Biological hydrogen production was investigated in continuous acidogenic reactors fed with sucrose at 30 °C without pH control. In the first experimental phase, three reactors were compared: a structured fixed-bed (FB), a granular UASB (UG) and a flocculent UASB (UF-1). They were run at 3.3 h HRT and 33 gCOD Ld OLR. Hydrogen production occurred throughout the experimental period with an average effluent pH of only 2.8. The FB, UG and UF-1 reactors presented volumetric hydrogen production rates (VHPR) of 95 ± 69, 45 ± 37 and 54 ± 32 mLH Lh, respectively; and H yields (HY) of 1.5 ± 0.8, 0.8 ± 0.6 and 1.2 ± 0.7 molH mol sucrose, respectively. The UF-1 reactor showed intermediate VHPR and HY, but no declining trend, contrary to what was observed in the FB reactor. Thus, aiming at continuous and long-term H production, a flocculent UASB was applied in the second experimental phase. In this phase, the HRT of the acidogenic reactor, which was named UF-2, was raised to 4.6 h, resulting in an OLR of 25 gCOD Ld. The VHPR and the HY increased considerably to 175 ± 44 mLH Lh and 3.4 ± 0.7 molH mol sucrose, respectively. These improvements were accompanied by greater sucrose removal, higher suspended biomass concentration, less production of lactate and more of acetate, and high ethanol concentration. Contradicting the current published literature data that reports strong inhibition of H production by dark fermentation at pH less than 4.0, the UF-2 reactor presented stable, long-term H production with satisfactory yields at pH 2.7 on average. 16 S rDNA sequencing revealed that two sequences assigned as Ethanoligenens and Clostridium accounted for over 70% of the microbiota in all the reactors. The non-necessity of adding alkalizing agents and the successful H production under very acid conditions, demonstrated in this study, open a new field of investigation in biological hydrogen production by dark fermentation towards a more sustainable and feasible technology.

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