Selective Photochemical Oxidation of Reduced Dissolved Organic Sulfur to Inorganic Sulfate

The chemical nature and stability of reduced dissolved organic sulfur (DOSRed) have implications on the biogeochemical cycling of trace and major elements across fresh and marine aquatic environments, but the underlying processes governing DOSRed stability remain obscure. Here, dissolved organic matter (DOM) was isolated from a sulfidic wetland, and laboratory experiments quantified dark and photochemical oxidation of DOSRed using atomic-level measurement of sulfur X-ray absorption near-edge structure (XANES) spectroscopy. DOSRed was completely resistant to oxidation by molecular oxygen in the dark and underwent rapid and quantitative oxidation to inorganic sulfate (SO42–) in the presence of sunlight. The rate of DOSRed oxidation to SO42– greatly exceeded that of DOM photomineralization, resulting in a 50% loss of total DOS and 78% loss of DOSRed over 192 h of irradiance. Sulfonates (DOSSO3) and other minor oxidized DOS functionalities were not susceptible to photochemical oxidation. The observed susceptibility of DOSRed to photodesulfurization, which has implications on carbon, sulfur, and mercury cycling, should be comprehensively evaluated across diverse aquatic environments of differing DOM composition.


Section S1. Description of Experimental Steps and Analyses a. DOM Sample Collection and Extraction
shown. 1 Pore water (30 L) was collected 10 cm below the sediment-water interface at a rate of 100 mL min -1 using a Teflon sipper connected to Teflon tubing and a peristaltic pump. Pore water temperature, conductivity (Orion four-cell conductivity electrode), pH (Orion ROSS Ultra™ electrode), dissolved oxygen (DO) concentration (Orion RDO optical probe), and oxidation-reduction potential (ORP; Orion ORP Triode electrode) were measured using a flow-through cell (Geotech; 40 mL dead volume) and multiparameter meters (Orion Star™ A329, Beckman Coulter pHi 410). Electrodes were calibrated using manufacturer specifications prior to field deployment. All measurements collected during the sampling are reported in an associated data release. 2 The sipper was repositioned laterally by approximately 0.5 m every 10 min to not deplete sediment pore waters. The pore water was field filtered (0.45 µm polysulfone capsule filters; Geotech) directly into 2 L polyethylene terephthalate bottles with no head space. The sample was stored at 4 °C and shipped on ice to the USGS (Boulder, Colorado) for DOM isolation.
In the laboratory, care was taken to minimize exposure of the pore water sample to atmospheric oxygen during storage and DOM isolation. First, residual inorganic sulfide (H2S/HS -; pKa = 7.0) was removed (to <0.3 µM) from the sample by adjusting to pH 4.0 with trace-metal grade HCl and purging with helium for 1 h. Sulfide removal was confirmed by preserving a subsample with 50% volume/volume sulfide anti-oxidant buffer and measuring S 2− by ion-elective electrode (Orion silver/sulfide; calibrated using 0.3 -310 µM calibration standards). Next, the hydrophobic organic acid (HPOA) fraction of DOM was isolated on XAD-8 resin with precautions taken to minimize possible oxidation of organic sulfur. All solutions used during the SPE were degassed with helium for 1 hour prior to use. During isolation, the sample was back-eluted with deaerated 0.1 M NaOH directly through proton-saturated cation exchange resin. Experimental results provided in Section S2 of the Supporting Information demonstrate that the isolation of DOM by XAD-8 resin using deaerated solutions did not result in measurable oxidation of organic sulfur compared to XAD-8 isolation using solutions at equilibrium with the ambient atmosphere or an alternative SPE procedure that uses a methanol elution ( Figure S2). 3 The HPOA sample accounted for 54% of the dissolved organic carbon (DOC) in the whole water sample and was stored for 21 days (pH 3.5, under nitrogen, 4 °C) until use in laboratory oxidation experiments.

b. Laboratory Oxidation Experiments
First, the purified DOM sample was diluted with deaerated high-purity water (≥18 MΩ cm; Barnstead GenPro UV) to a DOC concentration of 37.9 mg L -1 and adjusted to pH 7 with deaerated 0.1 M NaOH solution ( Figure S1). Differences in ionic strength are not expected to influence rates of DOS oxidation, 4 and therefore no other inorganic salts were added to the starting DOM solution. A subsample (1 L volume) was immediately preserved for analysis (termed t = 0 (initial)) to characterize the initial experimental conditions. Inorganic sulfate was quantified at 1.0 µM and accounted for approximately 3% of total sulfur at the start of the experiment. The following four treatments were performed in 2 L quartz round bottom flasks filled with 1.0 L of DOM solution.
1. A dark anoxic control treatment (n = 1, termed Dark, Anoxic Control) was stored in the dark (vessel covered in aluminum foil), under nitrogen at 22±2 °C for 14 d to identify changes in sulfur speciation during dark storage or DOM re-isolation on XAD-8 resin.
2. A dark O2 purge treatment (n = 1, termed Dark, O2 Purge) was purged in the dark (vessel covered in aluminum foil) with zero-grade air (20.5% oxygen, 79.5% nitrogen) at 30 mL min -1 at 22±2 °C for 192 h. This treatment quantified the susceptibility of reduced organic sulfur species in DOM to oxidation by molecular oxygen, and the purge duration was equivalent to the duration of the longest light treatment (Light 5).
3. Light treatment (n = 5, termed Light 1-5) were performed in a temperature-controlled solar simulator at 30 °C (Suntest XLS; calibrated prior to use). The Light 3 treatment was conducted in duplicate to assess experimental reproducibility. The irradiance was 500 W m −2 (300 -800 nm irradiance range) at the sample shelf of the solar simulator (spectrum provided in Figure S3). The solution surface was 10.5 cm above the sample shelf. The photon absorption rate could not be used to determine the photo-irradiance due to changes in DOM absorption 4 over long light experiment. Immediately prior to irradiation, experimental solutions were purged with zero-grade air for 15 minutes to ensure dissolved oxygen saturation (75 mL min -1 ). The dissolved oxygen concentration was measured at 7.1 mg L -1 (98% saturation; Orion RDO optical probe) prior to the start of the experiment. Independent vessels were irradiated for the following times: 1.3 h, 5.3 h, 24 h, 78 h, and 192 h. For Light treatments of 24, 78, and 192 h experimental duration, solutions were purged with zero-grade air every 12 hours for 15 minutes (75 mL min -1 ) to ensure oxygen saturation. The rationale was to maintain consistent experimental conditions, due to previous observations of dissolved oxygen depletion during DOM photolysis. 4 At the end of oxidation experiments, solutions were sampled for aqueous measurement of DOC concentration, DOM absorption and fluorescence spectra, inorganic sulfate (SO4 2-), and thiosulfate (S2O3 2-). Aliquots for DOC concentration, DOM absorption and fluorescence spectra, and inorganic sulfate were stored at 4 °C in pre-baked (450 °C for 4.5 h) amber borosilicate glass vessels with Teflon®lined caps. Aliquots for thiosulfate measurement were stored in high-density polyethylene bottles preserved with zinc acetate (1% volume/volume Zn acetate) and NaOH (1% volume/volume 1 M NaOH).
Next, solutions were deaerated by purging with nitrogen for 1 hour, stored under nitrogen, and DOM was re-isolated by SPE on XAD-8 resin taking precautions to minimize oxidation (as described above).
The average recovery of DOC during the re-isolation was 85%; there was no evidence of DOM fractionation based on spectroscopic measurements of the DOM before and after re-isolation on XAD-8 resin. DOM extracts were lyophilized and stored under nitrogen. Inorganic SO4 2was confirmed to be below the detection limit (<0.50 µM) in DOM extracts (as describe below in Section S1d), confirming that S XANES spectra strictly measured DOS species.

c. DOC Concentration and DOM Optical Analyses
DOC concentration was determined by persulfate oxidation (OI Analytical, model 700). 5 UV-vis absorption spectra were measured from 190-800 nm using a spectrophotometer (Agilent Technologies, model 8453) and a 1 cm quartz cuvette. Sample UV-vis spectra were measured with respect to a blank spectrum containing high-purity water (≥18 MΩ cm; Barnstead GenPro UV). Decadic absorbance coefficients at 254 nm ( 254 )and 400 nm ( 400 ) were quantified using Equation S2, where is the is the absorbance, and is the path length (cm).
The specific ultraviolet absorbance at 254 nm (SUVA254), a proxy for DOM aromaticity, 6 where ( ) is the absorption coefficient at the specified wavelength, ( ) is the absorption coefficient at the reference wavelength, and S is the slope fitting parameter. 7

e. Sulfur XANES Spectra Acquisition and Processing
The sulfur and carbon contents of freeze-dried DOM samples, used to quantify the atomic sulfur-tocarbon ratio (atomic S/C), were determined by Huffman Hazen Laboratories (Golden, CO). Sulfur K-edge XANES spectra were collected on DOM samples (n = 12) on beamline 9-BM-B of the Advanced Photon organosulfate (DOSSO4), were calculated with a precision estimated at ≤ 1.6% by measurement of DOM samples from the IHSS. 10 Accuracies of atomic fractions of reduced (DOSExo, DOSHetero) and oxidized sulfur functionalities (DOSSO2, DOSSO3 , DOSSO4) are estimated at 8% and 4%, respectively. 10 Figure S5 presents S XANES spectra of 4 model reduced S compounds with differing S functionalities, including cysteine (e.g., thiol), methionine (e.g., thioether), cystine (e.g., disulfide), and dibenzo thiophene (e.g., thiophene). Due to challenges in resolving the exact species of DOSRed, a product of the differences in X-ray absorption peak locations and features (e.g., doublets and shoulders), this study presents the total DOSRed, defined as the summation of DOSExo and DOSHetero.

Section S2. Evaluation of the DOM Solid Phase Extraction
Three solid phase extraction (SPE) procedures were compared on a single water sample to assess if changes in organic sulfur speciation may occur as a result of the DOM isolation, specifically during the base-elution step during XAD-8 resin isolation. The DOM samples were analyzed for elemental composition (C, H, S content) and S speciation by S-kedge XANES spectroscopy (Tables S1-S2, Figure S2). Negligible differences were observed between the spectra ( Figure S2) and distribution of sulfur functionalities (Table S1) between samples isolated on XAD-8 resin using solutions under ambient atmosphere versus deaerated. The DOM sample isolated using deaerated solutions had a modest 6% higher sulfur content relative to carbon (atomic S/C), but differences in the atomic fractions of sulfur functionalities were within the precision of spectral fits estimated previously to be ≤ 1.6% for a given functionality. 1 Table S1. Parameter values for Gaussian curve fitting (GCF) and sulfur atomic fractions (%) of measured spectra of DOM isolated using three separate solid-phase extraction (SPE) procedures. Measured spectra of these three samples are presented in Figure S2.   a Total reduced sulfur is defined as the summation of exocyclic reduced (DOSExo) and heterocyclic reduced sulfur (DOSHetero). Table S3. Parameter values for Gaussian curve fitting (GCF) and sulfur atomic fractions (%) of measured spectra of the hydrophobic organic acid (HPOA) fraction of DOM from laboratory oxidation experiments. GCF curves are shown in Figure S6. a values in bold were co-varied and values in bold and underlined were fixed during Gaussian curve fitting. b calculated using generic calibration curve. 10 c precisions of atomic fractions are estimated to be ≤ 1.6%; the accuracy of the atomic fraction of each reduced and oxidized sulfur functionality is estimated at 8% and 4%, respectively. 10 d the normalized sum-squared residual (NSS) is the normalized difference between experimental and fit spectra. Table S4. Parameter values for Gaussian curve fitting (GCF) and sulfur atomic fractions (%) of measured spectra of the hydrophobic organic acid (HPOA) fraction of DOM from laboratory Light treatments (GCF curves in Figure S8). a values in bold were co-varied and values in bold and underlined were fixed during Gaussian curve fitting. b calculated using generic calibration curve. 10 c precisions of atomic fractions are estimated to be ≤ 1.6%; the accuracy of the atomic fraction of each reduced and oxidized sulfur functionality is estimated at 8% and 4%, respectively. 10 d The normalized sum-squared residual (NSS) is the normalized difference between experimental and fit spectra.