Optimization and Scale-Up of Tuber spp. Growth on Olive Mill Wastewater and Expired Glucose Syrup Substrates

The present study investigates the potential of olive mill wastewater (OMW), supplemented with expired commercial glucose syrup, as a sustainable substrate for the submerged cultivation of Tuber spp. wild mushrooms. OMW contains considerable quantities of phenolic compounds, making it both a challenging pollutant and a promising nutrient source. To assess fungal performance under increasing phenolic stress, culture media were prepared with varying OMW concentrations (0–75% v / v on agar; 0–50% v / v in liquid media), while glucose was adjusted to ~30 g/L using expired glucose syrup. A sequential experimental approach was followed, beginning with Petri dish screenings on substrate/strain selection (measuring the mycelial growth rate; Kr, mm/day), progressing to 25-day shake flask fermentations and subsequently scaling up the most promising strain ( Tuber mesentericum ) in a controlled stirred-tank bioreactor. Throughout cultivation, substrate consumption (glucose, phenolics), pH evolution and decolorization were evaluated, while the resulting biomass was analyzed for polysaccharides, β-glucans, proteins, lipids, fatty acids, antioxidants, phenolic acids and triterpenoids content. Results showed that increasing OMW concentration enhanced tolerance and metabolic activity in selected Tuber species, with T. mesentericum exhibiting the highest resilience and achieving comparable or higher biomass yields in OMW-based media than in glucose (control). Phenolic removal exceeded 60% in flasks and 50% in the bioreactor, confirming simultaneous bioremediation capacity. Bioreactor cultivation demonstrated efficient substrate utilization and biomass production, while OMW-grown biomass presented high lipid content, enriched with unsaturated fatty acids, high β-glucan levels and increased antioxidant and phenolic profiles. Overall, this study demonstrates that OMW (supplemented with expired glucose syrup) can serve as a cost-effective and environmentally beneficial substrate for Tuber biomass production with dietary and antioxidant properties, offering an alternative source to mushroom carposomes, as well as supporting the circular bioeconomy strategies within olive oil processing industries.