The transport and fate of nitrate (NO 3 − ) to in the top 15 cm of a streambed has been well-documented, but an understanding of greater depths is limited. This work examines the transport and fate of nitrate (NO 3 − ) at depths of 30 cm, 60 cm, 90 cm, and 150 cm below the stream-streambed interface. Concentrations of nitrate as nitrogen (NO 3 -N) and chloride (Cl − ) were measured in the waters from the streambed, the stream water, and the groundwater. Mixing models predicted values of ∆NO 3 -N, the difference between measured NO 3 -N and theoretical NO 3 -N. At a 30-cm depth, the mean ∆NO 3 -N value was −0.25 mg/L, indicating a deficit of NO 3 -N and the removal of NO 3 -N from the system. At deeper levels, the values of ∆NO 3 -N began to approach zero, reaching a mean value of −0.07 mg/L at 150 cm. The reduction of NO 3 -N does not appear to be controlled by vegetation, as it was not correlated to either temperature or visible light. Larger negative ∆NO 3 -N values (more removal) occur when stream NO 3 -N concentrations are higher and organic matter is present.Hydrology 2018, 5, 55 2 of 17 Spatial variability in the distribution and composition of microbial communities, the concentrations of dissolved oxygen (DO), the concentration of organic matter (OM), and the concentration and species of nitrogen within the streambed control the rate of N removal [35,[39][40][41][42][43]. Longer residence time of the waters in the streambed correlate to enhanced reduction of NO 3 -N concentrations [44]. Seasonal variation of NO 3 -N concentrations in midwestern streams has been observed [45,46]; the variations are attributed to precipitation, fertilizer application, rate of stream water discharge, and the concentration of dissolved organic carbon in pore water within the streambed [3,4,6]. Concentrations of NO 3 -N tend to be higher during early spring following the application of fertilizers and when more frequent and higher magnitude precipitation events increase runoff. Nitrate concentrations are typically lowest during summer, when there is a limited source of NO 3 -N and there is increased uptake from growing plants [38,45]. The rate of denitrification, which is lowest during the winter months (November to March) and highest in early spring and summer (April to July), influences the seasonal variation of NO 3 -N concentrations in streams [4,47]. Moreover, the decrease in winter denitrification rates is attributed to a decrease in temperature and in microbial activity. Elevated denitrification rates during spring and summer are correlated to increased NO 3 -N entering the system and to increased amounts of decaying foliage entering the stream, providing OM for the denitrifying microbes.Nitrate removal in stream ecosystems is thought to occur disproportionately in zones with long residence times that facilitate the contact of reactive solutes with high biotic capacity for biogeochemical processing [42,48]. Despite studies indicating that significant microbial processes occur up to several meters below the streambed, the...