Dataset title: Physical and microbial processing of dissolved organic nitrogen (DON) (Photodegradation Experiment) along an oligotrophic marsh/mangrove/estuary ecotone (Taylor Slough and Florida Bay) for August 2003 in Everglades National Park (FCE), South Florida, USA Dataset ID: ST_ND_Jaffe_004 Research type: Short-term Dataset Creator Name: Dr. Rudolf Jaffe Position: Lead Principal Investigator Organization: Florida Coastal Everglades LTER Program Address: Florida International University University Park OE 148 Miami, Florida 33199 USA Phone: 305-348-2456 Fax: 305-348-4096 Email: jaffer@fiu.edu URL: http://serc.fiu.edu/sercindex/index.htm Metadata Provider Organization: Florida Coastal Everglades LTER Program Address: Florida International University University Park OE 148 Miami, FL 33199 USA Phone: 305-348-6054 Email: fcelter@fiu.edu URL: http://fcelter.fiu.edu Dataset Abstract A better understanding of the biogeochemical cycling of nutrients entering Florida Bay is a key issue regarding the restoration of the Everglades. In addition to precipitation, the other major source of freshwater to Florida Bay is from Taylor Slough and the C-111 Basin in the northeast section of the Bay. While it is known that these areas deliver significant amounts of N to the Bay, a significant portion of this is in the form of dissolved organic N (DON). The sources, environmental fate and bioavailability to microorganisms of this DON are however, not known. Should this DON be readily available, any increased load as a function of restoration changes might have an impact on internal phytoplankton bloom dynamics. No significant flocculation or precipitation of DOM occurred with increase in salinity, meaning that terrestrial DOM does not get trapped in the sediments but stays in the water column where it subjected to photolysis and advective transport. Sunlight has a significant effect on the chemical characteristics of DOM. While the DOC levels did not change significantly during photo-exposure, the optical characteristics of the DOM were modified. The environmental implications of this are conflicting: photo-induced polymerization may stabilize the DOM by reducing its bioavailability while photolysis may make the DOM more labile. Overall, DON bioavailability was relatively low in this region. Even though the amount of DON loaded to the bay may be significant, the fraction of DON available for microbial cycling is much smaller. The amount of N supplied by recycling may be a significant portion of the total DIN pool. All this must be considered in context with the proposed CERP modifications to flows. As of the latest initial Comprehensive Everglades Restoration Project (CERP) update, the flows to Taylor Slough and C-111/Panhandle Basis are not predicted to change very much from base conditions. Therefore we do not expect any great increases in TN loading in this region. In contrast, the proposed flow increases to the Shark River Slough are large and may have significant effects on transport of DOM to the Southwest Florida Shelf. We believe that future efforts in DON characterization and bioavailability should be concentrated in this area. Geographic Coverage Study Extent Description The Study Extent of this dataset includes the FCE Taylor Slough and Florida Bay research sites within Everglades National Park, South Florida Bounding Coordinates Geographic description: Samples were collected in the Taylor Slough and Shark River Slough, within Everglades National Park, South Florida. West bounding coordinate: -80.649 East bounding coordinate: -80.490 North bounding coordinate: 25.404 South bounding coordinate: 25.177 Geographic description: Florida Coastal Everglades LTER Study Area: South Florida, Everglades National Park, and Florida Bay West bounding coordinate: -81.078 East bounding coordinate: -80.490 North bounding coordinate: 25.761 South bounding coordinate: 24.913 FCE LTER Sites: TS/Ph2, TS/Ph6a, and TS/Ph9 All Sites Geographic Description:FCE LTER Site TS/Ph2 Longitude:-80.607 Latitude:25.404 Geographic Description:FCE LTER Site TS/Ph6a Longitude:-80.649 Latitude:25.214 Geographic Description:FCE LTER Site TS/Ph9 Longitude:-80.490 Latitude:25.177 Temporal Coverage Start Date: 2003-08-04 End Date: 2003-08-04 Data Table Entity Name: ST_ND_Jaffe_004 Entity Description: Physical and microbial processing of dissolved organic nitrogen (DON) along an oligotrophic marsh/mangrove/estuary ecotone (Taylor Slough and Florida Bay) in Everglades National Park, South Florida, USA Object Name: ST_ND_Jaffe_004 Data Format Number of Header Lines: 1 Attribute Orientation: column Field Delimiter: , Number of Records: Attributes Attribute Name: SITENAME Attribute Label: sitename Attribute Definition: Name of LTER site Storage Type: text Measurement Scale: Name of LTER site Missing Value Code: Attribute Name: Date Attribute Label: date Attribute Definition: Collection date Storage Type: datetime Measurement Scale: Missing Value Code: Attribute Name: Incubation_Days Attribute Label: Incubation days Attribute Definition: Incubation days Storage Type: Incubation days Measurement Scale: Incubation days Missing Value Code: Attribute Name: DOC Attribute Label: Dissolved organic carbon Attribute Definition: Dissolved organic carbon concentration Storage Type: data Measurement Scale: Units: milligramsPerLiter Precision: 0.01 Number Type: real Missing Value Code: -9999.00 (Value will never be recorded ) Attribute Name: Max_WL Attribute Label: Maximum Wavelength Attribute Definition: Emission wavelength that gives maximum emission intensity at a fixed excitation of 313 nm Storage Type: data Measurement Scale: Units: nanometer Precision: 1 Number Type: real Missing Value Code: -9999 (Value will never be recorded ) Attribute Name: Max_I Attribute Label: Maximum Intensity Attribute Definition: Emission intensity of maximum emission wavelength at a fixed excitation of 313 nm. Storage Type: data Measurement Scale: Units: QSUPerMilligramPerLiter Precision: 1 Number Type: real Missing Value Code: -9999 (Value will never be recorded ) Attribute Name: FI Attribute Label: Fluorescence Index Attribute Definition: Ratio of emission intensities at 450 and 500 nm at a fixed excitation wavelength of 370 nm. Storage Type: data Measurement Scale: Units: dimensionless Precision: 0.01 Number Type: real Missing Value Code: -9999.00 (Value will never be recorded ) Attribute Name: %285 Attribute Label: %285 Attribute Definition: Obtained from synchronous fluorescence spectrum at a constant offset of 30 nm (excitation wavelengths = 285, 350, 385, 460 nm). Percentage of the first peak intensity (285 nm). Storage Type: data Measurement Scale: Units: percent Precision: 0.001 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Peak 1 Attribute Label: Peak 1 Attribute Definition: Fluorescence intensity at an excitation of 285 nm in synchronous scan. Storage Type: data Measurement Scale: Units: QSUPerMilligramPerLiter Precision: 0.01 Number Type: real Missing Value Code: -9999.00 (Value will never be recorded ) Attribute Name: A_254 Attribute Label: Absorbance at 254nm Attribute Definition: UV absorbance at 254 nm. Storage Type: data Measurement Scale: Units: microgramsPerMilliliter Precision: 0.01 Number Type: real Missing Value Code: -9999.00 (Value will never be recorded ) Attribute Name: S_value Attribute Label: S value Attribute Definition: The slope of the UV-Vis spectrum Storage Type: data Measurement Scale: Units: dimensionless Precision: 0.01 Number Type: real Missing Value Code: -9999.00 (Value will never be recorded ) Attribute Name: SUVA254 Attribute Label: Specific UV Absorbance Attribute Definition: UV absorbance at 254nm divided by the DOC concentration Storage Type: data Measurement Scale: Units: milligramsPerLiter Precision: 0.01 Number Type: real Missing Value Code: -9999.00 (Value will never be recorded ) Methods Sampling Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6a) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Battin, T J 1998. Dissolved organic materials and its optical properties in a blackwater tributary of the upper Orinoco River, Venezuela. Organic Geochemistry, 28: 561-569. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Bell, R T 1993. Estimating production of heterotrophic bacterioplankton via incorporation of tritated thymidine. Lewis Publishers, Boca Raton, Florida, 8 pp. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Boyer, J N 1996. Bioavailability of water extractable organic carbon fractions in forest and agricultural soil profiles. Soil Biology and Biochemistry, 28: 783-790. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Coleman, A W 1980. Enhanced detection of bacteria in natural environments by fluorochrome staining of DNA. Limnology and Oceanography, 25: 948-951 . Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation De Souza Sierra, M M 1994. Fluorescence spectroscopy of coastal and marine waters. Marine Chemistry, 47: 127-144. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation De Souza Sierra, M M 1997. Spectral identification and behavior of dissolved organic fluorescent materials during estuarine mixing processes. Marine Chemistry, 58: 51-58. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Frankovich, T A 1998. A rapid, precise, and sensitive method for the determination of total nitrogen in natural waters. Marine Chemistry, 60: 227-234. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Hashimoto, S K 1985. Relationship between alkaline phosphatase activity and orthophosphate in the present Tokyo Bay. Journal of Environmental Science and Health, Part A 20A: 781-908. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Hoppe, H G 1983. Significance of exoenxymatic activities in the ecology of brackish water: measurements by means of methyllumbeliferyl-substrates. Marine Ecology Progress Series, 11: 299-308. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Jaffe, R 2004. Source characterization of dissolved organic matter in a subtropical mangrove-dominated estuary by fluorescence analysis. Marine Chemistry, 84: 195-210. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Lu, X 2003. Molecular characterization of dissolved organic matter in freshwater wetlands of the Florida Everglades. Water Research, 37: 2599-2606. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation McKnight, D M 2001. Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity. Limnology and Oceanography, 46: 38-48. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Porter, K G 1980. The use of DAPI for identifying and counting aquatic microflora. Limnology and Oceanography, 25: 943-948. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Servais, P 1989. Simple method for determination of biodegradable dissolved organic carbon in water. Applied Environmental Microbiology, 55: 2732-2734. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Stepanauskas, R 1999. Bioavailability of wetland-derived DON to freshwater and marine bacterioplankton. Limnology and Oceanography, 44: 1477-1485. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Method Step Description The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment. Citation Weishaar, J L 2003. Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon. Environmental Science and Technology, 37: 4702-4708. Instrumentation Whatman 0.7um glass fiber filers Millipore 0.2um hydrophilic membranes (Millipore Co.) Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.) Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC) Shimadzu TOC 5000 Analyzer ANTEK 7000N Nitrogen Analyzer Gilford Fluoro IV Spectrophotometer Quality Control Fluorescence measurements are corrected for internal absorbance quenching. Fluorescence spectra are corrected for internal instrument configuration using excitation and emission correction factors. For DOC we create calibration curves with standards and then graph the data. Distribution Online distribution: http://fcelter.fiu.edu/perl/public_data_download.pl?datasetid=ST_ND_Jaffe_004.txt Intellectual Rights These data are classified as 'Type II' whereby original FCE LTER experimental data collected by individual FCE researchers to be released to restricted audiences according to terms specified by the owners of the data. Type II data are considered to be exceptional and should be rare in occurrence. The justification for exceptions must be well documented and approved by the lead PI and Site Data Manager. Some examples of Type II data restrictions may include: locations of rare or endangered species, data that are covered under prior licensing or copyright (e.g., SPOT satellite data), or covered by the Human Subjects Act, Student Dissertation data and those data related to the FCE LTER Program but not funded by the National Science Foundation (NSF) under LTER grants #DEB-9910514, and # DBI-0620409. Researchers that make use of Type II Data may be subject to additional restrictions to protect any applicable commercial or confidentiality interests. All publications based on this dataset must cite the data Contributor, the Florida Coastal Everglades Long-Term Ecological Research (LTER) Program and that this material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DEB-1237517, #DBI-0620409, and #DEB-9910514. Additionally, two copies of the manuscript must be submitted to the Florida Coastal Everglades LTER Program Office, LTER Program Manager, Florida International University, Southeast Environmental Research Center, OE 148, University Park, Miami, Florida 33199. For a complete description of the FCE LTER Data Access Policy and Data User Agreement, please go to FCE Data Management Policy at http://fcelter.fiu.edu/data/DataMgmt.pdf and LTER Network Data Access Policy at http://fcelter.fiu.edu/data/core/data_user_agreement/distribution_policy.html. Dataset Keywords FCE Florida Coastal Everglades LTER ecological research long-term monitoring Everglades National Park biogeochemical cycling Taylor Slough Shark River Slough Florida Bay DON dissolved organic nitrogen Flocculation of DOM Precipitation of DOM Bioavailable DON dissolved organic matter fluorescence salinity emissions organisms nitrogen photodegradation incubation days incubation Data Submission Date: 2005-09-23 Maintenance This is a short-term DOM dataset. This dataset replaces the original version named ST_ND_Jaffe_004. The FCE program is discontinuing its practice of versioning data as of March 2013. Dataset Contact Position: Information Manager Organization: LTER Network Office Address: UNM Biology Department, MSC03-2020 1 University of New Mexico Albuquerque, NM 87131-0001 USA Phone: 505 277-2535 Fax: 505 277-2541 Email: tech-support@lternet.edu URL: http://www.lternet.edu Position: Information Manager Organization: Florida Coastal Everglades LTER Program Address: Florida International University University Park OE 148 Miami, FL 33199 USA Phone: 305-348-6054 Fax: 305-348-4096 Email: fcelter@fiu.edu URL: http://fcelter.fiu.edu Dataset Submission Date 2005-09-23 Information Management Notes This is a short-term DOM dataset. This dataset replaces the original version named ST_ND_Jaffe_004. The FCE program is discontinuing its practice of versioning data as of March 2013.