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April 26, 2026Open Access

Dark co-fermentation of food waste and agro-industrial wastewaters: A water-saving approach fostering industrial symbiosis and local circularity for renewable hydrogen production

Key Points

This research aims to assess the continuous dark co-fermentation of a ternary mixture for renewable hydrogen production.
Lab-scale continuous stirred-tank reactor operated for 39 days under mesophilic conditions at controlled pH (6.5) and stirring (300 rpm).
Evaluated four hydraulic retention times (HRTs): 16, 12, 9, and 6 hours under pseudo-steady-state conditions.
Conducted microbial analysis and PCA to evaluate the performance of H2 production.
At 9 h HRT, the volumetric H2 production rate reached 5.4 ± 0.5 NL H2/L-d and the yield was 48.7 ± 2.1 NmL H2/g VS added.
Dominance of Lactobacillus and Megasphaera was noted at optimal HRT, influencing hydrogen production.
Excessive reduction to 6 h HRT shifted metabolism towards acetate and propionate, lowering H2 production rates.

Abstract

Dark co-fermentation of mixed wastes delivers the benefits of multi-feed processing and enables co-production of renewable hydrogen (H 2 ) and value-added organic acids. This study investigated the continuous dark co-fermentation of a ternary mixture not previously explored for H 2 production, consisting of 25% food waste, 25% cheese whey, and 50% brewery wastewater (v/v), selected to obtain a feed with a volatile solids content close to 5%, in a lab-scale continuous stirred-tank reactor. The reactor was operated for 39 days under mesophilic conditions, at controlled pH (6.5) and with stirring at 300 rpm. Four hydraulic retention times (HRTs), namely 16, 12, 9, and 6 h, were evaluated under successive pseudo-steady-state conditions. Reducing the HRT from 16 to 9 h increased the volumetric H 2 production rate to 5.4 ± 0.5 NL H 2 /L-d and the H 2 yield to 48.7 ± 2.1 NmL H 2 /g VS added . Efficient operation at 9 h HRT aligned with an acetate-butyrate profile, whereas further reduction to 6 h shifted metabolism toward acetate and propionate and markedly reduced butyrate concentrations. Microbial analysis revealed a lactate producer-consumer consortium dominated by Lactobacillus and Megasphaera , whose relative contribution shifted with HRT. PCA, correlation analysis, and a soluble COD balance indicated that H 2 performance at 9 h HRT was associated with lactate-supported activity and butyrate-linked pathways, whereas excess lactate accumulation and the acetate-propionate route governed performance losses at 6 h HRT. These findings demonstrate the feasibility of high-rate H 2 production with this ternary mixture while partially substituting freshwater during feed preparation. • Unified cheese whey-brewery wastewater-food waste enabled high-rate H 2 production • The highest H 2 production rate (HPR), 5.4 NL H 2 L-d, was obtained at 6 h HRT • High-rate H 2 production co-occurred with Lactobacillus-Megasphaera dominance • Excessively short HRT (6 h) increased acetate/propionate and lowered HPR

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