Influence of Zn$^{2+}$ and Oxygen Supply on Malic Acid Production and Growth of Aspergillus oryzae

Malic acid is a valuable platform chemical traditionally derived from fossil-based resources. Microbial cultivation with Aspergillus oryzae offers a sustainable alternative based on renewable feedstocks. In this study, a well-established minimal medium for malic acid production, commonly used in previous research to ensure reproducibility, was employed. Despite its widespread use, respiration monitoring combined with stepwise nutrient pulsing unexpectedly revealed a nutrient depletion after 8 h of cultivation. Zn$^{2+}$ limitation was identified through a combination of respiration monitoring and systematic stepwise nutrient pulsing. Supplementation of Zn$^{2+}$ increased oxygen consumption, leading to hypoxic conditions. This induced hypoxia enhanced malic acid production and influenced the overall organic acid profile. Different dynamic oxygen concentration strategies were tested to evaluate their effect on malic acid productivity, showing that allowing growth into hypoxia and maintaining hypoxia throughout the production phase resulted in the best performance. By combining Zn$^{2+}$ supplementation, maintaining a culture pH of 7.00 and Zn$^{2+}$-induced hypoxia, final malic acid concentrations were elevated from 31.44 g L$^{-1}$ to 45.28 g L$^{-1}$, with a yield of 0.61 g malic acid per g of glucose and an average productivity of 0.19 g L$^{-1}$ h$^{-1}$.