Effect of process mode, nitrogen source and temperature on L-malic acid production with Aspergillus oryzae DSM 1863 using acetate as carbon source

Abstract

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.

Optimization of Rhodococcus erythropolis JCM3201T Nutrient Media to Improve Biomass, Lipid, and Carotenoid Yield Using Response Surface Methodology

“…Complex nitrogen sources provide additional nutrients, including amino acids, polypeptides, vitamins, and trace elements. Increased yield of biomass with complex nitrogen sources was achieved for a variety of micro-organisms, including C. oleaginosus and A. oryzae [ 27 , 33 ]. When cultivating the R. erythropolis strains MTCC1548, 2794, and 3951 using different nitrogen sources, inorganic nitrogen sources (ammonium sulfate and ammonium phosphate), as well as urea, resulted in very low biomass compared to complex organic nitrogen sources (yeast extract and meat peptone) [ 29 ].…”

Integrated fermentative production and downstream processing of L-malic acid by Aspergillus wentii using cassava peel waste

“…The A. oryzae, A. flavus, and A. wentii strains produced high yields of L-MA, using glucose as the substrate. A. oryzae is a widely studied species for L-MA production (Kövilein et al, 2022), and the production of aflatoxins delimits the intensive applications of A. flavus despite its high L-MA titers (Dai et al, 2018).…”

“…Similar behavior has been reported for other MA-producing microorganisms. For instance, a media pH of 5.5 has been reported as optimal for L-MA production in studies conducted using A. oryzae (Kövilein et al, 2022). Zambanini et al (2016) showed that for efficient malate synthesis by Ustilago trichophora TZ1, a pH higher than 5.4 is required.…”

“…In the quest for exploring alternative carbon sources for L-MA production, most studies have been limited to crude glycerol which showed substantial results (Bharathiraja et al, 2022;Selvakumari et al, 2021). Some reports demonstrated the use of renewable raw materials like acetate and xylose in their pure form (Kang et al, 2022;Kövilein et al, 2022). Only a few research have focussed on the use of readily available agroindustrial substrates to achieve low-cost L-MA production with waste valorization (Ding and Ye, 2023;Wu et al, 2022).…”