Light–dark conditions drive variability in phosphorus and ammonium uptake by epilithic biofilms along the main stem of a Mediterranean river

Abstract Benthic biofilms on cobbles (i.e., epilithic biofilms) control the uptake of soluble reactive phosphorus (SRP) and ammonium (NH 4 + ) in headwater streams. However, nutrient uptake by biofilms in higher‐order rivers, particularly those affected by anthropogenic nutrient inputs and hydrological regulation, remains poorly understood. To address this gap, we examined the spatial and temporal variability of SRP and NH 4 + uptake by epilithic biofilms, as well as their main drivers under light and dark conditions, along the main stem of a fifth‐order human‐regulated Mediterranean river. In situ light–dark incubations were conducted in open recirculating chambers at six sites across four dates, using dual nutrient additions to quantify gross uptake ( U ) of SRP and NH 4 + . Uptake of NH 4 + was consistently higher than U SRP across sites and dates, particularly under light conditions, with both nutrients showing rates similar to those reported in previous reach‐scale studies. Uptake of SRP was consistent under light and dark conditions, with spatial and temporal variability contributing similarly to the explained variation in uptake. In contrast, U NH4 differed markedly between light and dark conditions and exhibited site‐specific temporal patterns, suggesting a stronger dependence on photoautotrophic demand and greater influence on local environmental conditions. Finally, U SRP was primarily associated with biofilm metabolic activity, while U NH4 was more strongly related to ambient nutrient concentrations. Overall, this study expands our understanding of spatial and temporal variability in biofilm nutrient uptake and highlights the importance of considering light availability to better understand the role of biofilms in P and N retention.