A Comparison of Static and Shake-Flask Cultures for Filamentous Fungal Biomass and L-Carnitine Production in Rum Stillage

Abstract

This study investigated the potential of Bundaberg rum stillage as a low-cost medium for cultivating two food-related filamentous fungi: Aspergillus oryzae and Rhizopus oligosporus. Traditional shake-flasks and a novel static-liquid culture fermentation approach were used to assess biomass and L-carnitine synthesis. Stillage was assessed to determine the optimal concentration for use, and this was applied throughout. Growth was benchmarked against a complex synthetic medium containing glucose, yeast extract and malt extract. The highest biomass yields were obtained in the stillage supplemented with yeast extract, Aspergillus oryzae static culture (5.84 g/L), while R. oligosporus favoured agitated culture (4.82 g/L). Aspergillus oryzae produced the highest yield of L-carnitine (3.71 mg/g) in the stillage supplemented with ammonium in an agitated culture, while R. oligosporus synthesised 3.39 mg/g of L-carnitine under an agitated culture in the unsupplemented stillage. Volumetric yields of L-carnitine underscored the importance of aeration and nitrogen availability for metabolite synthesis, where both A. oryzae (15.8 mg/L) and R. oligosporus (12.7 mg/L) performed best in agitated ammonium-supplemented stillage. These findings demonstrate that the optimal cultivation strategy is product-specific, with static cultivation and yeast extract supplementation favouring fungal biomass production, while better aeration and an ammonium supplement improved L-carnitine yield. The results provide a basis for developing tailored bioprocesses for the commercial valorisation of rum stillage, enabling the sustainable production of fungal biomass and value-added metabolites while supporting circular bioeconomy approaches for distillery wastewater management.