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Characterization of dissloved organic nitrogen in an oligotrophic subtropical coastal ecosystem (Taylor Slough and Shark River Slough) for December 2001 in Everglades National Park (FCE), South Florida, USA


At a Glance


Authors: Rudolf Jaffe
Time period: 2001-12-01 to 2001-12-01
Package id: knb-lter-fce.1106.2
Dataset id: ST_ND_Jaffe_005

How to cite:
Jaffe, R.. 2006. Characterization of dissloved organic nitrogen in an oligotrophic subtropical coastal ecosystem (Taylor Slough and Shark River Slough) for December 2001 in Everglades National Park (FCE), South Florida, USA. Environmental Data Initiative. https://doi.org/10.6073/pasta/cc9f23891b8bb977eaf5d7eb6f76005f. 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: 25.410, S: 25.025, E: -80.607, W: -81.078

    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
    SRS4, SRS6, TS/Ph2, TS/Ph7a and TS/Ph10

    All Sites
    Geographic Description
    Bounding Coordinates
    FCE LTER Site SRS4
    N: 25.410, S: 25.410, E: -80.964, W: -80.964
    FCE LTER Site SRS6
    N: 25.365, S: 25.365, E: -81.078, W: -81.078
    FCE LTER Site TS/Ph2
    N: 25.404, S: 25.404, E: -80.607, W: -80.607
    FCE LTER Site TS/Ph7a
    N: 25.191, S: 25.191, E: -80.639, W: -80.639
    FCE LTER Site TS/Ph10
    N: 25.025, S: 25.025, E: -80.681, W: -80.681
  • Attributes
    • Data Table:   Characterization of dissolved organic nitrogen in Taylor Slough and Shark River Slough from December 2001
      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:
      datetime
      Attribute Definition:
      Collection date
      Storage Type:
      datetime
      Measurement Scale:
      Missing Value Code:
       

      Attribute Name:
      Max_WL
      Attribute Label:
      Max_WL
      Attribute Definition:
      Emission wavelength that gives maximum intensity at a fixed excitation of 313 nm
      Storage Type:
      data
      Measurement Scale:
      Units: nanometer
      Precision: 1
      Number Type: real
      Missing Value Code:
      -9999 (Value will never be recorded )

      Attribute Name:
      Max_I
      Attribute Label:
      Max_I
      Attribute Definition:
      Maximum emission intensity at a fixed excitation of 313 nm
      Storage Type:
      data
      Measurement Scale:
      Units: QSUPerMilligramPerLiter
      Precision: 1
      Number Type: real
      Missing Value Code:
      -9999 (Value will never be recorded )

      Attribute Name:
      FI
      Attribute Label:
      FI
      Attribute Definition:
      Fluorescence Index
      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:
      SUVA 254
      Attribute Definition:
      UV Absorbance at 254 nm normalized for 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:
      S_Value
      Attribute Label:
      S Value
      Attribute Definition:
      Slope of the log of absorbance
      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:
      A
      Attribute Label:
      A
      Attribute Definition:
      Intensity UV humic-like fluorescence
      Storage Type:
      data
      Measurement Scale:
      Units: QSU
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      C
      Attribute Label:
      C
      Attribute Definition:
      Intensity of visible humic-like fluorescence
      Storage Type:
      data
      Measurement Scale:
      Units: QSU
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      M
      Attribute Label:
      M
      Attribute Definition:
      Intensity of visible marine humic-like fluorescence
      Storage Type:
      data
      Measurement Scale:
      Units: QSU
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      B
      Attribute Label:
      B
      Attribute Definition:
      Intensity of Tyrosine-like fluorescence
      Storage Type:
      data
      Measurement Scale:
      Units: QSU
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      T
      Attribute Label:
      T
      Attribute Definition:
      Intensity of Tryptophan-like fluorescence
      Storage Type:
      data
      Measurement Scale:
      Units: QSU
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      A:C
      Attribute Label:
      A:C
      Attribute Definition:
      Ratio of UV humic-like fluorescence and visible humic-like fluorescence
      Storage Type:
      data
      Measurement Scale:
      Units: dimensionless
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      C:N
      Attribute Label:
      C:N
      Attribute Definition:
      Carbon to nitrogen ratio
      Storage Type:
      data
      Measurement Scale:
      Units: dimensionless
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Delta15_N
      Attribute Label:
      delta15N
      Attribute Definition:
      Nitrogen stable isotope ratio
      Storage Type:
      data
      Measurement Scale:
      Units: perMil
      Precision: 0.01
      Number Type: real
      Missing Value Code:
      -9999.00 (Value will never be recorded )

      Attribute Name:
      Delta13_C
      Attribute Label:
      delta13C
      Attribute Definition:
      Carbon stable isotope ratio
      Storage Type:
      data
      Measurement Scale:
      Units: perMil
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      %AA-N
      Attribute Label:
      Percent Amino Acid nitrogen
      Attribute Definition:
      Percentage of Amino Acid nitrogen
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      %AA-C
      Attribute Label:
      Percent Amino Acid carbon
      Attribute Definition:
      Percentage of Amino Acid carbon
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.1
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Aromatic_N
      Attribute Label:
      Aromatic N
      Attribute Definition:
      Percentage of aromatic nitrogen
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Peptide_Bond_N
      Attribute Label:
      Peptide Bond N
      Attribute Definition:
      Percentage of peptide bond N
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Primary_Amine_N
      Attribute Label:
      Primary Amine N
      Attribute Definition:
      Percent primary amine nitrogen
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Aspartic_Acid
      Attribute Label:
      Aspartic Acid
      Attribute Definition:
      mole percentage of Aspartic Acid
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 1
      Number Type: real
      Missing Value Code:
      -9999 (Value will never be recorded )

      Attribute Name:
      Glutamic_Acid
      Attribute Label:
      Glutamic Acid
      Attribute Definition:
      mole percentage of Glutamic Acid
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Serine
      Attribute Label:
      Serine
      Attribute Definition:
      mole percentage of Serine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Histidine
      Attribute Label:
      Histidine
      Attribute Definition:
      mole percentage of Histidine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Glycine
      Attribute Label:
      Glycine
      Attribute Definition:
      mole percentage of Glycine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Threonine
      Attribute Label:
      Threonine
      Attribute Definition:
      mole percentage of Threonine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Arginine
      Attribute Label:
      Arginine
      Attribute Definition:
      mole percentage of Arginine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Beta-Alanine
      Attribute Label:
      Beta-Alanine
      Attribute Definition:
      mole percentage of Beta-Alanine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Alanine
      Attribute Label:
      Alanine
      Attribute Definition:
      mole percentage of Alanine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Tyrosine
      Attribute Label:
      Tyrosine
      Attribute Definition:
      mole percentage of Tyrosine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Gamma-Aminobutyric Acid
      Attribute Label:
      Gamma-Aminobutyric Acid
      Attribute Definition:
      mole percentage of gamma-Aminobutyric Acid
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Valine
      Attribute Label:
      Valine
      Attribute Definition:
      mole percentage of Valine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Phenylalanine
      Attribute Label:
      Phenylalanine
      Attribute Definition:
      mole percentage of Phenylalanine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      L_Isoleucine
      Attribute Label:
      L-Isoleucine
      Attribute Definition:
      mole percentage of L-Isoleucine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      D_Isoleucine
      Attribute Label:
      D-Isoleucine
      Attribute Definition:
      mole percentage of D-Isoleucine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Leucine
      Attribute Label:
      Leucine
      Attribute Definition:
      mole percentage of Leucine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Ornithine
      Attribute Label:
      Ornithine
      Attribute Definition:
      mole percentage of Ornithine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (Value will never be recorded )

      Attribute Name:
      Lysine
      Attribute Label:
      Lysine
      Attribute Definition:
      mole percentage of Lysine
      Storage Type:
      data
      Measurement Scale:
      Units: percent
      Precision: 0.10
      Number Type: real
      Missing Value Code:
      -9999.0 (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
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Blough, N V 1995. Spectroscopic characterization and remote sensing of nonliving organic matter. p. 23-45 in Zepp, R G , (eds). Role of nonliving organic matter in the earth's carbon cycle. John Wiley and Sons Ltd., London, 22 pp.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Coble, P G 1996. Characterization of marine and terrestrial DOM in seawater using excitation-emission spectroscopy. Marine Chemistry, 51(4): 325-346.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Coble, P G 1993. Fluorescence contouring analysis of DOC intercalibration experiment samples: a comparison of techniques. Marine Chemistry, 41: 173-178.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Cook, R L 1996. A modified cross-polarization magic angle spinning 13C NMR procedure for the study of humic materials. Analytical Chemistry, 68: 3979-3986.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Cowie, G L 1992. Sources and reactivities of amino acids in a coastal marine environment. Limnology and Oceanography, 37(4): 703-724.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Dauwe, B 1999. Linking diagenetic alteration of amio acids and bulk organic matter reactivity. Limnology and Oceanography, 44(7): 1809-1814.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    De Souza Sierra, M M 1994. Fluorescence spectroscopy of coastal and marine waters. Marine Chemistry, 58: 127-144.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    De Souza Sierra, M M 1997. Spectral identification and behavior of dissolved organic fluorescent material during estuarine mixing processes. Marine Chemistry, 58: 51-58.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Donard, O F 1989. High-sensitivity fluorescence spectroscopy of Mediterranean water using a conventional or a pulsed laser excitation source. Marine Chemistry, 27(117-136): 127-144.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Hedges, J I 1984. Carbon and nitrogen determinations of carbonate-containing solids. Limnology and Oceanography, 29: 657-663.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Hedges, J I 1994. Origins and processing of organic matter in the Amazon River as indicated by carbohydrates and amino acids. Limnology and Oceanography, 39(4): 743-761.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Lorrain, A 2003. Decarbonation and preservation method for the analysis of organic C and N contents and stable isotope ratios of low-carbonated suspended particulate material. Analitica Chimica Acta, 491: 125-133.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    McKnight, D M 2001. Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity. Limnology and Oceanography, 46(1): 38-48.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    Citation
    Peersen, O B 1993. Variable-amplitude cross-polarization MAS NMR. Journal of Magnetic Resource, 104: 334-339.

    Instrumentation
    Whatman 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Method Step

    Description
    Surface water samples were collected in 25 L white low density polyethylene bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned beforehand by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted GF/F glass fiber filters (nominal pore size, 0.7 um) and concentrated (see Maie et al. 2005 for detail). During ultrafiltration the samples were cooled in iced water. In the case of saline water (TS/Ph10), diafiltration was conducted as follows: one liter of Milli-Q water was added to the concentrated sample and then re-concentrated to 100 mL. This was repeated a total of three times to eliminate salts. The concentrated samples were freeze-dried and powdered with an agate mortar and pestle in preparation for analysis. UDOM solutions of approximately 5 mgC L-1 were prepared in a 0.05 M Tris (Hydroxymethyl) aminomethane (THAM) buffer solution adjusted to pH 7.0 with phosphoric acid. Since the Florida Bay sample (TS/Ph10) exhibited considerably weaker absorbance, it was measured at 20 mgC L-1. After dissolution, all UDOM solutions were filtered through precombusted GF/F filters. The UV-Vis absorption spectra were measured 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 UV spectral slope (S). 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 S parameter is obtained by fitting the absorption data to a simple exponential equation (Blough and Green, 1995). The S parameter is known to be sensitive to baseline offsets; therefore, to correct for this, the average absorbance from 700 to 800 nm was subtracted from each spectrum (Green and Blough, 1994). Fluorescence spectra were measured. As a quick, simple means of distinguishing organic matter source changes, 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 0.5 nm increments 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 and maximum wavelength (Fmax) were determined (Donard, et al., 1989; 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 DOM derived from marine/microbial and terrestrial/higher plant origin. Originally, McKnight et al. (2001) introduced the fluorescence index as a ratio of emission intensities at 450 and 500 nm at an excitation wavelength of 370 nm. However, we noticed that after fully correcting fluorescence intensity values (including instrument bias corrections) there was a shift of emission maximum to longer wavelengths. Thus we modified the fluorescence index and used the ratio of fluorescence intensities at 470 and 520 nm, instead of 450 and 500 nm. Similar modifications are being considered by McKnight (pers. comm. 2004). Since comparison of FI values among published data was difficult due to inconsistent spectrum correction, in this study, interpretation was conducted based on the comparison within our sample set. For three dimensional fluorescence spectra, excitation emission matrix (EEM) measurements were collected in a 1 cm quartz cuvette. Forty emission scans were acquired at excitation wavelengths between 260 and 455 nm at 5nm intervals. The emission wavelengths were scanned from fex plus 10 nm to fex plus 250 nm at 2 nm intervals (Coble, 1996; Coble et al., 1993). The individual spectra were concatenated to form a three-dimensional excitation-emission matrix (EEM). Carbonates in UDOM samples were removed prior to analyses using acid vapor decarbonation (Hedges and Stern 1984). Briefly, samples were weighed in duplicate (around 2-5 mg) into silver capsules and exposed to hydrochloric acid vapor for 4 h. Acid vapor was removed under vacuum until there was no noticeable acid smell (Hedges and Stern 1984). Decarbonation of samples before measuring concentration and isotopic ratio of C is necessary and analytical artifacts due to decarbonation on the concentration and stable isotope ratio measurement of N are negligible (Lorrain et al. 2003). Organic carbon and total nitrogen concentrations were measured. C and N stable isotopic analyses were measured. Isotopic ratios are reported in the standard delta notation. Results are presented with respect to the international standards of atmospheric nitrogen (AIR N2) and Vienna Pee Dee belemnite (V-PDB) for carbon. Duplicate measurements were conducted and reproducibility was =/- 1.5ppm for delta15N and +/- 0.2ppm for delta13C on average. Solid state cross polarization magic angle spinning (CPMAS) 15N NMR spectra were obtained using a ramped pulse sequence (Peersen et al 1993; Cook et al., 1996), a rotation frequency of 5.5 kHz, a contact time of 1 ms, and a pulse delay of 200 ms. Between 1.1 - 4.6 x 105 single scans were accumulated and line broadenings of 50-160 Hz were applied. The chemical shift was referenced to nitromethane scale (= 0 ppm) and was adjusted with 15N-enriched glycine (-347.6 ppm). XPS-N1s spectra were recorded using Mg K non-monochromatic radiation with an analyzer pass energy of 32 eV, an electric current of 30 mA, and a voltage of 10 kV. A finely powdered sample (ca. 1 mg) was fixed on the surface of a metallic sample block by means of Scotch double-sided nonconducting tape. Spectra were recorded for each visible line with 0.05 eV per step. The time for one scan was set at 298 ms, and between 126-160 scanned data were accumulated. Amino acid analysis was performed according to Hedges et al (1994). Greater detail can be found in Cowie and Hedges (1992). Briefly, amino acids were analyzed by high-performance liquid chromatography. Samples of UDOM were spiked with a mixture of acidic, basic, and neutral nonprotein amino acids (charge-matched recovery standards) and then hydrolyzed with 6 N HCl under N2 for 70 min at 150 degrees C. The hydrolysate mixture was dried, dissolved, filtered, and converted to fluorescent o-phthaldialdehyde (OPA) derivatives, which were analyzed on a 15 cm x 4.6-mm-i.d. column (xxxx) operated in reverse-phase mode with 5-m C18 packing. The amino acids reported with this method are referred to as total hydrolysable amino acids (THAA). The precision of this method is approximately 10%. A total of 19 individual amino acids were analyzed, 15 of which were protein amino acids (aspartic acid, Asp; glutamic acid, Glu; serine, Ser; histidine, His; glycine, Gly; threonine, Thr; arginine, Arg; alanine, Ala; tyrosine, Tyr; methionine, Met; valine, Val; phenylalanine; Phe; isoleucine, Ile; leucine, Leu; lysine, Lys) and 4 non-protein amino acids (-alanine (BALA), -aminobutyric acid (GABA), -aminobutyric acid (AABA), and ornithine (Orn)). A degradation index (DI), which was introduced by Dauwe et al. (1999) to evaluate the diagenetic state of POM by using amino acid composition, was calculated for our UDOM samples. The concept of DI is that when natural organic matter samples with different diagenetic histories are subjected to principal component analysis (PCA) based on their amino acid composition (mol%), the first principal component (PC1) represents the degree of diagenesis, therefore the score plot of PC1 is referred to as the DI.

    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 0.7m glass fiber filers Nalgene 25-L brown polyethylene bottles Pellicon 2 Mini Tangential Flow Ultrafiltration System (Millipore Co., Billerica, MA, USA) Jobin Yvon Horiba Spex Fluoromax-3 Fluorometer Shimadzu UV-2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) ThermoFinnigan Delta C Isotope Ratio Mass Spectrometer Elemental Analyzer Bruker DMX 400 Spectrometer (Bruker SioSpin GmbH, Rheinstetten, Germany) X-Ray Photoelectron Spectrometer (ESCA-3300, Shimadzu)

    Quality Control
    Data was processed using Excel (ver. 11 Microsoft) and SigmaPlot (ver.7 SPSS Inc.).All the fluorescence spectra were acquired in ratio mode whereby the sample and reference signals are collected and the ratio is calculated. Several post-acquisition steps were involved in the correction of the fluorescence spectra. First, an inner filter correction was applied to the fluorescence data according to McKnight et al. (2001). After inner filter corrections, the sample EEM underwent spectral subtraction of the buffer solution to remove most of the effects due to Raman scattering. Corrections for instrument bias related to wavelength dependent efficiencies of the specific instrument's optical components (gratings, mirrors, etc) were then made by applying multiplication factors, supplied by the manufacturer, for both excitation and emission wavelengths for the range of observations. Finally, fluorescence intensity was converted to quinine sulfate unit (QSU) to facilitate inter-laboratory comparisons (Coble et al., 1993). For X-Ray photoelectron spectroscopy, correction of binding energy was made relative to the C-C/C-H signal at 385.0 eV in the C1s spectra measured simultaneously. The spectra were deconvoluted into three major N species depending on its binding energy (solid lines), by applying three Gaussian curves with peak centers at 399.0 plus or minus 0.1 eV (aromatic N including imine, pyridine, aromatic amine, and NH in guanidine), 400.4 plus or minus 0.1 eV (peptide bond N including other amides, pyrrole, secondary and tertiary amines, and imide); and 402.3 plus or minus 0.1 eV (primary amine N including protonated amine; Abe and Watanabe, 2004). The proportions of three N groups in total N were estimated from the relative areas surrounded by Gaussian curves and base line with respect to the spectral area.
  • Distribution and Intellectual Rights
    Online distribution
    http://fcelter.fiu.edu/perl/public_data_download.pl?datasetid=ST_ND_Jaffe_005.txt
    Data Submission Date:  2005-09-26

    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, nutrients, DON, dissolved organic nitrogen, oligotrophic, biogeochemical cycling, emissions, amino acids, fluorescence, organisms, nitrogen, water, carbon, emissions, marine, acidic
  • 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:  Characterization of dissolved organic nitrogen in Taylor Slough and Shark River Slough from December 2001

    Entity Name:
    ST_ND_Jaffe_005
    Entity Description:
    Characterization of dissolved organic nitrogen in Taylor Slough and Shark River Slough from December 2001
    Object Name:
    ST_ND_Jaffe_005
    Number of Header Lines:
    1
    Attribute Orientation:
    column
    Field Delimiter:
    ,
    Number of Records:
    5