At each site during each visit, we measured denitrification and nitrogen fixation rates on rocks and sediment and collected a set of standard water chemistry data to quantify the CPA values at each site. Primary data collected included the following:
Denitrification was measured using acetylene block assays (Smith & Tiedje, 1979). C, N, and chloramphenicol (to prevent bottle effects) were added to each chamber for a final concentration of 34mg/L sucrose and sodium nitrate and 114 mg/L chloramphenicol. Initial and fial gas samples were taken after ~2 h insitu incubation period. The gas samples were analyzed for N2O using the SRI8610C Gas Chromatograph with Hayesep D column, electron capture detector, and column oven set to 80 C ramping to 180 C after 5 min with helium or ultra-high purity nitrogen (for samples analyzed after February 2019) carrier gas. A 1000 ppm N2O standard was used to convert peak height to nmol of N2O following Dodds et al. (2017).
Nitrogen fixation rate was measured using acetylene reduction assays following (Eberhard et al. 2018; Capone, 1993). Initial and final samples were taken over a ~2 h in situ incubation. Samples were analyzed for ethylene concentration using a SRI 8610C Gas Chromatograph with Hayesep T column, flame ionization detector, and column oven set at 40 C ramping to 110 C after 2.5 min running with hydrogen carrier gas. A 100-ppm ethylene standard was used to convert peak height to nmol of ethylene in the gas sample.
Rock area was estimated by tracing each rock onto paper, then cutting those tracings out and weighing them, and comparing those weights to a standard curve created by weighing squares of paper with known areas (Bergey & Getty, 2006).
Surface area of sediment was determined by the surface area of the corer opening.
Chlorophyll a concentration was measured according to APHA, 2005. Rocks were scrubed and 50mL subsumes filtered through pre-ashed filters Filters were later extracted in 95% ethanol for 8-24 h. Using a spectrophotometer, absorbances were measured at 664, 665, and 750nm. The samples were acidified with 0.1 N HCl and absorbance was measured again (APHA, 2005; Nusch, 1980). Chlorophyll concentration was scaled up from subsample to total water volume to determine chlorophyll concentration for the chamber, then normalized by dividing by rock surface area.
Ash free dry mass (AFDM) was determined by first drying filters with remaining extract and sediment from fthe chambers in a 60°C oven for 48 hours, then ashed at 500°C for 4 h to measure ash free dry mass. AFDM was scaled up from subsample to total water volume to determine AFDM for the chamber.
Water chemistry was analyzed for each sampling date according to APHA (2005). Water was filtered through 0.45 μm membrane filters and stored on ice until return to the lab where the samples were frozen. Analysis included soluble reactive phosphorus (SRP; µg/L), total dissolved phosphorus (TDP; µg/L), soluble reactive phosphorous (SRP-; µg/L), nitrate (NO3-/NO2-; µg/L), ammonium (NH4; µg/L), total dissolved nitrogen (TDN, µg/L, and dissolved organic carbon (DOC; mg/L). NO3-+NO2 and SRP were analyzed using a SEAL AQ2 discrete water analyzer. NO3-+NO2 used AQ2 method EPA-127-A Rev. 9, SRP used AQ2 method EPA-155-A Rev. 0. Filtered water samples were acidified to pH < 2 and sent to Michigan Tech’s Laboratory for Environmental Analysis of Forests (LEAF) core facility which used a Shimadzu TOC-VCSN with a total N module TNM-1 (Shimadzu Scientific Instruments, Columbia, Mary- land) for DOC and TDN analysis. Dissolved Organic Nitrogen (DON) was calculated by subtracting NO3-++NO2 and NH4+ concentrations from TDN concentrations. TDP concentration was analyzed using molybdenum—antimony method.
Discharge was determined using USGS installed gauge (USGS 04043016 Pilgrim River at Paradise Road Near Dodgeville, MI; May 2017- June 2018) or by measuring using a Marsh McBirney Flo-mate (May 2017 - May 2019). Flow rate was measured at 10 equidistant points on a transect of the stream perpendicular to shore. The Flo-mate was attached to a wading rod to measure velocity (m s-1) at 0.6*stream depth (m) at each point along a 10 point transect. The area of each segment was determined by multiplying segment width by segment depth. Flow in each segment was determined by multiplying velocity by segment area. The flow in all segments was added together to get discharge (m3/ s). Canopy cover was measured using a spherical densitometer (Lemmon, 1956). Photosynthetically active radiation was retrieved from the Upper Great Lakes Observing System station located at Michigan Tech's Great Lakes Research Center (glos.us), which is approximately ~3 km from the study site. A MiniDO2T logger from PME was deployed to continuously measure O2 and temperature at the site, and open water metabolism, which includes gross primary production (GPP) and ecosystem metabolism (ER), was modeled using the StreamMetabolizer software package (github.com/USGS-R/streamMetabolizer).
Methods citations:
AMERICAN PUBLIC HEALTH ASSOCIATION (APHA). 2005. Standard methods for the examination of water and wastewater, 21st edition. American Public Health Association, American Water Works Association, and Water Environment Federation, Washington, DC. 1,368 pgs.
BERGEY, E. A., & GETTY, G. M. (2006). A review of methods for measuring the surface area of stream substrates. Hydrobiologia, 556(1), 7–16.
CAPONE, D. G. (1993). Determination of nitrogenase activity in aquatic samples using the acetylene reduction procedure. In Handbooks of Methods in Aquatic Microbial Ecology (pp. 621–631).
DODDS, W. K., BURGIN, A. J., MARCARELLI, A. M., & STRAUSS, E. A. (2017). Nitrogen transformations. In Methods in stream ecology (pp. 173-196). Academic Press.
EBERHARD, E. K., MARCARELLI, A. M., & BAXTER, C. V. (2018). Co-occurrence of in-stream nitrogen fixation and denitrification across a nitrogen gradient in a western U.S. watershed. Biogeochemistry, 139(2), 179–195.
NUSCH, E. A. 1980. Comparison of different methods for chlorophyll and phaeopigment determination. Archiv für Hydrobiologie–BeiheftErgebnisse der Limnologie 14, 14-36.
Smith, M. S., & Tiedje, J. M. (1979). Phases of denitrification following oxygen depletion in soil. Soil Biology and Biochemistry, 11(3), 261–267.
