Urban effluents override drying legacy in stream biofilm bacterial communities

Bacterial communities in stream biofilms play a fundamental role in the ecological functioning of fluvial ecosystems, contributing to nutrient cycling, organic matter decomposition, and primary production, while also serving as indicators of ecosystem health. In Mediterranean streams, these communities are increasingly exposed to multiple sequential stressors of varying intensity, such as drying and chemical pollution, yet their combined effects remain poorly understood. Here, we conducted a field experiment in four streams by colonizing artificial substrates, which were subsequently subjected to contrasting drying regimes (intermittent vs. continuous) and later translocated to reaches receiving urban effluents. Bacterial communities were characterized using high-throughput sequencing of the 16S rRNA gene. Both stressors significantly modified community diversity and composition, with consistent responses across streams. Continuous drying acted as a strong environmental filter, reducing diversity and favoring stress-tolerant taxa such as Bacilli and Actinobacteria while decreasing the relative abundance of Cyanobacteria. In contrast, effluent exposure promoted community homogenization and recolonization by effluent-derived taxa, largely dominated by Cyanobacteria and Gammaproteobacteria. Across both intermittent and continuous regimes, sequential exposure to drying followed by urban effluents further shaped bacterial communities. However, legacy effects of drying were generally weak and largely overridden by effluent inputs. Although some early-colonizing, stress-tolerant taxa persisted, community reorganization was mainly driven by effluent-derived colonization and local environmental conditions. Overall, these results show that the sequence and intensity of disturbances, together with initial community composition, regulate microbial resilience and community reorganization, with clear implications for the functioning of river ecosystems.