Adaptive mechanisms to Cr(VI) stress: Investigation on terrestrial/freshwater algae isolated in a chromingenous waterspring on Mount Prinzera (PR, Italy)

Sulfate metabolism plays a crucial role in enhancing cellular defense against heavy metals, through the production of cysteine (Cys), reduced glutathione (GSH), and phytochelatins. These sulfur-containing molecules significantly contribute to the onset of the phenomenon known as Sulfur Enhanced Defense (SED) (Rausch and Watcher 2005), both by directly chelating metal ions, including Cr(VI), and by scavenging metalinduced oxidative damage. In previous studies, we observed that different sulfate accumulation and cysteine production underlie the varying Cr(VI) sensitivity in two strains of Scenedesmus acutus (Gorbi et al. 2007, Marieschi et al. 2015, Sardella et al. 2019, Ferrari et al. 2022). To verify if the tolerance mechanisms observed in S. acutus could represent a common adaptive mechanism to Cr(VI), we isolated microalgae from a naturally contaminated habitat. Our study focused on the watersprings of Mount Prinzera, an ophiolitic metalliferous mountain near Parma, Italy, in which, depending on water flow, an average annual level of hexavalent chromium equal to 12 μg/l is recorded. This environment is therefore a good candidate for isolating organisms potentially adapted to Cr(VI) stress. From the water of Mount Prinzera we isolated 28 algal colonies and identified them through DNA barcoding, as belonging to 3 strains of Eterokontophyta (2 Eustigmatales and 1 Naviculales) and 6 strains of Chlorophyta (4 Sphaeropleales, 1 Chlorellales and 1Ulvales). Some other diatoms were presumably lost during selection. Among Chlorophyta, we tested the Spheropleales Neocystis brevis, Bracteacoccus polaris, and Chromochloris zofingiensis for Cr(VI) tolerance, and verified that all of them showed tolerance to Cr(VI) concentrations at least 10 times higher than the average Cr(VI) concentration found in the Mount Prinzera waterspring. However, many of the species we identified are commonly found in metal-contaminated soils (Patova and Dorokhova 2008; Trzcińska and Pawlik- Skowrońska 2008; Redkina and Shalygina 2021;), suggesting that they may be extremophiles rather than neutral species adapted to local conditions. For further analysis we thus focused on the strain identified as Chromochloris zofingiensis since the availability of a commercial strain CCAP 211/14 and the fully annotated genome could allow the comparison with an adequate control. Despite the unequivocal identification of the strain Prinzera 38 as Chromochloris zofingiensis this strain showed significant differences from the commercial strain, particularly in growth rate and average cell size. The analysis conducted on the two strains demonstrates that this species has a natural high level of Cr(VI) tolerance, maybe related to its reproductive modality. Analyzes are underway to evaluate the effect of sulfate metabolism on chromium tolerance in this alga and on the activity of enzymes involved in the cysteine synthesis pathway.