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Enriched activated sludge that can effectively convert crude glycerol into bio-hydrogen was selected by an eco-biotechnological approach, in very strict conditions, using biodiesel-derived glycerol as the only carbon source. The thus obtained functional consortium was characterized by the genera Klebsiella, Escherichia/Shigella and Cupriavidus. During enrichment, the dominant metabolic end-product shifted from a 1,3 propanediol to ethanol, with a concomitant increase of the hydrogen yield from 0.18 ± 0.003 to 0.66 ± 0.06 mol/mol and an almost five-fold increase of the hydrogen production. Glycerol degradation efficiency showed an increase of around 50%. In optimized and upscaled conditions it was possible to obtain a hydrogen production rate of 2960 mL H₂/L/day ± 185 at a near stoichiometric yield (of 0.90 mol/mol ± 0.01), with a carbon recovery of almost 90%, both in sterile and non-sterile conditions …