Phosphorus (P) entrapment by biofilms can be distinguished into the intracellular P entrapment (the P uptake by microbial cells) and the extracellular P entrapment via the extracellular polymeric substances. It is unknown how these two P entrapment pathways behave in natural ecosystems in which gradients of environmental drivers such as P, labile dissolved organic matter (DOM) and light occur. Another key aspect in P dynamics at the sediment-water interface is microbial activity. Microbially mediated mineralization of DOM and microbial activity in general is suspected to be a driver of internal P mobilization from the sediments. Here we brought such expectations into a real-world context by analysing the P entrapment patterns of benthic biofilms and the P release potential from the sediments along a longitudinal gradient in a third order river in front of the background of key microbial metabolic variables. The gradient consists of increasing P availability, DOM lability and light availability.</p> <p>We found a gradual shift in the dominance of P entrapment from higher intracellular P entrapment in the upstream biofilms to higher extracellular P entrapment in the downstream biofilms. This shift towards dominance of extracellular P entrapment was accompanied by an increase in the P release potential from the sediment. The increasing P release potential was also connected to high extracellular enzyme activity of alkaline phosphatase, an enzyme involved in the mineralization of P from organic compounds. We further found that a balanced ratio between intracellular and extracellular P was connected to a higher C metabolic diversity.</p> <p>All this evidence suggests an influence of the benthic biofilms on P dynamics at the sediment-water interface. This research advocates for a more integrated perspective that accounts for both intracellular and extracellular biofilm-mediated processes.