Trace nutrients act as cofactors in multiple enzymes that are used in pathways for nitrogen (N) and phosphorus (P) acquisition within stream biofilms. However, when examining streams, biofilms are typically thought to be limited by N and/or P, but trace nutrients may alleviate this limitation. In the 2021-2022 summers, we deployed nutrient diffusing substrate assays (NDS) with N, P, and trace nutrient treatments in 43 streams across Michigan, Ohio, Virginia, and South Carolina. In streams that were P limited, we hypothesized treatments with trace nutrients important in P acquisition enzymes (i.e., zinc) would show positive responses (response ratio > 0.387) while in N limiting streams treatments metals important in N acquisition (i.e., molybdenum, iron, and nickel) would show positive responses. We classified streams as N limited, P limited, or NP co-limited based on NDS growth (response ratio>0.365). The majority of streams sampled were N limited (13), 10 were NP co-limited, 3 were P limited, while 12 streams were not limited by either. There was a range of responses in the N limited streams with positive responses to molybdenum (23% of streams), nickel (7.7%), zinc (7.7%) and iron (30.8%); in P limited streams we found positive responses to molybdenum (33.3%), nickel (33.3%), zinc (33.3%), and iron (33.3%); and for NP co-limited streams we found positive responses to zinc (30%) and iron (70%). These positive responses may be due to increased trace nutrient concentrations supplied by NDS allowing for an increase in enzyme activity within biofilms and greater acquisition of either N, through enzymes such as nitrogenase or urease, or P, through enzymes such as alkaline phosphatase. These initial results show that trace nutrients may play a role in alleviating nutrient limitation and further work is planned to assess the enzymatic mechanisms by which trace nutrient additions influence nutrient acquisition pathways.