Microbial communities and nutrient-cycling potential in epilithic biofilms differ between light- and dark-exposed sides of cobbles in a Mediterranean river

Light exposure is a key driver of biofilm development in river ecosystems, yet microbial communities inhabiting the underside of cobbles (dark-side epilithic biofilms) remain largely overlooked, and their functional potential for phosphorus (P) and nitrogen (N) cycling is still poorly understood. Here, we investigated how microbial community composition and predicted functional potential for P and N cycling differ between light-exposed and dark-side epilithic biofilms along a Mediterranean river. Using 16S rRNA gene sequencing combined with phylogenetic functional prediction, we assessed spatial, temporal, and cobble-side (light vs dark) patterns in taxonomic and functional composition. Microbial communities were strongly structured by spatial and temporal gradients; however, differences in light exposure between cobble sides exerted a consistent influence on taxonomic composition and predicted nutrient-cycling potential. Light-exposed biofilms showed higher relative abundances of Cyanobacteria, whereas dark-side biofilms exhibited greater evenness and diversity. For N-cycling genes, cobble-side differences explained more variation than temporal factors, highlighting the ecological relevance of fine-scale spatial heterogeneity. Notably, nitrification showed the strongest light-dark differentiation, with higher predicted potential in dark-side biofilms. In contrast, P-cycling gene responses to light-dark differences were more heterogeneous and did not translate into consistent process-level differentiation. Despite these contrasts, structure-function coupling remained consistent between cobble sides and nutrients. Overall, our results indicate that incorporating dark-side communities increases the functional diversity captured and enhances the representation of key biogeochemical processes, underscoring the need to integrate both cobble sides when evaluating microbial contributions to nutrient cycling in river ecosystems.