Stillage valorization is a key aspect of cerealbased bioethanol production due to its significant economic value to first-generation biorefineries. In this study, whiterot fungi (wrf) were used to increase the nutritional characteristics of stillage from distiller's dried grains with solubles (DDGS), a by-product of the bioethanol industry. A ligninolytic fungus was selected from a screen of nine wrf strains, resulting in a 32 % increase in the protein content after secondary fermentation. Additionally, an important ligninolytic enzyme was produced from this treatment. A study of operational conditions, including the percentage of stillage in the culture medium, type of fermentation (agitated or static culture), and the scale-up from Erlenmeyer flasks to a 4 L bioreactor, was also conducted. © Springer Science+Business Media B.V. 2012.