Ancient low–molecular-weight organic acids in permafrost fuel rapid carbon dioxide production upon thaw

Significance

To our knowledge, this study is the first to directly link rapid microbial consumption of ancient permafrost-derived dissolved organic carbon (DOC) to CO₂ production using a novel bioreactor. Rapid mineralization of the freshly thawed DOC was attributed to microbial decomposition of low–molecular-weight organic acids, which were completely consumed during the experiments. Our results indicate that substantial biodegradation of permafrost DOC occurs immediately after thaw and before downstream transport occurs. We estimate that, by 2100, between 5 to 10 Tg of DOC will be released from Yedoma soils every year given the most recent estimates for projected thaw. This represents 19–26% of annual DOC loads exported by Arctic rivers, yet it is so far undetectable likely due to rapid mineralization in soils and/or headwater streams.

Abstract

Northern permafrost soils store a vast reservoir of carbon, nearly twice that of the present atmosphere. Current and projected climate warming threatens widespread thaw of these frozen, organic carbon (OC)-rich soils. Upon thaw, mobilized permafrost OC in dissolved and particulate forms can enter streams and rivers, which are important processors of OC and conduits for carbon dioxide (CO₂) to the atmosphere. Here, we demonstrate that ancient dissolved organic carbon (DOC) leached from 35,800 y B.P. permafrost soils is rapidly mineralized to CO₂. During 200-h experiments in a novel high–temporal-resolution bioreactor, DOC concentration decreased by an average of 53%, fueling a more than sevenfold increase in dissolved inorganic carbon (DIC) concentration. Eighty-seven percent of the DOC loss to microbial uptake was derived from the low–molecular-weight (LMW) organic acids acetate and butyrate. To our knowledge, our study is the first to directly quantify high CO₂ production rates from permafrost-derived LMW DOC mineralization. The observed DOC loss rates are among the highest reported for permafrost carbon and demonstrate the potential importance of LMW DOC in driving the rapid metabolism of Pleistocene-age permafrost carbon upon thaw and the outgassing of CO₂ to the atmosphere by soils and nearby inland waters.

Northern permafrost soils contain an estimated 1,700 Pg of organic carbon (OC) that has been isolated from the modern carbon cycle since the Pleistocene (1, 2). Yedoma permafrost soils, deposited as windblown silts in the late Pleistocene across Siberia and Alaska and frozen in place, account for a significant portion of the northern permafrost OC pool. It is estimated that these frozen, organic-rich silts contain anywhere from 210 (2, 3) to 456 Pg (2, 4) of OC. Rising global temperatures are expected to enhance thawing of this vast reservoir of carbon. Regions of discontinuous permafrost, including much of the Yedoma complex, are particularly vulnerable to thaw (5). Upon thaw, this stored carbon is subject to mineralization and emission to the atmosphere as carbon dioxide, CO₂, and methane, CH₄ (6, 7), and to export to aquatic systems (8). Historically, it has been assumed that aged OC is substantially degraded relative to modern OC and therefore relatively less biodegradable (9). In the case of aged permafrost OC, this assumption has been challenged by recent studies that demonstrate high biodegradability (10–13). If a significant portion of the OC released from these thawing soils is indeed labile, either to microbial decomposition or photooxidation, it represents a vast potential source of greenhouse gas emissions to the atmosphere. These emissions would contribute to further warming and thereby accelerate permafrost thaw, a process known as the “permafrost climate feedback” (6, 11).

Thawed permafrost OC can be respired in situ in soils, or dissolved and mobilized in soil pore water, meltwater, or precipitation. Once dissolved and mobilized, permafrost-derived dissolved OC (DOC) can undergo mineralization within soil pore water or at any given point during subsurface or surface water transport to inland waters (14, 15). Aquatic pathways may be particularly important for permafrost soil-derived OC turnover following the physical disconnection of OC and decomposer communities occurring in the soil matrix (16). Evidence supporting these aquatic pathways is nearly ubiquitous around the world, and it has been widely demonstrated that fluvial systems are important processors of terrestrial OC and significant sources of CO₂ to the atmosphere (17–19). Once permafrost DOC is mineralized in a fluvial environment, the byproducts (aged CO₂ and CH₄) are emitted from surface waters to the atmosphere over large spatial areas, depending on the rate of gas emission (19, 20). The potential for, and pathways of, translocation of permafrost DOC from source (thawed soil) to fate (downstream transport or outgassing) pose challenges for quantifying and constraining the amount of thawed permafrost OC entering the atmosphere via mineralization in both terrestrial and aquatic environments (15).

Although recent investigations into the aquatic pathway in Siberia and Alaska demonstrate export of highly biodegradable DOC from thawing Yedoma soils to fluvial systems (11, 21), they have focused on stream and river DOC that has already undergone transport and potentially extensive mineralization in soils and in headwater stream networks. To complicate matters, the labile component of DOC is often a small and quickly cycling subset of the overall OC pool (22), which means that most river biodegradation measurements quantify the biolability of the remaining bulk fraction (23) and not of the original source DOC. To most accurately estimate the quantity of CO₂ released from thawing permafrost soils, the biodegradability of all DOC components must be assessed immediately upon thaw, before significant mobilization and/or mineralization occurs. To that end, we investigate three important unknowns regarding Yedoma-derived DOC decomposition in Arctic ecosystems: (i) What is the biodegradability of this DOC immediately upon soil thaw? (ii) How does the chemical composition of the DOC relate to its susceptibility to microbial decomposition? (iii) What proportion of DOC leached from Yedoma soils will be converted to CO₂ for eventual emission to the atmosphere? Answering these questions is key to understanding where on the landscape thawing Yedoma-derived DOC is likely to be decomposed, and to explaining observed and projected CO₂ and DOC patterns in Arctic watersheds.

To address these questions, we collected Yedoma permafrost soils for DOC biodegradability experiments from a newly excavated extension of the Fox Permafrost Tunnel, operated by the Cold Regions Research and Engineering Laboratory in Fairbanks, Alaska. We thawed the soils at low temperature (2 °C) and immediately upon thaw leached them for DOC for incubation and chemical analysis. We measured DOC biodegradability using a novel incubation method with high-resolution subsampling and in situ instrumentation (Fig. S1), which allowed simultaneous characterization of changes in the DOC pool and tracking of the mineralization to dissolved inorganic C (DIC). Five replicate incubations were conducted at room temperature (20 °C), which is comparable to maximum summer temperatures in Arctic fluvial ecosystems (24, 25).

Fig. S1.

Schematic of DOC biodegradation chamber and components.

Results and Discussion

The permafrost soil had a radiocarbon age of 35,800 y B.P. (±200 y B.P.). The permafrost leachates yielded an average of 38.4 ± 3.69 μmol C as DOC per gram soil. Leached DOC had a radiocarbon age of 33,700 y B.P. (±270 y B.P.). The average C content of the permafrost soil was 1.36% by weight and thus the average DOC yield per gram soil C was 3.39%. Leachates were diluted for incubation with carbon-free Milli-Q water (>18 MΩ⋅cm), having an average initial diluted DOC concentration of 1,520 ± 181 μmol⋅L⁻¹ (Table 1). Over the 200-h incubations, DOC concentrations decreased by an average of 53.5 ± 4.1%, with the majority of loss occurring in the first 100 h (Table 1 and Fig. 1A). We attribute DOC loss entirely to microbial uptake given the absence of other consumptive processes (i.e., photooxidation, sorption) in the closed incubation system. No DOC flocculation was observed. DOC mineralization resulted in a 7.4-fold average increase in DIC concentration (Fig. 1B; DIC = dissolved gaseous CO₂ plus dissolved bicarbonate plus headspace gaseous CO₂), which accounted for an average of 50% of the reduction in DOC concentration. Microbial biomass growth was estimated to account for the majority of the residual DOC loss (∼40%), although a small fraction of the loss may be due to DOC adsorption to biofilm produced over the course of the incubations (Materials and Methods). Assuming that the majority of the DOC loss was due to bacterial assimilation (BA) [equivalent to the sum of bacterial production (BP) and bacterial respiration (BR)], the bacterial growth efficiencies (BGE = BP/BA) were estimated to be 30–40%, which is relatively high for freshwater ecosystems (26). This range of BGE is approximate, because confounding factors such as secondary respiration or DOC adsorption would either increase or decrease growth efficiency, respectively. Dissolved oxygen concentrations decreased simultaneously with DIC production and DOC loss, starting at an average of 60.5% saturation and dropping to an average 53.6% by the end of the experiment (Fig. 1B). The biodegradable fraction of Yedoma permafrost DOC was respired to CO₂ extremely rapidly (<100 h). The average DOC consumption rate was 114 ± 12.5 μmol C⋅L⁻¹⋅d⁻¹ and was relatively consistent across experiments.

Table 1.

DOC, DIC, acetate, butyrate, and pH at 0 and 200 hours for all experiments

	DOC		DIC		Acetate		Butyrate		pH
Experiment	0 h	200 h	0 h	200 h	0 h	200 h	0 h	200 h	0 h	200 h
1	1,630	753 (−53.7%)	63.9	463 (+624%)	417	1.67 (−99.6%)	390	<4.55 (greater than −98.8%)	6.56	7.05
2	1,500	627 (−58.2%)	77.6	481 (+520%)	272	3.33 (−98.8%)	425	<4.55 (greater than −98.9%)	6.63	7.04
3	1,330	600 (−54.7%)	52.4	441 (+743%)	291	9.17 (−96.8%)	436	<4.55 (greater than −99.0%)	6.34	6.94
4	1,770	813 (−54.1%)	71.1	550 (+674%)	292	5.00 (−98.3%)	432	<4.55 (greater than −98.9%)	6.46	6.92
5	1,380	733 (−46.9%)	49.5	402 (+713%)	253	3.33 (−98.7%)	382	<4.55 (greater than −98.8%)	6.27	6.76
Mean	1,520	705 (−53.5%)	62.9	468 (+655%)	305	4.17 (−98.4%)	413	<4.55 (greater than −98.9%)	6.45	6.94

Data other than pH are reported in micromoles per liter (percentage change). Acetate and butyrate data are reported in micromoles of carbon per liter. Average values reported as mean (mean percentage change between 0 and 200 hours) in micromoles per liter.

Fig. 1.

Dissolved organic carbon (DOC) (A, yellow circles), specific UV absorbance at 254 nm (SUVA₂₅₄) (A, green squares), dissolved inorganic carbon (DIC) (B, red circles), dissolved oxygen (B, blue squares), and pH (C) over 200 h for five incubation replicates. Dark lines represent mean values at each time point, and shaded areas represent 1 SD from the mean.

Yedoma leachate contained high initial average concentrations of the low–molecular-weight (LMW) organic acids acetate and butyrate (accounting for 20% and 27% of initial DOC on a molar basis, respectively). Over the course of the incubations, we measured near-complete losses of these LMW organic acids (Table 1). Consumption of both acetate and butyrate accounted for an average of 87% of the DOC consumed across experiments. Average DOC specific UV absorbance at 254 nm (SUVA₂₅₄) was initially very low (0.58 L⋅mg C⁻¹⋅m⁻¹) and increased to 1.29 L⋅mg C⁻¹⋅m⁻¹ during the incubation (Fig. 1A). The time series of fluorescence excitation–emission matrices (EEMs) measured during the incubations indicate decreasing intensity of a LMW peak at emission of 310 nm/excitation of 275 nm (Fig. S2).

Fig. S2.

Fluorescence excitation/emission matrices (EEMs) for a representative permafrost DOC incubation. EEMs measured at 0 h (A), 70 h (B), and 200 (C) are shown on the Left, whereas subtraction EEMs illustrating the change in fluorescence after 70 h (D) and change after 200 h (E) are on the Right. Note the loss of the peak at emission of 310/excitation of 275 over time.

The DOC biodegradability we measured, on a percent basis, is among the highest recorded for DOC derived from permafrost soils (10, 11, 21). A direct comparison of DOC biodegradability across studies using different incubation methods (e.g., static bottles, plug-flow bioreactors, circulating bioreactors) is difficult given the variety of physicochemical conditions they produce (27, 28). We designed our method to produce high-resolution data that allowed for tracking of the organic and inorganic C pools to address the key question of how much CO₂ is generated from Yedoma permafrost DOC biodegradation. The biodegradation method in this study likely resulted in relatively conservative degradation rates compared with other methods, such as those that use bioreactors containing beads (27, 29), due to the relatively low surface area-to-volume ratio. Additionally, our method excluded soil particles and their associated microbes. Nevertheless, biodegradation rates were potentially accelerated at the incubation temperature we chose compared with average soil and surface water temperatures (30), although the incubation temperature is comparable to the reported maximum summer temperatures of Arctic fluvial ecosystems (24, 25). Although temperature can control the rate of respiration via bioenergetic constraints, it does not control which compounds are respired (i.e., the biodegradable fraction). Furthermore, decomposition of inherently biolabile DOC, such as LMW organic acids, is known to be less sensitive to temperature than more stable DOC (31). In other permafrost studies, bioenergetic constraints from lower temperature incubation did not alter the age or fraction of the used DOC (13). In light of these temperature dynamics and the fact that acetate and butyrate were exhausted over 200 h, percent loss is the preferred metric for both comparisons across incubation methods and for the determination of potential biodegradability in situ.

The measured 53% reduction in DOC concentration and corresponding 7.4-fold increase in DIC suggests that the freshly leached DOC collected immediately upon thaw is highly biodegradable relative to other studies and that we captured the maximum potential biodegradable fraction. Incubations of DOC in soil pore waters and surface waters likely miss a significant portion of this highly biodegradable pool (32) and should therefore be considered relatively conservative compared with the original source material (23). If we apply our average Yedoma permafrost soil DOC yield to the estimated thawed Yedoma soil OC pool by 2100 (2), we would expect between 260 and 480 Tg of rapidly biodegradable DOC released upon thaw by 2100 (Table 2), about one-half of which would be rapidly converted to DIC (130–240 Tg) for release as CO₂ to the atmosphere.

Table 2.

Estimated DOC yields from Yedoma soils by 2100

Category	Carbon stock, Pg C	DOC yield per g C, Tg C	DOC export, Tg C⋅y−1
Total permafrost	1,330–1,580*	—	—
Yedoma complex	210–456*	—	—
Total release by 2100	37–174*	—	—
Yedoma release by 2100	14.5–26.6†	493–900	—
DOC exported by Arctic rivers	—	—	30.1–40.4‡
Yedoma DOC increase by 2100	—	—	5.8–10.6

^*Schuur et al. (2).

^†Calculated using average of 92-Pg total permafrost C release.

^‡Spencer et al. (48).

The large DOC loss was fueled by rapid microbial consumption of LMW DOC, as evidenced by the complete loss of acetate and butyrate over the incubation, accounting for 87% of the total DOC loss. As observed in previous studies, the loss of these organic acids had a slight titration effect on water pH (33). Over 200 h, pH increased by an average of ∼0.5 units as a result of acetate and butyrate consumption (Fig. 1C). High acetate concentrations have been reported in other Yedoma permafrost soils (34), suggesting that the concentrations we measured are not unusual. Our study, however, is the first (to our knowledge) to directly link high concentrations of LMW organic acids to Yedoma permafrost soil-derived DOC biodegradability. In general, LMW organic acids such as acetate and butyrate are highly biodegradable (23, 35–37), explaining why they are largely absent from temperate soils (38).

Given the sedimentary history of Yedoma soils, we suggest that the LMW organic acids observed in this study were formed as the byproducts of fermentation by anaerobic bacteria (39). Little is known about the specific oxidation states of Yedoma sediments as they formed. One possibility is that rapid burial rates and saturated conditions led to anoxia and reducing conditions in these depositional environments. Under these conditions, fermenters would have been able to hydrolyze and metabolize complex carbohydrates into LMW compounds, which are known intermediates of methanogenesis (40). It is not clear why these LMW acids such as acetate would not undergo full conversion to methane, but it has been suggested that low pH can interrupt the progression from acetogenesis to acetoclastic methanogenesis (41). Regardless, the subsequent freezing of these soils would have preserved these highly biodegradable LMW organic compounds from microbial utilization. It is not known what percent of the standing Yedoma permafrost carbon stock is composed of LMW compounds, but if they exist in similar concentrations as observed in this study, a sizable (3.3–7.1 Pg) yet elusive pool of organic carbon is poised for turnover immediately upon thaw.

The chromophoric dissolved organic matter data from the incubations are consistent with the measured LMW organic acid consumption. Average DOC SUVA₂₅₄ was initially low relative to typical fluvial systems (42) and increased during the incubation, indicating a relative increase in the aromaticity of the DOC pool (43). Because the average UV₂₅₄ absorbance did not change significantly over the incubation (P > 0.5), the increase in SUVA₂₅₄ was driven solely by the loss of nonchromophoric, LMW DOC. The fluorescence time series shows the loss of a peak in what is typically denoted as the “protein-like” region (44). Compounds that fluoresce in this region can be simple amino acids and have been shown to be biodegradable (32, 44). Although changes in fluorescence cannot be used to ascertain quantitative changes in the DOC pool, the loss of the peak at emission of 310/excitation of 275 provides some insight as to the composition of the remaining 13% of the DOC loss not accounted for by acetate and butyrate. Overall, the increase in SUVA₂₅₄ and the loss of fluorophores commonly associated with biodegradable compounds are consistent with LMW organic compounds as the dominant substrate for microbial respiration.

Current and future thawing of Pleistocene Yedoma permafrost soils will mobilize highly biodegradable LMW organic acids that fuel microbial metabolism and the production of CO₂. Rapid decomposition rates in the presence of indigenous permafrost microbes suggests that the majority of these biodegradable LMW organic acids will be metabolized soon after thaw in soil pore waters, and promptly emitted as CO₂ from soils or immediately upon export to adjacent surface waters before significant downstream translocation can occur. Furthermore, the rapid turnover of ancient DOC to CO₂ observed in this study helps to explain the absence of a highly aged Yedoma DOC signature (12, 13, 45–47) at the mouths of large Arctic rivers, because any Yedoma permafrost-derived DOC was likely metabolized in soil pore waters and headwaters upstream of sample collection sites on major rivers. We estimate that thawing Yedoma soils may release 5.8–10.6 Tg of DOC⋅y⁻¹ by 2100 across Siberia and Alaska (Table 2), which is equivalent to 19–26% of all DOC currently exported by Arctic rivers (48, 49). The majority of this Yedoma permafrost-derived DOC will not reach the main stem of major Arctic rivers and export to the Arctic Ocean, given the high biodegradability we measured here. This is consistent with research on streams and rivers that drain Yedoma soils in Siberia demonstrating the loss of an aged DOC permafrost signature downstream, with almost no trace present in the river’s main stem (12, 13).

An important consequence of this rapid mineralization is that it creates a blind spot with regard to assessing total DOC turnover to CO₂. Recent work suggests that as much as 70–95% of DOC in Arctic inland waters may be processed via photochemical oxidation, with microbial processes playing a much smaller role (50). This likely misses the microbial removal of highly biodegradable DOC in soil pore waters that does not persist long enough to be measurable in receiving waters. The majority of the DOC leached from Yedoma permafrost soils is nonabsorbing within UV wavelengths and has high relative contributions of LMW organic acids (as evidenced by low SUVA₂₅₄ values, Fig. 1A). Therefore, microbial processing of DOC in Arctic systems is indeed highly important, and large predominantly nonchromophoric DOC sources such as Yedoma permafrost soils (this study) and some fresh vegetation leachates (32) are primarily mineralized through microbial processes rather than photochemical processes. We assert that the continuum of DOC oxidation starts immediately upon leaching from source material, be it vegetation, litter, or soil, and that it is important to recognize the inherent influence of processing that occurs before export to surface waters.

Permafrost thaw increases flow pathlengths, water residence times, and microbial mineralization of DOC to DIC in the soil active layer (14). Incorporating data from this study, and related studies, we present an updated conceptual model for the mobilization of permafrost-derived DOC (Fig. 2) where highly biodegradable permafrost DOC is respired to the atmosphere directly from soils or headwater streams, and more stable DOC is exported downstream or oxidized by photochemical (50) or microbial (30) processes. The majority of mineralized permafrost DOC is emitted to the atmosphere as CO₂ high in the watershed, after relatively short residence times. Generally, low-order streams exhibit higher concentrations of CO₂ and emit CO₂ more rapidly than larger rivers (19, 51).

Fig. 2.

Conceptual model for the mobilization and mineralization of permafrost-derived DOC.

Pore and headwater processing of permafrost-derived DOC poses a challenge for accurately quantifying and predicting the flux of permafrost C to the atmosphere upon thaw. Headwater ecosystems in the Arctic are generally inaccessible, are chemically and physically heterogeneous, and are distributed widely across the landscape. Furthermore, headwater ecosystems respond rapidly to hydrologic events and extremely detailed temporal studies are required to truly capture carbon fluxes from these systems (52, 53). New research into catchment-scale carbon cycling, including the integration of aquatic and terrestrial budgets, will help inform methods and target these important interface zones. Specifically, we suggest prioritizing headwater ecosystem sampling for detection of aged (¹⁴C-depleted) DOC and DIC. Additionally, more information is needed as to the ubiquity of highly biodegradable LMW organic acids in permafrost soils across the Arctic, as well as the conditions that produce and concentrate them. Such work will provide crucial information as to how different permafrost-dominated areas will respond to ongoing climate change in northern ecosystems.

Materials and Methods

Study Site.

Frozen permafrost soils were collected from a newly excavated extension of the Fox Permafrost Tunnel, which is operated by the Cold Regions Research and Engineering Laboratory in Fairbanks, Alaska. In total, 250 kg of frozen soil was collected, homogenized, and split into five different samples. Homogenization consisted of loosely mixing roughly 12 kg of frozen permafrost from different shipping containers by hand, removing any large woody debris that would impact the DOC yield ratios, and ensuring general uniformity before thaw.

Leachate Preparation.

For each of the five analytical replicates, 600 g of frozen Yedoma permafrost soil was leached into 2.8 L of deionized water (>18 MΩ⋅cm) for 24 h at 2 °C, with intermittent agitation. After 24 h, a 40-mL sample of leachate was collected and filtered with a 1.6-μm prerinsed Whatman GF/A 13-mm syringe filter for use as the inoculant. The remainder of the leachate was decanted through a sieve and centrifuged at 8,000 rpm Sorvall Superspeed RC2-B Automatic Refrigerated Centrifuge, National Research Project, US Geological Survey, Boulder, CO for 25 min at low temperature (∼10 °C) to remove fine particulates and suspended clays. Two liters of the centrifuged leachate were then filtered through a 0.45-μm prerinsed GeoTech capsule filter.

Experimental Setup.

The bioreactor consisted of a gas-tight, 12-L stainless-steel container with inlet and outlet ports (Fig. S1). A Masterflex peristaltic pump drew water from the bioreactor and pumped it through a gas-tight exterior flow-through system at a rate of 1.1 L⋅min⁻¹ to ensure continuous mixing (complete turnover time, 10 min). The flow-through system was equipped with a YSI Professional Plus Multimeter, and a Liqui-Cel MiniModule membrane gas equilibrator connected to a PP Systems EGM-4 CO₂ Analyzer for continuous in-line measurements of dissolved oxygen, pH, temperature, conductivity, and dissolved CO₂. Discrete water samples were collected from an in-line sample port with a three-way valve. A system blank was performed, and no DOC was found to have leached from the incubation system. Five replicate incubations were carried out under identical conditions. In each experiment, 2 L of filtered leachate were added to the bioreactor and were diluted to a total volume of 11 L with carbon-free MilliQ water (>18 MΩ⋅cm). The leachate was inoculated with the 40 mL of 1.6-μm-filtered water, and mixed by shaking for 1 min. Incubations were run at 20 °C. Initial water samples (t0) were collected, and the bioreactor was sealed and connected to the flow-through system. Fifty-milliliter water samples were collected at 1, 3, 6, and 12 h followed by every 12 h for a total of 21 samples over 204 h. Each time a water sample was collected, 50 mL of CO₂-free air was added to the headspace to maintain constant pressure. Data from the CO₂ Analyzer and the YSI were collected continuously for the duration of the experiment. Discrete water samples were immediately filtered with a 0.45-μm syringe filter and analyzed immediately for UV absorbance and fluorescence (t0, t70, and t200). Sample splits were preserved with 30 μL of H₃PO₄ for DOC measurements, stored refrigerated in the dark until analysis, and frozen for LMW organic acids analysis (t0 and t200).

Analyses.

Soil radiocarbon ages were measured on homogenized, oven-dried (50 °C) samples, and DOC radiocarbon ages were measured on initial filtered soil leachates. ¹⁴C-DOC samples were freeze-dried and subsequently acidified under HCl vapors to remove inorganic carbon before combustion. All radiocarbon samples were analyzed at the Accelerator Mass Spectrometry Facility at the Woods Hole Oceanographic Institute on triplicate samples. Soil organic carbon content was measured using standard techniques on an elemental analyzer (Costech ECS 4010) (54). All incubation experiments were carried out at the US Geological Survey (USGS) National Research Program laboratories in Boulder, Colorado. UV and visible light absorbance were measured using a Hewlett-Packard model 8453 photodiode array spectrophotometer (wavelength, 200–800 nm) and a 1-cm pathlength quartz cell. Change of UV₂₅₄ absorbance over time was tested using a paired t test of significance. DOC samples were analyzed using an O.I. 700 TOC Analyzer via the platinum-catalyzed persulfate wet oxidation method (55). Three-dimensional fluorescence EEMs were generated for t0, t70, and t200 time points using unacidified water extracted via the discrete sampling port and analyzed on a Jobin-Yvon Horiba Fluoromax 3 fluorometer. Acetate, butyrate, and formate concentrations were analyzed on a Dioxnex DX-600 Ion Chromatograph using established protocols (e.g., ref. 34). Formate was not detected in any samples.

Carbon Balance Calculations.

We tracked the speciation of carbon among the organic and inorganic pools using our time series measurements of DOC concentration, dissolved CO₂ concentration, pH, temperature, and system volumes. Concentrations of dissolved gaseous CO₂, bicarbonate (HCO₃⁻), and headspace CO₂ were derived from the CO₂ concentration measured in the aqueous phase, pH, and the volumes of both the water and the headspace. Total DIC in the system is the sum of the dissolved and gaseous inorganic C constituents. Because the water and headspace were assumed to be in equilibrium over the course of the incubation, any CO₂ exchanged to the headspace originated as dissolved gaseous CO₂ or bicarbonate. For this reason, calculated headspace CO₂ concentrations were included in the reported total DIC. Particulate organic C (POC) (i.e., microbial biomass) concentrations were assumed to be negligible at the beginning of each experiment because the leachates were filtered at 0.45 μm. At the end of the experiments, final POC concentrations were estimated by scraping and suspending microbial biomass formed as a biofilm on the bioreactor walls, vigorously shaking the bioreactor, and filtering the water through preweighed 0.45-μm precombusted glass fiber filters. The filters were air-dried and reweighed, and the difference was attributed to microbial biomass. We assumed that 50% of the microbial biomass was C (56). DOC adsorption to biofilms was assumed to play a minor role in overall DOC loss (or POC production) given the lack of observed flocculation, the absence of a substrate (i.e., soil particles) for adsorption, the simultaneous increase in DIC, and that the DOC lost was composed largely of biolabile and monovalent LMW organic acids, which are known to weakly adsorb to a soil’s solid phase (57). However, any small fraction of DOC lost to adsorption to biofilms would mean that our estimated BGEs are somewhat elevated, because we assumed all DOC loss was due to bacterial utilization.