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.
References:
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.
Coble, P G 1996. Characterization of marine and terrestrial DOM in seawater using excitation-emission spectroscopy. Marine Chemistry, 51(4): 325-346.
Coble, P G 1993. Fluorescence contouring analysis of DOC intercalibration experiment samples: a comparison of techniques. Marine Chemistry, 41: 173-178.
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.
Cowie, G L 1992. Sources and reactivities of amino acids in a coastal marine environment. Limnology and Oceanography, 37(4): 703-724.
Dauwe, B 1999. Linking diagenetic alteration of amio acids and bulk organic matter reactivity. Limnology and Oceanography, 44(7): 1809-1814.
De Souza Sierra, M M 1994. Fluorescence spectroscopy of coastal and marine waters. Marine Chemistry, 58: 127-144.
De Souza Sierra, M M 1997. Spectral identification and behavior of dissolved organic fluorescent material during estuarine mixing processes. Marine Chemistry, 58: 51-58.
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.
Hedges, J I 1984. Carbon and nitrogen determinations of carbonate-containing solids. Limnology and Oceanography, 29: 657-663.
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.
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.
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.
Peersen, O B 1993. Variable-amplitude cross-polarization MAS NMR. Journal of Magnetic Resource, 104: 334-339.
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.
