| Summary: | Dark fermentation (DF) is a promising process for mitigating environmental pollution and producing “green” H<sub>2</sub>. 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 H<sub>2</sub> production kinetics confirmed the significant potential of using VLA in enhancement of dark fermentative H<sub>2</sub> production. The maximum potential H<sub>2</sub> yield (202.4 mL H<sub>2</sub>/g COD or 3.4 mol H<sub>2</sub>/mol hexose) was obtained after pretreatment in VLA for 45 s and was 45.8% higher than the control. The maximum H<sub>2</sub> production rate after 5 and 45 s of pretreatment was 256.5 and 237.2 mL H<sub>2</sub>/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 Fe<sup>2+</sup> and Fe<sup>3+</sup> and negatively with the lag phase, indicating a positive effect of pretreatment in VLA on the start of H<sub>2</sub> production.
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