As global energy demands increase, there is a requirement to decrease our dependency on fossil fuels due to their finite supply and negative environmental impacts. Alternative sources of energy are required that offer sustainability, reduced cost and environmental benefits. Second generation biofuels remove the 'food vs fuel' drawback of the first generation. They utilise lignocellulosic biomass, providing cheap and abundant starting materials for energy production. The major biotechnological challenge associated with lignocellulosic processing is the natural recalcitrance of the substrate to sugar conversion (saccharification). This recalcitrance is largely associated with lignin, an aromatic heteropolymer that encases cellulose. To improve bioethanol yields, there is a need for cost effective and environmentally friendly pretreatment methods that can remove lignin.An enzymatic pretreatment strategy was investigated using laccase from the fungus Trametes versicolor (TvL). Expansion of the laccase substrate range towards non-phenolic substrates was explored by screening of a panel of synthetic and naturally derived phenolic compounds as potential redox mediators with laccase. Both groups enabled decolourisation of the recalcitrant dye (RB-5) to varying degrees, which laccase alone was unable to achieve. In the case veratryl alcohol, a lignin model substrate, synthetic compounds 1-HBT, ABTS and violuric acid proved effective laccase mediators. On this basis, TvL with 1-HBT was selected as the most successful laccase mediator system (LMS) and was further explored as a biomass pretreatment method.The effects of LMS treatments towards the saccharification of acid hydrolysed wheat straw were extensively investigated. Optimisation revealed that when both TvL and a TvL LMS of synthetic origin (1-HBT, violuric acid) were applied, saccharification was improved. The observed increase in glucose release was only detected when a second lignin removing technique was applied in succession. Both alkaline-peroxide and organosolv extractions were successfully used to demonstrate the role of laccase/LMS in saccharification improvement, with improvements reaching up to 44.6%. The effect was further demonstrated with additional substrates (corn and sorghum stover) and additional laccases (Pleurotus ostreatus, Agaricus bisporus and Rhus vernicifera). Further studies using β-O-4 structures, py-GC/MS and FTIR analyses provide further information on the structural actions of an LMS towards lignin, including strong evidence for Calpha-Cβ cleavage and Calpha hydroxyl oxidation mechanisms.