Dataset: Changes in dissolved organic carbon, stable carbon isotopes, pH, condensed aromatic carbon, and carboxyl-rich alicyclic molecules during laboratory photo-irradiation of vanillin

Vanillin (Acros, 99%), a lignin monophenol, was chosen as a representative monomeric unit of lignin, a major component of tDOM (Opsahl and Benner, 1998). A solution of 20 mg-C of vanillin in 1 L ultrapure MilliQ water was prepared to serve as a stock solution in photochemical experiments. This concentration was chosen because it is representative to carbon contents in upstream terrestrial aquatic environments such as rivers and lakes (Gonsior et al., 2009).

Photo-irradiation. Approximately 350 mL of vanillin stock solution was aliquoted into a pre-combusted 500 mL quartz round-bottom flask capped with a pre-combusted glass cap. An aliquot of the stock solution was immediately wrapped with aluminum foil and stored at room temperature as a dark control. Another aliquot of the stock solution was frozen to evaluate the stability of the dark control at room temperature. Up to three 500 mL quartz flasks were placed in a flatbed SUNTEST CPS+ solar simulator equipped with a daylight UV glass filter (ATLAS Cat. No. 56052371, cutoff at ~290 nm) for simulating solar radiation outdoors. Irradiation was performed under controlled chamber air temperature of 35 °C in 24-hour cycles using power of 250 W/m² yielding ~21,600 kJ/m² per 24 hours. Flasks were irradiated for 24 hours and then opened for 30 minutes during which they were aerated and gently stirred. During this aeration step, a 20-, 40-, or 50-mL subsample was taken out for analyses. To keep the same total volume throughout the irradiation (350 mL), the same amount of ultrapure water was added back. Multiple irradiation experiments were performed to yield sufficient sample volumes for the different analytical methods employed for the characterization of the photo-products. 

Dissolved organic carbon. Dissolved organic matter was quantified as dissolved organic carbon (DOC) equivalents (in mg-C/L) on a Shimadzu TOC-VCPH instrument following standard operating procedures. Briefly, samples were acidified and sparged with air for 3 minutes to remove any inorganic carbon species (dissolved CO, CO2, HCO3-, CO32-) or other dissolved volatile gases (e.g., CH4). Then, samples were injected in a high temperature combustion reactor (720 ˚C) to oxidize the DOM to CO2. Detection of CO2 is achieved using a non-dispersive infrared detector. DOC concentrations were quantified using standard calibration curves made with potassium hydrogen phthalate (Nacalai Tesque, JIS special grade).

Stable carbon isotopic composition (δ13C). Photo-irradiated solutions of vanillin were aliquoted to Falcon® tubes that were pre-aged in 2 % (w/w) NaOH for one month to remove any leachable carbon. Samples were freeze-dried and resuspended in ultrapure water to yield concentrated solutions necessary for analysis on a spooling-wire microcombustion (SWiM) device (Sessions et al. 2005) interfaced with an isotope-ratio mass spectrometer (IRMS) to determine the ratio of 13C to 12C atoms expressed in a delta notation (δ13C) and referenced to Vienna Pee Dee Belemnite (Camin et al. 2025).

The SWiM system used has been employed previously to measure δ¹³C values of aqueous samples (Mohr et al. 2014). The SWiM interface is connected to a ThermoFinnigan DeltaPlus Advantage IRMS via an open split. Reference CO₂ is controlled using a Conflo III, and data are collected and analyzed using Isodat v3.0.  Each analytical run begins with three pulses of standard CO₂ reference gas and ends with two pulses of standard CO₂ reference gas. Samples were injected in 1 μL aliquots, separated by 30 sec; 3 to 5 injections are made of each sample.  All data are corrected assuming linear drift of the m/z 45/44 ratio over the duration of the run. Each run includes samples (unknowns) and standard dilution series of two laboratory reference materials (dextrose, -11.2‰, and nor-leucine, -27.7‰). Empty tube blanks containing ultrapure water are also measured. Directly measured blank δ¹³C values were smaller than values inferred from the dilution series, determined by the intersection of δ¹³C versus Area-1 for the two dilution series, which intersect at Area-1 and δ¹³C of the blank. All samples were subsequently blank-corrected using the intersection of these two lines.

pH. pH measurements were performed using a Thermo Orion Star Series benchtop conductivity meter on unirradiated and irradiated vanillin solutions. The pH meter was calibrated daily with pH 4.01 buffer solution (YSI 3821), pH 7.00 buffer solution (YSI 3822), and pH 10.00 buffer solution (YSI 3823).

Condensed aromatic carbon (ConAC). Exactly 40 mL of vanillin control and photochemically processed samples were freeze-dried. The obtained residue was redissolved in 2 mL of 1:1 methanol:water and transferred to 2-mL glass ampules. The solvent was evaporated at 60 °C and the residue was mixed with 0.5 mL concentrated nitric acid (65 %). Ampoules were flame-sealing and then samples were digested in an oven at 160°C for 6 hours. After this thermal digestion, the nitric acid was evaporated at 70°C in a sand bath under a stream of ultrapure nitrogen. The residue was redissolved in 0.5 mL of 0.6 mol L⁻¹ H₃PO₄ and filtered through a 0.2 µm syringe filter (Thermo Scientific™ Titan3™ PTFE). Benzenepentacarboxylic acid (B5CA, C₁₁H₆O₁₀) and benzenehexacarboxylic acid (B6CA, C₁₂H₆O₁₂) were quantified using an Agilent 1100 high-performance liquid chromatography system (Wagner et al. 2017). Their concentrations were then converted to carbon concentrations (i.e., B6CAC and B5CAC) and used to estimate the ConAC content of the original sample prior to digestion by multiplying by a scaling factor of 7.04 (Bostick et al., 2018) followed by normalization to organic carbon content. The molar ratio of B6CA to B5CA was also calculated to serve as an indicator of the degree of condensation of ConAC. 

Carboxyl-rich alicyclic molecules (CRAM). Carboxyl-rich alicyclic molecules (CRAM) were quantified using liquid-state 1H NMR spectroscopy. Analyses were performed at room temperature (24.9 ± 0.1 °C) on a 400 MHz (9.4 Tesla) Bruker BioSpin AVANCE III spectrometer fitted with a double-resonance broadband inverse (BBI) probe at the College of Sciences Major Instrumentation Cluster (COSMIC) facility of Old Dominion University (Norfolk, VA). Instrument control and data processing were performed using the TopSpin software. Analyses were performed without pre-concentration (e.g., via solid-phase extraction) or filtering on aliquots of samples taken at different time points. Samples were prepared by mixing a 516-µL aliquot of the sample with 60 µL deuterated water (Acros Organics, 99.9% D, CAN RN 7789-20-0) to produce a 90:10 H2O:D2O solution necessary for obtaining a deuterium lock. Additionally, sodium 2,2,3,3-tetradeutero-3-trimethylsilylpropanoate was added (24 µL of 25 µM solution; Acros Organics, 98% D) to serve as an internal standard. Once all aliquots were added to an NMR tube, they were mixed using a vortex mixer. NMR tubes were pre-cleaned by soaking in aqua regia for 2+ weeks. Prior to use, each NMR tube is rinsed with ~250 mL of ultrapure water and dried at 60 °C to ensure no contamination.

One-dimensional (1D) quantitative 1H spectra were acquired using the PEW5shapepr pulse program, which utilizes Perfect Echo - WATERGATE and shaped presaturation techniques for water suppression (Adams et al., 2013, Whitty et al., 2019). The employment of this pulse sequence results in quantitative spectra, with the water resonance at 4.7 ppm being suppressed. The relaxation delay (D1) employed for all quantitative analyses was 13.5 s, which was determined after an inversion-recovery experiment. For each individual sample the 90° excitation pulse width (P1) and transmitter offset (O1) were optimized to ensure quantitativeness of the signals. The obtained free-induction decay of 4096 summed transients was zero-filled to a 16k-sized dataset and apodized using a 10-Hz Lorentzian window function. 

Additional Data 

Additional spectroscopic and mass spectrometric data is available from the authors upon request.