Quality of the granular sludge developed during the start-up of anaerobic up-flow sludge bed reactors is of crucial importance to ensure the process feasibility of treating industrial wastewater such as those containing solvents. In this study, the microbial granule formation from suspended-growth biomass was investigated in two chitosan-assisted reactors. These reactors operated mimicking industrial sites working with night closures treating a mixture of ethanol, ethyl acetate, and 1-ethoxy-2-propanol. Each reactor operated under different hydrodynamic regimes typical from UASB (R1: <0.15 m h−1) and EGSB (R2: 3 m h−1). High soluble COD removal efficiencies (>90%) accompanied by rapid formation of robust anaerobic granules were achieved at both up-flow velocity levels. After three weeks from the start-up, mean size diameters of 475 µm and 354 µm were achieved for R1 and R2, respectively. The performance of the process was found to be stable for the whole operational period of 106 days treating intermittent OLR up to 13 kg COD m−3 d−1. A memory dose of chitosan at day 42 was beneficial to guarantee good quality of the granules by offsetting the negative impact of intermittent water supply on the granular size. Methanocorpusculum was identified as the dominant archaea at both up-flow velocities. Acetobacterium, Geobacter and Desulfovibrio bacteria were also abundant, demonstrating its role on the degradation of light-oxygenated solvents.