Biohydrogen production from lactate derived via co-fermentation of cassava starch wastewater and glycerol in a continuous multiple tube reactor

This study evaluated biohydrogen production in a continuous multiple tube reactor (CMTR) using a lactic acid-rich substrate derived from the co-fermentation of cassava starch wastewater (CSW) with glycerol. The process had two stages: (i) lactic acid (LA) production in an anaerobic sequential batch reactor (ASBR); and (ii) use of the LA-rich substrate in the CMTR at different organic loading rates (OLRs): 48, 72, and 96 g COD L⁻¹ d⁻¹, with a fixed hydraulic retention time of 4 h. The lactic fermentation produced a homogeneous substrate with 41% LA and 52% glycerol, suitable for hydrogen generation. CMTR performance varied with OLR: the highest OLR (96 g COD L⁻¹ d⁻¹) resulted in the greatest volumetric hydrogen production rate (1,960.3 mL H₂ L⁻¹ d⁻¹), biogas flow (9,360.9 mL d⁻¹), and COD removal (41.8%). The intermediate OLR (72 g COD L⁻¹ d⁻¹) achieved the highest hydrogen yield (8.4 mmol H₂ g⁻¹ COD), along with 95% lactic acid and 65% glycerol conversion. Metabolite profiling reinforced LA's role as a strategic substrate in promoting efficient fermentative routes, indicating a selective shift toward the butyric pathway, where lactic and acetic acids are converted into butyric acid and hydrogen. Overall, the results demonstrate that lactic pre-fermentation of CSW and glycerol produces a viable substrate for biohydrogen production, enabling the application of elevated OLRs and maintaining a pH favourable to hydrogenogenic microbial activity. The CMTR proved to be a promising system for agro-industrial waste valorisation through sustainable hydrogen generation.