Background Microbial malic acid production is currently not able to compete economically with well-established chemical processes using fossil resources. The utilization of inexpensive biomass-based substrates containing acetate could decrease production costs and promote the development of microbial processes. Acetate is a by-product in lignocellulosic hydrolysates and fast pyrolysis products or can be synthesized by acetogens during syngas fermentation. For the fermentation of these substrates, a robust microorganism with a high tolerance for biomass-derived inhibitors is required. Aspergillus oryzae is a suitable candidate due to its high tolerance and broad substrate spectrum. To pave the path towards microbial malic acid production, the potential of acetate as a carbon source for A. oryzae is evaluated in this study. Results A broad acetate concentration range was tested both for growth and malic acid production with A. oryzae. Dry biomass concentration was highest for acetic acid concentrations of 40–55 g/L reaching values of about 1.1 g/L within 48 h. Morphological changes were observed depending on the acetate concentration, yielding a pellet-like morphology with low and a filamentous structure with high substrate concentrations. For malic acid production, 45 g/L acetic acid was ideal, resulting in a product concentration of 8.44 ± 0.42 g/L after 192 h. The addition of 5–15 g/L glucose to acetate medium proved beneficial by lowering the time point of maximum productivity and increasing malic acid yield. The side product spectrum of cultures with acetate, glucose, and cultures containing both substrates was compared, showing differences especially in the amount of oxalic, succinic, and citric acid produced. Furthermore, the presence of CaCO3, a pH regulator used for malate production with glucose, was found to be crucial also for malic acid production with acetate. Conclusions This study evaluates relevant aspects of malic acid production with A. oryzae using acetate as carbon source and demonstrates that it is a suitable substrate for biomass formation and acid synthesis. The insights provided here will be useful to further microbial malic acid production using renewable substrates.

Malic acid, mainly used as acidulant and taste enhancer in the food industry, is currently produced from fossil resources. In this study, microbial L-malate production with the filamentous fungus A. oryzae using the carbon source acetate was evaluated. Acetate is for example contained in biomass-derived substrates such as lignocellulosic hydrolysates and condensates of fast pyrolysis, thus avoiding competition with food production. Since research on malic acid synthesis from acetate is limited and reported productivities and yields are low, this work aimed to improve the process. First, different cultivation temperatures were tested. This parameter was found to affect the ratio between malic and succinic acid, which is the major by-product of organic acid production with A. oryzae. At 32°C, the malate share was highest (53.7 ± 1.6%), while it was lowest at 38°C (43.3 ± 1.1%) whereas succinate represented the main product (51.5 ± 1.0%). Besides the temperature, the type of nitrogen source was also found to affect malate synthesis as well as biomass production. In the pre-culture, the biomass concentration was increased by a factor of 3.4–3.9, and germination started earlier with the complex nitrogen sources yeast extract, casein hydrolysate and peptone compared to the defined nitrogen source (NH4)2SO4. Especially with yeast extract, malate synthesis in the main culture was accelerated and the titer obtained after 48 h was about 2.6 times higher than that quantified with (NH4)2SO4. To reduce substrate inhibition in acetate medium, fed-batch and repeated-batch processes were evaluated using (NH4)2SO4 or yeast extract as nitrogen source. In the fed-batch process, the period of malate production was extended, and the maximum product concentration was increased to 11.49 ± 1.84 g/L with (NH4)2SO4 and 12.08 ± 1.25 g/L with yeast extract. In the repeated-batch process, the total acid production was highest within the first 240 h of fermentation, but optimization is required to maintain high production rates in later cycles. The lessons learned in this study will help in the development of further process strategies to maximize malate production using acetate as alternative substrate to the commonly used glucose.