Oxidative stress and toxicology of Cu²⁺ based on surface areas in mixed cultures of green alga and cyanobacteria: The pivotal role of H₂O₂

The toxicity of heavy metals in algal monocultures is well studied and is mediated by reactive oxygen and nitrogen species (ROS/RNS). However, little is known about the toxicity of heavy metals and the mechanisms involved in mixed cultures. Here we examine the oxidative stress and toxic effects of Cu²⁺ on the green alga Dunaliella salina (DS) and the cyanobacteria Synecochoccus elongatus (SE) in both mono- and mixed cultures. We find that both species benefit in mixed cultures and acquire higher resistance to Cu²⁺ toxicity, with a particularly marked effect on SE. DS has a larger surface area than SE, so increases in the number of DS cells compared to SE diminishes the proportion of SE surface area exposed to Cu²⁺, and contributes to increasing cyanobacterial resistance in mixed cultures. However, these mixed cultures also display as an unexpected property an increased resistance of DS in mixed cultures. SE and DS cells showed significant differences on the kinetics of H₂O₂ production and antioxidant capacities. The integrated (overall) redox response of mixed cultures, in terms of total amount of H₂O₂ produced, was proportional to the total surface area of algal species exposed to Cu²⁺, independent of algal composition in mixed systems. However, mixed cultures display emergent properties, as the time course of H₂O₂ accumulation is not a simple function of the composition of the mixed cultures. Emergent properties are also observed in the speed of membrane lipid oxidation by the two species, as measured using mixed cultures in which only one of the two species is labeled using the membrane oxidation indicator C₁₁-BODIPY^581/591. We suggest that, in addition to H₂O₂¸ other redox signals (e.g. NO) and allelochemicals (auxins, cytokinins, etc.) may be used to construct a complex inter-species communication network. This could allow mixed algal systems, whatever their composition, to integrate their cellular responses and perform as a coherent unit against toxic Cu²⁺ ions.