Algal biofilms play an essential role in ecosystem functioning, but their physiological and biochemical changes during salt stress remain poorly understood. This study aimed at investigating how biofilm communities react to different salinity in a microcosm experiment and to offer insights into the potential effects of future freshwater salinization. Biofilm samples collected from Iriri River (Drâa basin, Southern Morocco) were exposed to water gradually enriched with sodium chloride to obtain three final concentrations (1, 10, and 30 g/L) over 14 days, and the changes were compared with control. Several biofilm responses were analyzed throughout the experiment, including biofilm biomass, changes in community structure and composition via eDNA metabarcoding, physiological feedback, biochemical parameters, antioxidant activities, non-enzymatic salt stress markers, osmoprotection indicators, and nitrogen assimilation. High salt concentrations significantly altered biofilm communities' composition, structure, and function, leading to an overall decrease in alpha diversity and a decrease in diatom and cyanobacterial biomass, while green algae remained unchanged. Chlorophyll a content increased in biofilm exposed to low salt concentration (1 g/L) and decreased at high salt concentrations while carotenoid significantly increased even at high salinity (10 g/L). Biofilm also showed an increase in carbohydrates and a decrease in protein content under high salinities (10 and 30 g/L). In addition, salt stress-induced ROS generation in the biofilm, with an increase in H2O2 content, caused oxidative damage and lipid peroxidation with an increase in MDA levels. To prevent this damage, elevated proline content was recorded, and additionally, the biofilm exhibited antioxidant defense mechanisms by rising activity of ROS-scavenger enzymes such as SOD, CAT, and POD. Our experimental results highlighted the salinity effect on algal biofilm as well as physiological and biochemical responses. This study could help predict the consequences of freshwater salinization on river biofilms and its potential impact at the ecosystem level.
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