between alkali and ultrasound in the combined pretreatment approach enhances the accessibility of cellulose in sugar cane bagasse and Enset fiber, thereby increasing the production of glucose The third and last goal of this research study was to investigate the effect of sequential ball milling (500 rpm, 10 min) followed by deep eutectic solvents (DESs) (choline chloride: ethylene glycol (ChCl:EG) and choline chloride: lactic acid (ChCl:LA)) pretreatment on the extent of delignification and enzymatic saccharification of Enset fiber. Alkaline retreatment using 6% w/v NaOH solution was carried out for the purpose of comparison. It was found that DESs alone IX could not increase the glucose yield from Enset fiber. The maximum glucose yields were 149 and 185 g/kg biomass for the pretreatments with ChCl:EG and ChCl:LA solvents, respectively, compared with 490 g/kg biomass obtained from NaOH pretreated Enset fiber. However, the sequential planetary ball milling and DESs pretreatment increased the glucose yield more efficiently than the DESs pretreatment alone. The glucose yields obtained were 309 and 453 g/kg biomass for the BM-ChCl:EG and BM-ChCl:LA pretreatments, respectively. Therefore, the sequential pretreatment of BM-ChCl:LA was proposed as an alternative to the conventional alkaline pretreatment, allowing higher glucose recovery comparable to NaOH pretreatment. Therefore, the sequential pretreatment of BM-ChCl:LA has been proposed as an alternative to the conventional pretreatment, enabling glucose recovery comparable to NaOH pretreatment (6% w/v). The result will provide useful information on the intensification of the biorefinery process for efficient utilization of Enset fiber. In general, integrated pretreatment methods lead to significant physical and chemical changes in the agro-industrial residues. Dry chemomechanical pretreatment is superior to ultrasoundassisted alkaline pretreatment to induce significant changes in crystallinity, morphology, and particle size. These changes greatly improved hydrolysis performance and glucose yield during enzymatic hydrolysis. At lower NaOH concentration pretreatment, 34% more glucose was produced from enzymatic saccharification of dry chemomechanical pretreated Enset fiber compared to that produced from ultrasound-assisted alkaline pretreated Enset fiber. Moreover, dry chemomechanical pretreatment has 13.5 times higher energy efficiency than ultrasoundassisted alkaline pretreatment. Furthermore, the amount of water required to rinse the dry chemomechanical pretreated Enset fiber was smaller than that required by ultrasound-assisted alkaline pretreated samples, therefore it reduced the consumption of water and the generation of wastewater. In conclusion, dry chemomechanical pretreatment is a promising technique for the scale-up and commercialization of bioethanol production from lignocellulosic materials with minimized operating costs, reduced environmental impacts, and improved performance. Keywords: Bioethanol, Crystallinity, Deep eutectic solvents, Lignocellulosic biomass, Morphology, Planetary ball milling, Ultrasonication