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Physical and microbial processing of dissolved organic nitrogen (DON) (Salinity Experiment) along an oligotrophic marsh/mangrove/estuary ecotone (Taylor Slough and Florida Bay) for August 2003 in Everglades National Park (FCE), South Florida, USA


At a Glance


Authors: Rudolf Jaffe
Time period: 2003-08-04 to 2003-08-04
Package id: knb-lter-fce.1104.2
Dataset id: ST_ND_Jaffe_003

How to cite:
Jaffe, R.. 2006. Physical and microbial processing of dissolved organic nitrogen (DON) (Salinity Experiment) along an oligotrophic marsh/mangrove/estuary ecotone (Taylor Slough and Florida Bay) for August 2003 in Everglades National Park (FCE), South Florida, USA. Environmental Data Initiative. https://doi.org/10.6073/pasta/07272b339cff887abca38b8676789a56. Dataset accessed 2020-08-08.

Geographic Coverage


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Detailed Metadata


  • 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
    Samples were collected in the Taylor Slough and Shark River Slough, within Everglades National Park, South Florida.
    N: 25.404, S: 25.214, E: -80.607, W: -80.649

    Florida Coastal Everglades LTER Study Area: South Florida, Everglades National Park, and Florida Bay
    N: 25.761, S: 24.913, E: -80.490, W: -81.078

    FCE LTER Sites
    TS/Ph2 and TS/Ph6a

    All Sites
    Geographic Description
    Bounding Coordinates
    FCE LTER Site TS/Ph2
    N: 25.404, S: 25.404, E: -80.607, W: -80.607
    FCE LTER Site TS/Ph6a
    N: 25.214, S: 25.214, E: -80.649, W: -80.649
  • Attributes
    • Data Table:   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
      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:
      Salinity
      Attribute Label:
      Salinity
      Attribute Definition:
      Salinity
      Storage Type:
      data
      Measurement Scale:
      Units: dimensionless
      Precision: 1
      Number Type: real
      Missing Value Code:
      -9999 (Value will never be recorded )

      Attribute Name:
      DOC
      Attribute Label:
      DOC
      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 wavelength of 313nm.
      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:
      Maximum emission intensity at a fixed excitation wavelength of 313nm.
      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 obtained at a fixed excitation 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 with a synchronous fluorescence scan and is used to determine the amount of proteinaceous material present in a water sample.
      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 maximum intensity
      Attribute Definition:
      Maximum emission intensity of the first peak of the sychronous 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 254 nm
      Attribute Definition:
      UV absobance at 254 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:
      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:
      SUVA254
      Attribute Definition:
      UV absorbance at 254 nm normalized for 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 and Intellectual Rights
    Online distribution
    http://fcelter.fiu.edu/perl/public_data_download.pl?datasetid=ST_ND_Jaffe_003.txt
    Data Submission Date:  2005-09-23

    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.

  • 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
  • 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

    • Name: Rudolf Jaffe 
    • Position: Project Collaborator
    • 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

    • 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

  • Data Table and Format
    Data Table:  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

    Entity Name:
    ST_ND_Jaffe_003
    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_003
    Number of Header Lines:
    1
    Attribute Orientation:
    column
    Field Delimiter:
    ,
    Number of Records:
    20