BackgroundCarboxylates like volatile fatty acids (VFAs) can be produced by acidogenic fermentation (AF) of dairy wastes like cheese whey, a massive residue produced at 160.67 million m3 of which 42% are not valorized and impact the environment. In mixed‐culture fermentations, selection pressures can favor AF and halt methanogenesis. In this study, inoculum pre‐treatment was evaluated as a selective pressure for AF demineralized cheese whey in batches. Alkaline (NaOH, pH 8.0, 6 h) and thermal (90°C for 5 min, ice‐bath until 23°C) pre‐treatments, were tested with batch operations runs at initial pH 7.0 and 9.0, food‐to‐microorganism (F/M) ratios of 0.5 to 4.0 g COD g‐1 VS, and under pressurized and non‐pressurized headspace, in experiments duplicated in two different research institutes.ResultsAcetic acid was highly produced on both Unicamp and TU Delft samples (1.36 and 1.40 g CODAcOH L‐1, respectively), at the expense of methanogenesis by combining a thermal pre‐treatment of inoculum with a non‐pressurized batch operation started at pH 9.0. Microbial communities comprised of VFAs and alcohol producers, such as Clostridium, Fonticella, and Intestinimonas, and fermenters such as Longilinea and Leptolinea. Communities also presented the lipid‐accumulating and bulk and foaming Candidatus Microthrix and the metanogenic Methanosaeta regardless of no methane production. An F/M ratio of 0.5 g COD g‐1 VS led to the best VFA production of 1,769.4 mg L‐1.ConclusionOverall, inoculum thermal pre‐treatment, initial pH 9.0, and non‐pressurized headspace acted as a selective pressure for halting methanogenesis and producing VFAs, valorizing cheese whey via batch acidogenic fermentation.This article is protected by copyright. All rights reserved.