The conversion of agro-industrial byproducts, residues and microalgae, which are representative or adapted to the Mediterranean climate, to hydrogen (H-2) by C. butyricum was compared. Five biomass types were selected: brewery's spent grain (BSG), corn cobs (CC), carob pulp (CP), Spirogyra sp. (SP) and wheat straw (WS). The biomasses were delignified and/or saccharified, except for CP which was simply submitted to aqueous extraction, to obtain fermentable solutions with 56.2-168.4 g total sugars L-1. In small-scale comparative assays, the H-2 production from SP, WS, CC, BSG and CP reached 37.3, 82.6, 126.5, 175.7 and 215.8 mL (g biomass)(-1), respectively. The best fermentable substrate (CP) was tested in a pH-controlled batch fermentation. The H-2 production rate was 204 mL (L h)(-1) and a cumulative value of 3.9 L H-2 L-1 was achieved, corresponding to a H-2 production yield of 70.0 mL (g biomass)(-1) or 1.6 mol (mol of glucose equivalents)(-1). The experimental data were used to foresight a potential energy generation of 2.4 GWh per year in Portugal, from the use of CP as substrate for H-2 production. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.