Alterations in nitrogen metabolism caused by heavy metals in the acid-tolerant microalga Coccomyxa onubensis

The microalga Coccomyxa onubensis is an extremophile microorganism with a unique ecosystem (Río Tinto, Huelva, Spain) that contains high amounts of contaminants, including heavy metals, sulphates, and nitrates, in acidic environments (pH 2.5). The current work presents an evaluation of the capacity of Coccomyxa onubensis to assimilate different nitrogen sources under Cu²⁺, Cd²⁺, AsO₃³⁻, AsO₄³⁻ and Hg²⁺ stress, and the metabolic implications of these stressors. The results showed that ammonium consumption was less affected than nitrate consumption when microalgae were cultivated with heavy metals (except cadmium). The activities of enzymes involved in nitrogen metabolism, such as nitrite reductase (NiR; EC:1.7.7.1), glutamine synthetase (GS; EC:6.3.2.1) and glutamate dehydrogenase (GDH; EC:1.4.1.2) were characterised to determine the Michaelis-Menten constant (K_m) and optimal temperature and pH values, being 45, 40 and 60 °C and pH values of 7.5, 6.0 and 9.0 for NiR, GS, and GDH, respectively. The effects of different heavy metals on these enzymes were assessed at multiple levels, and the results showed that the enzymatic activity of NiR was downregulated, specially under copper stress, maintaining 23 % of control NiR activity at 2 mM Cu²⁺. The enzymatic activity of GS was upregulated at low concentrations under cadmium and mercury stress (115–120 % of control cultures GS activity at 25 μM Cd²⁺ and 50 nM Hg²⁺, respectively) and downregulated at high concentrations of these elements. GDH activity was upregulated in the presence of Cu²⁺, Cd²⁺, and Hg²⁺, with increases up to 192, 155 and 154 % at 1 mM Cu²⁺, 300 μM Cd²⁺, and 250 nM Hg²⁺, respectively. These results provide a better explanation of the effects of heavy metal stress on N metabolism in Coccomyxa onubensis, which may be used as a model eukaryotic organism of the Tinto River acidophilic ecosystem.