This article presents the results of the start-up of continuous production of biohydrogen from cheese whey (CW) in an anaerobic filter (AF) and anaerobic fluidized bed (AFB) with a polyurethane carrier. Heat and acid pretreatments were used for the inactivation of hydrogen-scavengers in the inoculum (mesophilic and thermophilic anaerobic sludge). Acid pretreatment was effective for thermophilic anaerobic sludge to suppress methanogenic activity, and heat treatment was effective for mesophilic anaerobic sludge. Maximum specific yields of hydrogen, namely 178 mL/g chemical oxygen demand (COD) and 149 mL/g COD for AFB and AF, respectively, were obtained at the hydraulic retention time (HRT) of 4.5 days and organic load rate (OLR) of 6.61 kg COD/(m³ day). At the same time, the maximum hydrogen production rates of 1.28 and 1.9 NL/(L day) for AF and AFB, respectively, were obtained at the HRT of 2.02 days and OLR of 14.88 kg COD/(m³ day). At the phylum level, the dominant taxa were Firmicutes (65% in AF and 60% in AFB), and at the genus level, Lactobacillus (40% in AF and 43% in AFB) and Bifidobacterium (24% in AF and 30% in AFB).

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.

Spatial separation into acidogenic and methanogenic stages is considered a viable option to ensure process stability, energy efficiency and the better control of key anaerobic digestion (AD) parameters. The elucidation of the optimal modes of two-stage AD for the maximization of the recovery of biofuels (H₂ and CH₄) is still an urgent task, the main optimization criteria being the highest energy yield (EY) and energy production rate (EPR). In this work, a response surface methodology was used for an optimization of energy production from the two-stage mesophilic–thermophilic AD of cheese whey (CW). Three dilution rates of CW, providing values of 10.9, 14.53 and 21.8 g for the chemical oxygen demand (COD)/L in the influent and three hydraulic retention times (HRTs) (1, 2 and 3 days) in methanogenic biofilters at a constant HRT in an acidogenic biofilter of 0.42 days, were tested to optimize the EY and EPR. The desirability approach produced combined optimum conditions as follows: the dilution rate of the CW provided 17.58 g COD/L (corresponding to OLR of 6.5 g COD/(L·day)) in the influent and a HRT in the methanogenic biofilter of 2.28 days, both of which provided a maximum EPR of 80.263 kJ/(L·day) and EY of 9.56 kJ/g COD, with an overall desirability value of 0.883.

For the first time, the effect of thickened digestate treatment in a vortex layer apparatus (VLA) prior to its recirculation into anaerobic reactor on the efficiency of anaerobic bioconversion of an organic substrate was investigated. At the hydraulic retention time of the recirculated digestate in VLA for 30 seconds, of the substrate in the anaerobic bioreactor for 8 days and the recirculation coefficient of 1.5, the average specific yield of biogas increased by 75.2%, and the average volumetric methane production – by 16.8%. There was no increase in methane concentration in biogas, while the average concentration of hydrogen sulfide in biogas decreased by 8 times.