| Summary: | 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 (H<sub>2</sub> and CH<sub>4</sub>) 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.
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