mass spectrometry

Journal of Analytical and Applied Pyrolysis 62 (2002) 249– 258

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Comparing bioavailability in five Arctic soils by pyrolysis-gas chromatography/mass spectrometry X.Y. Dai a,*, D. White b, C.L. Ping a a

Uni6ersity of Alaska Fairbanks, Agricultural & Forestry Experiment Station, 533E Fireweed, Palmer, AK 99645, USA b College of Science, Engineering and Mathematics, Uni6ersity of Alaska Fairbanks, Fairbanks, AK 99775 -5900, USA Received 6 September 2000; accepted 16 January 2001

Abstract The bioavailability of soil organic matter (SOM) from five Arctic soils was studied using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and laboratory incubation techniques. Soil samples were incubated concurrently at 4 and 25 °C for approx. 4 months. Py-GC/MS was used to determine the relative percentage of a suite of chemical components in the SOM before and after incubation at both temperatures. Following incubation at 4 °C, the relative percentage of polysaccharides and phenols in SOM decreased compared to that of amino carbohydrates and lipids. The change in the relative percentage of lignin was uncorrelated to any other fraction of the soil organic matter. Following incubation at 25 °C, the relative percentage of polysaccharides and lignin in SOM decreased compared to that of lipids. The changes in relative percentage of amino carbohydrates and phenols were uncorrelated to any other fraction of the SOM. The cumulative mass of carbon dioxide respired during incubation was found to be strongly correlated to the initial relative abundance of polysaccharides for incubations at 4 °C. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Soil organic matter; Chemical composition; Bioavailability; Py-GC/MS

* Corresponding author. E-mail address: [email protected] (X.Y. Dai). 0165-2370/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 3 7 0 ( 0 1 ) 0 0 1 2 3 - 1

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1. Introduction Soil organic matter (SOM) includes many different organic compounds ranging from plant residues and microbial biomass to highly stable humus. All organic matter is subject to chemical, physical, or biological transformation. Not all SOM participates equally in the transformation processes, however [1]. It is assumed that the bioavailability of organic matter to microbes depends on the chemical composition of SOM [2]. Laboratory incubations were conducted on five soils from Arctic Alaska. Cumulative CO2 evolved was used as an index of bioavailable carbon (C). Prolysis-gas chromatography/mass spectrometry (Py-GC/MS) was conducted on the SOM before and after incubation to evaluate its potential as a bioavailability test. Py-GC/MS was conducted to evaluate the relative proportion of certain chemical components of SOM in an attempt to estimate SOM quality. Py-GC/MS served as a valuable analytical technique because pyrolysis products could be separated by gas chromatography and detected by mass-spectrometry [3]. Due to quantitative limitations of Py-GC/MS [4], only relative abundance analysis was used to compare selected classes of compounds in each sample. In this study, a laboratory incubation method was used to determine the bioavailability of SOM in five different soils. Py-GC/MS was then used to compare the relative compositional changes in SOM before and after incubation at two temperatures, and to determine the relationship between the initial relative proportion of selected compounds quantified by Py-GC/ MS and the bioavailability measured by laboratory incubation.

2. Materials and methods

2.1. Soil samples Soils from three NSF-LAII-Flux study sites were sampled for this study in July 1995. The location and characteristics of the sampling sites are presented in Table 1. At each sampling site, a 1×1 m2 soil pit was open to 1 m deep using spade or gas-powered jackhammer when permafrost was encountered. All soils were described and classified according to Soil Taxonomy [5]. All soil samples were kept in a cooler in the field then frozen before shipping. The soil samples were thawed, homogenized by hand before incubation and visible roots were picked out. Soil subsamples were homogenized by hand and air-dried before total C&N analysis. Total carbon and nitrogen were determined on a LECO 1000 CHN analyzer.

2.2. Incubation of samples A 5 g (dry weight) sample of fresh soil was placed in a 250 ml jar and the pH was adjusted to 7 with phosphate buffer. Twenty-five millilitres of distilled (DI) water was added to the samples. The microcosms were incubated in the dark on an orbital shaker rotating at 60 rpm at 4 and 25 °C for 4 months. The incubation jars

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were kept aerated with the caps loosely fitted, and then sealed for 24 h before CO2 reading was taken. The initial CO2 (equilibrated with air) in each jar was taken by syringe immediately after the jar was sealed and determined by Perkin-Elmer 8500 gas chromatography, and CO2 was determined again after 24 h. The respired CO2 from the soil sample was calculated from the difference between the two readings. Moisture content was maintained by adding DI water to keep the same weight as the beginning every 3 days. CO2 was determined three times in the first week, once a week in the first month, and twice a month in the last three months.

2.3. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) Soil samples with a mass of 25 mg were placed in quartz tubes (2 cm in length, 2 mm inside diameter) and quantified using a Mettler microbalance. Py-GC/MS was conducted as described by White and Beyer [6]. The samples were held in the quartz tube by plugs of quartz wool at each end. Py-GC/MS was conducted on each sample and used to identify as many compounds as possible. Py-GC/MS was conducted with a CDS Model 1000 pyrolyzer and a Model 1500 GC interface. The interface temperature was set at 280 °C. During pyrolysis, the sample was heated from 280 to 700 °C in 0.1 s and held at 700 °C for 9.9 s. The 700 °C maximum temperature is a nominal set point. The temperature in the center of the sample is expected to approach this temperature. Our experience has shown that a 700 °C set point provides optimal pyrolysis yield for organic soils. The pyrolysis reactor was mounted on a gas chromatograph, HP 5890 Series II, with a Hewlett Packard HP-1 column (cross-linked methyl-siloxane) 25 m× 0.2 m× 0.33 mm film thickness. The GC temperature program was 35 °C for 15 min, 2 °C min − 1 ramp to 250 °C and hold for 10 min. The GC was plumbed directly to an HP 5971A Series Mass Selective Detector in electron impact mode. The MS scanned mass units 45–650. All mass spectra were compared to the Wiley 138 mass spectral library. Helium was used as the carrier gas at 0.5 cm3 min − 1. The sample was injected with a split ratio of 1:50. Table 1 Location, classification and site characteristics of samples used in the study Site ID

Area

Land cover classa, soil classificationb

Landform, Microrelief

Site 1

Prudhoe Bay, AK Sagwon Hills, AK

Moist nonacidic tundra, Euic Sapric Glacistels Moist acidic tundra, Fine-loamy, mixed Ruptic-Histic Aquaturbel Moist acidic tundra, Loamy, mixed Ruptic-Histic Aquaturbel

Coastal Plain, 35 Flat-Polygon Center Hills, Tussock tundra 40

Site 2

Site 3

a b

Toolik Lake, AK

(Adapted from Auerbach and Walker [13]). (Adapted from Soil Survey Staff [5]).

Hills, Tussocks,Mid-slope

Active layer (cm)

35

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Table 2 Biochemical classes selected to represent soil organic materials Biochemical classes

Post pyrolysis compounds

Polysaccharides

2,4-Dimethylfural, furancarboxyaldehyde, benzofuran Phenol, methylphenol, ethylphenol Methoxyacetaphenone, unresolved methoxyphenolic Unresolved amino sugar

Phenol (protein and phenol precursors) Lignin Amino carbohydrates (amino sugar and N-acetyl amino sugars) Lipids

Unresolved PAH, unresolved alkene, unresolved alkane

Following Py-GC/MS, many compounds were identified and a suite of compounds was selected from the biochemical classes: polysaccharides, amino sugars and N-acetyl amino sugars (hereafter referred to as amino carbohydrates), lignins, phenol precursors and proteins (hereafter referred to as phenols), and lipids (see Table 2) [7]. Specific compounds were chosen to represent each class that are widely reported in soil pyrolysis literature and known to be derived from specific fractions of soil organic matter. Comprehensive lists of all compounds in each biochemical class are published elsewhere [7–9]. Although these lists were consulted for the purposes of this work, only the dominant peak(s) in each class were chosen for analysis. The dominant peaks could be easily identified and were not subject to complications caused by chromatographic co-elution. In other sets of samples using this method we have observed a 0.96– 0.99 self-correlation for three replicates. The relative percentage of each selected compound was calculated according to peak height above baseline. The percentage of each compound reported herein, therefore, is the relative percentage of that compound among the compounds identified, not the percentage of that compound compared to the total SOM. The results are not intended to represent the SOM composition in its entirety, but rather provide an index by which bioavailablility can be evaluated.

3. Results and discussion Sample collection sites and soil classifications are presented in Table 1. Selected properties of the soils and cumulative 4-month CO2 evolution data are presented in Table 3. Site 1 soils are from the arctic coastal plain. The landform is characterized by frost polygons and thaw lakes and drainage is poor to very poor. Sites 2 and 3 soils are from the arctic foothills, which are glaciated uplands characterized by rolling hills with imperfect drainage.

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3.1. Respiration of SOM in whole soils Cumulative CO2 evolution from whole soils incubated for 4 months is reported in Table 3. Temperature had an appreciable influence on the amount of CO2 evolved, indicating that microbes were more active at 25 °C than 4 °C. On average, for a temperature increase from 4 to 25 °C, the CO2 evolution increased roughly 67%. The result was consistent with that of Flanagan and Veu´ m [10] who indicated that most organisms in tundra were cold-tolerant mesophiles. For example, fungi able to respire heterotrophically to − 6.5 °C had an optimum growth temperature between 20– 30 °C.

3.2. The relati6e proportion of the selected chemical compounds 6ersus the CO2 e6olution Correlation coefficients for the relative percentage of selected chemical compounds (Table 4, Fig. 1) to the CO2 evolution are reported in Table 5. In the soils incubated at 4 °C, the relative percentage of polysaccharides was strongly correlated to CO2 evolution (r =0.96). That is to say, the soils with the highest relative proportion of polysaccharides produced the most CO2. At 25 °C, however, the correlation coefficient between polysaccharides and cumulative CO2 (r=0.74) was not as strong as it was at 4 °C. In general, polysaccharides are considered the most readily degradable constituent of SOM [11]. If, on average, the polysaccharides are degraded fastest and to the greatest extent, the correlation coefficient between polysaccharides and cumulative CO2 produced should be greatest in the incubations exhibiting the greatest respiration rate. This was not observed, however, when comparing incubations from 4 °C and 25 °C. Even though the soils incubated at 4 °C produced only one-third of the CO2 as the soils incubated at 25 °C, the CO2 evolution was more closely correlated to polysaccharides in the former. This observation could be accounted for if the incubation at 4 °C were considered an

Table 3 Some properties and the cumulative CO2 evolution of soilsa Site ID

Hor.

Depth (cm)

pH 1:1

TOC (%)

TON (%)

C/N

CO2-C mg g C−1 4 °C 25 °C

Site 1 Prudhoe Bay Site 2 Sagwon Hills Site 3 Toolik Lake a

Oa1 Oa2 Oe O/A Cf

10–22 22–50 8–16 37–50 30–100

(Adapted from Dai et al. [14]).

5.7 6.6 5.2 5.8 5.4

14.2 19.9 36.0 11.9 6.9

0.9 1.3 1.4 0.8 0.4

15.4 15.8 25.0 15.8 18.1

6.3 4.4 9.0 10.1 11.1

23.6 12.8 32.8 31.5 23.3

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Fig. 1. Examples of pyrograms of Site 1 soil Oa1 horizon (a) and Site 2 soil Oe horizon (b).

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Table 4 Distribution of the five classes compounds in the whole soil Sample c

Polysaccharides

Lignin

Amino carbohydrates

Lipids

Phenols

Oa1 Oa2 Oe O/A Cf Oa1 4 Ca Oa2 4 C Oe 4 C O/A 4 C Cf 4 C Oa1 25 Cb Oa2 25 C Oe 25 C O/A 25 C

8 8 15 21 17 10 6 19 15 10 9 7 17 12

17 19 19 19 5 15 18 21 19 4 30 24 15 15

7 6 6 3 4 6 6 4 2 6 6 3 4 3

37 26 22 16 24 35 33 20 25 41 32 24 28 29

30 32 30 40 50 34 42 23 34 38 23 38 27 36

a b

Soil samples incubated at 4 °C. Soil samples incubated at 25 °C.

earlier stage of decomposition by the same microbial consortium as that active at 25 °C. If the organisms incubated at 25 °C consumed all available polysaccharides, the correlation between CO2 evolved and relative proportion of polysaccharides would decrease with respiration of additional substrates.

3.3. The change of chemical composition of SOM in the whole soil after incubation at 4 and 25 °C The correlation coefficients for the change in distribution of organic compounds in the SOM before and after incubation are shown in Table 6. Strong correlations were observed for changes in polysaccharides compared to lipids, phenols compared to lipids, and phenols compared to amino carbohydrates before and after incubation at 4 °C. That is to say, with a decrease in the relative proportion of

Table 5 Correlation coefficients between the CO2 evolution and the relative percentage of each class of compounds in whole soils at both temperatures Relative percentage of each class of compounds

CO2 incubated at 4 °C CO2 incubated at 25 °C

Polysaccharides Lignin Phenol Amino carbohydrates Lipids

0.96 −0.38 0.49 −0.87 −0.67

0.74 0.09 −0.05 −0.46 −0.46

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Table 6 Correlation coefficients between the changes of each class of compounds before and after incubation Soils

Polysaccharides

Before and after incubation at 4 °C Polysaccharides Phenols 0.7 Amino carbohydrates −0.65 Lipids −0.97 Lignin 0.41 Before and after incubation at 25 °C Polysaccharides Phenols −0.5 Amino carbohydrates −0.42 Lipids −0.68 Lignin 0.43

Phenols

0.7 −0.87 −0.85 −0.09 −0.5 −0.52 0.49 −0.52

Amino carbohydrates

Lipids

Lignin

−0.65 −0.87

−0.97 −0.85 0.78 − −0.32

0.41 −0.09 −0.19 −0.32

−0.68 0.49 −0.39

0.43 −0.52 −0.02 −0.92

0.78 −0.19 −0.42 −0.52 −0.39 −0.02

−0.92

polysaccharides and phenols, there was an increase in the relative proportion of lipids and amino carbohydrates. For incubations at 25 °C, strong correlations were observed between changes in lipids compared to lignin and polysaccharides. That is to say, with a decrease in the relative proportion of polysaccharides and lignin, there was an increase in the relative proportion of lipids. Correlation between the change in polysaccharides compared to lipids after incubation at 25 °C (r= − 0.68), was not as strong as it was at 4 °C (r= −0.97). The correlation coefficient for the change in lignin compared to lipids, however, was much stronger for the incubation at 25 °C (r= −0.92) than at 4 °C. This result suggested that at 25 °C, microbes used more recalcitrant substances, such as lignin, as an energy and C source, likely in place of depleted polysaccharides. This is consistent with the hypothesis proposed previously which stated that the organisms incubated at 4 °C were earlier in the decomposition process and more reliant on polysaccharides than substrates such as lignin. Another explanation for the observed differences in degradation at the two temperatures is the enrichment of a different microbial consortium. Other important correlations were observed between the relative abundance of chemical compounds before and after incubation at 4 °C. The change in polysaccharides was positively correlated to the change of phenols (r= 0.7) suggesting that the relative abundances of polysaccharides and phenols decreased proportionately. The change of amino carbohydrates was positively correlated to the change of lipids (r= 0.78), demonstrating that the relative abundance of amino sugars and lipids increased proportionately. This is not to say that lipids are necessarily produced during the incubation, but that the relative abundance of lipids and amino carbohydrates increased compared to the other classes of compounds. The same correlations were not found for the soils incubated at 25 °C.

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These results indicated that temperature played an important role in the ability of microorganisms to use different substrates during the incubation. At 4 °C, the decrease in relative abundance of polysaccharides was significantly correlated to the increase in the relative abundance of the lipids. In other words, polysaccharides were degraded in the SOM while lipids remained. However, at 25 °C, the strongest correlation was between lignin and lipids where a decrease in the relative abundance of lignin strongly correlated to the increase in relative abundance of lipids. One plausible explanation is that at higher temperature, the microorganisms could use lignin in addition to polysaccharides as their energy and carbon source. However, at 4 °C, the lignin was not [or could not] be decomposed by the microorganisms. Instead, polysaccharides were the only energy and carbon sources for the active microbial consortium. This result is consistent with that of Nadelhoffer et al. [12] who suggested that decomposition processes operating at low versus high temperature in arctic soils were functionally different. At temperatures above 10 °C, enzymatic degradation of cellulosics and simple organic compounds both increased with temperature. Below 10 °C, however, only simple compounds were degraded and decomposition of cellulosics and other more recalcitrant compounds decomposition was limited (both by low number of cellulose microbes and by greater thermodynamic constraints on cellulase activities). 4. Conclusions Although Py-GC/MS was only used to look at a limited suite of organic compounds, it exhibited some potential for evaluating the relative quality of SOM. Together with the laboratory incubation method, Py-GC/MS was used to compare the changes of relative proportion of compounds before and after incubation, to identify the chemical composition of SOM, and to study correlation between the relative proportion of chemical composition to bioavailability of SOM. Acknowledgements The authors gratefully acknowledge the financial support provided by NSF ARCSS-LAII program (OPP-97-32731), the field assistance of Dr J. M. Kimble of USDA-NRCS, and the laboratory staff of the Palmer Research Center. The pyrolysis-GC/MS was conducted at the Water and Environmental Research Center. The authors are grateful for the assistance provided by D. Sarah Garland, Bill Schnabel and Huan Luong. References [1] H.H. Cheng, J.A.E. Molina, In search of the bioreactive soil organic carbon: The fractionation approaches, in: R. Lal, J. Kimble, E. Levine, B.A. Stewart (Eds.), Advances in Soil Science- Soil and Global Change, CRC Lewis, Boca Raton, FL, 1995, pp. 343 – 350.

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