Trace metal–macronutrient colimitation of algal biofilms in streams with differing ambient inorganic nutrients

The supply of nutrients in streams is an important driver of biofilm production, ecosystem process rates, and basal resource availability. Current understanding of bottom-up drivers of microbial processes derives from studies of N and P, even though algal biofilms require a much larger set of elements to sustain growth. Studies in marine and lake ecosystems demonstrate that trace metals like Fe, Zn, Ni, and Mo can limit the growth of primary producers, but it is not known if these patterns hold in streams. We used trace metal nutrient diffusing substrata to experimentally enrich biofilms with N, P, Fe, Zn, Ni, and Mo alone and in specific combinations to test for macronutrient (i.e., N and P) limitation and trace metal–macronutrient colimitation. We completed enrichment experiments in 5 low-macronutrient streams in the Upper Peninsula of Michigan, USA, and 5 high-macronutrient streams in northeast Ohio, USA. As expected, biofilm chlorophyll a was most frequently colimited by N and P (40% of streams), with macronutrient limitation more common in the Upper Peninsula streams. At least 1 trace metal was limiting or colimiting with a macronutrient in 9/10 study streams, including streams that showed no evidence of N or P limitation. Trace metal colimitation with macronutrients was more frequent in streams with low inorganic N and P surface-water concentrations. In 4 streams, we observed algal biomass responses consistent with biochemically dependent colimitation, in which a trace metal alleviates N or P limitation by increasing access to an alternative source (e.g., organic P, N2). In biochemically dependent, colimited biofilms, the growth enrichment was less for trace metals than the inorganic nutrient (<15%), which suggests a substantial energy trade-off when relying on alternate nutrient sources. Overall, we demonstrated that trace metals are critical nutrients for stream primary producers, and that trace metal limitation may be an overlooked bottom-up driver that can have unexplored consequences for the structure and function of streams.