Arctic Permafrost Active Layer Detachments Stimulate Microbial

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Environ. Sci. Technol. 2010, 44, 4076–4082

Arctic Permafrost Active Layer Detachments Stimulate Microbial Activity and Degradation of Soil Organic Matter BRENT G. PAUTLER,† ´ J. SIMPSON,† ANDRE DAVID J. MCNALLY,† SCOTT F. LAMOUREUX,‡ AND M Y R N A J . S I M P S O N * ,† Department of Chemistry, University of Toronto, Toronto, Ontario, M1C 1A4 Canada, and Department of Geography, Queen’s University, Kingston, Ontario, K7L 3N6 Canada

Received December 4, 2009. Revised manuscript received April 22, 2010. Accepted April 26, 2010.

Large quantities of soil organic carbon in Arctic permafrost zones are becoming increasingly unstable due to a warming climate. High temperatures and substantial rainfall in July 2007 in the Canadian High Arctic resulted in permafrost active layer detachments (ALDs) that redistributed soils throughout a small watershed in Nunavut, Canada. Molecular biomarkers and NMR spectroscopy were used to measure how ALDs may lead to microbial activity and decomposition of previously unavailable soil organic matter (SOM). Increased concentrations of extracted bacterial phospholipid fatty acids (PLFAs) and large contributions from bacterial protein/peptides in the NMR spectra at recent ALDs suggest increased microbial activity. PLFAs were appreciably depleted in a soil sample where ALDs occurred prior to 2003. However an enrichment of bacterial derived peptidoglycan was observed by 1H-13C heteronuclear multiple quantum coherence (HMQC) and 1H diffusion edited (DE) NMR and enhanced SOM degradation was observed by 13C solid-state NMR. These data suggest that a previous rise in microbial activity, as is currently underway at the recent ALD site, led to degradation and depletion of labile SOM components. Therefore, this study indicates that ALDs may amplify climate change due to the release of labile SOM substrates from thawing High Arctic permafrost.

Introduction Arctic ecosystems have been accumulating large quantities of organic carbon in permafrost zones and hold almost twice as much carbon than that present in the atmosphere (1–3). Soil organic matter (SOM) in these zones is a complex mixture of natural organic molecules that includes labile constituents such as fresh vascular plant material and microbes to more complex refractory components that have accumulated over time (2, 4). SOM composition in Arctic regions has not been studied to the same extent as temperate regions; however, it has been suggested that physical constraints such as permanently frozen/wet ground and short thaw season have * Corresponding author phone: 1-416-287-7234; fax: 1-416-2877279; e-mail: [email protected]. † University of Toronto. ‡ Queen’s University. 4076

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led to a large accumulation of labile SOM constituents that may be susceptible to climate change due to their lower metabolic activation energies (5, 6). Recent studies have focused on quantification of climatic controls and turnover of SOM by measuring CO2 respiration (3, 6–9). Heating during the short thaw season in the Arctic results in the thawing of the uppermost layer of soil (referred to as the active layer) giving microbes the opportunity to turnover some of the accumulated SOM in permafrost regions, although the decomposition mechanisms and feedbacks to climate change are very complex (10). Interpretation of SOM responses to warming temperatures is complicated by the mixture of SOM ages, the different reaction rates of recalcitrant and labile components and limited microbial productivity in Arctic ecosystems (7). A recent study using radiocarbon dating identified that “old carbon” was respired from tundra ecosystems over the past 15 years and determined that the loss of SOM from permafrost thaw in Arctic regions will act as a large source of atmospheric CO2 with rising temperatures (3). In addition to ongoing respiration, physical disruption to the SOM from Arctic warming may amplify or perhaps become an even greater contributor to degradation and CO2 flux (10). Permafrost disturbances that lead to mass movements of soil along the base of the seasonally thawed active layer are known as active layer detachments (ALDs) and occur when saturated overburdened material slides over a frozen substrate, resulting in the mixing of the surface active layer with the uppermost permafrost and rapid down slope movement of that material up to hundreds of meters over low slope angles (11). These ALDs in conjunction with the thickening of the active layer removes nutrient, water, oxygen, and carbon substrate constraints for native soil microbes, further promoting degradation and depletion of labile SOM (12, 13). Such events may provide an additional atmospheric CO2 source in Arctic ecosystems by enhanced degradation of previously unavailable SOM. The objective of this study was to characterize changes in Arctic SOM composition induced by ALDs using organic geochemical biomarkers and modern NMR spectroscopy. Arctic ecosystems are currently experiencing some of the fastest rates of warming (14), particularly during the summer months (15), therefore understanding SOM at the molecularlevel will aid in assessment of carbon turnover and microbial activity in a changing climate. Unprecedented warm temperatures during July 2007 in the Canadian High Arctic resulted in ALDs (16) and provided a unique opportunity to measure alterations of freshly disrupted Arctic SOM. Biomarkers are organic compounds that can be used as environmental tracers because their carbon skeleton is indicative of their natural product precursor (17). Changes in microbial activity as the result of ALDs were measured by phospholipid fatty acids (PLFAs) biomarkers that are characteristic of living microorganisms (18, 19) and by identification of distinct microbial protein/peptide signals in SOM humic extracts by solution-state NMR techniques (20, 21). In addition, solid-state 13C NMR was employed for a total assessment of the overall composition and degradation of Arctic SOM. This study reports the SOM structural changes by the combination of several complementary molecularlevel techniques to determine the fate of Arctic SOM in a rapidly changing environment.

Experimental Section Study Site. Soil samples were collected from the Cape Bounty Arctic Watershed Observatory located on the south-central 10.1021/es903685j

 2010 American Chemical Society

Published on Web 05/11/2010

FIGURE 1. Biomarker and organic carbon concentrations for the west and east watershed samples. (A) Organic carbon (OC) content, (B) bacterial, fungal, and actinomycetes phospholipid fatty acid (PLFA) concentrations, (C) PLFA substrate availability stress ratios, (D) Labile plant lipid concentrations (C20-C32). W1, W4, E1, and E2 are samples from undisturbed sites; W2 was sampled from an area with a historic active layer detachment (ALD), and W3 was sampled from a recent ALD. Error bars represent the standard error of duplicate measurements. coast of Melville Island, Nunavut, Canada (74°54′N, 109°35′W; Figure S1) in August 2008. The site contains two adjacent watersheds, referred to as West (8 km2) and East (11.6 km2) which drain into similar small lakes. The soil consists of a homogeneous raised marine sediment parent material with an organic horizon of 1-5 cm while the landscape is characterized by simple drainage patterns, sparse tundra vegetation, and continuous permafrost (22). Soils were collected from the mesic and polar desert communities with little variation in vegetation distribution that is dominated by prostrate dwarf shrub graminoids and mosses (23). Persistent warm temperatures (average 10.6 °C) during July 2007 (compared to 2003-2006 mean July temperature of 4.0 °C) at Cape Bounty together with a heavy rainfall event (10.8 mm) in late July deepened the active layer and resulted in widespread ALDs across the West watershed (Supporting Information (SI) Figure S2 and ref 16). Four study locations were selected for sampling in the West watershed and two samples from the East (SI Figure S1). W3 represents an area of the watershed recently affected by a 2007 ALD and was chosen to measure the impact that ALDs had on SOM composition whereas W2 represents an area where there were clear signs of a historic ALD, which happened prior to 2003 when the comprehensive watershed monitoring network was established (16). Two undisturbed samples were collected from the West catchment in close proximity to these sites to represent SOM of natural, undisturbed locations of the watershed (W1 and W4) along with two intact East watershed samples (E1 and E2) to test for variations between the watersheds. Undisturbed sample locations throughout the watershed have a similar macrosoil structure with intermediate density, productivity and consistent drainage

while locations affected by ALDs experience an increase in hydrological activity, drainage and vegetation disturbance (16). Six soil samples (top 5 cm) were collected from each location (within 2 m) and placed in Whirl-paks to generate one representative sample from each location. Samples were kept in the dark and frozen for the remainder of the field season (ca. 2 weeks). After sampling, the samples were freezedried and stored at -20 °C prior to analysis. Organic Carbon and Biomarker Extractions. Total carbon contents, organic carbon (OC) and inorganic carbon (IC) were determined by combustion with a LECO analyzer. For all samples, IC was not detected, therefore the total carbon is equal to the OC content. Lipid biomarkers were extracted by sonication of soil (∼20 g) sequentially in 30 mL of CH2Cl2, CH2Cl2:CH3OH (1:1 v/v) and CH3OH followed by filtration, concentration by rotary-evaporation and drying under N2 (24). Total solvent-extracts were redissolved in 300 µL of hexane and separated using silica column chromatography into alkane, aromatic and polar fractions by elution with 5 mL of hexane, CH2Cl2 and CH3OH, respectively. PLFAs were isolated by extracting soil (∼12 g) with CHCl3 followed by fractionation into neutral lipids, glycolipids, and polar lipids with 10 mL CHCl3, 20 mL acetone, and 10 mL CH3OH by silica column chromatography respectively (18). The polar lipid fraction containing the phospholipids was evaporated to dryness under N2, and converted to fatty acid methyl esters (FAMEs) by a mild alkaline methanolysis reaction. The FAMEs were recovered with a hexane:CHCl3 mixture (4:1 v/v). The solvents were evaporated under a stream of N2. Biomarkers were quantified by gas chromatography/mass spectrometry (GC/MS) and normalized to OC content. Microbial substrate availability was tested by the application of stress proxies for VOL. 44, NO. 11, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. 13C CP-MAS NMR spectra of HF-treated soils. Major integration areas and functional group peaks are highlighted (5, 37–39). W2 (historic ALD) has the highest Alkyl/O-Alkyl ratio (Table 1) suggesting SOM is more degraded than at the other sites. individual PLFA biomarkers (9, 25). GC/MS methods and PLFA nomenclature/origin are listed in the SI. NMR Spectroscopy. Only samples from the West Watershed were analyzed by NMR because biomarker results did not reveal any major differences between undisturbed East and West sites (Figure 1). Soil samples were repeatedly treated with HF (0.3 M), rinsed with deionized water and then freeze-dried to concentrate SOM and remove any paramagnetic minerals (26, 27). Overall SOM structure is not significantly altered by HF treatment (28). Solid-state 13C cross-polarization-magic angle spinning (CP-MAS) NMR spectra were acquired on a 500 MHz Bruker Avance III system using a spinning speed of 13 kHz and a ramp-CP contact time of 1 ms (full experimental details are listed in the SI). Soil humic materials were exhaustively extracted from HFtreated soils from with NaOH (0.1 M) under nitrogen, filtered through a 0.2 µm Teflon filter, cation exchanged with Amberjet 1200 H ion-exchange resin and freeze-dried. Humic extract samples (100 mg) were dissolved in D2O/NaOD (1 mL) and transferred to 5 mm NMR tubes for analysis. The 1 H PURGE NMR was employed for water suppression (29) and diffusion edited (DE) 1H NMR was employed to isolate signals from macromolecules and/or rigid components (30). Heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy experiments were completed to provide 1H-13C bond correlations to resolve overlapping signals (31). All solution-state experiments were acquired on a 500 MHz Bruker Avance III system (full experimental details are listed in the SI). A relative quantitative approach was taken to compare the HMQC spectra of samples. The integration values of particular functional groups were normalized to the total NMR signal intensity of all peaks in the spectrum. All samples were acquired under identical experimental conditions. However, absolute quantification of a HMQC NMR spectrum is complicated by relaxation processes and the use of a single average 1J C-H coupling constant during acquisition. Chemical shift assignments were based on previous studies (20, 21, 32–34) and confirmed by spectral prediction (21, 35).

Results and Discussion Differences in soil OC concentrations (1.0-2.7%) indicate heterogeneity and variability in OC content between sample 4078

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locations suggesting variation in surface mixing from permafrost processes. To account for such variations the biomarker concentrations were normalized to the OC% (Figure 1). A high concentration of PLFAs was observed at W3 where the ALDs occurred in 2007 (Figure 1B). More than double the concentration of PLFAs were observed for bacteria (gram-negative bacteria: 16:1ω7, cy17:0, 18:1ω7, and cy19:0; + gram positive bacteria: i14:0, i15:0, a15:0, i16:0, i17:0, a17: 0, i18:0, and a18:0), fungi (18:2ω6), and actinomycetes (10Me18:0; Figure 1B) than in the undisturbed sites (W1, W4, E1, E2; Figure 1 and SI Figure S3). Low PLFA concentration was observed at W2 where historic ALDs have taken place. PLFAs are only detected in viable cells because they decay within 2-4 days after cell death (36) and therefore are useful for assessing changes in microbial activity (19). An increase in cyclopropane PLFA-to-monoenoic precursor biomarkers (ratios of cy17:0/16:1ω7 and cy19:0/18:1ω7; Figure 1C) has been observed with declining substrate availability (9, 25). The ratio of cy17:0/16:1ω7 was four times higher in W2 than the remaining samples suggesting that microbes at this location have limited substrates available. W3 however, had the lowest ratio but the highest total PLFA biomarker concentrations suggesting that the ALDs may have introduced additional nutrients that has allowed native microbes to flourish (Figure 1C). The differences in cy19:0/18:1ω7 ratios between W2 and W3 were less prevalent, but are consistent with higher microbial substrate constraints at W2. Concentrations of solvent-extractable labile plant lipids (C20-C32: n-alkanes, n-alkanols, n-alkanoic acids) for both W3 and W2 are lower than the undisturbed samples (W1, W4, E1, E2) which may result from microbial activity and preferential mineralization of labile constituents at W3 (Figure 1D). Plant lipids from W2 are the most depleted, which in conjunction with the PLFA biomarker stress proxies suggests a previous increase in microbial activity and degradation that depleted the some of the labile components, leading to a lower OC content. 13 C CP-MAS NMR analysis provides general structural information about SOM constituents (37). Chemical shift regions arising from alkyl C (0-50 ppm), O-alkyl C (50-110 ppm), aromatic C (110-165 ppm) and carboxylic C (165-210 ppm) are labeled in Figure 2 with integration values listed in Table 1 (37). The 13C NMR integration results suggest a

TABLE 1. 13C NMR Integration Results and Calculated Alkyl/O-Alkyl Ratios (37, 39) relative percentage of 13C NMR signal sample W3 W2 W4 W1 a

alkyl C (0-50 ppm) O-alkyl C (50-110 ppm) aromatic C (110-165 ppm) carboxylic C (165-210 ppm) alkyl/O-alkyl a

(recent ALD ) (historic ALDa) (undisturbed) (undisturbed)

30 33 30 34

51 38 51 49

12 20 11 11

7 9 8 6

0.59 0.87 0.59 0.69

ALD, active layer detachment.

FIGURE 3. 1H NMR spectra for SOM humic extract from W1 (undisturbed site). (A) Conventional 1H NMR spectrum acquired with PURGE NMR (29) and (B) The analogous diffusion edited (DE) 1H NMR. The application of DE 1H NMR identifies major signatures of microbial peptide/protein contributions to OM (20, 21, 33). PG denotes the peak from the N-acetyl functional group of peptidoglycan (21). Spectra for the remaining samples (W2-W4) are shown in SI Figure S4. depletion of carbohydrates in W2 in comparison to the undisturbed sites (W1, W4) and W3. Carbohydrates at W3 are likely a combination of newly synthesized structures (38), along with “old” preserved carbohydrates released from permafrost (3, 5, 10) by ALDs. In contrast, the carbohydrate concentration in W2 is substantially lower, likely from labile structure catabolism into the more stable alkyl fraction (38). This suggests that the increase in microbial activity from ALDs, as observed by PLFAs, results in the degradation of labile (O-alkyl) SOM which results in the enrichment of more recalcitrant forms of carbon, such as alkyl and aromatic carbon (Table 1). O-alkyl and acetal carbon (anomeric carbon constituents) are considered to serve as substrates for a large number of bacteria and fungi in SOM (5). In addition, increased microbial activity in soils results in the synthesis of new carbohydrates, initially increasing the labile SOM concentration (38). Over time, the catabolism of O-alkyl carbon by microbes leads to a net alkyl carbon accumulation and SOM degradation by the synthesis of new alkyl carbon structures (38) as observed at W2, which has the highest concentration of alkyl carbon (Table 1). The relative degree

of SOM degradation has been estimated from the alkyl/Oalkyl ratio which has been observed to increase with degradation (5, 37, 39). The calculated alkyl/O-alkyl ratios (Table 1) range from 0.59 to 0.87, indicating that SOM at W2 is the most degraded (Table 1) further suggesting that labile SOM components were depleted due to enhanced microbial activity. 1 H NMR and 1H DE NMR were employed to assess the contributions of microbial populations in soil humic extracts (20). The 1H NMR and 1H DE NMR spectra of the SOM humic extract from W1 are shown in Figure 3 (spectra for the remaining samples are shown in SI Figure S4). The spectra show that 1H nuclei from CH2 aliphatic signals are mainly attenuated suggesting that this component of the SOM is comprised mainly of smaller aliphatic compounds such materials derived from plant cuticles and/or lipids (40, 41). Contributions from carbohydrates and protein/peptide side chain residues are observed in the DE spectrum (Figure 3B). These large protein/peptide signals are believed to originate from microbial species (20). Some carbohydrates may stem from microbial cells and catabolism as discussed previously VOL. 44, NO. 11, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. HMQC NMR spectrum of the W1 (undisturbed site) SOM humic extract. Major assignments pertaining to microbial proteins and carbohydrates are as follows: 1, anomeric protons (carbohydrates); 2, other CH in carbohydrates; 3, CH2 in carbohydrates; 4, r-protons in peptides and proteins; 5, N-acetyl functionality in peptidoglycan; 6, CH3 from peptides with a small contribution from terminal CH3 in lipids (21, 35). The peak contours represent the overall intensity and therefore contributions of particular chemical functional groups to the entire sample. The calculated normalized peak intensity integrations of each region the samples are listed in Table 2. Region 6 is not integrated due to overlapping resonances from proteins/peptides and lipids (21, 35). Spectra for the remaining samples are presented in SI Figure S5. (20), but likely do not represent all carbohydrates observed as some may be from plant-derived SOM (5, 6). The broad peak at 2.03 ppm (N-acetyl functional group) which remains in the DE spectrum, suggests the presence of peptidoglycan (21) but cannot be assigned with certainty from the 1H NMR spectra alone. Alternatively, HMQC NMR spectroscopy provides 1H-13C bond correlations which helps resolve overlapping signals from 1H NMR data (31). Figure 4 shows the HMQC NMR spectrum for W1 (spectra for the remaining samples can be found in SI Figure S5). Both CH and CH2 functionalities observed originate from carbohydrates, possibly originating from plant cellulose or hemicellulose (20, 21, 32). R-CH groups are likely from microbial peptides/ proteins (20) and the N-acetyl functional group from peptidoglycan, a principle component of microbial cell walls (21), were also prominent in all HMQC NMR spectra. The increases/decreases in the normalized area ratios between the samples were used as a proxy for assessment of changes in microbial populations from ALDs (Table 2). Due to peak

overlap of CH3 functional groups from plant lipids and proteins/peptides, region 6 in Figure 4 was not included in the integration analysis of the 2-D NMR (35). Carbohydrate CH and CH2 groups have the largest normalized ratio in W3 and lowest at W2, confirming observations from solid-state 13 C NMR and biomarker analyses (Table 1, Figures 1 and 2) which suggest the release of labile carbon substrates from permafrost (5, 6). The R-CH groups, which mainly originates from microbial peptides/proteins (20), have the smallest normalized ratio in W2, suggesting the smallest microbial population which is consistent with the observed low concentrations of microbial PLFA biomarkers (Figure 1). The differences in normalized ratios of R-CH groups between W3 and undisturbed samples (W1 and W4), though agree with the observed trends from PLFA biomarkers and solidstate 13C NMR are less apparent. An inverse trend was observed for the signal of the N-acetyl group of peptidoglycan. Peptidoglycan is a major component of bacterial cell walls and has been used to estimate bacterial concentrations (21, 42). W2 contained the largest normalized ratio of N-acetyl functional groups suggesting that a large amount of bacterial cell walls are present. Peptidoglycan can be protected from microbial degradation after cell death in soil by copolymerization reactions and transformation, substantially adding to the refractory nitrogen pool in SOM (43–45). Therefore it is plausible that the observed peptidoglycan originates from both living and dead bacteria. This knowledge in conjunction with the low concentration of extracted PLFA biomarkers at W2 suggests that the majority of the detected peptidoglycan is from dead cells that have been protected, providing further support of higher microbial population at W2 from previous ALDs. The peptidoglycan normalized ratio (Table 2) is lower in W3 than the in the samples from the undisturbed sites (W1, W4) despite having the highest PLFA concentration (Figure 1B). This suggests a possible dilution effect from the release of labile SOM from ALDs. As time progresses, the microbes at W3 may continue to degrade the available labile SOM, and may result in increased peptidoglycan relative to the remaining SOM area (similar to W2). Permafrost ALDs results in changes to microbial activity and suggests that the biogeochemical cycling rates of SOM previously stabilized under the harsh Arctic climate conditions are altered, perhaps leading to an overall SOM priming effect (SI Figure S6). SOM priming has been observed with the addition of labile carbon compounds to subarctic soils (46), therefore it is possible that ALDs induced by warmer temperatures may provide the available labile nutrients and optimal conditions that may stimulate microbial activity (W3); but as time progresses the decomposition of Arctic SOM is enhanced (W2). The precise mechanism by which ALDs could affect the overall mineralization of previously locked carbon from permafrost has many complicating factors (10), however it appears that ALDs in conjunction with rising temperatures result in the destabilization of frozen SOM, thereby potentially increasing carbon substrate availability and exposure to water

TABLE 2. Summary Comparison of Normalized HMQC NMR Ratios of Carbohydrate and Microbial Associated Functional Groups (20, 21, 32–35) HMQC NMR normalized ratios for specific function groups sample W3 W2 W4 W1 a

4080

r 1H-13C from proteins (4.0-5.0, 48-58 ppm)

carbohydrate (CH + CH2) (4.1-5.6, 96-106 ppm +3.1-4.5, 58-90 ppm)

peptidoglycan (N-acetyl) (2.0-2.1, 18-22 ppm)

2.09 1.86 1.96 2.07

4.51 3.27 3.75 3.32

0.96 1.16 0.98 1.01

(recent ALDa) (historic ALDa) (undisturbed) (undisturbed)

ALD, active layer detachment.

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and oxygen. This may release more CO2 into the atmosphere, which will further amplify the climate change impact on the Arctic, potentially leading to more ALDs, microbial decomposition, and SOM priming. The net result is a positive feedback loop to climate change, leading to a depletion of the SOM previously unavailable thereby providing an additional release of CO2 from Arctic ecosystems. The methods employed in this study collectively suggest that Arctic permafrost ALDs stimulate microbial activity and degradation of SOM however, these results should be confirmed in other areas of the High Arctic and with an expanded number of samples to confirm the observed trends.

Acknowledgments We thank four anonymous reviewers for their comments which greatly improved the quality of this manuscript. The Government of Canada International Polar Year program, Polar Continental Shelf Project and Natural Resources Canada are thanked for their support. MJS thanks the Natural Science and Engineering Research Council (NSERC) of Canada for support via Discovery Grant. BGP thanks NSERC for the Canada Graduate Scholarship. Long term research at Cape Bounty has been supported by NSERC and ArcticNet awards to S.F.L.

Supporting Information Available Detailed biomarker extraction/quantification procedure and NMR experimental details along with additional figures. This material is available free of charge via the Internet at http:// pubs.acs.org.

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