Carbon-Source Dependent Interplay of Copper and Manganese Ions Modulates the Morphology and Itaconic Acid Production in Aspergillus terreus

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.

The Role of Metal Ions in Fungal Organic Acid Accumulation

“…We thus consider it possible that-besides inhibiting manganese transport-Cu 2+ can induce the same symptoms of oxidative stress in the presence of manganese that manganese deficiency would cause. In line with this, it was recently described that the ratio of Cu 2+ to Mn 2+ -rather than their respective absolute concentration-determines micro-and macro-morphology of the A. terreus biomass and modulates itaconic acid production yield [32] on the glycolytic carbon sources D-glucose and D-fructose. Inclusion of the Cu 2+ ion concentration amongst the process variables even led to the formulation of growth media for the production of itaconic acid without the need to remove surplus Mn 2+ in advance [58].…”

“…Although it is-as with iron and zinc-required as an essential trace element in many biochemical reactions, it rapidly becomes toxic at concentrations where Fe, Zn and Mn do not, since it inactivates other metalloenzymes by metal displacement, and generates reactive oxygen species by the Fenton reaction [27][28][29] This toxicity of copper is also a means for the competition between organisms, e.g., the innate phagocyte utilizes copper for defence against microbes [30,31]. Sándor et al [32] have recently shown that copper toxicity in A. terreus is dependent on the carbon source and the concentration of Mn.…”

Bioreactor as the root cause of the “manganese effect” during Aspergillus niger citric acid fermentations

“…A high-yield citric acid fermentation (shake-flask or bioreactor alike) was defined as a culture reaching a Y p/s of >0.8 (Karaffa et al, 2021). Three aspects of mycelial morphology were monitored by microscopy: 1) swollen, yeast-like hyphae; 2) filamentous hyphae; and 3) pellet-like mycelial clumps (aggregates) (Bartoshevich et al, 1990;Paul and Thomasn, 1998;Sándor et al, 2021). To improve image contrast, lactophenol cotton blue (Fluka Chemie, Buch, Switzerland) at a final concentration of 10% (v/v) was added to the samples.…”

Recent Advances in Biotechnological Itaconic Acid Production, and Application for a Sustainable Approach

“…Several strains are much more efficient in producing AI after a genetic modification. Therefore, these strains, even if they are not originally IA producers, with the introduction of the relevant gene expressions can produce a high IA quantity from various substrates, not just from glucose but also from acetate, lignin, cellobiose, xylose, glycerol (biodiesel waste), and several other substrates [ 41 , 91 ].…”

“…This unsaturated organic acid is an advantageous platform chemical suitable for monomer or during monomer synthesis [ 39 , 40 ]. Important chemical properties can be indexed to the methylene groups’ conjugated double bonds that enable polymerization through condensation and esterification with distinct co-monomers by the carboxylic groups [ 41 , 42 ]. Owing to its multifaceted utilization ( Figure 1 ), these are enclosed in the leading 12 building block chemicals acquired from renewable biomass, agriculture-based wastes, food by-products, municipal solid wastes, or industrial by-products (i.e., glycerol) according to the US Department of Energy [ 39 ].…”