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Wastewater from the yeast production industry (WWY) is potentially harmful to surface water due to its high nitrogen and organic matter content; it can be used to produce compounds of higher commercial value, such as polyhydroxyalkanoates (PHA). PHA are polyester-type biopolymers synthesized by bacteria as energy reservoirs that can potentially substitute petrochemical-derived plastics. In this exploratory work, effluent from WWY was used to produce PHA, using a three-step setup of mixed microbial cultures involving one anaerobic and two aerobic reactors. First, volatile fatty acids (VFA; 2.5 g/L) were produced on an anaerobic batch reactor (reactor A) fed with WWY, using a heat pretreated sludge inoculum to eliminate methanogenic activity. Concurrently, PHA-producing bacteria were enriched using synthetic VFA in a sequencing batch reactor (SBR, reactor C) operated for 78 days. Finally, a polyhydroxybutyrate (PHB)-producing reactor (reactor B) was assembled using the inoculum enriched with PHA-producing bacteria and the raw and distilled effluent from the anaerobic reactor as a substrate. A maximum accumulation of 17% of PHB based on cell dry weight was achieved with a yield of 1.2 g PHB/L when feeding with the distilled effluent. Roche 454 16S rRNA gene amplicon pyrosequencing of the PHA-producing reactor showed that the microbial community was dominated by the PHA-producing bacterial species Paracoccus alcalophilus (32%) and Azoarcus sp. (44%). Our results show promising PHB accumulation rates that outperform previously reported results obtained with real substrates and mixed cultures, demonstrating a sustainable approach for the production of PHA less prone to contamination than a pure culture.