DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set.
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