Second generation biofuels such as biohythane, a mixture of biohydrogen (H2) and methane (CH4), produced through two-stage H2 / CH4 anaerobic processes represent a promising alternative to fossils fuels. However, lignocellulosic substrates contain recalcitrant structures that require a pretreatment step before anaerobic fermentative processes. In the present study, the impact of alkaline pretreatment (55°C, 24h, 4% NaOH) before a two-stage H2(batch)/CH4(continuous) process was investigated. Alkaline pretreatment did not enhance hydrogen yield probably due to the low solubilization of holocelluloses into soluble sugars. However, alkaline pretreatment enhanced the methane yield resulting in a 30 % increase of the total energy produced compared to the two-stage H2 (batch)/ CH4 (continuous) with no pretreatment.