Wastewater discharge is a global threat to freshwater resources. Streams, in particular, are receiving waterbodies that are directly impacted chemically and biologically due to effluent discharge. However, it is largely unknown how wastewater serves as a subsidy or a stressor to aquatic biodiversity, particularly microbiota, over space. Nutrient-diffusing substrata (NDS) were deployed; NDS release nutrients through diffusion into the water column into a wastewater-dependent stream across three reaches. We used N, P, and N + P treatments for the measurement of single nutrient and co-nutrient limitation, and a no-nutrient control. Both algal and total biofilm biomass was measured and the 16S ribosomal RNA genes via targeted amplicon sequencing was used to assess bacterial/archaeal community diversity. Data indicated that total organic matter in biofilms differs spatially with the greatest organic matter (OM) concentrations in the confluence downstream of wastewater inputs. Biofilm OM concentrations were greatest in P and N + P treatments in the confluence site relative to control or N-only treatments. This indicates heterotrophic microbial communities-likely bacteria that dominate stream biofilms-are P-limited in this ecosystem even with upstream wastewater inputs. In conjunction, bacteria/archaeal communities differed the greatest among nutrient treatments versus spatially and had several indicator taxa belonging to Flavobacterium spp. in N treatments relative to controls. Collectively with historical water quality data, we conclude that this wastewater-fed stream is primarily N-enriched but potentially P-limited, which results in significant shifts in biofilm bacterial communities and likely their overall biomass in this urban watershed.

Importance: Streams in arid and semi-arid biomes are often dependent on their flow from municipal sources, such as wastewater effluent. However, wastewater has been shown to contain high concentrations of nutrients and chemical pollutants that can potentially harm aquatic ecosystems and their biota. Understanding if and the type of microorganisms that respond to pollution sources, specifically effluent from wastewater treatment facilities, in regions where flow is predominantly from treatment facilities, is critical for developing a predictive monitoring approach for eutrophication or other ecological degradation states for freshwaters.