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Environmental Processes

Molecular Insights into Arctic Soil Organic Matter Degradation under Warming Hongmei Chen, Ziming Yang, Rosalie K. Chu, Nikola Toli#, Liyuan Liang, David E Graham, Stan D. Wullschleger, and Baohua Gu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05469 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018

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Environmental Science & Technology

Molecular Insights into Arctic Soil Organic Matter Degradation under Warming

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Hongmei Chen1,¶, Ziming Yang1,2,¶, Rosalie K. Chu3, Nikola Tolic3, Liyuan Liang3, David E. Graham4, Stan D. Wullschleger1, Baohua Gu1,*

4 5 6 7 8 9 10 11 12 13 14

1

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States 2

Department of Chemistry, Oakland University, Rochester, Michigan 48309, United States

3

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, United States 4

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States

15 16 17 18 19 20 21 22 23 24 25 26 27

¶

These authors contributed equally

*

Corresponding Author: email: [email protected]; phone: (865)-574-7286

1

ACS Paragon Plus Environment


28 29

ABSTRACT

30

Molecular composition of the Arctic soil organic carbon (SOC) and its susceptibility to

31

microbial degradation are uncertain due to heterogeneity and unknown SOC compositions. Using

32

ultrahigh resolution mass spectrometry, we determined the susceptibility and compositional

33

changes of extractable dissolved organic matter (EDOM) in an anoxic warming incubation

34

experiment (up to 122 days) with a tundra soil from Alaska, United States. EDOM was extracted

35

with 10 mM NH4HCO3 from both the organic and mineral-layer soils during incubation at either

36

–2 or 8°C. Based on their O:C and H:C ratios, EDOM molecular formulas were qualitatively

37

grouped into nine biochemical classes of compounds, among which lignin-like compounds

38

dominated both the organic and mineral soils and were the most stable, whereas amino sugars,

39

peptides and carbohydrate-like compounds were the most biologically labile. These results

40

corresponded with shifts in EDOM elemental composition, in which the ratios of O:C and N:C

41

decreased, while average C content in EDOM, molecular mass, and aromaticity increased after

42

122 days of incubation. This research demonstrates that certain EDOM molecules, such as amino

43

sugars, peptides, and carbohydrate-like compounds, are disproportionately more susceptible to

44

microbial degradation than others in the soil, and these results should be considered in SOC

45

degradation models to improve predictions of Arctic climate feedbacks.

46

2


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48

INTRODUCTION

49

Terrestrial soils and permafrost in the northern circumpolar region of almost 19 million

50

square kilometers store approximately half of the global belowground organic carbon (~1,700 Pg

51

C),1, 2 accumulated over thousands of years due to slow microbial decomposition and turnover

52

under freezing conditions. Because of rapid climate warming, Arctic soil organic carbon (SOC)

53

in permafrost is experiencing unprecedented thawing and accelerated microbial decomposition,3,

54

4

55

(e.g., CO2 and CH4) to the atmosphere.2,

56

understanding of the global carbon cycle and climate feedback concerns the dynamics of SOC

57

degradation (both decomposition and production) in warming water-saturated tundra. A

58

significant knowledge gap is the extent to which DOM composition and mineral protection

59

influence microbial metabolism during permafrost thaw. SOC stored in permafrost is generally

60

considered to be vulnerable because it has not undergone significant decomposition and thus is

61

prone to microbial degradation.11-13 The C chemistry in permafrost is complex, since the stored

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carbon may come from many different sources including, but are not limited to, the decomposed

63

or undecomposed plant materials, animal residues, and microbial biomass. Global climate

64

models therefore view belowground SOC as occupying several interconnected pools with

65

differing intrinsic decomposition rates.3, 4 However, the physical and chemical differences among

66

these SOC pools are poorly defined,14, 15 due to extreme heterogeneity of the C sources and their

67

varied compositions and characteristics.14-18 Without definition, these pools cannot be reliably

68

measured to parameterize climate models and assess their predictions.

releasing large quantities of dissolved organic matter (DOM) to rivers 5, 6 and greenhouse gases 7-10

However, a large source of uncertainty in

3



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Field and laboratory short-term studies often show an initial rapid release of CO2 and

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CH4 within first few weeks upon warming, followed by declined rates of C loss.2, 13, 16, 19-21 It is

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suggested that specific biochemical classes of SOC compounds are preferentially degraded in

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permafrost soils, promoting rapid release of CO2 and CO4 upon thawing.2, 14, 21 Our recent studies

73

confirm that low-molecular-weight DOM compounds, such as reducing sugar, ethanol, and

74

acetate, are among the most labile compounds that largely account for the initial rapid release of

75

CO2 and CH4 through anaerobic metabolism.21,

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initial, rapid degradation is related to, among other factors, SOC’s potential for decomposition,2

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although at the molecular level it is unclear which SOC molecules are more susceptible to rapid

78

breakdown with increasing temperature.2,

79

biochemical composition is a critical factor determining DOM degradation potential or

80

vulnerability,2, 14, 21, 24-27 whereas others argue that environmental properties (e.g., temperature,

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microbial community, and organo-mineral association), other than intrinsic molecular

82

recalcitrance, determine DOM degradation potential.18,

83

understanding of SOC biogeochemistry limits our ability to develop process-based models to

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predict global carbon cycling and climate change. Consequently, the research community has

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highlighted SOC biochemistry as a critical area to elucidate C, N, and nutrient cycling in the

86

Arctic.15, 17, 30

23

22

Conceptual models also suggest that this

Numerous studies have indicated that DOM

28, 29

This lack of mechanistic

87

The overall goal of this study was to apply ultrahigh resolution Fourier transform ion

88

cyclotron resonance mass spectrometry (FTICR-MS) to assess DOM molecular composition and

89

its dynamic changes during a simulated soil warming experiment and to provide molecular-level

90

insights into how DOM composition influences its response to microbial degradation. This is a

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companion work to our previous studies of soil C degradation and microbial processes in the

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Arctic,21, 22 as described above. Here, using the same set of soil samples, DOM was extracted

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(hitherto referred to as “extractable DOM” or EDOM) with NH4HCO3 at pH 7.3. We aimed to

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determine: (1) which EDOM molecules or C pools are most vulnerable to rapid breakdown under

95

warming, and (2) what are the resulting degradation products and dynamics (e.g., decomposition

96

vs production of new DOM molecules) in both organic and mineral soil layers. The remaining

97

solid-phase or particulate organic matter (POM) after extraction was not analyzed due to

98

limitations of FTICR-MS. By focusing on EDOM molecular changes and dynamics during

99

anaerobic metabolism, we find that EDOM biochemical composition is one of the key factors

100

controlling SOC transformation in Arctic soils.

101 102

MATERIALS AND METHODS

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Laboratory incubation studies of both the organic and the mineral soil layers have

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previously been described.21 The soil samples after incubation were used to extract DOM in this

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study for FTICR-MS analysis. In brief, a frozen soil core (3˝ diameter × 36˝ length) was

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collected in April 2012 (average temperature, –15 °C) from the trough area of a high-center

107

polygon at the Barrow Environmental Observatory (BEO) in northern Alaska, United

108

States.16,31,32 It was taken in sterilized PVC liners and kept frozen during shipment and storage

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(–20 °C) to minimize potential disturbance until they were processed. The organic soil layer (8-

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20 cm below surface) and the mineral soil layer (22-45 cm below surface) of this aquiturbel soil

111

were separated, and then thawed (within 3 h) and homogenized under saturation (not sieved).

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Subsamples (~150 g) from each soil layer were incubated at –2 °C or 8°C in N2-purged glass

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bottles (600 mL) to mimic near-freezing and thawing conditions for up to 122 days under dark

114

and anoxic conditions.21 Triplicate samples per soil type per incubation temperature were

5



115

conducted although limited EDOM samples were analyzed by FTICR-MS due to limited

116

instrument time. At pre-determined time intervals, an aliquot of the incubated soil (1-2 g) was

117

taken and immediately stored in –20 oC freezer until DOM extraction. DOM and SOC were

118

measured using a Shimadzu L-TOC analyzer and a LECO TruSpec CN analyzer, respectively.

119

CO2 and CH4 production were measured during the incubation,21 and the headspace of glass

120

bottles was evacuated by a vacuum pump and re-filled with N2 after each sampling event. Prior

121

to FTICR-MS analysis, soil samples were defrosted and thawed at room temperature and then

122

equilibrated with 10 mM NH4HCO3 (pH ~ 7.3) solution for 6 h. Here NH4HCO3, instead of KCl,

123

was used to increase the yield of EDOM, while minimizing the formation of Cl-adducts that

124

would suppress DOM signals in FTICR-MS analysis.33 Samples were centrifuged for 15 min at

125

6500×g, and the supernatants (containing EDOM) were collected and filtered through 0.45-µm

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Nylon membrane filters before analysis.

127

The EDOM was analyzed using a 15T FTICR-MS (Bruker SolariX, Billerica, MA) fitted

128

with a standard electrospray ionization (ESI) interface.33,

34

129

methanol (1:1 v/v) to a concentration of 10 mg C/L and directly infused using a Hamilton

130

syringe at a flow rate of 2 µL/min. Instrumental blanks were measured using a mixture of

131

ultrapure water and methanol (1:1 v/v) in the same manner as the samples. The coated glass

132

capillary temperature was set to 180 °C and the electrospray voltages were optimized to keep the

133

ion current constant for each sample.33, 35 Both negative and positive ESI mode data (ESI– and

134

ESI+) were acquired. Negative(–) or positive(+) ions were generated by setting the voltages to

135

either +4.5 kV or –4.5 kV, respectively, during the electrospray process.36 The ion accumulation

136

time was set to 0.1 s, and the time of flight was 0.65 ms, with 144 scan averages co-added in

137

broadband mode between 100 and 1000 m/z. Prior to sample analysis, the instrument was

6


All samples were diluted with

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externally calibrated with Agilent ESI-L low concentration tune mix (Agilent Technologies,

139

Santa Clara, CA), and the syringes and transfer lines were flushed with 50/50 methanol/water

140

(v/v) between samples.

141

Mass spectra were internally calibrated using a series of reference organic acids

142

commonly found in soil.35 Instrumental blank peaks were removed from the sample peak list

143

before formula calculations were applied using a molecular formula calculator (Molecular

144

Formula Calc v.1.0 NHMFL) developed at the national High Magnetic Field Laboratory in

145

Tallahassee, FL.37, 38 Both ESI- and ESI+ mode formulas were calculated for peaks with a signal

146

to noise (S/N) ratio ≥ 7, a m/z range between 100 and 800, and a mass error less than 1.0 ppm.

147

The following criteria were used for molecular formula assignments: C2-50, H2-100, N0-7, O0-30, S0-

148

2,

149

formulas (both H+ and Na+ forms) identified by ESI+ mode, only the H+-form formulas were

150

used and the Na+-form was removed from the final dataset. Molecular formulas were screened by

151

searching

152

nih.gov/search). We matched each assigned molecular formula with PubChem database entries

153

to validate identified compounds or isomers. Formulas with no chemical structures identified in

154

the Pubchem database were excluded from the final formula assignment. The majority of

155

assigned formulas agreed with the measured masses within 0.5 ppm error. The average mass

156

resolution was approximately 500,000 at m/z 500. Replicate soil samples (~20% of the total

157

samples) were prepared and analyzed in the same manner, and they shared common mass peaks

158

in the range of 73-88%, as is commonly observed.39-41

P0-1, and Na0-1 (for ESI+ mode only), 0.33 < H:C ≤ 2.5, and O:C ≤ 1.2.33, 35 For duplicate

chemical

structures

in

the

Pubchem

database

(https://pubchem.ncbi.nlm.

159

Both ESI– and ESI+ FTICR-MS data were acquired for each DOM sample at the same

160

DOM concentration (10 mg C L-1) and pH (7.3), and additional details and discussion were

7



161

provided in Supporting Information (SI). ESI– mode favors detection of molecules with acidic

162

functional groups that deprotonate, such as carboxylic acids, whereas ESI+ mode favors

163

detection of molecules with basic functional groups such as those containing heteroatoms

164

(CHON and CHONS) or amines and amino sugars in EDOM.42-44 The two mode data are highly

165

complementary and were combined for the analysis of EDOM molecular composition or

166

compositional changes during incubation. For duplicate formulas identified by both modes, only

167

the ESI– mode data were retained, and ESI+ mode data were removed from the final dataset.

168

Principal component analysis (PCA) was performed to examine the relative enrichment

169

of certain molecular formulas in the EDOM before and after warming incubation, using

170

previously established methods.36,

171

intensities calculated by dividing the peak intensity of each individual DOM formula by the total

172

peak intensity within each soil sample.37, 45 In situations where a DOM formula was absent in a

173

given soil sample, the peak intensity of this formula was set to zero. The output results were

174

given in a biplot, showing the score of each soil sample taken at different incubation times and

175

the loading of each variable, i.e., the relative peak intensity of each molecular formula.

44

The PCA data matrix was created using relative peak

176 177

RESULTS AND DISCUSSION

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EDOM degradation and molecular compositional changes after warming incubation

179

We previously showed that anoxic warming incubation of both the organic and mineral

180

soil layers at 8 °C resulted in an initial rapid release of CO2 and CH4 through anaerobic

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fermentation, although the CO2 production rate was about an order of magnitude higher in the

182

organic than in the mineral soil.21 Lower production rates of CO2 and CH4 were observed at –2

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ºC, which were 0.2 and 0.001 µmol g-1 dwt. soil day-1, respectively, compared to 3.0 and 0.1 8


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µmol g-1 dwt. soil day-1 at 8 ºC (SI Table S1). Interestingly, however, the EDOM concentrations

185

remained relatively constant during incubation and were 400±20 and 50±8 µmol C g-1 in the

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organic and mineral soils, respectively (SI Table S1). This observation was attributed in part to

187

slow release of EDOM from the solid-phase SOC and a high organic C content in the organic

188

(22.5 mmol C g-1 dwt.) and the mineral soil (11.6 mmol C g-1 dwt.) since the degradation

189

represented 60%) after 122 days of incubation at 8 °C (Figure 2c).

251

By grouping the formulas based on their elemental composition (Figure 2d), we further

252

demonstrate that, although less abundant (with only 130 formulas), CHONS molecules were the

11



253

most labile with > 65% of them having disappeared after 122 days. This trend is followed by

254

CHON formulas (1635 total), with ~ 45% of them degraded in the same period. As expected,

255

CHO molecules were the most abundant (with 2419 formulas) and stable, with >70% of the

256

formulas remaining after 122 days. These observations provide additional evidence that amino

257

sugars and peptides were the most susceptible for degradation at 8 °C in the organic soil (Figure

258

2c), suggesting biochemical composition or DOM substrate quality was important in influencing

259

the rate and extent of soil organic carbon degradation. Using conventional high performance

260

liquid chromatography (HPLC) and ion chromatography (IC) analyses, our previous studies were

261

unable to identify the abundance and vulnerability of N-containing amino sugars and peptides,

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but showed that free-reducing sugar, ethanol, and some low-molecular-weight organic acids

263

(e.g., acetate, formate, propionate) were the most vulnerable under 8 °C warming conditions (SI

264

Table S1).21 The degradation of simple sugar and alcohol compounds largely accounted for

265

initial rapid release of CO2 and CH4 through anaerobic fermentation, whereas the fermentation

266

products, acetate and formate, were subsequently utilized as primary substrates for

267

methanogenesis.21

268

Examination of the time-dependent degradation of biochemical classes of compounds

269

also revealed that most amino sugars and carbohydrates were degraded in the first 10 days of

270

incubation at 8 °C (Figure 3a), whereas most of the peptides degraded after 34 days of

271

incubation. This result is consistent with rapidly decreased reducing sugar and alcohol

272

compounds during the first month of incubation, as previously described,21 and confirmed by

273

rapid decrease in CHONS and CHON formulas over time (Figure 3b). On the other hand, EDOM

274

formulas in the classes of condensed aromatics, tannins, lignin, and other aliphatics increased

275

with incubation time (Figure 3a), and this observation is also shown by increased numbers of

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CHO and CHOS formulas (Figure 3b). Furthermore, analyses of average molecular mass,

277

elemental compositions and ratios (Table 1) indicate that average C number and C content (%) in

278

EDOM formulas, molecular mass, double bond equivalent (DBE), and aromaticity index

279

(AIMOD)50 all increased following 122 days of incubation at 8 °C. However, the total number of

280

EDOM formulas, the average O, N, and S contents, as well as O:C, N:C, and S:C ratios, all

281

decreased. These observations again demonstrate that EDOM molecules such as amino sugars,

282

peptides, and carbohydrates were preferentially decomposed, leading to increase in average

283

molecular weight, DBE, and aromaticity. The results are in general agreement with the microbial

284

degradation potentials of different biochemical classes of organic compounds.17, 21, 31, 33

285

A similar trend, although to a lesser extent, was observed during incubation with the

286

organic soil at –2 °C (Table 1, Figure 3c,d). Average C number and C content (%) in EDOM

287

formulas, molecular mass, DBE, and aromaticity index all slightly increased following 122 days

288

of incubation at –2 °C (Table 1), whereas the total number of EDOM formulas, the average N

289

and S contents, as well as N:C and S:C ratios, slightly decreased. Similarly, EDOM formulas

290

within the biochemical classes of amino sugars, peptides, and carbohydrates decreased with

291

incubation time, whereas lignin, condensed aromatics, and tannins increased slightly (Figure 3c).

292

This result is consistent with gradual decrease in CHONS formulas with time (Figure 3d), but the

293

trends in other elemental groups were not as clear as those observed at 8 oC. This observation

294

was attributed to lower microbial activity and slower degradation at –2 °C than 8 oC, as

295

previously reported.21,

296

observed at the –2 °C incubation.22

297

Molecular characterization of extractable DOM in the mineral soil

22

No significant changes in the microbial community structure were

13



298

A larger percentage of EDOM molecules (68%) remained unchanged in the mineral soil

299

(Figure 4a,b) than that in the organic soil (56%) (Figure 2b) after 122 days at 8 °C. While this

300

observation may be attributed to the partial degradation of labile EDOM as it leaches down to the

301

mineral layer, mineral sorption of EDOM molecules could also make them less extractable by

302

NH4HCO3. Nonetheless, similar decreases in major biochemical classes (i.e., peptides ②,

303

carbohydrates ④, and amino sugars ③ were observed in the mineral soil, albeit to a lesser

304

extent than in the organic soil (Figure 4c). Lignin ⑦, lipids ①, and other aliphatics ⑤ were

305

among the most stable and remained in the soil. Again, amino sugars ③ and carbohydrates ④

306

appeared the most labile, and ~45% of them degraded after 122 days. This was further

307

demonstrated in the elemental group, in which CHONS and CHON formulas decreased the most

308

(Figure 4d). Conversely, lignin and CHO formulas were the most abundant, with 70–80% of the

309

formulas remaining after 122 days. Analyses of elemental compositions also indicate that the

310

total number of EDOM formulas, the average O, N, and S contents, as well as O:C, N:C, and S:C

311

ratios, decreased following 122 days of incubation (Table 1). However, the average molecular

312

mass, C number and C content in EDOM formulas, DBE, and aromaticity index all increased due

313

to preferential degradation of EDOM molecules, such as amino sugars and carbohydrates.

314

These trends generally also hold for the mineral soil incubated at –2 °C (Table 1, SI

315

Figure S3). The result further supports the observation that EDOM formulas in the biochemical

316

classes of amino sugars, peptides, and carbohydrates were disproportionately more susceptible to

317

degradation than other organic components, resulting in decreases in O and N contents but

318

increases in H content, molecular mass, and aromaticity. Therefore, biochemical composition of

319

DOM could play an important role in influencing soil C degradation, and this result is consistent

14


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with observations that DOM in mineral soils is usually more reduced than that in the upper

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organic soil in the Arctic.13, 32

322

However, the trend for time-dependent degradation of biochemical molecules in the

323

mineral soil (SI Figure S3) was not as clear as that observed in the organic soil. This result was

324

partially attributed to relatively slow degradation rates of organic C in the mineral soil compared

325

to the organic soil, as previously described.21 The mineral soil released nearly an order of

326

magnitude less CO2 than organic soil incubated at 8 °C (SI Table S1).16, 21 Although organo-

327

mineral interactions likely slow the degradation of soil organic carbon and release less

328

extractable EDOM,13, 18 DOM compositional differences between the organic and mineral soils

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are hypothesized to be partially responsible for the observed differences. We therefore compared

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compositional differences between the initial organic and the mineral soils at Day 0. The van

331

Krevelen plot (Figure 5a, SI Table S3) shows that the formulas present only in the mineral soil

332

have a much lower O:C ratio than the organic soil. Amino sugar is the smallest component with

333

only 60 formulas identified, mostly from the organic soil. Peptides are the major N-containing

334

compounds identified, and the organic soil showed slightly more peptide formulas than the

335

mineral soil. However, EDOM in the mineral soil had a higher number of formulas that fell into

336

the biochemical classes of unsaturated hydrocarbons, aliphatics, and lipids than the organic soil,

337

consistent with high C and H contents but low O:C ratio in the mineral soil (Table 1). These

338

observations may again be attributed to the degradation of N-containing compounds, as DOM

339

leaches down the soil profile, and to potential sorption of these compounds onto minerals.

340

Lignin-like compounds are again the most abundant biochemical component in both the organic

341

and mineral soils (Figure 5c). Elemental composition analysis results also indicate that most

342

EDOM molecules contained CHON or CHONS in their formulas, with the organic soil

15



343

containing slightly more N-containing formulas than the mineral soil (Figure 5d). On the other

344

hand, the mineral soil contained relatively higher numbers of CHOS and Others elemental

345

groups. Compared with the organic soil, EDOM molecules in the mineral soil also exhibited

346

higher average molecular mass, H:C ratio, and aromaticity, but lower O%, N%, O:C, N:C, and

347

DBE (SI Table S3).

348

Importantly we note that, although N is a minor component of EDOM (2–3% w/w, Table 1),

349

about 40% of the identified EDOM formulas contains one or more N atoms (Figure 5d), of

350

which 633 formulas (~ 30% of N-containing formulas) were identified as peptides (Figure 5c). If

351

only the peptides were considered, the average N:C ratio in the organic soil (0.229) would be

352

about twice that in the mineral soil (0.107) (SI Figure S4). This result again indicates that these

353

N-containing compounds may be preferentially degraded during transport from the upper organic

354

soil to the lower mineral soil and/or sorbed onto minerals. Consequently, the unique formulas in

355

the mineral soil showed lower average N:C ratios than those in the organic soil (SI Table S3).

356

The result suggests that N limitation in the mineral soil may be another factor governing

357

microbial community and partially responsible for the observed slower decomposition of SOC.

358

Arctic soils are generally depleted in inorganic N, and any changes in soil organic N cycling

359

could have the potential to alter heterotrophic soil microbes and hence processes related to C

360

cycling.25 These findings correspond well with previous observations that soil C quality and

361

composition could significantly influence N mineralization and emission of N2O, CH4, and

362

CO2,25, 51-54 and thus should be considered in global C models.27

363

SOC biodegradation index and implications for modeling

364

Using ultrahigh-resolution FTICR-MS analysis, we addressed the question of whether

365

DOM molecular composition or biochemical classes of compounds influence soil C degradation 16


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in Arctic tundra. In general, microbial C degradation decreased elemental ratios of O:C, H:C, and

367

N:C in DOM. Molecular formulas in the biochemical classes of amino sugars, carbohydrates,

368

and peptides were among the most susceptible to degradation, and amino sugars and

369

carbohydrates had mostly disappeared after about one month of incubation in the organic soil at

370

8 °C. Conversely, lignin, tannins, condensed aromatics, and other aliphatics were identified as

371

the most stable EDOM components and mostly remained following 122 days of incubation.

372

Lignin formulas were the dominant component in all soil samples, and their number percentages

373

generally increased with incubation time and were higher in the mineral layer than the organic

374

layer. These results clearly indicate that certain EDOM components were disproportionately

375

more susceptible to microbial degradation compared to other biochemical classes within the soil

376

C pool.

377

Understanding changes in DOM molecular composition, in addition to physicochemical

378

and biological properties that determine soil C degradation in Arctic tundra, is essential for

379

predicting how C fluxes may respond to global climate change. A large source of uncertainty

380

exists in current model predictions because soil C pools are poorly defined.14, 15, 20 While it is

381

impractical to incorporate thousands of soil C molecules in those models, a Biodegradation

382

Index, i.e., the ratio of EDOM labile C components (e.g., amino sugars, carbohydrates, and

383

peptides) to lignin-like compounds (the most stable component), is proposed and may be used as

384

a proxy to describe soil C degradation potential. The Biodegradation Index shows a decreasing

385

trend following incubation of the organic and mineral soils at both incubation temperatures (SI

386

Figure S5), and was higher in the initial soil than that incubated after 122 days, as expected.

387

Similarly, the index was higher in the initial organic soil layer than the initial mineral soil layer,

388

since the organic soil contains higher amounts of labile EDOM compounds such as amino sugars

17



389

and peptides (Figure 5). These results indicate that EDOM in the mineral soil was partially

390

degraded or aged as organic matter was transported from the top organic soil to the bottom

391

mineral soil layer under field conditions. The Biodegradation Index may be potentially

392

incorporated into the current soil C decomposition cascade models, such as the Enzyme model,23

393

in which microbial community responds to changes of soil C composition and availability. This

394

in turn affects extracellular soil enzymatic processes.55, 56 The index may also be parameterized

395

into future fine-scale ecosystem models30 by considering the dynamic interactions between soil C

396

substrates, microbial processes, and sorptive mineral surfaces to better predict C fluxes and

397

cycling in Arctic soils.

398 399

ASSOCIATED CONTENT

400

Supporting Information

401

The Supporting Information is available free of charge on the ACS Publications website at

402

http://pubs.acs.org.

403

Methods of ESI- and ESI+ mode characterization; Table S1, Summary of EDOM

404

characteristics. Table S2, Summary of all detected and formula-assigned mass peaks. Table

405

S3, Summary of the identified EDOM molecular formulas and characteristics. Figure S1,

406

Comparisons of EDOM formulas identified by ESI- and ESI+ modes. Figure S2, Elemental

407

analysis and grouping of molecular formulas. Figure S3, Number percentages of the

408

identified EDOM molecular formulas in different biochemical classes and elemental groups.

409

Figure S4, van Krevelen diagram showing peptide molecular formulas. Figure S5,

410

Biodegradation index.

411

18


Page 18 of 32

Page 19 of 32

412


ACKNOWLEDGEMENTS

413

We thank Xiangping Yin and Wei Fang for technical assistance and sample analysis. The

414

Next Generation Ecosystem Experiments (NGEE-Arctic) project is supported by the Office of

415

Biological and Environmental Research in the DOE Office of Science. All data are available in

416

an online data repository (NGEE-Arctic Data Portal, DOI: 10.5440/1410297).

417

National Laboratory is managed by UT-Battelle LLC for DOE under contract DE-AC05-

418

00OR22725. The FTICR-MS analysis was performed at Environmental Molecular Science

419

Laboratory (EMSL), a DOE Office of Science User Facility sponsored by BER at Pacific

420

Northwest National Laboratory.

421

The authors declare no competing financial interest.

Oak Ridge

422 423

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424 425 426

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562 563

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575 576

50. Koch, B. P.; Dittmar, T. From mass to structure: an aromaticity index for high-resolution mass data of natural organic matter. Rapid Commun. Mass Spectrom. 2006, 20, 926-932.

577 578

51. Steudler, P. A.; Bowden, R. D.; Melillo, J. M.; Aber, J. D. Influence of nitrogen fertilization on methane uptake in temperate forest soils. Nature 1989, 341, 314-316.

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52. Adamsen, A. P. S.; King, G. M. Methane consumption in temperate and subarctic forest soils: rates, vertical zonation, and responses to water and nitrogen. Appl. Environ. Microbiol. 1993, 59, 485-490.

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53. Schnell, S.; King, G. M. Mechanistic analysis of ammonium inhibition of atmospheric methane consumption in forest soils. Appl. Environ. Microbiol. 1994, 60, 3514-3521.

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56. Burns, R. G.; DeForest, J. L.; Marxsen, J.; Sinsabaugh, R. L.; Stromberger, M. E.; Wallenstein, M. D.; Weintraub, M. N.; Zoppini, A. Soil enzymes in a changing environment: current knowledge and future directions. Soil Biol. Biochem. 2013, 58, 216234.

594 595

23



596

FIGURE LEGENDS

597 598 599 600 601 602 603

Figure 1. (a) Principal component analysis in the organic soil incubated at 8 oC. The blue-box area represents molecular formulas enriched in the initial samples (Day 0), where D0_A and D0_B represent experimental duplicates. The red-box area represents formulas enriched in the final samples after incubation (at Day 88 and 122). Purple triangles and labels are sample IDs with incubation days. (b) Elemental analysis and grouping of EDOM formulas enriched in the initial and final samples. Elemental group “Others” represents a combination of CH, CHN, CHS, CHNS, and P-containing formulas.

604 605 606 607 608 609 610 611 612 613 614 615 616

Figure 2. Identification and comparison of EDOM molecular formulas at Day 0 and Day 122 at 8 o C in the organic soil. (a) van Krevelen diagram showing unique EDOM formulas identified at Day 0 (blue square), common formulas at both Day 0 and 122 (yellow diamond), and unique formulas at Day 122 (red triangle). Shaded areas with numbers indicate different biochemical classes of identified EDOM formulas:45-47 ①=lipids, ②=peptides, ③=amino sugars, ④=carbohydrates, ⑤=other aliphatics (including area of ① with no oxygen, and areas of ② and ③ with no nitrogen), ⑥=unsaturated hydrocarbons, ⑦=lignin, ⑧=tannins, and ⑨=condensed aromatics. (b) Number percentages of EDOM formulas identified only at Day 0 (blue), Common (yellow) at both Day 0 and Day 122, and only at Day 122 (red). (c) Bar chart showing the percentages of identified EDOM formulas by biochemical classes defined in (a). The number of EDOM formulas in each class is also shown on the X-axis. (d) Bar chart showing percentages of identified EDOM formulas by elemental groups. The number of formulas identified in each group is shown on the X-axis.

617 618 619

Figure 3. Number fractions of the identified EDOM molecular formulas in different biochemical classes (a, c) and elemental groups (b, d) at various incubation times in the organic soil. Refer to Figure 2 legend for the classification of biochemical classes and elemental groups.

620 621 622 623 624 625 626 627 628 629

Figure 4. Identification and comparison of EDOM molecular formulas at Day 0 and Day 122 at 8 o C in the mineral soil. (a) van Krevelen diagram showing unique EDOM formulas identified at Day 0 (blue square), common formulas at both Day 0 and 122 (yellow diamond), and unique formulas at Day 122 (red triangle). (b) Percentages of EDOM formulas identified only at Day 0 (blue), Common (yellow) at both Day 0 and Day 122, and only at Day 122 (red). (c) Bar chart showing the percentages of identified EDOM formulas by biochemical classes. The number of EDOM formulas in each class is also shown on the X-axis. (d) Bar chart grouping the percentages of identified EDOM formulas by elemental groups, and the number of EDOM formulas in each group is shown on the X-axis. Additional details are provided in Figure 2 legend.

630

24


Page 24 of 32

Page 25 of 32


631 632 633 634 635 636 637 638 639 640

Figure 5. Identification and comparison of EDOM molecular formulas in the initial organic and mineral soils at Day 0. (a) van Krevelen diagram showing unique EDOM formulas identified at Day 0 (blue square), common formulas at both Day 0 and 122 (yellow diamond), and unique formulas at Day 122 (red triangle). (b) Percentages of EDOM formulas identified only in the organic soil (blue), common in both organic and mineral soils (yellow), and only in the mineral soil (red). (c) Bar chart showing the percentages of identified EDOM formulas by biochemical classes. (d) Bar chart showing the percentages of identified EDOM formulas by elemental groups. Additional details are provided in Figure 2 legend.

641

25



Page 26 of 32

642 643 644 645 646

Table 1. Summary of the identified EDOM molecular formulas, their average elemental compositions and ratios, double bond equivalences (DBE), and aromaticity indexes (AIMOD) for both organic and mineral soils incubated at either 8 °C or –2 °C. Organic Soil

647

Mineral Soil

Total formulas Averaged parameters: Mass C% H% O% N% S% P%

Day0 3949

Day122 (8 °C) 3269

Day122 (-2 °C) 3775

Day0 3785

Day122 (8 °C) 3446

Day122 (-2 °C) 3060

369 58.53 6.49 30.66 3.191 1.025 0.105

376 60.54 6.42 30.10 2.075 0.768 0.093

378 58.99 6.19 31.40 2.561 0.786 0.073

375 61.47 6.99 27.35 2.941 1.116 0.135

380 62.25 7.06 26.96 2.538 1.058 0.126

381 62.35 7.23 26.69 2.518 1.069 0.143

C# O:C H:C N:C S:C P:C DBE AI MOD

18.1 0.426 1.328 0.053 0.007 0.001 7.637 0.222

19.0 0.402 1.256 0.033 0.005 0.001 8.329 0.297

18.7 0.430 1.249 0.042 0.006 0.001 8.448 0.274

19.3 0.362 1.358 0.046 0.008 0.001 7.580 0.236

19.8 0.349 1.352 0.039 0.007 0.001 7.717 0.244

19.9 0.346 1.385 0.039 0.008 0.001 7.467 0.229

Note: DBE = 1 + C – H/2 + N/2 + P/2; AIMOD = (1 + C – O/2 – S – H/2) / (C– O/2 – S – N – P).

648 649 650

26


48

Page 27 of 32


(a)

(b) 0.8

0.036

D10

D34 0.012 0

D122

D0_A

-0.012

D88 -0.024 -0.036 -0.022

Number Fraction

PC2 (18%)

0.024

D0_B

0.7

Enriched in the intial

0.6

Enriched in the final

0.5 0.4 0.3 0.2 0.1 0

-0.011

0

0.011

0.022

CHO

PC1 (44%)

CHON CHOS CHONS Others Elemental Group

651 652 653 654 655 656 657 658 659 660

Figure 1. (a) Principal component analysis in the organic soil incubated at 8 oC. The blue-box area represents molecular formulas enriched in the initial samples (Day 0), where D0_A and D0_B represent experimental duplicates. The red-box area represents formulas enriched in the final samples after incubation (at Day 88 and 122). Purple triangles and labels are sample IDs with incubation days. (b) Elemental analysis and grouping of EDOM formulas enriched in the initial and final samples. Elemental group “Others” represents a combination of CH, CHN, CHS, CHNS, and P-containing formulas.

661

27



Page 28 of 32

662

(a)

2.5

(b)

⑤

678 15%

2.0

H:C

①

②

③

④

1.5 1.0

⑥

⑦

1358 29%

⑧

Common = “remained” Only at Day0 = “degraded” Only at Day122 = “produced”

⑨

0.5 0.0 0.0

0.2

0.4

0.6 O:C

0.8

1.0

1.2

0%

0%

486

20% 311

20%

1933

40%

108

40%

641

60%

239

80%

60%

53

(d) 100%

80%

544

(c) 100%

312

2591 56%

2419 CHO

① ② ③ ④ ⑤ ⑥ ⑦ ⑧ ⑨ Biochemical Class

1635 341 130 102 CHON CHOS CHONS Others

Elemental Group

663 664 665 666 667 668 669 670 671 672 673 674 675 676 677

Figure 2. Identification and comparison of EDOM molecular formulas at Day 0 and Day 122 at 8 oC in the organic soil. (a) van Krevelen diagram showing unique EDOM formulas identified at Day 0 (blue square), common formulas at both Day 0 and 122 (yellow diamond), and unique formulas at Day 122 (red triangle). Shaded areas with numbers indicate different biochemical classes of identified EDOM formulas:45-47 ①=lipids, ②=peptides, ③=amino sugars, ④=carbohydrates, ⑤=other aliphatics (including area of ① with no oxygen, and areas of ② and ③ with no nitrogen), ⑥=unsaturated hydrocarbons, ⑦=lignin, ⑧=tannins, and ⑨=condensed aromatics. (b) Number percentages of EDOM formulas identified only at Day 0 (blue), Common (yellow) at both Day 0 and Day 122, and only at Day 122 (red). (c) Bar chart showing the percentages of identified EDOM formulas by biochemical classes defined in (a). The number of EDOM formulas in each class is also shown on the X-axis. (d) Bar chart showing percentages of identified EDOM formulas by elemental groups. The number of formulas identified in each group is shown on the X-axis.

678

28



0.5

0.15

0.10

0.10

0.05

0.00

0.00

Organic soil (8 °C)

0.4 0.2

Day0 Day10 Day34 Day88 Day122

0.06 0.04

0.6

Organic soil (-2 °C)

0.4 0.2


0.06 0.04 0.02

0.02 0.00


0.15

0.05

0.6

Organic soil (-2 °C)

0.4

Number Fraction

Number Fraction

0.4

Number Fraction

0.5


Organic soil (8 °C)

Number Fraction

Page 29 of 32

CHO

CHON

CHOS

CHONS

0.00

Others

CHO

CHON

CHOS

CHONS

Others

679 680 681 682 683

Figure 3. Number fractions of the identified EDOM molecular formulas in different biochemical classes (a, c) and elemental groups (b, d) at various incubation times in the organic soil. Refer to Figure 2 legend for the classification of biochemical classes and elemental groups.

684

29



Page 30 of 32

685

(a)

2.5

(b)

⑤

512 12%

2.0

H:C

①

②

③

④

851 20%

1.5 1.0

⑥

⑦

2934 68%

⑧ Common = “remained” Only at Day0 = “degraded” Only at Day122 = “produced”

⑨

0.5 0.0 0.0

0.2

0.4

0.6 O:C

0.8

1.0

1.2

(c) 100%

(d) 100%

80%

80%

60%

60%

331

162

1876

151

699

137

0%

32

20%

0%

501

40%

20%

408

40%


2183 CHO


Elemental Group

686 687 688 689 690 691 692 693 694 695 696 697 698 699

Figure 4. Figure 4. Identification and comparison of EDOM molecular formulas at Day 0 and Day 122 at 8 oC in the mineral soil. (a) van Krevelen diagram showing unique EDOM formulas identified at Day 0 (blue square), common formulas at both Day 0 and 122 (yellow diamond), and unique formulas at Day 122 (red triangle). (b) Percentages of EDOM formulas identified only at Day 0 (blue), Common (yellow) at both Day 0 and Day 122, and only at Day 122 (red). (c) Bar chart showing the percentages of identified EDOM formulas by biochemical classes. The number of EDOM formulas in each class is also shown on the X-axis. (d) Bar chart grouping the percentages of identified EDOM formulas by elemental groups, and the number of EDOM formulas in each group is shown on the X-axis. Additional details are provided in Figure 2 legend.

700 701 30


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702

(a)

(b)

2.5

⑤ 2.0

H:C

①

②

③

1093 22%

④

1.5 1.0

⑥

⑦

0.5

1257 25%

⑧ Common Only in organic soil Only in mineral soil

⑨

0.0 0.0

0.2

0.4

2692 53%

0.6 O:C

0.8

1.0

1.2

(c) 100%

(d) 100%

80%

80%

60%

60%

457

288

2082

154

708

264

0%

60

20%

0%

633

40%

20%

396

40%


2470 CHO


Elemental Group

703 704 705 706 707 708 709 710 711 712 713 714

Figure 5. Identification and comparison of EDOM molecular formulas in the initial organic and mineral soils at Day 0. (a) van Krevelen diagram showing unique EDOM formulas identified at Day 0 (blue square), common formulas at both Day 0 and 122 (yellow diamond), and unique formulas at Day 122 (red triangle). (b) Percentages of EDOM formulas identified only in the organic soil (blue), common in both organic and mineral soils (yellow), and only in the mineral soil (red). (c) Bar chart showing the percentages of identified EDOM formulas by biochemical classes. (d) Bar chart showing the percentages of identified EDOM formulas by elemental groups. Additional details are provided in Figure 2 legend.

715 716

31



TOC graphic 45x25mm (600 x 600 DPI)


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Molecular Insights into Arctic Soil Organic Matter ... - ACS Publications

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