Permafrost soil cores were collected on the North Slope of Alaska during the ice-free summer months of June-August 2018 near the Toolik Field Station. Soil cores were collected from within the permafrost layer (at 85 cm below the surface) of Imnavait Creek wet sedge tundra and Toolik Lake tussock tundra soils. In June 2022, soil was sampled from a thermokarst failure on the shore of Lake LTER 395 on the North Slope of Alaska, where an abrupt collapse of thawing soil exposed deeper permafrost soil. Soil was sampled from the permafrost layer exposed in the headwall of the thermokarst failure (\> 80 cm below the surface) using MilliQ-rinsed pickaxes. The permafrost and thermokarst soil samples were collected as previously described in detail (Bowen et al., 2020), including precautions to minimize radiocarbon (¹⁴C) contamination by rinsing gloves and tools with deionized water prior to soil collection and storing soil samples in ¹⁴C-free facilities and freezers. These protocols were shown to result in no detectable ¹⁴C contamination of soils (Bowen et al., 2020). All soils were stored in freezers at the Toolik Field Station until overnight shipment to Woods Hole Oceanographic Institution (WHOI), where soils were stored in ¹⁴C-free freezers until further use.
Dissolved organic carbon (DOC) was leached from the permafrost and thermokarst soils as previously described (Bowen et al., 2020). Briefly, frozen soil and MilliQ water were mixed in 5-gallon, MilliQ-rinsed HDPE buckets and allowed to leach in the dark for up to 48 hours at 4 °C. Both the soil-to-water ratio of soil leachates and the leaching time were adjusted to achieve a final concentration of ~1500 μM DOC in the leachates, as estimated from the absorbance of chromophoric dissolved organic matter at 305 nm (a₃₀₅). All leachates were passed through MilliQ-rinsed 60 μm mesh screens to remove the largest particulates and then through MilliQ-rinsed 5 μm high-capacity Whatman cartridge filters. Subsamples of each leachate for light exposure experiments were then filtered through 0.2 μm high-capacity cartridge filters. Additional subsamples of each leachate were prepared as inoculum for biological incubations by passing the 5-μm-filtered water through 1.2 μm glass-fiber filters (hereafter referred to as the inoculum). Soil leachates for light exposure experiments and biological incubations were stored at 4 °C until further use (less than 48 hours for light exposure experiments, and less than 1 week for biological incubations).
Each 0.2-μm-filtered soil leachate was allowed to warm from the 4 °C storage temperature to room temperature for 12-24 hours prior to the start of dark or light treatments in light exposure experiments. Each soil leachate was then placed in six precombusted, 500 mL quartz flasks with ground glass stoppers without headspace. For each soil leachate, duplicate quartz flasks were exposed to 305 nm (UV) and 405 nm (visible) LED light treatments using custom built 10x1 LED chip arrays maintained at 30 °C using heat sinks and cooling fans (Bowen et al., 2020; Ward et al., 2021). Duplicate dark controls were run alongside light treatments at room temperature (23 °C) in the dark. The amount of DOC oxidized to CO₂ during LED light exposures was estimated from photochemical O₂ consumption, assuming 1 mol DOC completely oxidized to CO₂ per mol O₂ consumed (Cory et al., 2014; Ward & Cory, 2020). The durations of light exposures were chosen to achieve complete oxidation of ~5-10% of the initial DOC at each wavelength of light and ranged from 20 to 120 hours depending on the leachate. The duplicates of soil leachates from each dark or light treatment were composited in precombusted glass bottles and stored overnight in the fridge at 4 °C. This storage period allowed for decay of reactive oxygen species produced upon exposure of DOC to UV and visible light (Andrews et al., 2000; Cory et al., 2010; Page et al., 2014; White et al., 2003).
Each soil leachate treatment (dark, UV, visible) was mixed with the respective inoculum from each site to achieve 20% inoculum by volume (Cory et al., 2013). From each inoculated dark and light-exposed leachate, replicates for ¹⁴C and ¹³C analysis of the CO₂ produced by microbial respiration were filled in precombusted 125 mL borosilicate bottles with greased glass stoppers and no headspace. For each dark or light-exposed leachate, there were duplicate viable and killed treatments to quantify the C isotopic signatures of the CO₂ produced during respiration. The viable treatment was unamended, and the killed treatment was amended with saturated mercuric chloride (all preservations had 1% HgCl₂ by sample volume). The amount of DOC respired from each dark and light-exposed leachate was quantified as O₂ consumption and CO₂ production by microbial respiration. O₂ consumed and CO₂ produced by respiration were quantified as the difference in dissolved O₂ and dissolved inorganic carbon (DIC), respectively, between viable and killed treatments from a split of each inoculated dark and light-exposed leachate placed in precombusted, gas-tight 12 mL soda glass exetainers with no headspace. For each dark and light-exposed leachate, there were triplicate viable and killed treatments for analysis of O₂ and DIC. The viable treatment was unamended, and the killed treatment was amended with saturated mercuric chloride. All viable and killed inoculated soil leachates were incubated in the dark at 10 °C for between 17 to 30 days. The incubation duration was chosen for each set of dark and light-exposed soil leachates from a soil site so that respiration produced at least a 10% increase in the total dissolved inorganic carbon (DIC) of the water compared to the start of the incubation. At the end of the incubations, all viable leachate treatments in 125 mL borosilicate bottles and 12 mL exetainers were preserved by addition of saturated mercuric chloride. These preserved samples were stored at 4 °C in the dark until ¹⁴C and ¹³C analysis (up to 2 months) or until analysis of O₂ consumption and CO₂ production by respiration (less than 2 weeks) as previously described (Bowen et al., 2020; Cory et al., 2014).
A 75 mL sample of each soil leachate after dark or light treatment and inoculation was 0.22-μm Sterivex filtered and frozen for ¹⁴C and ¹³C analysis of the DOC present at the start of the biological incubations. The Δ¹⁴C and δ¹³C of the DOC were quantified at the National Ocean Sciences Accelerator Mass Spectrometry (NOSAMS) Facility at WHOI, using previously described methods (Xu et al., 2021). Briefly, soil leachates were acidified to pH < 2 with UVC-oxidized trace-metal grade phosphoric acid (85%) and stripped of dissolved inorganic carbon (DIC) with high-purity helium gas in the dark. The DOC was then oxidized with UVC light to DIC, and the resultant CO₂ was extracted cryogenically. A subsample of the CO₂ was analyzed for ¹³C using a VG Prism-II or Optima stable isotope ratio mass spectrometer, and the δ¹³C (‰) was calculated as follows:
δ¹³C = (¹³R<sub>sample</sub>/¹³R<sub>standard</sub> – 1)
where ¹³R is the isotope ratio of a sample or standard (VPDB), as defined by:
¹³R = (¹³C/¹²C)
The remaining CO₂ was reduced to graphite with H₂ and an iron catalyst, and then analyzed for ¹⁴C isotopic composition using an accelerator mass spectrometer at the NOSAMS facility (Longworth et al., 2015). The Δ¹⁴C (‰) and radiocarbon age of DOC were calculated from the fraction modern using the oxalic acid I standard (NIST-SRM 4990).
To characterize the isotopic composition of the CO₂ produced by respiration of permafrost DOC by microbes, the Δ¹⁴C and δ¹³C of dissolved inorganic carbon (DIC) were quantified in duplicate at the National Ocean Sciences Accelerator Mass Spectrometry (NOSAMS) Facility at WHOI from the viable and killed treatments of each dark and light-exposed leachate at the end of the incubation, following procedures previously described for quantification of the Δ¹⁴C and δ¹³C of CO₂ produced from photomineralization of permafrost DOC (Bowen et al., 2020). Briefly, water samples were acidified to pH < 2 with trace-metal grade phosphoric acid (85%) and stripped of DIC using high-purity nitrogen gas. The resultant CO₂ was trapped and purified cryogenically. The ¹³C of the CO₂ was analyzed at the NOSAMS facility and converted to Δ¹⁴C and δ¹³C values as described in the previous paragraphs.
An original set of dark control samples for the thermokarst soil leachates was compromised due to substantial microbial respiration consuming DOC in the dark control leachate as it sat at room temperature during light exposure experiments and prior to biological incubations. To replace the compromised dark control thermokarst waters, a second leachate was prepared from the thermokarst soil on a different date using the same soil and leaching conditions. The original thermokarst soil leachate was used for the UV and visible light treatments and subsequent biological incubation, while the second thermokarst soil leachate was used for the dark treatment and subsequent biological incubation. For all other soils, no detectable microbial respiration occurred in either the dark controls or the light-exposed leachates during light exposure experiments, and thus the same soil leachate was used for both the dark and light treatments.
Separately, samples of the CO₂ from microbial respiration in the dark and visible light-exposed tussock tundra leachate were lost during preparation for isotopic analysis at NOSAMS. Therefore, a second soil leachate was prepared from the tussock tundra soil using the same soil and leaching conditions, and all of the dark and light treatments and biological incubations for this soil leachate were repeated.
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