Dark fermentation (DF) is a promising process for mitigating environmental pollution and producing “green” H2. However, wider implementation and scaling of this technology is hampered by insufficient process efficiency. In this work, for the first time, the effect of innovative pretreatment of cheese whey (CW) in a vortex layer apparatus (VLA) on characteristics and DF of CW was studied. Pretreatment in VLA resulted in a heating of the CW, slight increase in pH, volatile fatty acids, iron, and reduction in fat, sugar, and chemical oxygen demand (COD). The biochemical hydrogen potential test and analysis of H2 production kinetics confirmed the significant potential of using VLA in enhancement of dark fermentative H2 production. The maximum potential H2 yield (202.4 mL H2/g COD or 3.4 mol H2/mol hexose) was obtained after pretreatment in VLA for 45 s and was 45.8% higher than the control. The maximum H2 production rate after 5 and 45 s of pretreatment was 256.5 and 237.2 mL H2/g COD/d, respectively, which is 8.06 and 7.46 times higher than in the control. The lag phase was more than halved as a function of the pretreatment time. The pretreatment time positively correlated with the total final concentration of Fe2+ and Fe3+ and negatively with the lag phase, indicating a positive effect of pretreatment in VLA on the start of H2 production.
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