It has been proposed that the microbial bioproduction of chemical precursors could reduce societal overreliance on the petrochemical industry. However, economic implementation remains elusive due to a lack of understanding of the mechanisms involved. The current study evaluated malic acid production using a novel immobilized Aspergillus oryzae fungal bed bioreactor. The experimental procedures involved two different nitrogen sources (urea and (NH4)2SO4), two‐phase growth/production and simultaneous single‐step growth/production runs, the presence and absence of added CO2, the presence and absence of pH control, and the use of glucose and dl‐malate as the carbon sources. The experimental malic acid titers were remarkably poor (between 1.7 and 3.3 g.L−1) in comparison with CaCO3 buffered malic acid titers from literature (concentrations in excess of 100 g.L−1 have been reported reported). In contrast, citric acid was observed to be the main metabolite. The dl‐malate carbon source was consumed with concomitant growth, indicating A. oryzae′s ability to utilize both d and l enantiomers of malic acid. Detailed metabolic flux analyses predicted partial consumption of urea during growth, while (NH4)2SO4 was fully utilized. During the urea runs, significant amounts of glycogen were accumulated in the biomass – which were utilized as substrate during the production run. Nitrogen limitations induced significant lipid production, corresponding to citric acid accumulation in the media; the mechanism for fungal lipid biosynthesis involves citric acid excretion at the expense of malic acid. These results indicate a possible calcareous trigger for malic acid production as opposed to the traditionally accepted nitrogen starvation mechanism – nitrogen starvation resulted in lipid accumulation with corresponding malate loss. © 2022 The Authors. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd.
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