We collected water samples from 10 estuary stations to represent a gradient from river to lake on 13 dates between April 2012 and September 2013. Stations 1-5 represented upper estuary sites, while stations 6-10 were lower. Stations were situated near the thalweg, but were shifted laterally to avoid traffic within the shipping channel. Sampling occurred approximately monthly during the open water season when sites were accessed by boat, and once during winter ice cover when a subset of sites were visited on foot. In addition to the core 10 stations, we sampled an additional 20 sites, four times over the two-year study during a high flow and baseflow period. These sites include 7 end members (Saint Louis River, Nemadji River, Bluff Creek, Kinsbury Creek, Pokegama River, and Lake Superior) and an additional 15 in-estuary sites (i.e., stations 16-30). Additional sites were occasionally visited and geographic locations to all stations are provided in SLRESitesTable.Physical LimnologyWe used a YSI EXO2 or 6-Series sonde (Yellow Springs, OH) to measure temperature, specific conductivity, dissolved oxygen, pH, turbidity, and algae fluorescence. Briefly, the sonde was lowered to appr. 0.5 m depth and allowed to stabilize. The sonde was calibrated in the lab that morning according to Lake Superior National Estuary Research Reserve (LSNERR) protocols.Light extinction was determined by lowering a photosynthetically active radiation (PAR) sensor (Licor model 192 or 193) attached to a light meter (Licor model 250A) through the water column. The sensor was allowed to stabilize at 0.25 m depth intervals. We linearly regressed the natural log of the measured light intensity against depth. The slope of this regression is the negative light extinction coefficient (k). Briefly k values closer to zero indicate clearer waters that transit more light.Water ChemistrySurface water from each station was collected into an HDPE carboy and processed in the lab within 10 h of collection. We processed samples in the lab (instead of on the boat) to expedite sample collection so that all stations could be visited within a single day (or within 2 days for spatial intensive surveys). Integrated water samples were taken from 0-2 m using a peristaltic pump or an integrated water sampler and stored in a cooler to maintain ambient temperature. Samples for dissolved solute analysis were filtered through a 0.45 microm Geotech capsule filter. All samples were refrigerated, frozen, or acidified (dependent on the analysis in question) within 12 h of collection. See meta data for SLREWaterChemTable for specifics regarding lab responsible for analyses.Samples for major cations (Calcium (Ca), Iron (Fe), Potassium (K), Sodium (Na), Magnesium (Mg), and Manganese (Mn)) were filtered upon collection into 60 mL acid-washed HDPE bottles, acidified to 1 percent ultrapure hydrochloric acid (HCl) and stored at room temperature until analysis (within 6 months). Cations were analyzed simultaneously on an optical inductively-coupled plasma emission on a Perkin-Elmer model 4300 DV ICP spectrophotometer according to methods outlined at the North Temperate Lakes- Long Term Ecological Research site.Samples for major anions (Chloride (Cl) and sulfate (SO4)) were filtered into a new 20 mL HDPE scintillation vials and stored at 4degree C until analysis (within 3 months). Anion samples were analyzed simultaneously by Ion Chromatography, using a hydroxide eluent determined by a Dionex model ICS 2100 using an electro-chemical suppressor.Samples for dissolved organic carbon (DOC) and dissolved inorganic carbon (DIC) were analyzed on a Shimadzu TOC analyzer. DOC and DIC samples were filtered into acid-washed 24 mL glass vials and capped with septa, leaving no headspace. DOC samples were acidified with 100 microL of 2 M HCL upon collection. Both DOC and DIC were stored at 4 degreeC, and then analyzed within three weeks at the University of Minnesota-Twin Cities. Both DOC and DIC were collected in duplicate and reported as means.Samples for UV absorbance were filtered into ashed 40 mL glass amber vials and stored at 4degree C until analysis (within 2 months). We measured UV absorbance at 254 nm (Abs254) using a spectrophotometer (Cary 50 UV-Vis Spectrophotometer, Varian, Palo Alto, CA). Specific UV absorbance at 254 nm (SUVA254) was then calculated by dividing Abs254 by the DOC concentration of the water sample.Nitrate plus nitrite nitrogen (referred to as NO3-N), ammonium plus ammonia nitrogen (referred to as NH4-N), and soluble reactive phosphorus (SRP) were analyzed colormetrically. Samples were filtered into new 20 mL plastic scintillation vials and frozen within 8 h of collection. Samples were thawed within 4 months and were analyzed in parallel by automated colorimetric spectrophotometry, using an Astoria-Pacific Astoria II segmented flow autoanalyzer. NO3-N was determined using the automated cadmium reduction method with absorption monitored at lambda=520 nm. NH4-N was determined using the Berthelot Reaction, producing a blue colored indophenol compound, where the absorption was monitored at lambda=660 nm. SRP was determined by forming a phosphoantimonymoledbeun complex and was measured as lambda=880nm.Samples for total and dissolved nitrogen and phosphorus analysis were collected together and in-line filtered (dissolved nitrogen and phosphorus only) into 60 ml LDPE bottles and acidified to a 1 percent HCl. Once acidified, the samples were stored at room temperature until analysis, which occurred within one year. The samples were first prepared for analysis by adding a NaOH–Persulfate digestion reagent and heated for 1 h at 120 degreeC and 18-20 pounds per square inch (psi) in an autoclave. The samples were analyzed for total nitrogen and total phosphorus simultaneously by automated colorimetric spectrophotometry, using a segmented flow autoanalyzer. Total nitrogen is determined by utilizing the automated cadmium reduction method where the absorption is monitored at 520 nm; total phosphorus is determined using ascorbic acid-molybdate method where the absorption is monitored at 880 nm. Both are described in LTER standard methods.We determined dual isotopic natural abundance of nitrate (NO3) and water (H2O) from a subset of collected water samples. Samples for delta18O-NO3 and delta15N-NO3 were filtered into acid-washed 60 mL HDPE bottles and frozen within 8 h of collection. Nitrate isotope samples were analyzed using the denitrifier method at the Colorado Plateau Stable Isotope Laboratory. delta18O-NO3 and delta15N-NO3 isotopes were reported as the per mil (per-mille) deviation from Vienna Standard Mean Ocean Water (VSMOW) and air standards, respectively. Samples for isotopes of water (delta18O-H2O and delta2H-H2O) were collected without headspace in glass vials and measured using isotope ratio infrared spectroscopy at the University of Minnesota – Biometeorology lab. Six replicates were run per sample, and delta18O-H2O and delta2H-H2O were determined relative to VSMOW.Chlorophyll ALaboratory analysis of chlorophyll A (ChlA) uses the Turner Designs model 10-AU fluorometer, following improvements described in Welschmeyer (1994). In this method, ChlA in 90percent acetone is separated from other pigments by the use of specialized optical filters. ChlA samples were preserved within 24 h of water sampling, by collecting filtrand on a 0.2 microm cellulose nitrate filter, placing the filter in a 15 mL falcon tube, and freezing it. Between 200 and 1000 mL of sample was based through the filter until the filter was moderately stained and filtering speed slowed. Within three weeks of collection, filters were thawed, and 12.0 mL of acetone was added to tube, which was allowed to steep for 18-24 h in the dark at 4 degreeC. After steeping, samples were centrifuged at high speed in Sorvall GLC-2B centrifuge for 20 min and warmed to room temperature. Sample fluorescence was then measured on a calibrated Turner Designs model 10-AU fluorometer (excitation 436 nm, emission 680 nm). Sample fluorescence was then converted to a water column concentration by multiplying by the extract volume (i.e., 12 mL) and divided by the volume of water that passed through the filter (i.e., 200-1000 mL).ParticulatesSimilar to ChlA, particulate carbon, nitrogen, and phosphorus samples were collected by passing 200-1000 mL of water through a pre-combusted 0.7 glass fiber filter (GFF) and analyzing the filtrand. Filters were frozen immediately after filtration, and then dried at 60 degreeC for at least 48 hours. Particulate carbon and nitrogen was measured using a Thermo Fisher Flash 2000 elemental analyzer. Particulate phosphorus was determined from a separate filter. Filters were digested in 5 mL potassium persulfate and phosphorus was analyzed spectrophotometrically using the ascorbic acid-molybdate method (Menzel and Corwin 1965).NitrificationWater column nitrification rates were determined on 30 July 2013 for a subset of the water chemistry sampling stations (n = 15) that represented the full spatial extent and previously observed NH4-N range of the estuary. Water from each station was transferred to 333 mL polycarbonate bottles within 10 h of collection and spiked with 15NH4Cl to achieve a concentration of 0.03 micromol 15NH4 L-1. Samples were incubated at ambient temperature (20 degreeC ) in a dark cooler for 20 h. Pre- and post-incubation samples were filtered through 0.45 microm filters and analyzed for NO3-N, NH4-N and delta15N-NO3. Nitrification rates were determined based on changes in NO3-N, NH4-N, and delta15N-NO3 according to methods outlined in Small et al. (2013). Analysis for each station was performed in duplicate and reported as the mean.SedimentsSediments were collected on 8 of the water chemistry survey dates from stations 2-9 to determine spatial and temporal patterns of denitrification and sediment organic content. We also collected a single sediment sample from additional lower (n = 17) and upper (n = 6) stations on 19 June 2012 and 24 June 2013, respectively, to increase the spatial extent of our survey. In total, 56 and 42 individual sediment collections were made in 2012 and 2013, respectively. Sediments were collected from the upper 5-20 cm of the benthic zone using an Ekman dredge. At least 500 mL of benthic material was transferred to 1-L widemouth Nalgene containers and used in denitrification rate experiments. Fifteen mL of the uppermost sediment layer was transferred into sterile 100 mL disposable plastic screw-top containers to be analyzed for sediment composition content. Sediments were stored in a cooler while on the boat and transferred to 4 degreeC within 6 h.To assess the effects of sediment composition on denitrification, dry:wet ratios, bulk density, particle size distributions, loss-on-ignition (LOI), percent carbon, and percent nitrogen were determined from the 15 mL sediment subsamples. Sediments were weighed before and after drying at 60 degreeC for at least 48 h to determine dry:wet ratios and bulk density. Sediment particle size composition was determined optically using a Coulter LS-10 particle size analyzer and sizes were binned into percent clay (0-2.0 microm), silt (2.0-63.0 microm) and sand (63-2000 microm) (Scheldrick and Wang 1993). LOI was determined by the loss in mass of 2.0plus/-0.2 g dried homogenized sediment combusted at 550 degree Celsius for 4 h. Sediments were ground and analyzed for percent carbon and nitrogen using a Thermo Fisher Flash 2000 elemental analyzer.Sediment denitrificationWe determined actual (DeN) and potential (DEA) sediment denitrification rates in the laboratory using the acetylene block technique modified from Groffman et al. (1999) within 48 h of collection. We incubated 40±2 g of wet sediment saturated with 40±0.1 mL of estuary water in 125 mL glass Wheaton bottles at 20 degreeC. DEA incubations were spiked with glucose and NO3-N to a final concentration of 40 mg C L-1 and 100 mg N L-1, respectively; DeN incubations were given no amendments. All incubations were augmented with 10 mg L-1 chloramphenicol to inhibit microbial proliferation (Smith and Tiedje 1979). Samples were capped with rubber septa, flushed with helium (He) for 5 min to remove oxygen (O2), and injected with 10 mL acetylene. We allowed the acetylene 30 min to fully diffuse into the sediment slurry before taking the initial headspace sample (T0). Samples were placed on a shaker table in the dark for 2.6 h then sampled the final headspace (T1). The change in headspace N2O partial pressures (pN2Ofinal - pN2Oinitial) was used to determine the denitrification rate using the Bunsen correction and the ideal gas law. For both T0 and T1 samples, 10 mL of headspace was withdrawn from incubation bottles and injected into a He-flushed 12 mL gas-tight glass vials (Exetainers) sealed with rubber septa. We determined pN2O and pO2 in parallel on a gas chromatograph equipped with an electron capture detector (ECD) and thermal conductivity detector (TCD) using methods outlined in Spokas et al. (2005). Gas samples with O2 concentrations greater than 5percent were removed from analysis due to potential gas leakage. Denitrification rates were standardized to sediment dry mass. Samples collected on or before 6 June 2013 were incubated in triplicate; samples collected after were incubated in duplicate.Denitrification controls were further investigated by amending sediments with combinations of NO3-N and two types of organic carbon: glucose and natural organic matter (NOM; supplied by the International Humic Substance Society). On two dates in 2013, we incubated sediments from five of our core stations that spanned a gradient of sediment organic content with the following amendments: NO3-N only, NO3-N and glucose (DEA), NO3-N and NOM, glucose only, NOM only, and no amendments (DeN). The two carbon treatments were intended to test for possible effects of carbon quality, with NOM representing a recalcitrant, humic-rich carbon source similar to allochthonous materials in the SLRE to contrast the labile glucose treatment. Both carbon sources were amended to 40 mg C L-1, and NO3-N was amended to 100 mg N L-1. Sediments were incubated in parallel (see above).Breakdown Dissolved Organic Carbon (bDOC)Breakdown of DOC (bDOC) was determined from core stations (1-10) from water collected on 23 April and 30 July 2012. Briefly, 250 mL of estuary water was filtered through a 0.45 microM Geotech flow-through filter using a peristaltic pump into sealable glass jars. 25 mL of 2.0 microm filtered water from a common estuary source was added to the filtered jars. DOC samples were collected after 0, 1, 2, 4 ,8, 16, and 32 d and analyzed for DOC (see above). A linear model was fit between time since inoculation and DOC concentration to determine the breakdown of DOC from water column microbes.ReferencesMeyers PA, Teranes JL. 2001. Sediment organic matter. Pages 239-269, In: Track Enviornmental Change Using Lake Sediments Vol 2 Phys Geochemical Methods. Dordrecht: Kluwer Academic Publishers.Groffman, Peter M, Holland EA, Myrold DD, Robertson GP, Zou X. 1999. Denitirification. Pages 272-288 in Standand Soil Methods Long-Term Ecological Research, Oxford University, New York.Menzel DW, Corwin N. 1965. The measurement of total phosphorus in seawater based on the liberation of organically bound fractions by persulfate oxidation. Limnol and Oceanogr 10: 280–282.Scheldrick HB, Wang C. 1993. Particle size distribution. Pages 499-512 In: Soil Sampling and Methods of Analysis. Boca Raton: CRC Press LLC.Small GE, Bullerjahn GS, Sterner RW, Beall BFN, Brovold S, Finlay JC, McKay RML, Mukherjee M. 2013. Rates and controls of nitrification in a large oligotrophic lake. Limnol Oceanogr. 58:276–86.Smith MS, Tiedje JM (1979) Phases of denitrification following oxygen depletion in soil. Soil Biol Biochem 11:261-267Spokas K, Wang D, Venterea R. 2005. Greenhouse gas production and emission from a forest nursery soil following fumigation with chloropicrin and methyl isothiocyanate. Soil Biol Biochem. 37:475–85.Welschmeyer, N.A. 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments. Limnol Oceanogr 39:1985-1992.