Dataset title: 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 Dataset ID: ST_ND_Jaffe_005 Research type: Short-term Dataset Creator Name: Dr. Rudolf Jaffe Position: Lead Principal Investigator Organization: Florida Coastal Everglades LTER Program Address: Florida International University University Park OE 148 Miami, Florida 33199 USA Phone: 305-348-2456 Fax: 305-348-4096 Email: jaffer@fiu.edu URL: http://serc.fiu.edu/sercindex/index.htm Metadata Provider Organization: Florida Coastal Everglades LTER Program Address: Florida International University University Park OE 148 Miami, FL 33199 USA Phone: 305-348-6054 Email: fcelter@fiu.edu URL: http://fcelter.fiu.edu Dataset Abstract A better understanding of the biogeochemical cycling of nutrients entering Florida Bay is a key issue regarding the restoration of the Everglades. In addition to precipitation, the other major source of freshwater to Florida Bay is from Taylor Slough and the C-111 Basin in the northeast section of the Bay. While it is known that these areas deliver significant amounts of N to the Bay, a significant portion of this is in the form of dissolved organic N (DON). The sources, environmental fate and bioavailability to microorganisms of this DON are however, not known. Should this DON be readily available, any increased load as a function of restoration changes might have an impact on internal phytoplankton bloom dynamics. No significant flocculation or precipitation of DOM occurred with increase in salinity, meaning that terrestrial DOM does not get trapped in the sediments but stays in the water column where it subjected to photolysis and advective transport. Sunlight has a significant effect on the chemical characteristics of DOM. While the DOC levels did not change significantly during photo-exposure, the optical characteristics of the DOM were modified. The environmental implications of this are conflicting: photo-induced polymerization may stabilize the DOM by reducing its bioavailability while photolysis may make the DOM more labile. Overall, DON bioavailability was relatively low in this region. Even though the amount of DON loaded to the bay may be significant, the fraction of DON available for microbial cycling is much smaller. The amount of N supplied by recycling may be a significant portion of the total DIN pool. All this must be considered in context with the proposed CERP modifications to flows. As of the latest initial Comprehensive Everglades Restoration Project (CERP) update, the flows to Taylor Slough and C-111/Panhandle Basis are not predicted to change very much from base conditions. Therefore we do not expect any great increases in TN loading in this region. In contrast, the proposed flow increases to the Shark River Slough are large and may have significant effects on transport of DOM to the Southwest Florida Shelf. We believe that future efforts in DON characterization and bioavailability should be concentrated in this area. Geographic Coverage Study Extent Description The Study Extent of this dataset includes the FCE Taylor Slough and Florida Bay research sites within Everglades National Park, South Florida Bounding Coordinates Geographic description: Samples were collected in the Taylor Slough and Shark River Slough, within Everglades National Park, South Florida. West bounding coordinate: -81.078 East bounding coordinate: -80.607 North bounding coordinate: 25.410 South bounding coordinate: 25.025 Geographic description: Florida Coastal Everglades LTER Study Area: South Florida, Everglades National Park, and Florida Bay West bounding coordinate: -81.078 East bounding coordinate: -80.490 North bounding coordinate: 25.761 South bounding coordinate: 24.913 FCE LTER Sites: SRS4, SRS6, TS/Ph2, TS/Ph7a and TS/Ph10 All Sites Geographic Description:FCE LTER Site SRS4 Longitude:-80.964 Latitude:25.410 Geographic Description:FCE LTER Site SRS6 Longitude:-81.078 Latitude:25.365 Geographic Description:FCE LTER Site TS/Ph2 Longitude:-80.607 Latitude:25.404 Geographic Description:FCE LTER Site TS/Ph7a Longitude:-80.639 Latitude:25.191 Geographic Description:FCE LTER Site TS/Ph10 Longitude:-80.681 Latitude:25.025 Temporal Coverage Start Date: 2001-12-01 End Date: 2001-12-01 Data Table 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 Data Format Number of Header Lines: 1 Attribute Orientation: column Field Delimiter: , Number of Records: Attributes Attribute Name: SITENAME Attribute Label: SITENAME Attribute Definition: Name of LTER site Storage Type: text Measurement Scale: Name of LTER site Missing Value Code: Attribute Name: Date Attribute Label: 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 Online distribution: http://fcelter.fiu.edu/perl/public_data_download.pl?datasetid=ST_ND_Jaffe_005.txt Intellectual Rights These data are classified as 'Type II' whereby original FCE LTER experimental data collected by individual FCE researchers to be released to restricted audiences according to terms specified by the owners of the data. Type II data are considered to be exceptional and should be rare in occurrence. The justification for exceptions must be well documented and approved by the lead PI and Site Data Manager. Some examples of Type II data restrictions may include: locations of rare or endangered species, data that are covered under prior licensing or copyright (e.g., SPOT satellite data), or covered by the Human Subjects Act, Student Dissertation data and those data related to the FCE LTER Program but not funded by the National Science Foundation (NSF) under LTER grants #DEB-9910514, and # DBI-0620409. Researchers that make use of Type II Data may be subject to additional restrictions to protect any applicable commercial or confidentiality interests. All publications based on this dataset must cite the data Contributor, the Florida Coastal Everglades Long-Term Ecological Research (LTER) Program and that this material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DEB-1237517, #DBI-0620409, and #DEB-9910514. Additionally, two copies of the manuscript must be submitted to the Florida Coastal Everglades LTER Program Office, LTER Program Manager, Florida International University, Southeast Environmental Research Center, OE 148, University Park, Miami, Florida 33199. For a complete description of the FCE LTER Data Access Policy and Data User Agreement, please go to FCE Data Management Policy at http://fcelter.fiu.edu/data/DataMgmt.pdf and LTER Network Data Access Policy at http://fcelter.fiu.edu/data/core/data_user_agreement/distribution_policy.html. Dataset Keywords FCE Florida Coastal Everglades LTER ecological research long-term monitoring Everglades National Park biogeochemical cycling Taylor Slough nutrients DON dissolved organic nitrogen oligotrophic biogeochemical cycling emissions amino acids fluorescence organisms nitrogen water carbon emissions marine acidic Data Submission Date: 2005-09-26 Maintenance This is a short-term DOM dataset. This dataset replaces the original version named ST_ND_Jaffe_005. The FCE program is discontinuing its practice of versioning data as of March 2013. Dataset Contact Position: Information Manager Organization: LTER Network Office Address: UNM Biology Department, MSC03-2020 1 University of New Mexico Albuquerque, NM 87131-0001 USA Phone: 505 277-2535 Fax: 505 277-2541 Email: tech-support@lternet.edu URL: http://www.lternet.edu Position: Information Manager Organization: Florida Coastal Everglades LTER Program Address: Florida International University University Park OE 148 Miami, FL 33199 USA Phone: 305-348-6054 Fax: 305-348-4096 Email: fcelter@fiu.edu URL: http://fcelter.fiu.edu Dataset Submission Date 2005-09-26 Information Management Notes This is a short-term DOM dataset. This dataset replaces the original version named ST_ND_Jaffe_005. The FCE program is discontinuing its practice of versioning data as of March 2013.