Abstract
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.
Highlights
Itaconic acid (2-methylenesuccinic acid, 1-propene-2,3dicarboxylic acid) is an unsaturated, weak diprotic acid
We investigated the influence of Cu2+ ions on the growth, morphology and itaconic acid formation in the high-titer itaconic acid producer Aspergillus terreus NRRL1960 on D-glucose, D-xylose, L-arabinose and an alternative glycolytic carbon source, D-fructose
To ensure that the major physical parameters of the two cultivation systems were essentially identical, and that fermentation kinetics would be independent of the vessel type, protocols for monitoring pH and Dissolved oxygen (DO) were applied as previously described by us (Kolláth et al, 2019)
Summary
Itaconic acid (2-methylenesuccinic acid, 1-propene-2,3dicarboxylic acid) is an unsaturated, weak diprotic acid. In Aspergilli, manganese deficiency transforms filamentous hyphal morphology to one dominated by “yeast-like cells” (branches of short, swollen forms) on the micro-morphology-, and small compact (75%) specific molar itaconic acid yields (Yp/s) occur only when cultures overwhelmingly comprise such morphology ( referred to as “overflow-associated morphology”), improving the rheology of the culture broth, which in turn results in increased oxygen transfer throughout the fermentation (Kuenz et al, 2012; Karaffa et al, 2015)
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