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Sugarcane stillage is an abundant wastewater from ethanol production. It has drawn considerable interest as a potential feedstock for biotechnological processes aiming at the recovery of energy and value-added products. In this study we explored the use of stillage for the production of polyhydroxyalkanoates (PHA), which are microbial bioplastics with potential to replace conventional plastics in specific applications. As stillage is nutrient deficient, we investigated the impact of external nutrient supplementation on the selection of PHA-producing mixed cultures. Cultures selected under four different total-chemical-oxygen-demand-to-nitrogen ratios (COD.t/N) (15, 30, 40 and 80) were evaluated according to their PHA production performance and microbial composition. The experimental results demonstrated that the most favorable PHA production was achieved for the biomass selected under strict carbon-limiting conditions, with a COD.t/N ratio of 15. This resulted in an overall PHA volumetric productivity of 2.10 g PHA L−1 d−1, a maximum intracellular PHA content of 0.65 g PHA g VSS−1 and a PHA storage yield of 0.71 g COD PHA g COD H.Org −1. The resulting PHA was a copolymer of 3-hydroxybutyrate (73 %mol) and 3-hydroxyvalerate (27 %mol) monomers. The microbial consortium was enriched by members of the Brevundimonas genus, known PHA producers, with a relative abundance of 26.4 %. • Optimal biomass enrichment occurred under strict carbon-limiting conditions. • Nutrient supplementation increased volumetric PHA productivity by 4.5-fold. • Nutrient supplementation increased HV monomer content in the PHA. • The microbial consortium was enriched in Brevundimonas genus (26.4 % abundance). [ABSTRACT FROM AUTHOR]