Itaconic acid is a five-carbon dicarboxylic acid with an unsaturated alkene bond, frequently used as a building block for the industrial production of a variety of synthetic polymers. It is also one of the major products of fungal "overflow metabolism" which can be produced in submerged fermentations of the filamentous fungus Aspergillus terreus. At the present, molar yields of itaconate are lower than those obtained in citric acid production in Aspergillus niger. Here, we have studied the possibility that the yield may be limited by the oxygen supply during fermentation and hence tested the effect of the dissolved oxygen concentration on the itaconic acid formation rate and yield in lab-scale bioreactors. The data show that a dissolved oxygen concentration of 2% saturation was sufficient for maximal biomass formation. Raising it to 30% saturation had no effect on biomass formation or the growth rate, but the itaconate yield augmented substantially from 0.53 to 0.85 mol itaconate/mol glucose. Furthermore, the volumetric and specific rates of itaconic acid formation ameliorated by as much as 150% concurrent with faster glucose consumption, shortening the fermentation time by 48 h. Further increasing the dissolved oxygen concentration over 30% saturation had no effect. Moreover, we show that this increase in itaconic acid production coincides with an increase in alternative respiration, circumventing the formation of surplus ATP by the cytochrome electron transport chain, as well as with increased levels of alternative oxidase transcript. We conclude that high(er) itaconic acid accumulation requires a dissolved oxygen concentration that is much higher than that needed for maximal biomass formation, and postulate that the induction of alternative respiration allows the necessary NADH reoxidation ratio without surplus ATP production to increase the glucose consumption and the flux through overflow metabolism.

Itaconic acid is used as a bio-based, renewable building block in the polymer industry. It is produced by submerged fermentations of the filamentous fungus Aspergillus terreus from molasses or starch, but research over the efficient utilization of non-food, lignocellulosic plant biomass is soaring. The objective of this study was to test whether the application of two key cultivation parameters for obtaining itaconic acid from D -glucose in high yields – Mn 2+ ion deficiency and high concentration of the carbon source – would also occur on D -xylose, the principal monomer of lignocellulose. To this end, a carbon and energy balance for itaconic acid formation was established, which is 0.83 moles/mole D -xylose. The effect of Mn 2+ ions on itaconic acid formation was indeed similar to that on D -glucose and maximal yields were obtained below 3 μg L –1 Mn 2+ ions, which were, however, only 0.63 moles of itaconic acid per mole D -xylose. In contrast to the case on D -glucose, increasing D -xylose concentration over 50 g L –1 did not change the above yield. By-products such as xylitol and α-ketoglutarate were found, but in total they remained below 2% of the concentration of D -xylose. Mass balance of the fermentation with 110 g L –1 D -xylose revealed that >95% of the carbon from D -xylose was accounted as biomass, itaconic acid, and the carbon dioxide released in the last step of itaconic acid biosynthesis. Our data show that the efficiency of biomass formation is the critical parameter for itaconic acid yield from D -xylose under otherwise optimal conditions.

The effects of the interplay of copper(II) and manganese(II) ions on growth, morphology and itaconic acid formation was investigated in a high-producing strain of Aspergillus terreus (NRRL1960), using carbon sources metabolized either mainly via glycolysis (D-glucose, D-fructose) or primarily via the pentose phosphate shunt (D-xylose, L-arabinose). Limiting Mn2+ concentration in the culture broth is indispensable to obtain high itaconic acid yields, while in the presence of higher Mn2+ concentrations yield decreases and biomass formation is favored. However, this low yield in the presence of high Mn2+ ion concentrations can be mitigated by increasing the Cu2+ concentration in the medium when D-glucose or D-fructose is the growth substrate, whereas this effect was at best modest during growth on D-xylose or L-arabinose. A. terreus displays a high tolerance to Cu2+ which decreased when Mn2+ availability became increasingly limiting. Under such conditions biomass formation on D-glucose or D-fructose could be sustained at concentrations up to 250 mg L–1 Cu2+, while on D-xylose- or L-arabinose biomass formation was completely inhibited at 100 mg L–1. High (>75%) specific molar itaconic acid yields always coincided with an “overflow-associated” morphology, characterized by small compact pellets (<250 μm diameter) and short chains of “yeast-like” cells that exhibit increased diameters relative to the elongated cells in growing filamentous hyphae. At low concentrations (≤1 mg L–1) of Cu2+ ions, manganese deficiency did not prevent filamentous growth. Mycelial- and cellular morphology progressively transformed into the typical overflow-associated one when external Cu2+ concentrations increased, irrespective of the available Mn2+. Our results indicate that copper ions are relevant for overflow metabolism and should be considered when optimizing itaconic acid fermentation in A. terreus.

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