Sample collection
Active layer and permafrost samples were collected from exposed bluffs near Drew Point during August 10-14, 2019. Three active layer (i.e. seasonally thawed) samples, three shallow permafrost samples, and three deeper permafrost samples were sampled from a young (approximately 500 yr BP) drained thermokarst lake basin (Hinkel et al., 2003; Jones et al., 2012). An additional three active layer samples were sampled from primary material that has never been reworked by thaw-lake cycles. Previous work has shown that the upper, organic rich permafrost horizon at Drew Point consists of Holocene terrestrial soils and/or lacustrine sediments, and that the lower mineral permafrost horizon consists of late-Pleistocene relict marine sediment (Bristol et al., 2021).
Samples from the drained lake basin were collected by scraping thawed material from exposed bluff before coring horizontally with a 7.5 cm diameter SIPRE core auger. Due to tall (~6m), steep bluffs at the primary surface site, active layer samples were collected by drilling vertically from the tundra surface to a depth of approximately 35 cm; these cores were subsampled to capture a similar depth range as horizontal cores. The SIPRE core barrel was wiped clean in between samples, and the average depth of the sample was measured from the tundra surface. Core sections were stored in clean Ziplock bags inside coolers packed with ice or frozen permafrost and transported back to the Barrow Arctic Research Center in Utqiagvik, Alaska by helicopter or float plane. Samples were stored in at -20C in a freezer at the Barrow Arctic Research Center until transported frozen back to the University of Texas Marine Science Institute. Seawater used for the experiment was collected from Beaufort Sea surface waters near Kaktovik, AK during August 2018, run through pre-combusted Whatman GF/F (0.7 um) filters, transported frozen to the University of Texas Marine Science Institute, and stored at -20C. The seawater had a salinity of 31.
Dissolved Organic Carbon Leaching
Active layer and permafrost samples were thawed at 4C and gently homogenized. Then, subsamples were taken to measure soil/permafrost organic carbon (OC) content (see "Bulk Geochemical Analyses") and water content. After subsampling, the homogenized soil/sediments were refrozen so that soil/permafrost OC could be quantified before leaching. Slurries were formed by combining thawed soil/permafrost samples with 700 mL GF/F filtered seawater in pre-combusted beakers. We normalized the soil/permafrost OC added to each slurry with a target concentration of 5 g OC L-1. This was equivalent to ~75-550 g wet soil/sediment L-1, depending on OC and water content, and resulted in high DOC concentrations. To act as a control, one beaker contained only seawater. The slurries and seawater control were covered and stored in the dark, gently stirring three times throughout the leaching period. After 24 hours, the slurries and seawater control were decanted and vacuum filtered using pre-combusted GF/F filters.
Biodegradable Dissolved Organic Carbon
To facilitate comparison with other studies, we generally followed the standardized biodegradable dissolved organic carbon (BDOC) protocol suggested by Vonk et al. (2015). This lability assay measures DOC loss throughout an incubation, often 28 days at room temperature, in the dark to prevent photochemical reactions or primary production. Initial samples are 0.7 um filtered before the incubation, removing particulate matter but allowing some microbial biomass to pass through. At the end of the incubation period, samples are re-filtered (0.7 um) to remove any bacterial aggregates that may have formed. Therefore, the DOC loss represents DOC that was respired and/or incorporated into microbial biomass.
Immediately after filtering the slurries, each leachate and the seawater control were subsampled for initial chemical analyses (i.e., DOC concentrations, CDOM, FT-ICR MS). Acid washed, pre-leached polycarbonate bottles were used throughout the experiment. Each leachate and the seawater control were incubated in triplicate in the dark on a shaker table in an environmental chamber and sampled at 26 and 90 days. The incubation temperature averaged 19C. Bottles were sealed to prevent evaporation but had a 1:1 solution to headspace ratio and were uncapped and aerated for 10 minutes weekly. After 26 and 90 days, triplicate sets of bottles were re-filtered to remove large microbial biomass aggregates using pre-combused GF/F filters and analyzed for DOC concentrations.
Bulk Geochemical Analyses
Subsamples for soil/sediment TOC (%) analysis were acidified with 10% ultrapure HCl in silver capsules before carbon mass was analyzed with a Thermo Fisher EA-Isolink-CNSOH elemental analyzer. Concentrations of DOC were measured with a Shimadzu TOC-V CSH analyzer. Samples were acidified to a pH of 2 with concentrated HCl (ACS reagent grade; JT Baker) immediately after filtering and DOC concentrations were analyzed within 24 hours.
Subsamples for CDOM were stored at 4C and measured within 24 hours using an Ocean Optics UV-visible light absorbance spectrophotometer. Ultrapure water blanks were run approximately every 5 samples and used to correct sample spectra. Parameters S275-295 (log-transformed spectral slope between 275 and 295 nm) and SR (275-295 nm slope:350-400 nm slope) were calculated from CDOM spectra according to Helms et al. (2008). The specific ultra-violet absorbance at 254 nm (SUVA254; decadic absorbance at 254 nm normalized by DOC concentration in mg L-1) was calculated according to Weishaar et al. (2003). All bulk chemical analyses were completed at the University of Texas Marine Science Institute.
Ultra-High Resolution Mass Spectrometry
Leachate DOM samples for FT-ICR MS analyses (t0 only) were solid phase extracted onto reverse phase BondElut PPL cartridges (100 mg; Agilent) following an established protocol (Dittmar et al., 2008). Briefly, leachate subsamples were acidified to a pH of 2 with HCl, passed through pre-conditioned PPL cartridges, rinsed with acidified water, and eluted with 1 mL methanol for a target concentration of 40 ug C mL-1. ACS reagent grade HCl (JT Baker) and LC/MS grade methanol and water (Fisher Chemical) were used throughout.
Extracts were stored in methanol at -20 degrees C until analysis on a custom-built hybrid linear ion trap 21 T FT-ICR MS at the National High Magnetic Field Laboratory in Tallahassee, Florida (Hendrickson et al., 2015; Smith et al., 2018) using negative electrospray ionization. For each spectrum, 100 time domain acquisitions were co-added. Mass spectra were phase-corrected (Xian et al., 2010) and molecular formulae assigned to peaks that had >6σ root-mean-square baseline noise (Behnke et al., 2021; O'Donnell et al., 2016) with PetroOrg © TM software (Corilo, 2015). Formulae were assigned using elemental constraints of C1-45H1-92O0-35N0-4S0-2 and with a mass accuracy less than or equal to 300 ppb (Kellerman et al., 2018). The modified aromaticity index (AImod) was calculated from the molecular formulae to measure the degree of aromaticity (Koch & Dittmar, 2006, 2016). Elemental ratios and AImod were used to assign the following compound classes to the molecular formulae: polyphenolics (0.5 < AImod > 0.66); condensed aromatics (AImod > 0.66); highly unsaturated and phenolics (HUPs; AImod<=0.5, H/C < 1.5, O/C <= 0.9); aliphatic (1.5 <= H/C <= 2.0, O/C <= 0.9 and N = 0); sugar-like (O/C > 0.9); and peptide-like (1.5 <= H/C <=2.0, and N > 0) (Behnke et al., 2021). Each assigned molecular formula may contain multiple isomers, and compound structure cannot be assessed from FT-ICR MS data. The relative abundance of each formula was calculated by normalizing each peak magnitude to the sum of all peak magnitudes assigned in each sample. The relative contributions (expressed as percentages) of each compound class and elemental composition grouping (e.g., CHO, CHON) were then calculated as the sum of all the relative abundances of all the peaks in each compound class divided by the sum of all the assigned formulae abundances in each sample.
Data Analyses
Results from samples incubated in triplicate were averaged. DOC leaching yields were calculated using the t0 leachate concentrations normalized by both soil/permafrost dry weight and OC content accounting for DOC in the seawater (1.2 mg-C L-1). Here, we report the absolute loss of DOC as well as the percent loss of DOC (i.e., biodegradable DOC; BDOC) at each timepoint. There was no detectible change in seawater control DOC concentrations throughout the incubation experiment indicating there was no biodegradable DOC in the seawater. Therefore, BDOC reported here is the percent loss ofleached DOC where the leached DOC concentration is equivalent to the measured leachate DOC concentration minus the measured seawater control DOC concertation.
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