characterization of organic matter in a northern hardwood forest soil by 13c nmr spectroscopy and...

Characterization of organic matter in a northern

hardwood forest soil by 13C NMR spectroscopy

and chemical methods

David A.N. Ussiri, Chris E. Johnson*

Department of Civil and Environmental Engineering, Syracuse University, 220 Hinds Hall, Syracuse,

NY 13244, USA

Received 20 March 2002; received in revised form 13 August 2002; accepted 30 August 2002

Abstract

Soil organic matter plays an important role in soil properties and influences ecosystem cycles of

C, N, Al, Fe, and other major and trace elements. We examined spatial variations in the structure and

chemistry of soil organic matter at the Hubbard Brook Experimental Forest in New Hampshire,

USA. Humic substances were extracted and isolated chromatographically into humic acid, fulvic

acid, and polysaccharide fractions. Chemical methods and solid-state 13C NMR spectroscopy were

used to determine structural chemistry. On average, extractable humic substances accounted for

nearly 50% of soil organic matter, with alkyl and O-alkyl C (carbohydrate) being the largest C

fractions in whole soils and isolated humic substances. Alkyl C ranged from 33% to 56% of C, while

O-alkyl C comprised 20–45% of C. Alkyl C increased, while O-alkyl C decreased with soil depth in

whole soils, humin, and humic acid. Aromatic C increased with soil depth in whole soils and humin,

while carbonyl C increased with depth in whole soils and fulvic acids. Fulvic acids were more acidic

than humic acids, and were less phenolic and aliphatic than humic acids. Carboxylic acidity

accounted for about 80% and 50% of total acidity in fulvic acid and humic acid, respectively. Soil

from higher-elevation sites exhibited greater alkyl C and lower O-alkyl and aromatic C in the Oa

horizon, suggesting a greater degree of decomposition of the organic matter in the Oa horizon of

these conifer-rich sites. Mineral soils in conifer-rich sites contained organic matter that was more

aromatic than in hardwood sites. Variations in humification processes, source materials, and transport

0016-7061/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0016 -7061 (02 )00257 -4

* Corresponding author. Fax: +1-315-443-1243.

E-mail addresses: [email protected] (C.E. Johnson).

www.elsevier.com/locate/geoderma

Geoderma 111 (2003) 123–149

of organic matter could account for variations in the structure and chemistry of organic matter in

these forest soils.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: CPMAS 13C NMR; Forest soil; Humic substances; Soil acidity; Soil organic matter; Spodosol

1. Introduction

Soil organic matter is a heterogeneous mixture of organic compounds of plant, animal,

and microbial origin in various stages of decomposition. Humic substances are the

amorphous, highly transformed, darkly colored component of organic matter. They consist

of a wide range of structures and functional groups (Stevenson, 1994; Hayes and Swift,

1978). Humic substances are ubiquitous in the environment, occurring in all soils, waters,

and sediments. In soils, humic substances comprise up to 80% of soil organic matter

(Hayes, 1998). Humic substances form an important pool of C in the global C budget.

According to Schlesinger (1997), the total mass of organic C stored in soils ranges from

1100� 1015 to 3000� 1015 g C, greater than atmospheric and living biomass C combined.

In addition to their role in C cycle, humic substances also influence soil fertility, soil

development, and various soil chemical properties, including cation exchange capacity

(CEC), buffer capacity, pH, acid–base chemistry, and metal transport (Petersen, 1980;

Buol et al., 1997), and serve as a source of energy for soil macro- and microorganisms

(Paul and Clark, 1987).

While chemical investigations of humic substances go back more than 200 years,

much remains unknown about their structure and properties. Research has shown that

humic acids and fulvic acids extracted from soils formed under the same environ-

mental conditions have broadly similar analytical characteristics and chemical struc-

tures (Griffith and Schnitzer, 1975; Schnitzer, 1977; Chen et al., 1978; Burch et al.,

1978). The chemical nature of the litter, composition of the microbial community, and

environmental factors such as temperature and moisture play important roles in

influencing the chemical and structural components found in humic substances. Forest

floors that develop under broadleaf or mixed forests are more conducive to decom-

position than those formed under conifers. For example, Prescott et al. (2000)

observed faster decomposition of broadleaf litter compared to needle litter. They

attributed this to the lower lignin and higher N concentrations in deciduous leaf litter.

Also, the degree of decomposition and vegetation type have been shown to influence

the chemical composition of SOM (Mathur and Farnham, 1985; Kogel et al., 1988;

Krøsshavn et al., 1992). Humic substances from similar geographic locations but

different depositional environments often have different chemical structures (Rasyid et

al., 1992).

The objective of this research was to examine spatial variations (or lack thereof) in the

chemical and structural characteristics of soil organic matter in a forest soil in New

Hampshire, USA. We employed chemical analysis methods and 13C NMR spectroscopy to

study both whole soils and isolated humic substances. Our goal was to address the

D.A.N. Ussiri, C.E. Johnson / Geoderma 111 (2003) 123–149124

following research questions: (1) How do chemistry and structural composition of organic

matter vary with soil depth? (2) What are the effects of forest composition and/or elevation

on soil organic matter? (3) How does the structural chemistry of isolated humic substances

compare with whole soil organic matter? (4) How do estimates of carboxyl functional

group content made by different methods compare?

2. Materials and methods

2.1. Site description

The Hubbard Brook Experimental Forest (HBEF) lies in the southern White

Mountain region of New Hampshire (43j56VN; 71j45VW). The Hubbard Brook

Valley is underlain by granite gneiss of the Rangley formation and the Kinsman

quartz monzonite, covered with glacial till derived primarily from the local bedrock

(Likens et al., 1967). Vegetation at the HBEF is mostly northern hardwoods,

dominated by American beech (Fagus grandifolia), sugar maple (Acer saccharum),

and yellow birch (Betula alleghaniensis). Pockets of conifer-rich stands, consisting of

red spruce (Picea rubens), balsam fir (Abies balsamea), and white birch (Betula

papyrifera) are found at the highest elevations and on exposed slopes (Whittaker et

al., 1974). Detailed descriptions of the climate, geology, vegetation, and hydrology at

HBEF have been published elsewhere (Federer et al., 1990; Likens and Bormann,

1995). This research was conducted in watershed 5 (W5), a 23-ha watershed which

was dominated by a 65-year-old second-growth northern hardwood forest in 1983

when the soils were sampled. The elevation extends from about 500 to 750 m. To

assess spatial variations in organic matter chemistry, we divided the watershed into

three subcatchments. The upper subcatchment (SFB) was characterized by an abun-

dance of spruce, fir, and white birch, with shallow soils and relatively flat topography.

The high hardwood (HH) zone was dominated by deciduous vegetation, steeper

slopes, and deeper soils compared to SFB. The low hardwood (LH) subcatchment

has deep soils underlain by dense glacial till and is dominated by hardwoods.

2.2. Soils

Soils on W5 are predominantly well-drained, coarse–loamy, mixed–frigid, Typic

Haplorthods with a 3- to 15-cm organic layer at the surface. These soils are acidic

(pH < 4.5) with low base saturation and CEC (Johnson et al., 1991b). Average depth

to C horizon (or bedrock) is about 60 cm (Johnson et al., 1991a). The soils used in

this study were sampled from W5 in 1983. Soil pits were excavated at 60 sites, and

samples were collected by depth and by genetic horizons. Samples were air-dried to a

constant weight and sieved through a 2-mm screen. The samples have been archived

in glass jars. A more detailed description of the sampling methodology can be found

elsewhere (Huntington et al., 1988; Johnson et al., 1991a,b).

Humic substances were extracted from samples of the Oa, E, Bh, Bs1, and Bs2

horizons. Soils from 6 of 60 sampling locations were selected to reflect the range of

D.A.N. Ussiri, C.E. Johnson / Geoderma 111 (2003) 123–149 125

elevation and forest type in W5. The pits selected also exhibited the full suite of

horizons typically found in HBEF Spodosols (i.e., Oi +Oe, Oa, E, Bh, Bs1, and Bs2).

Selected properties of soils used in this study are shown in Table 1. The averages in

Table 1 are similar to the averages for all 60 pits reported in Johnson et al. (1991a,b).

2.3. Extraction, fractionation, and purification of humic substances

The procedures used for the extraction, fractionation, and purification of humic

substances are summarized in Fig. 1. Humic substances were extracted with 0.5 M NaOH

at a soil-to-solution ratio of 1:10 (mass:volume) under a N2 atmosphere (Stevenson, 1994).

The extracted humic substances were then separated into humic acid and fulvic acid

fractions by acidifying the extract to pH 2 using 6 M HCl. The extract was allowed to

settle overnight, and centrifuged to remove the humic acid, which was then purified by the

Stevenson (1994) method. Polysaccharides were isolated from the fulvic acid fraction by a

modification of the method described by Cheshire et al. (1992).

2.4. Elemental and acidic functional group analysis

The elemental composition of isolated and freeze-dried humic substances was deter-

mined by dry combustion, followed by gas chromatography using an elemental C, H, and N

analyzer (Carlo Erba model EA1108). The content of O was estimated as the ash-free mass

less C, H, andN. Ash content was determined by combustion overnight in amuffle furnace at

500 jC.The content of carboxylic functional groups was estimated by both direct and indirect

acid–base titration methods for comparison purposes. In the direct method, 20–30 mg of

freeze-dried humic or fulvic acid was suspended in DDI water and titrated to pH 10.5 under

Table 1

Average chemical properties of the soils used in this study

Horizon LOIa

(g kg� 1)

Total C

(g kg� 1)

Total N

(g kg� 1)

CECeb

(cmolc kg� 1)

Exchangeable

bases

(cmolc kg� 1)

Exchangeable

acidity

(cmolc kg� 1)

pHwc pHs

d

Oa 505

(399–602)

309

(227–364)

14.2

(9.4–19.6)

15. 6

(10.5–22.1)

8.4

(5.6–14.8)

7.1

(4.5–9.5)

3.8

(3.4–4.2)

3.2

(2.9–3.5)

E 37.2

(19. 1–53)

21.9

(9.3–31.1)

1.1

(0.4–1.6)

2.5

(1.4–3.7)

0.5

(0.1–0.6)

2.0

(0.9–3.5)

4.4

(3.8–5.0)

3.4

(2.9–3.9)

Bh 125

(73.9–186)

65.3

(35.9–105)

3.4

(2.1–5)

6.5

(4.2–9.3)

0.8

(0.43–1.8)

5.7

(3.6–8.8)

4.2

(3.8–4.6)

3.4

(2.9–3.9)

Bs1 127

(95.8–152)

61.9

(45.0–73.1)

2.8

(2.3–3.4)

6.7

(5.3–8.6)

0.5

(0.4–0.7)

6.2

(4.8–8.3)

4.1

(3.9–4.3)

3.6

(3.1–3.8)

Bs2 75.5

(42.7–98.3)

32.3

(17.5–47.9)

1.3

(0.7–2.0)

3.4

(1.8–5.8)

0.2

(0.1–0.2)

3.2

(1.6–5.6)

4.5

(4.4–4.7)

4.1

(3.8–4.3)

Values in parentheses represent ranges of the six samples for each horizon.a Loss on ignition (500 jC).b ‘‘Effective’’ CEC measured in unbuffered NH4Cl (1 M; Johnson et al., 1991b).c pH measured in DI water.d pH measured in 0.01 M CaCl.


N2 against 0.05 M NaOH using a Metrohm autotitrator (Brinkmann Instruments, Westbury,

NY).

Among the principal functional groups present in humic substances, carboxyl groups

have the lowest pKa values, generally less than 7.0 (Stevenson, 1994). Thus, it was assumed

that carboxyl groups were entirely dissociated at the equivalence point, while other weaker

functional groups (e.g., phenolic and alcoholic) remained protonated. The total carboxyl

group content was therefore estimated as the amount of NaOH required to reach the

equivalence point.

The calcium acetate and barium hydroxide methods (Schnitzer and Khan, 1972) were

used to determine the concentrations of carboxylic and total acidic functional groups,

respectively. The suspensions were filtered through 0.45-Am membrane filters as recom-

Fig. 1. Procedure for extraction, isolation, and purification of humic substances.


mended by Perdue (1985). Phenolic acidity was estimated as the difference between total

and carboxyl acidity.

2.5. Solid-state cross-polarization and magic angle spinning (CPMAS) 13C NMR analysis

The structural chemistry of whole soils, freeze-dried humic and fulvic acids, and

residual soils (i.e., the humin fraction) was characterized using solid-state 13C NMR

analysis with cross-polarization and magic angle spinning (CPMAS). Three replicates

each of whole soils and humin from the Oa, Bh, and Bs1 horizons were analyzed. In

addition, three replicates of freeze-dried humic acid and fulvic acid from the Oa, Bh, Bs1,

and Bs2 were analyzed by 13C NMR. Prior to analysis, whole soil samples from the Bh

and Bs1 horizons were treated with 2% HF to remove paramagnetic materials as described

by Dai and Johnson (1999). The treated samples were then freeze-dried, and ground to

pass a 400-Am screen. The Oa horizon samples were ground to pass a 400-Am screen

without HF treatment. The humin samples were rinsed with DI water until free of NaOH,

freeze-dried, ground, and sieved through a 400-Am screen. Semiquantitative solid-state

CPMAS 13C NMR analysis was conducted on a Bruker AMX 300 spectrophotometer at

75.47 MHz. Samples were spun at 5 kHz in a zirconia rotor within a MAS probe. The

number of transients required for an acceptable signal-to-noise ratio ranged from 3918 to

99,224. The contact time for each spectrum was 1 ms and recycle time was 1 s. Spectral

width was 33,112 Hz and the acquisition time and number of decay curves were 61 ms and

4096, respectively. Chemical shifts were externally referenced to glycine resonance at 176

ppm.

Spectra derived from CPMAS 13C NMR analyses of soil humic substances are known

to be semiquantitative, at best (Kinchesh et al., 1995a,b; Preston, 1996; Mathers et al.,

2000). Suboptimal quantitation arises from many reasons, most notably: (1) the presence

of paramagnetic compounds (e.g., Fe, Mn); (2) the presence of 13C atoms that are distant

from 1H atoms, reducing the efficiency of cross-polarization; and (3) variations in the rates

of magnetization and relaxation of C atoms in different functional groups. Wilson (1987)

found that the carbon detected by NMR analysis represented only 30–62% of the C in

various humic and fulvic acids. Similarly, Smernik and Oades (2000a,b) found that the

CPMAS 13C NMR signal intensities of whole soils, HF-treated soils, and density fractions

represented 27–63% of the C in the samples. While many researchers have proposed

methods for improving the quantitation of 13C NMR analyses (e.g., Kinchesh et al., 1995b;

Conte et al., 1997; Mao et al., 2000; Smernik and Oades, 2000a), these methods require

additional analyses and/or extended analytical time.

Our goal was to analyze as many samples as possible in order to assess the variability

in organic matter composition within the forest ecosystem. Therefore, we limited our

analyses to CPMAS 13C NMR. Studies of soils, peats, and humic substances have

suggested that the fraction of C in the aromatic and carbonyl regions of CPMAS 13C

NMR may be underrepresented (Mao et al., 2000; Smernik and Oades, 2000a,b).

Consequently, the distribution of C derived from a particular spectrum must be interpreted

with caution. As Kinchesh et al. (1995b) pointed out, however, it is reasonable to use

these results for the comparison of samples from different land uses, cover types, or soil

horizons.


3. Results

3.1. Humic substance concentrations

Humic substances accounted for 44–62% of the soil organic matter. The percentage of

OM extracted remained fairly constant regardless of the organic matter concentration in

each horizon. The concentrations of the individual fractions, however, varied significantly

among horizons. The concentration of humic acid ranged from 6.2% to 44.6% of the soil

organic matter, and decreased with increasing soil depth (Fig. 2). The concentration of

fulvic acid ranged from 4.7% to 39.4% of soil organic matter, and increased with

increasing soil depth (Fig. 2). The concentration of polysaccharides ranged from 1.2%

Fig. 2. Distribution of humic substance fractions in different subcatchments at the Hubbard Brook Experimental

Forest.


to 9.2% of soil organic matter. Humic substances extracted from the same horizons in

different subcatchments did not differ significantly (Fig. 2).

3.2. 13C NMR analysis of humic substances

3.2.1. Humic and fulvic acid

Solid-state CPMAS 13C NMR analysis was conducted on the humic acid and fulvic

acid fractions to determine their chemical composition. Example spectra of humic acid and

fulvic acid fractions extracted from different horizons are presented in Fig. 3. In general,

all spectra had peaks in the resonance areas of alkyl C (0–50 ppm), O-alkyl C (50–110

ppm), aromatic C (110–160 ppm), and carbonyl C (160–220 ppm). The spectra of humic

acids from the Oa horizon had large peaks at 30 ppm in the alkyl C (0–50 ppm) range.

This peak is likely due to aliphatic carbons in alkyl chains (Schnitzer and Preston, 1983,

1986; Breitmaier and Voelter, 1987). In the O-alkyl C (50–110 ppm) region, signals for

aliphatic C substituted by oxygen and nitrogen are usually observed. In this region, we

observed intense signals at 55, 72, and 107 ppm. The peak at 55 ppm is due to methoxyl C

(Hatcher, 1987) and is normally associated with lignin. The peaks at 72 and 107 ppm

generally arise from carbohydrates (Hatcher et al., 1980). The peak at 72 ppm dominates

the O-alkyl C region in Hubbard Brook soils, and it is attributed to ring C of

carbohydrates; the peak at 107 ppm includes anomeric C of carbohydrates. In the aromatic

region (110–160 ppm), there were peaks at 130 and 148 ppm. These peaks could be

Fig. 3. Solid-state 13C NMR spectra for fulvic and humic acid isolates from various soil horizons at the Hubbard

Brook Experimental Forest.


assigned to aromatic units contained in lignin (Hatcher, 1987; Baldock and Preston, 1995).

The peak at 173 ppm in the carbonyl C region is assigned to carboxyl C. Amides and

esters may contribute to this peak (Hatcher et al., 1980).

The peaks observed in the humic acid spectra from mineral horizons were generally

similar to those found in the Oa horizon spectra. However, the 13C spectra became much

broader with increasing depth, indicating some loss of identity of the original biochemical

components due to increased decomposition. Other notable differences were the decrease

and eventual disappearance of methoxyl, carbohydrate, and aromatic peaks at chemical

shifts 55, 72, 104, and 148 ppm, respectively, with soil depth.

The spectra of fulvic acid from the Oa horizon had a broad alkyl C peak at 28 ppm and

a small peak at 48 ppm, corresponding to aliphatic carbons in alkyl chains and alkyl

moieties in lignin, respectively. The small peak at 55 ppm is contributed by methoxyl C,

while the larger peaks at 71 and 102 ppm are mainly due to carbohydrate C (Breitmaier

and Voelter, 1987). As in the humic acid spectra, peaks at 127, 148, and 173 ppm are likely

due to C- and H- substituted aromatic C, O- and/or N-substituted aromatic C, and

carboxylic C, respectively. The fulvic acid spectra from the mineral horizons were similar

in form to the Oa. The most notable change observed in the mineral horizons was an

increase in the carboxyl C peak with soil depth.

Results from the integration of 13C NMR spectra of humic acids and fulvic acids are

presented in Fig. 4. Integration of the major regions of 13C resonance revealed that alkyl C

and O-alkyl C were the dominant C components in humic and fulvic acids in all horizons.

Humic acids, however, contained significantly more alkyl C (48–56% of total C) than

fulvic acid (33–36% of total C) ( p = 0.01, n = 24; Fig. 4). Our results are consistent with

the 13C NMR ranges compiled by Mahieu et al. (1999) for humic and fulvic acids from

forest soils. On average, signal intensities for alkyl C in humic acid did not differ

statistically among the Bh, Bs1, and Bs2 horizons, but were slightly lower in the Oa

horizon (Fig. 4a). In contrast, O-alkyl C was significantly greater in Oa humic acids

( p = 0.05, n = 12) and decreased with depth in mineral soil.

In fulvic acid, signal intensities for alkyl C were slightly higher in the Oa horizon

compared to the Bh and Bs horizons (Fig. 4b). However, O-alkyl C concentration in fulvic

acid did not vary with depth in the soil profile. This pattern is contrary to the patterns in

humic acid. On average, the signal intensities for aromatic C (110–160 ppm) decreased

slightly with soil depth in both humic acid and fulvic acid (Fig. 4). Carbonyl C (160–220

ppm) in fulvic acid increased significantly in the mineral soils ( p = 0.01, n = 12). A similar

trend was also observed for humic acid, although it was not statistically significant. Since

carboxylic C is the dominant fraction in the carbonyl C region, the spectroscopic data are

consistent with the chemical analysis data, which revealed similar trends (see Acidic

Functional Groups section).

3.2.2. Humin

Example spectra of the humin fraction are presented in Fig. 5. Spectra of the humin

from the mineral horizons were not well resolved, probably due to interferences from

paramagnetic materials—Fe in particular. The humin from the Oa horizon showed

relatively strong signals for lignin as suggested by peaks around 55 and 148 ppm, which

have been associated with lignin structures (Hatcher, 1987; Kogel et al., 1988; Baldock


and Preston, 1995). These signals declined with depth, which could indicate degradation

of lignin in the mineral soils. Peaks around 30 ppm, corresponding to aliphatic C in alkyl

chains, were present in all spectra. Peaks at 73 and 105 ppm, representing carbohydrates,

were most pronounced in the Oa horizon. The peak at 175 ppm, corresponding to carboxyl

C, also decreased with soil depth, suggesting that carboxylic C in the humin fraction

decreased with increasing soil depth. However, poor spectral quality, especially in the Bs1

horizon, may mask peaks in this range.

Integration of the major regions of 13C resonance indicated that the C components of

the humin fraction were mainly alkyl C (33–44% of total C) and O-alkyl C (30–42% of

total C) for all horizons (Fig. 6). The alkyl C fractions in the Bh and Bs1 horizons were

significantly greater than in the Oa horizon. In contrast, the O-alkyl C fraction was

significantly greater ( p = 0.01) in the Oa horizon. The aromatic C fraction increased with

Fig. 4. Distribution of C in humic substances based on 13C NMR analysis of (a) humic acid and (b) fulvic acid.

Error bars represent standard errors of the means.


soil depth, and carbonyl C decreased with increasing soil depth. Although statistically

significant, these results should be interpreted with caution because of the poor spectral

quality of the humin from the mineral soils. Overall, the spectral properties of humin were

similar to those observed for whole soils (see below).

3.3. 13C NMR analysis of whole soils

Examples of solid-state CPMAS 13C NMR spectra of whole soils from the Oa, Bh, and

Bs1 horizons are presented in Fig. 7, and the distribution of C among the major spectral

regions is presented in Fig. 8. The spectrum of the Oa horizon had a large peak at 31 ppm

corresponding to alkyl C (Kogel et al., 1988; Preston et al., 1994). In the O-alkyl C region,

Fig. 5. Solid-state 13C NMR spectra for humin fractions from various soil horizons at the Hubbard Brook

Experimental Forest.


peaks at 57, 73, and 104 ppm indicated methoxyl C in lignin structures (Hatcher, 1987)

and carbohydrate C (Breitmaier and Voelter, 1987; Kogel et al., 1988). The aromatic C

region (110–160 ppm) had peaks at 112, 128, and 131 ppm, suggesting unsubstituted

aromatic rings such as alkyl-benzenes (Hatcher et al., 1980; Schnitzer and Preston, 1986)

and aromatic C in lignin (Kogel et al., 1988). In the carbonyl C region (160–220 ppm),

there was a carboxyl C peak at 174 ppm.

The Bh and Bs1 horizon spectra were similar to the Oa, but had poorer signal-to-

noise ratios (Fig. 7). In the alkyl C region, the peak became broader, with shoulders at

32 and 49 ppm that were not visible in the Oa spectra. The broader alkyl C peaks

observed for Bh and Bs1 horizons (Fig. 7) suggest that a considerable loss of identity of

the original biochemical components has occurred in the alkyl C chains, revealing an

increase in decomposition with increasing soil depth. The broader alkyl peaks may also

be due to greater paramagnetic broadening in the spectra of the mineral soils. However,

the mineral soil samples in this study were pretreated with 2% HF to remove

paramagnetic interferences, so this effect should be minimal. In the O-alkyl C region,

the methoxyl C (57 ppm) and carbohydrate C (73 ppm) peaks were much smaller in the

B horizons. The disappearance with depth of the methoxyl C peak and that of C-

substituted aromatic C at 130 ppm also indicated the degradation of lignin in the mineral

soil.

Fig. 6. Distribution of C in humin based on 13C NMR analysis. Error bars represent standard errors of the means.


In all spectra, the alkyl C and O-alkyl C peaks dominated over the aromatic and

carbonyl C peaks (Fig. 8). Integration of the major regions of 13C resonance indicated

that the major C components in whole soil organic matter were alkyl (39–49% of

total C) and O-alkyl C (32–45% of total C). Together, alkyl C and O-alkyl C

accounted for as much as 82% of C in Hubbard Brook soils. The contributions from

aromatic C (10–15% of total C) and carbonyl C (5–7% of total C) were much

smaller in whole soils than in the isolated fractions (Figs. 4 and 6). These ranges are

consistent with the ranges for forest soils compiled by Mahieu et al. (1999). Signal

intensities for alkyl C increased with depth from 39% in the Oa horizon to 49% in

mineral soils. Aromatic C increased from 10% (Oa) to 14% (Bs1), while O-alkyl C

decreased from 45% in the Oa horizon to 32% in the Bs1 horizon (Fig. 8). Similar

patterns of C distribution have been reported for whole soils from a nearby watershed

(W1) at Hubbard Brook (Dai et al., 2001).

Fig. 7. Solid-state 13C NMR spectra for whole soils at the Hubbard Brook Experimental Forest.


3.4. Elemental composition of humic substances

The average elemental composition of the humic fractions is shown in Table 2. Humic

acids had significantly higher ( pV 0.001) C concentration than fulvic acids. Neither

elevation nor soil depth showed significant influence on C concentrations of humic acids.

In fulvic acid, however, slightly lower C concentrations were observed in Oa and E

horizons than in fulvic acid extracted from lower mineral horizons (Table 2). The carbon

concentration in humic and fulvic acid ranged from 488 to 617 and from 477 to 543 g

kg� 1, respectively. The mean and ranges of C concentration were generally consistent

with previously reported ranges for humic acids (Steelink, 1985; Rice and MacCarthy,

1991). However, the mean for fulvic acid was somewhat higher than the mean for soil

fulvic acid reported by Rice and MacCarthy (1991)—453 g kg� 1. This was probably due

to the isolation and purification procedure used in this study. In our procedure, the

polysaccharide fraction (which is 40% C) was isolated from the fulvic acid fraction. In

most other studies, polysaccharides have not been isolated from the fulvic acid fraction.

Removing polysaccharides, therefore, resulted in higher C concentration in the fulvic acid.

Fig. 8. Distribution of C in whole soils based on 13C NMR analysis. Error bars represent standard errors of whole

soil means.


Humic acid contained significantly higher N concentration ( pV 0.001) and had

significantly lower C:N ratio than fulvic acid. Humic acid extracted from the Bh and

Bs1 horizon contained greater N concentration. In contrast, fulvic acids extracted from Oa

and E horizons had greater concentration of N, and N concentration decreased with

increasing soil depth in lower mineral soils (Table 2). The concentration of N ranged from

23 to 43 and from 10 to 22 g kg� 1 for humic and fulvic acids, respectively. The C:N ratio

ranged from 18.3 to 26.2 and from 27.3 to 57.2 for humic and fulvic acids, respectively

(Table 2). The average nitrogen concentration of fulvic acid (14.4 g kg� 1) is lower than

previously reported N concentrations of soil fulvic acids (26 g kg� 1; Rice and MacCarthy,

1991).

Neither soil depth nor elevation/species showed any significant influence on H

concentration in humic acid. However, fulvic acid extracted from the Oa and E horizons

had greater H concentration and higher H:C ratio, suggesting that fulvic acids from the Oa

and E horizons are generally more aliphatic than those in lower mineral horizons.

The O concentration and O:C ratios in fulvic acids were significantly higher than in

humic acids. Neither elevation nor soil depth showed significant influence on O

concentration. The oxygen concentration ranged from 293 to 441 and from 403 to 481

g kg� 1 for humic acids and fulvic acids, respectively. The molar O:C atomic ratios ranged

from 0.36 to 0.68 and from 0.56 to 0.76 for humic and fulvic acids, respectively. The mean

Table 2

Average elemental concentration and selected molar ratios of purified soil humic substance fractions at the

Hubbard Brook Experimental Forest, New Hampshire

Horizon Subcatchment C (g kg� 1) N (g kg� 1) H (g kg� 1) O (g kg� 1) H:C O:C C:N

Humic acid

Oa All 579 29.8 47.0 344 0.97 0.45 23.0

E All 594 27.3 59.4 320 1.19 0.41 25.8

Bh All 577 36.9** 51.5 332 1.06 0.43 18.9**

Bs1 All 574 35.8** 49.2 341 1.02 0.45 19.0**

Bs2 All 573 30.8 55.5 340 1.15 0.45 22.2

All SF 576 29.0* 52.2 351 1.10 0.45 22.2*

All HH 583 33.4 52.8 348 1.10 0.43 20.7

All LH 580 32.0 49.0 364 1.01 0.45 20.6

All All 578 31.6 51.5 354 1.06 0.44 21.3

Fulvic acid

Oa All 503 16.1** 44.0** 437 1.04 0.65 37.1

E All 506 18.4** 39.0** 436 0.92 0.65 33.0

Bh All 512 14.4 35.2 439 0.82 0.65 41.9

Bs1 All 514 12.6 34.6 439 0.80 0.64 48.3

Bs2 All 513 11.7 34.9 441 0.90 0.65 52.2

All SF 509 14.1 38.7 438 0.91 0.65 44.6

All HH 503 15.3 36.9 439 0.88 0.67 39.5

All LH 509 14.1 37.8 445 0.88 0.65 43.8

All All 507 14.4 37.9 440 0.89 0.65 42.9

Concentrations are expressed on an ash-free basis.

*Significantly different ( pV 0.05) from HH and LH values.

**Significantly different ( pV 0.05) from the other horizons.


and ranges for O and O:C are consistent with reported values for soil humic and fulvic

acids (Steelink, 1985; Rice and MacCarthy, 1991).

3.5. Acidic functional groups

The concentrations of acidic functional groups in humic acid and fulvic acid,

determined by calcium acetate and barium hydroxide extraction, are presented in Table

3. In humic acids, total, carboxylic, and phenolic functional groups ranged from 393 to

895, from 238 to 402, and from 93 to 547 cmolc kg� 1, respectively. In fulvic acids, total,

carboxylic, and phenolic acidity ranged from 690 to 1053, from 503 to 784, and from 99 to

411 cmolc kg� 1, respectively. These ranges are consistent with ranges reported by

Schnitzer (1977) for cool acid soils. Generally, fulvic acids contained more acidic

functional groups than humic acids. On average, carboxylic functional groups accounted

for nearly 80% of the total acidic functional groups in fulvic acid, while in humic acid,

carboxylic acidity accounted for only 52% of total acidic functional groups. Overall,

humic acid contained significantly higher phenolic acidity than fulvic acid (Table 3).

Humic acids extracted from SFB soils had significantly higher concentrations of phenolic

functional groups than that from the HH and LH zones, while fulvic acid from the HH and

LH had higher carboxylic acidity than in the SFB subcatchment. Humic acid and fulvic

acid from the Oa and E horizons had significantly lower acidic functional group

concentrations than in lower mineral horizons.

4. Discussion

The composition of soil organic matter is controlled primarily by three factors: (1) the

chemical composition of the carbon inputs; (2) the nature and magnitude of decomposition

processes; and (3) solubilization and transport of organic matter within the profile. The

Table 3

Average concentrations of acidic functional groups in soil humic acid (HA) and fulvic acid (FA) at the Hubbard

Brook Experimental Forest, New Hampshire, determined by calcium acetate and barium hydroxide extraction

Horizon Elevation Total acidity COOH Phenolic

zoneHA

(cmol kg� 1)

FA

(cmol kg� 1)

HA

(cmol kg� 1)

FA

(cmol kg� 1)

HA

(cmol kg� 1)

FA

(cmol kg� 1)

Oa All 591 783 306 611 285 172

E All 505 895 292 641 212 254

Bh All 675* 928* 318 748* 358* 177

Bs1 All 779* 964* 341 758* 438* 206

Bs2 All 537 911 349 745* 188 166

All SFB 688** 959** 337 697 351** 261**

All HH 551 874 334 733 217 140

All LH 582 922 280 716 302 206

All All 611 904 316 705 296 199

*Significantly greater than Oa horizon ( p= 0.05).

**Significantly greater than HH and LH ( p= 0.05).


decomposition process may be controlled by the chemical composition of C inputs and the

type of microbial organisms, while the magnitude of decomposition and transport

processes will mainly be regulated by environmental conditions such as temperature,

moisture, pH, and redox potential.

4.1. Variation in organic matter chemistry with depth

Degradation of organic matter in the environment is a continuous process that

gradually and selectively modifies the chemical character of soils. Ultimately, organic

matter in soil reaches a dynamic equilibrium if conditions remain unchanged. As

decomposition proceeds, less reactive structures become more dominant in soil organic

matter. In Spodosols, translocation of organic matter from O horizons to B horizons

results in the accumulation of highly decomposed and relatively soluble organic matter

in mineral soils. The concentration patterns of the different fractions of humic

substances largely reflected the solubility properties of the fractions. Decomposition

processes in the forest floor result in the release of organic acids, which lowers the pH

of the Oa horizon. This tends to immobilize humic acid, causing it to accumulate in the

O horizon and precipitate earlier than fulvic acid (Fig. 2). In contrast, fulvic acid

remains soluble and tends to move downward with the percolating water, accumulating

in the lower horizons.

Results of other decomposition studies, especially those involving NMR, suggest that

aliphatic (alkyl C) structures are more recalcitrant, therefore increasing in abundance

relative to other fractions as decomposition proceeds (Wilson et al., 1983; Norden and

Berg, 1990; Baldock and Preston, 1995; Gressel et al., 1996; Dai et al., 2001). In our

study, alkyl C increased, while O-alkyl C decreased, with increasing depth in the soil

profile for whole soils, humic acid, and humin (Figs. 4, 6, and 8). Similar results have been

reported by Gressel et al. (1996), who observed increasing alkyl C and decreasing O-alkyl

C with depth from the Oi to A horizon in both whole soils and NaOH extracts. The

increase in alkyl C and decrease in O alkyl C with depth in whole soils are also consistent

with the findings of Preston et al. (1994), who observed similar patterns with depth in

organic horizons (L, F, H) underlying a Douglas-fir stand. Hatcher et al. (1983) suggested

that the increase in the proportion of alkyl C as decomposition proceeds is the result of the

utilization of easily decomposable carbohydrates by soil microbes, and the selective

preservation of more recalcitrant alkyl C associated with original plant biopolymers such

as cutin, suberin, and waxes. The decrease we observed in polysaccharide in the lower

mineral horizons (Fig. 2) is consistent with this hypothesis. In addition, microorganisms

are known to synthesize alkyl C structures as metabolic products of decomposition

(Harvey et al., 1989; Baldock et al., 1990). The transformation of these synthesized

structures and the residual organic matter to more recalcitrant forms may contribute to the

increase in alkyl C content during decomposition.

The decrease in O-alkyl C with soil depth can be attributed to preferential biological

degradation of carbohydrates, which is the principal component of the O-alkyl C fraction

(Kogel-Knabner, 1992, 1993). Baldock and Preston (1995) suggested using the ratio of

alkyl C to O-alkyl C (R) as an index of the degree of decomposition. In whole soils, humic

acid, and humin, R increased significantly from the Oa to Bh horizon, but not between the


Bh and Bs1 horizons (Fig. 9). In fulvic acids, there were no significant variations in this

decomposition index (Fig. 9).

The abundance of aromatic C may increase or decrease during decomposition,

depending on the interaction of plant composition, microbial community, and climatic

conditions (Baldock and Preston, 1995). The aromatic C fraction in whole soils and

humin at Hubbard Brook increased from 10% and 15% in the Oa horizon to 14% and

25% in the Bs1 horizon, respectively (Figs. 6 and 8), while in the humic and fulvic

acids, there were few significant variations (Fig. 4). These results suggest that

aromatics were preferentially preserved relative to other organic C forms, although

changes in structure occurred with increasing depth. Furthermore, accumulation of

aromatic C is occurring primarily in humin, the least soluble organic matter pool in the

soil. The increase in aromatic C with depth is consistent with the findings of Gressel et

al. (1996), who observed increases in aromatic C with depth from Oi to A horizons in

a forest soil in California. Increases in aromatic C with the extent of decomposition

have also been reported in litterbag decomposition studies (Wilson et al., 1983; Norden

and Berg, 1990). Increases in aromatic C have been observed in forest environments in

which bacteria and/or brown rot fungi dominate the decomposer community (Baldock

and Preston, 1995). In these systems, lignin decomposition is slow, resulting in

accumulation of aromatic C.

Fig. 9. Alkyl C/O-alkyl C ratio (R) of organic matter in fulvic acid (FA), humic acid (HA), humin (HN), and

whole soils (WSL). Error bars represent standard errors of the means.


4.2. Variation in organic matter chemistry by species/landscape position

In order to evaluate the influence of species composition and/or landscape position on

the chemical and spectroscopic properties of soil organic matter at the HBEF, the samples

were grouped into three subcatchments: spruce–fir birch (SFB), high elevation hardwoods

(HH), and lower elevation hardwoods (LH). We were particularly interested in the Oa

horizon in each subcatchment, since organic matter found in the Oa horizon is relatively

less decomposed compared to organic matter in the lower mineral horizons. At the HBEF,

it is difficult to separate species effects from landscape position because spruce and fir are

found almost exclusively at higher elevations.

Our results indicated that species and/or elevation did not significantly affect the

concentrations of the humic substances extracted (Fig. 2). Results of the elemental analysis

of humic and fulvic acids showed lower N concentrations and greater C:N ratio for both

humic and fulvic acids extracted from the SFB subcatchment (Table 2), suggesting that

humic and fulvic acid extracted from SFB subcatchment are slightly depleted in N. This is

consistent with lower N concentrations in spruce–fir needle litter compared to broadleaf

litter (Fyles and Fyles, 1993; Prescott et al., 2000). Humic and fulvic acid extracted from

SFB soils had more phenolic functional groups and were generally more acidic than

hardwood soils (Table 3).

The average spectroscopic properties of organic matter and humic substances in each

subcatchment are presented in Table 4. Overall, in all subcatchments, a comparison of Oa

Table 4

Percentage distribution of C in humic substances and whole soils in different subcatchments at the HBEF based

on solid-state CPMAS 13C NMR analysis

Subcatchment Alkyl C O-alkyl C Aromatic C Carbonyl C R

Oa Mineral

soil

Oa Mineral

soil

Oa Mineral

soil

Oa Mineral

soil

Oa Mineral

soil

Whole soils

SF 44 45 44 34 8 15 4 6 1.0 1.4

HH 41 48 44 33 10 14 6 6 0.9 1.5

LH 33 50 48 34 13 11 6 6 0.7 1.5

Humin

SF 35 36 35 31 19 26 11 7 1.0 1.2

LH 30 44 41 29 17 21 12 6 0.7 1.5

Humic acid

SF 54 55 21 20 15 13 10 12 2.5 2.9

HH 44 51 27 21 15 13 14 15 1.6 2.4

LH 46 56 28 20 16 13 11 11 1.6 2.7

Fulvic acid

SF 37 34 28 29 17 16 19 22 1.3 1.2

HH 38 36 29 28 17 16 16 20 1.3 1.3

LH 34 30 31 32 18 15 17 23 1.1 0.9


horizon and mineral horizon spectroscopic properties indicates that organic matter in

mineral horizons is more decomposed and more humified than in the Oa horizon. In whole

soils, humin, and humic acid, alkyl C was always lower and O-alkyl C was always greater

in Oa horizons than in the mineral soils. Hence, the index of decomposition R was up to

twice as large in mineral soils as in Oa horizons in all subcatchments (Table 4). In contrast,

fulvic acid showed almost no variation in composition between Oa and mineral horizons.

This absence of variation in fulvic acid composition is consistent with its highly mobile

character.

There were some notable differences in C composition among the subcatchments

(Table 4). For example, whole soils, humin, and humic acid in Oa horizons of the SFB

subcatchment had significantly higher alkyl C content, and lower O-alkyl C and aromatic

C contents than Oa horizon in the LH subcatchment. This may reflect a greater degree of

decomposition of organic matter in SFB Oa horizon as reflected in the higher value of R

(Table 4). The Oa horizon in the SFB subcatchment is significantly thicker and more

massive than in the hardwood subcatchments (Johnson et al., 2000), which is consistent

with the accumulation of highly decomposed organic matter. In contrast, whole soils,

humin, and humic acid from SFB mineral soils all contained less alkyl C and more

aromatic C than in hardwood mineral soils (Table 4). Wilson et al. (1983) monitored the

decomposition of beech leaves and pine needles for 2 years and found an increase with age

in the aromatic C in pine needles, while in beech leaves, only alkyl C increased with age.

The lack of accumulation of aromatic C in beech leaves indicated that beech lignin was

more susceptible to decomposition than pine lignin. Our observation of lower aromatic C

in the SFB Oa horizons and greater aromatic C in SFB mineral soils suggests that

decomposition in the forest floor of the coniferous SFB zone results in the mobilization of

more aromatic organic matter, which is then accumulated in mineral horizons.

4.3. Chemistry of humic substance fractions vs. whole soils

In soils and humic substance fractions, alkyl C and O-alkyl C were the most abundant

organic C fractions, followed by aromatic C and carbonyl C. Despite their general

similarities, there were important differences in composition among the humic fractions.

Humic and fulvic acids were considerably richer in carbonyl C than whole soils and humin

(Table 4), presumably reflecting a high content of carboxylic functional groups. The

abundance of highly polar carboxyl groups is largely responsible for their relatively high

solubility, compared to humin. The distribution of chemical structures in the humic and

fulvic acids is not representative of the whole soils (Table 4, Figs. 4 and 8). In addition to

greater carboxyl C, humic and fulvic acids have generally lower O-alkyl C content than

whole soils. The humin fraction is more aromatic than whole soils, but otherwise exhibits a

C distribution pattern similar to the whole soil samples. The greater aromatic C content of

humin may reflect the recalcitrant nature of aromatic structures—lignin in particular—in

soil organic matter.

Humic acid exhibited higher C, N, H, and H/C, and lower O, O/C, and C/N

compared to fulvic acids (Table 2). The higher H/C ratio in humic acids indicates that

soil humic acids at HBEF are more aliphatic than fulvic acid. This is further supported

by the higher alkyl C content in humic acid (Table 4, Fig. 4). This is contrary to the


findings of Rice and MacCarthy (1991), who suggested that soil fulvic acid is, in

general, more aliphatic than soil humic acid. This may be partly due to the procedure

used for the isolation of fulvic acid in this study, in which polysaccharides and other

neutral, low-molecular-weight compounds were isolated from the fulvic acid fraction.

Since polysaccharides contain high concentrations of H, they will tend to increase the H/

C atomic ratio if they are not isolated, making the fulvic acid fraction appear more

aliphatic. The polysaccharides isolated in this study had an average H concentration of

63.5 g kg� 1 and an H/C ratio of approximately 2 (data not shown), significantly greater

than both humic and fulvic acid.

The lower O content and O/C ratio in humic acid are a reflection of the lower

concentrations of carboxyl functional groups compared to fulvic acid (Table 3). This is

also consistent with the 13C NMR results, which indicate a lower carbonyl C content in

humic acid (Fig. 4). The lower carboxyl group content in humic acid relative to fulvic acid

has been found in soils throughout the world (Stevenson, 1994), and is consistent with

humic acid’s lower solubility.

Higher N contents and lower C/N ratios in humic acid probably reflect the presence of

protein or peptide fragments (Stevenson, 1994). This is supported by strong signals around

chemical shifts 50–60 ppm for humic acid (Fig. 3), which partly arise from amino acid

(Schnitzer and Preston, 1986). The C/N ratio of humic acid is similar to the C/N ratio

found in soils at HBEF (19.4–23.9; Johnson, 1995). Therefore, humic acid has retained N

in proportion to soil organic matter, whereas fulvic acid is depleted in N.

The organic matter recovery in the extracted soils ranged from 92% to 99%

(average = 95%). Therefore, to evaluate whether the composition of humic substance

fractions could be used to predict the composition of whole soils, we estimated the

whole soil C distribution by weighting the chemical compositions of the humic

substance fractions (humic acid, fulvic acid, polysaccharides, and humin) by the fraction

concentrations (Fig. 2). On the whole, the weighted fractions were similar to the whole

soil composition (Fig. 10). In all horizons, the alkyl and O-alkyl C contents of the whole

soils were somewhat larger than those estimated from the fractions, while estimates of

aromatic and carbonyl C were lower than those estimated from fractions. The loss of

low-molecular-weight organics through the dialysis membranes during purification of the

humic fractions could account for lower alkyl and O-alkyl C in the fractionated organic

matter. One possible explanation for lower aromatic and carbonyl C in the whole soils is

that paramagnetic elements such as Fe and Mn were bonded to phenolic and carboxylic

functional groups, masking the signals of aromatic and carbonyl C. However, we treated

the Bh and Bs1 horizon samples with 2% HF prior to 13C NMR analysis in order to

minimize this effect. Alternatively, the HF pretreatment may have selectively removed

carbonyl C from the whole soils. Despite several studies, the literature is unclear

regarding preferential loss of carbonyl C during HF treatment (Skjemstad et al., 1994;

Dai and Johnson, 1999; Mathers et al., 2002).

4.4. Comparison of COOH estimated by different methods

The concentration of carboxylic functional groups in fulvic acid and humic acid was

estimated by calcium acetate, direct titration of the freeze-dried humic substances, and


calculation from 13C NMR spectra. Using the 13C NMR data, the COOH content for a

sample was calculated as:

½COOH� ¼ fCOOHfc105

12

� �ð1Þ

where [COOH] is the carboxyl group concentration [cmol kg� 1], fCOOH is the fraction of

the NMR spectrum in the carboxyl C range (160–185 ppm), and fc is the mass fraction of

C in the humic substances.

Fig. 10. Distribution of C in whole soils, and predicted weighted average of the compositions of humic substance

fractions. Error bars represent 1 S.E.


Carboxyl content determined by direct titration and 13C NMR both exhibited linear

relationships with carboxyl content determined by calcium acetate (Fig. 11). In both cases,

the correlations were high:

½COOH�titration ¼ 0:81½COOH�acetate þ 30; R2 ¼ 0:98 ð2Þ

½COOH�NMR ¼ 0:64½COOH�acetate þ 166; R2 ¼ 0:88 ð3Þ

An analysis of variance comparing the carboxylic group concentrations determined by

these methods revealed that [COOH] was not significantly different among the three

methods of determination ( p = 0.74). Agreement among the methods was generally good,

except at the highest [COOH] values where direct titration and NMR yielded lower values

than calcium acetate (Fig. 11). Incomplete removal of the reacted humic matter during

filtration could contribute to higher carboxylic acid functional groups determined by the

calcium acetate method. This effect would be greatest in the high [COOH] samples— the

fulvic acids—which are more soluble and have lower molecular weight and are, therefore,

more difficult to remove completely by filtration. The general agreement between carboxyl

content estimated by NMR and titration indicates that solid-state 13C NMR can be used to

estimate carboxyl C in soils or humic substances. This is somewhat surprising because (1)

Fig. 11. Carboxyl (COOH) functional group concentration determined by direct titration and 13C NMR, plotted

against COOH concentration measured by the calcium acetate method.


moieties other than COOH can contribute to resonance observed in the carbonyl C region

of the 13C NMR spectra; and (2) very strong acid COOH groups (pKa < 2) would not be

titrated with the method we used. The agreement between NMR and titration suggests that

these factors are not significant, at least in Hubbard Brook soils.

5. Conclusions

Alkyl and O-alkyl C are the dominant C constituents in both whole soils and humic

substances at the Hubbard Brook Experimental Forest, New Hampshire. Both chemical

and spectroscopic analyses revealed that organic matter chemistry varied both spatially

and with depth in the soil profile. Decomposition of organic matter was characterized by

increasing alkyl and aromatic C, and decreasing O-alkyl C (carbohydrate C) with

increasing depth in the soil. This trend was observed in whole soils, humin, and humic

acid fractions. Fulvic acid differed from the other fractions, exhibiting increasing carbonyl

C with depth while O-alkyl and aromatic C did not change. The behavior of fulvic acid

was consistent with its greater mobility and solubility. Humin was somewhat more

aromatic, but otherwise similar in composition to whole soils.

Chemical and spectroscopic methods revealed differences in the composition of organic

matter and humic substances in different subcatchments. Humic and fulvic acids extracted

from the SFB subcatchment had higher total and phenolic acidity, lower N concentration,

and a higher C/N ratio. Organic matter from SFB Oa horizons contains less O-alkyl and

aromatic C and more alkyl C than Oa horizons in hardwood areas, perhaps reflecting a

greater degree of decomposition of coniferous litter.

The structural composition of soil organic matter, estimated by a weighted average of

the compositions of humic substances, was generally comparable to the composition

determined on whole soils. Therefore, it should be possible to infer changes in soil organic

matter from changes in the chemical composition of humic substances.

Acknowledgements

We thank Dave Kiemle of the State University of New York College of Environmental

Science and Forestry for performing the 13C NMR analyses. We are grateful to K’o Dai for

his help and advice. This work was supported by grants from the USDA-NRI Competitive

Grants Program andNSF Long-Term Ecological Research Program. This is a contribution of

the Hubbard Brook Ecosystem Study. The Hubbard Brook Experimental Forest is operated

by the USDA Forest Service Northeast Experiment Station (Newtown Square, PA).

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characterization of organic matter in a northern hardwood forest soil by 13c nmr spectroscopy and...

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