Advancing Malic Acid Production of Aspergillus oryzae: Combined Process Optimization and Application of Various Substrates

In light of climate change and growing resource scarcity, microbial production of organic acids offers a sustainable alternative to fossil-based chemical synthesis. In this study, malic acid production by Aspergillus oryzae was optimized through cultivation temperature adjustment and biotin supplementation, while organic acid formation from various carbon sources was systematically characterized. The process conditions applied during substrate screening, including pH control using Na2CO3 and NaOH, Zn2+ supplementation and hypoxia, were based on previously established strategies to stimulate malic acid production. Cultivation at 35°C increased respiratory activity compared to 32°C, resulting in an average productivity of 0.17 g L-1 h-1. Biotin supplementation enhanced productivity by 20% and increased the carbon yield, defined as the proportion of consumed carbon recovered in malic acid, by 5%. Under optimized cultivation conditions, the highest malic acid productivity was achieved in cultivation with glucose as substrate and Na2CO3 as pH-neutralizing agent, reaching 57.57 g L-1 malic acid with a yield of 0.66 g g-1 and an overall productivity of 0.24 g L-1 h-1, while fructose and glycerol resulted in substantially lower productivities. Furthermore, we demonstrate the ability to perform carbon balancing even in the presence of carbonate-based neutralizing agents. This is achieved by quantifying and subtracting the CO2 generated during neutralization reactions from the total emissions, enabling precise determination of microbial CO2 production and calculation of carbon yields. By systematically combining optimization strategies reported in previous studies, this work achieves productivity and carbon efficiency exceeding those of the individual approaches reported so far.