Enhanced biohydrogen production from cheese whey: integrating dark fermentation and microbial electrolysis

Background information Cheese whey (CW) is a byproduct of curd recovery during milk coagulation, generated at a rate of approximately 9–10 liters per kilogram of cheese produced. With a chemical oxygen demand (COD) often exceeding 100 gCOD/L, it poses significant environmental challenges. Dark fermentation is an anaerobic bioprocess that enables the conversion of CW sugars, such as lactose, and other compounds into volatile fatty acids (VFAs) and hydrogen (H₂). While H2 is a green energy carrier that can be directly converted into electrical power using fuel cells, VFAs can be further exploited for chemical syntheses, such as biopolymer production, or for additional H₂ generation in microbial electrolysis cells (MECs). Main results In this study, we investigated the feasibility of novel biotechnological CW treatment train consisting of two sequential steps: (i) an initial dark fermentation step, carried out in presence of electrically conductive materials (ECMs) to enhance VFA production, followed by the (ii) treatment of the fermented effluent in a MEC, where the bioelectrochemical oxidation of VFAs at the anode supplies electrons for H₂ generation at the cathode. To this end, undiluted CW was fermented at 35°C in 250-mL stirred anaerobic bioreactors in presence of different types of ECMs (i.e., biochar, magnetite, and graphite). Notably, a significant shift in product composition was observed: VFAs (mainly propionic acid) increased up to twenty-fold in presence of ECMs compared to the unamended controls, where lactic acid was the predominant product. ECMs likely promoted the reduction of lactic acid into propionate by facilitating interspecies electron transfer processes. The VFA-rich fermented effluent was then used to feed the anodic compartment (0.5 L) of a custom-designed dual-chamber continuous-flow bioelectrochemical reactor. Electrical current, VFAs concentration and H₂ production at the cathode were continuously monitored. Additionally, the potential use of the photosynthetic microorganism Rhodopseudomonas palustris sp. as a cathodic biocatalyst for light-driven H₂ production was also explored. Conclusions Overall, our findings indicate that this sequential treatment represents a promising strategy to maximize biohydrogen production from CW, towards a circular bioeconomy approach.