ORIGINAL ARTICLE
Chemically stabilized soil organic carbon fractions in a reclaimedminesoil chronosequence: implications for soil carbonsequestration
Sriroop Chaudhuri • Louis M. McDonald •
Eugenia M. Pena-Yewtukhiw • Jeff Skousen •
Mimi Roy
Received: 21 March 2012 / Accepted: 15 January 2013
� Springer-Verlag Berlin Heidelberg 2013
Abstract With adoption of appropriate reclamation
strategies, minesoils can sequester significant amount of
soil organic carbon (SOC). The objective of this study was
to isolate different SOC fractions and coal-C in a reclaimed
minesoil chronosequence and assess effects of increasing
time since reclamation on each SOC fraction and selected
soil properties. The chronosequence was comprised of four
minesoils with time since reclamation ranging between 2
and 22 years. Total SOC (TSOC, summation of all SOC
fractions), ranged between 20 and 8 g kg-1, respectively,
at the oldest (Mylan Park) and youngest (WVO1) minesite,
indicating increasing SOC sequestration along the chron-
osequence. The humin fraction accounted for about 43 and
7 % of TSOC, respectively, at Mylan Park and WVO1,
indicating increasing humification and biochemical stabil-
ization of SOC with increasing time since reclamation. At
WVO1,[60 % of TSOC was apportioned among the acid-
hydrolysable (labile) and mineral-bound SOC fractions.
Total soil carbon (TSC, TSOC ? coal-C) were signifi-
cantly (p \ 0.05) related to the humin fraction in older
minesoils, whereas with the acid-hydrolysable (labile)
fraction in the younger minesoils indicating that C stabil-
ization mechanisms differed substantially along the
chronosequence. Coal-C was unrelated to any SOC fraction
at all minesites indicating that SOC sequestration estima-
tions in this chronosequence was unaffected by coal-C. Soil
cation exchange capacity and electrical conductivity were
significantly (p \ 0.05) related to the humin fraction at
Mylan Park while to the acid-hydrolysable and mineral-
bound SOC fractions at WVO1 indicating that the relative
influences of different SOC fractions on soil quality indi-
cators differed substantially along the chronosequence.
Keywords Soil organic carbon sequestration � Reclaimed
minesoil � Humin � Coal � Sequential fractionation � Cation
exchange capacity
Abbreviations
AHSOC 65 % HNO3 Hydrolysable soil organic carbon
ARSC Residual soil carbon after 65 % HNO3
hydrolysis
BHSOC Base (0.5 M NaOH) hydrolysable soil organic
carbon
CEC Cation exchange capacity
EC Electrical conductivity
FBHSOC Final base (0.5 M NaOH) hydrolysable soil
organic carbon
FBRSC Residual soil carbon after final base hydrolysis
HFSOC 10 % HF Hydrolysable soil organic carbon
HFRSC Residual soil carbon after 10 % HF hydrolysis
RSC Residual soil carbon after each step of
sequential fractionation
SOC Soil organic carbon
SOM Soil organic matter
TSC Total soil carbon
TSOC Total soil organic carbon
S. Chaudhuri (&)
Texas A&M AgriLife Research and Extension Center,
P.O. Box 1658, Vernon, TX 76385, USA
e-mail: [email protected]
L. M. McDonald � E. M. Pena-Yewtukhiw � J. Skousen
Division of Plant and Soil Sciences, West Virginia University,
1102 Agricultural Sciences Building, P.O. Box 6108,
Morgantown, WV 26506-6108, USA
M. Roy
Department of Agronomy and Soils, Auburn University,
Auburn, AL 36849, USA
123
Environ Earth Sci
DOI 10.1007/s12665-013-2256-8
Introduction
Surface mining for coal in the Appalachian region leads to
drastic perturbation of soil systems and alters ecosystem
processes (Shrestha and Lal 2007; Chatterjee et al. 2009).
Environmentally adverse consequences of mining include
degradation of hydrologic systems and loss of aquatic
habitat, destruction of soil structure, increased soil erosion,
loss of soil organic matter (SOM), stunted plant growth,
and restricted nutrient cycling (Johnson and Skousen 1995;
Lal and Ussiri 2005). However, with the adoption of
appropriate reclamation strategies, minesoils can sequester
significant amounts of soil organic carbon (SOC) and
contribute to terrestrial C sequestration efforts (Akala and
Lal 2000, 2001; Lal 2004; Shukla et al. 2004, 2005;
Shrestha and Lal 2006; Ussiri et al. 2006). Researches have
shown that within 20–50 years of reclamation, SOC can
increase by 10–50 % with substantial improvement in
overall soil quality (Shukla et al. 2004; Ussiri et al. 2006;
Shrestha and Lal 2007; Chatterjee et al. 2009). Significance
of reclaimed minesoils in terrestrial greenhouse gas miti-
gation effort was shown by a recent estimate that indicated
that US minelands can sequester up 0.50–1.00 MgC
ha-1 year-1 through reclamation which in turn could
incorporate about 1.60–3.20 TgC year-1 into the soils and
counterbalance about 5.8–11.2 Tg CO2 year-1 emerging
from coal combustion (Lal 2004). Accumulation of SOC also
has beneficiary effects on several agro-ecological and envi-
ronmental processes (Stevenson 1994; Trumbore 1997).
Soil organic carbon (SOC) sequestration ensues from
SOC stabilization via: (i) soil aggregation (Six et al. 2004),
(ii) organo-mineral complex formation (Guggenberger and
Kaiser 2003) and (iii) inherent biochemical recalcitrance of
SOC (Tan et al. 2004; Paul et al. 2006). A variety of
chemical, isotopic, and molecular techniques have been
used to elucidate SOC stabilization patterns. Chemical
methods mainly include acid hydrolytic (Paul et al. 2006;
Helfrich et al. 2007), oxidative (Eusterhues et al. 2003;
Helfrich et al. 2007), and demineralization techniques
(Eusterhues et al. 2007; Helfrich et al. 2007) to isolate
different SOC fractions. Isotopic methods (14C) distinguish
between SOC fractions based on their mean residence
times (MRT) and turnover rates (Trumbore 2000; Bruun
et al. 2005). Molecular methods rely upon the structural
and compositional differences among the organic moieties
associated with SOM (Kogel-Knabner 2000; Fontain et al.
2007; Solomon et al. 2005, 2007).
Owing to extreme biochemical heterogeneity of SOM,
fractions isolated by different extractants, however, are
considered ‘‘operationally defined’’ (Herbert and Bertsch
1995). The dimensions and characteristics of SOC fractions
vary widely depending on soil physico-chemical charac-
teristics, biological processes, geographic location, climate,
and land management practices. For example, base
hydrolysis with 0.5 M NaOH fractionation can isolate up to
80 % of SOM, with higher fractionation efficiencies gen-
erally achieved for coarser textural classes (Stevenson
1994). Helfrich et al. (2007) found that demineralization
(10 % HF) followed by oxidation (sodium hypochlorite,
NaOCl) can isolate about 40–60 % of SOC. The oxidisable
SOC fraction can account for as much as 72 % of total
SOC (Siregar et al. 2005). Lee et al. (2009) found that
organo-mineral association stabilized [70 % of total SOC
while soil aggregation stabilized about 8–17 % of SOC.
Zimmerman et al. (2007) showed that about 63–91 and
35–66 % of total SOC, respectively, was isolated by oxi-
dation with sodium hypochlorite (NaOCl) and acid
hydrolysis (6 N HCl). Although no consensus exists
regarding the amount of SOC that can be isolated by acid
hydrolyses, in general, the acid-resistant (acid non-hydro-
lysable, NHC) carbon constitutes about 30–80 % of total
SOC, depending upon soil type, texture, and land man-
agement practices (Paul et al. 2006). The authors analyzed
about 1,100 records obtained from literature and found that
NHC accounted for about 48, 56, 55, and 56 % of total
SOC, respectively, under conventional tillage, no tillage,
forests, and grassland soils. Tan et al. (2004) found that
NHC constituted about 53, 37, and 39 % of total SOC,
respectively, in forest, conventional, and no till soils. Jen-
kinson and Rayner (1977) have shown that NHC could
represent 20–50 % of total SOC in the upper horizons in
the temperate zones. The NHC fraction in agricultural soils
in the US Midwest accounted for about 35–65 % of the
total SOC and was about 1,300–1,800 years older than the
bulk soil (Leavitt et al. 1996; Paul et al. 1997, 2001).
Unlike native soils, chemical isolation studies of different
SOC fractions are relatively rare in reclaimed minesoils.
Lorenz and Lal (2007) used oxidative (disodium perox-
odisulphate, Na2S2O8) removal of carbon followed by
demineralization (10 % HF) in minesoils reclaimed to
forest and pasture ecosystem. Results indicated that the
highest amounts of both SOC fractions were found at older
minesites while the least at the youngest. However, the
study did not account for coal-C and a rigorous chrono-
sequence based approach was recommended to identify
temporal changes in the dimensions of SOC fractions.
Although reclaimed minesoils can sequester significant
amount of SOC, quantification of SOC sequestration in
these disturbed ecosystems, however, is challenged by
presence of coal-C (Rumpel et al. 1998; Dick et al. 2006).
Distinction between coal-C and SOC has been achieved by
stable isotopic (d13C) (Ussiri and Lal 2007), radiocarbon
(14C) (Rumpel et al. 2005; Ussiri and Lal 2007) and ther-
mogravimetric (Siewert 2004; Maharaj et al. 2007a, b)
methods. Such methods, however, are expensive and
require significant amount of resources (Bruun et al. 2005).
Environ Earth Sci
123
In addition, studies documenting the relative contribution
of different SOC fractions to overall soil C budget and/or
SOC dynamics and temporal changes therein are still
lacking for reclaimed minesoils. The main objective of this
study was to isolate and quantify different SOC fractions in
a reclaimed minesoil chronosequence and assess their rel-
ative effects on selected soil quality parameters. We used
relatively inexpensive and easy-to-use chemical fraction-
ation scheme to (i) isolate different SOC fractions and
characterize temporal trends along a well-established
chronosequence, (ii) quantify coal-C, and (iii) evaluate
interrelationships between different SOC fractions and
selected soil quality parameters such as soil cation
exchange capacity (CEC) and electrical conductivity (EC).
Materials and methods
Study area
A chronosequence comprising four reclaimed minesoil
namely, Mylan Park (MP), New Hill (NH), WVSK, and
WVO1, was identified in Monongalia County (39�3704500N,
79�5702200W), West Virginia. Details of the minesoil
characteristics and reclamation methods are described
elsewhere (Chaudhuri et al. 2011, 2012a). The minesoils
had similar soil forming conditions except for time since
reclamation which ranged from 2 to 22 years along the
chronosequence (Table 1). At the time of sampling mine-
soil ages were 2, 4, 5 and 22 years for WV01, WVSK, New
Hill, and Mylan Park, respectively. Initial reclamation
work at WVSK was performed in the mid-1990s. However,
in 2004, the topsoil (0–3 cm) was scraped and pushed back
to the high wall situated along the rear margin which
effectively established its time since reclamation as
4 years. The minesoils were reclaimed to mixed grass–
legume pasture ecosystem. The predominant species were
orchard grass (Dactylis glomerata), alfalfa (Medicago
sativa), red clover (Trifolium pratense), white clover
(Trifolium repens), timothy (Phleum pratense L.), tall
fescue (Festuca arundinacea), and bird’s foot trefoil (Lotus
corniculatus). The reclaimed sites were owned by the same
company and were mined and reclaimed in similar manner
(Table 1), following the protocols established by the 1977
Surface Coal Mining Reclamation Act. Minesoils were
compacted and graded to adjoining contours so as to merge
uniformly with the regional topography. The regional
geology consisted of interbedded, limy and acidic gray
shale, siltstone, sandstone, coal, limestone, and thin beds of
limy red shale. The region is characterized by temperate
climate with average winter and summer temperatures
around 0 and 22 �C respectively. Thirty year average
annual precipitation amounts to about 104 cm.
Soil sampling and analysis
Each minesite was sampled over approximately 0.5 ha area
in the early summer (May–June) of 2008. Soil cores were
collected from the upper 6 cm using site-specific irregular
grids (7 9 5 m) based on the previous knowledge of spa-
tial variability of SOC (Chaudhuri et al. 2011). A total of
64, 83, 79, and 74 soil cores were collected from WVO1,
WVSK, New Hill, and Mylan Park, respectively. Soils
were air dried for 48 h, ground and passed through\2 mm
sieve before performing chemical analysis.
Before performing the SOC fractionation method, soil
samples were treated with 1M HCl to remove inorganic
carbonates (Midwood and Boutton 1998; Harris et al. 2000;
Komada et al. 2008). In brief, 10 g of soil was shaken with
40 mL of 1M HCL for 8 h (Midwood and Boutton 1998).
Following acid treatment, the soil slurries were centrifuged
and supernatants were decanted. Soil residues were rinsed
twice with deionized water (DI), freeze-dried for 48 h, and
gently ground to pass through \2 mm sieve. Soil carbon
in the acid-treated and untreated soil was determined by
complete combustion using an elemental analyzer unit. The
difference between pre- and post-acid treatment yielded the
amount of soil inorganic carbon. Residual carbon left in
soil after inorganic carbonate removal represented organic
carbon and was subjected to sequential fractionation using
acid and base hydrolyses reaction followed by thermal
oxidation to isolate different SOC fractions.
The complete combustion was performed with LECO
CNS-2000 analyzer, a non-dispersive, infrared, micro-
computer-based facility designed to measure total carbon,
nitrogen, and sulfur in solid samples (soil, plant tissue,
fertilizers, meat products, dairy products, seeds, food, res-
ins, and environmental wastes) with a nominal sample
requirement (200 mg). The instrument has a detection limit
of 0.02 mg C with a precision of about 0.4 % relative
standard deviation (RSD). The combustion involves oxi-
dation of SOM at 950 �C, in the presence of ultra-high pure
(UHP) helium, oxygen and low-moisture compressed air.
Oxidation of SOM yields CO2 gas which is detected by an
infrared sensor and expressed as %C on a soil dry weight
basis. Before carbon analysis, 10 blanks and five desic-
cated, pure primary standards were used to determine the
calibration factor and correct for drift in SOC estimates.
Quality control tests were performed by using (1) three
replicates and (2) one each of a primary standard and blank
after 15 unknowns (soil samples).
The detail of the sequential fractionation method is
available in Ussiri and Lal (2007). In brief, 2 g of car-
bonate-free soil was subject to sequential treatment with
0.5M NaOH (1:10 soil:solution ratio; 15 h shaking; twice),
followed by 60 % HNO3 (1:10 soil:solution ratio shaking;
15 min), 10 % HF (1:10 soil:solution ratio shaking; 4 h),
Environ Earth Sci
123
0.5M NaOH (1:10 soil:solution ratio; 30 min shaking;
twice) and thermal oxidation in muffle furnace (340 �C;
3 h) (Fig. 1). Following each fractionation step, soil resi-
dues were rinsed twice with DI, freeze-dried for 48 h, and
gently ground to pass through \2 mm sieve. Residual soil
carbon (RSC) in the soil residues after each fractionation
step and after the final thermal oxidation was determined
by complete combustion as outlined earlier. Hydrolysable
soil organic carbon (HSOC) isolated at any particular
fractionation step was determined by computing the dif-
ference in the RSC between two successive steps as
follows:
1. Base-hydrolysable fraction (BHSOC) = TSC – RSC
after base hydrolysis (BRSC).
2. Acid-hydrolysable fraction (AHSOC) = BRSC – RSC
after acid hydrolysis (ARSC).
3. HF-hydrolysable fraction (HFHSOC) = ARSC –
HFRSC.
4. Final base-hydrolysable fraction (FBHSOC) =
HFRSC – FBRSC.
5. Coal-C = RSC after thermal oxidation.
Where, BHSOC = Base (0.5 M NaOH) Hydrolysable soil
organic carbon; AHSOC = 65 % HNO3 Hydrolysable soil
organic carbon; HFSOC = 10 % HF hydrolysable carbon;
FBHSOC = Final base (0.5 M NaOH) hydrolysable soil
organic carbon; TSC = Total soil carbon; RSC = Resid-
ual soil carbon after each step of sequential fractionation;
ARSC = Residual soil carbon after 65 % HNO3 hydro-
lysis; HFRSC = Residual soil carbon after 10 % HF
hydrolysis; FBRSC = Residual soil carbon after final base
hydrolysis.
Total soil organic carbon (TSOC) was determined by
adding each HSOC fraction (BHSOC ? AHSOC ?
HFHSOC ? FBHSOC). Each SOC fraction was presented
as (1) absolute concentration (g kg-1) and (2) relative
proportion (%). Relative proportions of each SOC fraction
Table 1 General characteristics of the reclaimed minesites included in the study
Minesoil characteristics WVO1 WVSK New Hill Mylan Park
Time since reclamation (years) 2 4 5 22
Coal type Waynesburg
Mining method Contour mining, front end loaders
Overburden 70–80 % Sandstone, rest is shale
Reclamation method Backfilled, 300 topsoil, graded
Post-reclamation land management practice Grass-legume pasture
Soil C (g kg-1) 11.04 ± 1.66c 11.96 ± 3.84c 17.69 ± 4.62b 21.73 ± 5.97a
Bulk density (Mg m-3) 2.01 ± 0.33a 1.34 ± 0.22c 1.27 ± 0.18c 1.47 ± 0.31b
CEC (cmolc kg-1) 2.82 ± 0.06d 4.75 ± 0.07c 7.63 ± 1.38b 11.78 ± 2.35a
EC (dS m-1) 0.05 ± 0.001a 0.04 ± 0.001b 0.04 ± 0.001b 0.03 ± 0.01c
pH 6.68 ± 0.11b 5.54 ± 0.26c 6.67 ± 0.72b 7.43 ± 0.47a
Clay (g kg-1) 17.7 ± 2.52a 27.6 ± 2.85a 16.3 ± 2.69b 15.6 ± 2.37c
Sand (g kg-1) 29.5 ± 4.63c 32.5 ± 7.56b 36.7 ± 5.27a 20.8 ± 6.86d
Samples collected 64 83 79 74
Reported values for each soil property indicate mean ± standard deviation. Same letters for each soil property indicate no significant difference
at a = 0.05
Fig. 1 Sequential fractionation scheme to isolate SOC fractions and
coal-C (BHSOC base-hydrolysable SOC fraction, AHSOC Acid-hydro-
lysable SOC fraction, HFHSOC 10 % HF-hydrolysable SOC fraction,
FBHSOC final base-hydrolysable SOC fraction; TSOC = BHSOC ?
AHSOC ? HFHSOC ? FBHSOC) (modified from Ussiri and Lal 2007)
Environ Earth Sci
123
were expressed as percentages of the particular SOC
fraction with respect to the TSOC. Total soil carbon (TSC)
for each minesoil was determined by adding the TSOC
(g kg-1) and coal-C (g kg-1).
To test the effects of the chemical extractants on coal-C
the entire fractionation scheme was applied on artificial
coal:soil (w:w) mixture containing 100, 50, 10, and 5 %
coal. No statistically significant (p \ 0.05) differences
were observed in coal-C for any of the mixtures which
indicated suitability of procedure in selectively extracting
different soil C fractions without affecting coal-C.
Soil C recovery by the fractionation scheme was
assessed for each minesoil by computing the percentage of
(TSOC ? coal-C), determined by the fractionation proce-
dure with respect to the TSC (determined on un-fraction-
ated whole soil). Soil-C recovery by this fractionation
scheme ranged between 84 and 94 % among the minesoils
(Table 2). Relatively better soil-C recovery was observed
for the older minesoils. Lowest recovery was found at
WVO1 (84 %) which probably was due lack of adequate
time to initiate pedological processes that lead to accu-
mulation of pedogenic C (SOC).
Statistical analyses were performed with SAS v 9.1.3
(SAS 1990). Individual SOC fractions were compared
between minesoils by 1-way ANOVA using the MIXED
model approach and Fisher’s least significant difference
(LSD) at 95 % confidence level. Regression analyses were
conducted to assess the influence of each SOC fraction on
TSC and soil quality parameters.
Results and discussion
Distribution of total soil carbon (TSC; g kg-1)
along the chronosequence
Average TSC ranged from about 22 (Mylan Park) to
11 g kg-1 (WVO1) and decreased significantly (p \ 0.05)
along the chronosequence following the order Mylan
Park [ New Hill [ WVSK & WVO1 (Fig. 2). Average
TSOC ranged from about 20 (Mylan Park) to 8 g kg-1
(WVO1) and followed similar trend as TSC along the
chronosequence indicating significantly (p \ 0.05) higher
SOC sequestration in the older minesoils (Fig. 2). Time is a
major soil forming factor and a key driver of all pedolog-
ical processes. Owing to their well-defined ages (time since
reclamation) reclaimed minesoils are suitable for deter-
mining the rates and directions of changes in pedological
processes, including changes in soil C and soil quality
indicators (White et al. 2005). Overall, the results indicated
a progressive SOC sequestration trend over time which
corroborated with earlier findings (Chaudhuri et al. 2012a,
b). Inorganic carbonates accounted for\4 % of TSC in the
chronosequence, without statistically significant (p \ 0.05)
differences among the minesoils which indicated that
overall soil C budget in this chronosequence was dictated
by changes in organic carbon (SOC and coal-C).
Absolute concentrations (g kg-1) of SOC fractions
along the chronosequence
Average BHSOC fractions ranged between 4.9 (Mylan
Park) and 2.3 g kg-1 (WVSK), decreasing significantly
(p \ 0.05) following the order Mylan Park [ New Hill [WVO1 & WVSK along the chronosequence (Fig. 3a).
Base hydrolysis (e.g. 0.5 M NaOH) removes the ester-
bound biopolymers enriching the residual soil organic
matter (SOM) with alkyl-C species, such as alkanols,
alkanoic acids, hydroxyalkanoic acids (Nierop et al. 2003;
Zegouagh et al. 2004; Rumpel et al. 2005). There are two
main mechanisms involved in base hydrolysis reactions,
(i) replacement of proton bridges within SOM by Na? ions,
leading to stabilization and rearrangement of organic
associations, and (ii) competition between the OH- groups
of NaOH and other anionic moieties of the SOM for
adsorption sites via pH-induced changes (Piccolo 2002).
Table 2 Descriptive statistics for total C recovery (%) along the
chronosequence
Minesoil Mean Min Max
Mylan Park (22 year) 93.04a 88.48 94.51
New Hill (5 year) 89.98ab 81.01 92.07
WVSK (4 year) 85.49b 78.10 96.69
WVO1 (2 year) 83.77b 77.54 88.24
Same letters indicate no significant difference at a = 0.05
Fig. 2 Comparison of the average ± standard deviation values of
total soil carbon (TSC, analyzed on the whole soil before sequential
fractionation) and total soil organic carbon (TSOC, sum total of all
hydrolysable SOC fractions) for each reclaimed minesoil across the
chronosequence. Same letter for TSC and TSOC indicates no
significant difference between the minesoils at a = 0.05. Years inthe parenthesis indicate time since reclamation. (MP Mylan Park,
NH New Hill)
Environ Earth Sci
123
Average acid-hydrolysable fraction (AHSOC) ranged
from 4.9 (Mylan Park) to 2.2 g kg-1 (WVSK) and decreased
significantly (p \ 0.05) along the chronosequence following
the order Mylan Park & New Hill [ WVO1 & WVSK
(Fig. 3a). Acid-hydrolysis isolates the labile SOM compo-
nents such as proteins and polysaccharides (Barriuso et al.
1987; Ostle et al. 1999), thus enriching the RSC with rela-
tively recalcitrant biomolecules such as long-chain alkyls,
waxes, lignin, and aromatic C species (Schnitzer and Preston
1983; Haile-Mariam et al. 2000; Collins et al. 2000; Kiem
et al. 2000; Paul et al. 2000, 2001, 2006). In addition, acid
hydrolysis removes polyvalent cations which forms
‘bridges’ between soil particles which leads to soil aggre-
gation and in turn protect the organic molecules from
microbial enzymatic degradation (Oades 1984). The SOC
remaining after acid-hydrolysis have been related to rela-
tively older and resistant ‘passive’ SOC fractions (Leavitt
et al. 1996; Paul et al. 1997).
Average HFHSOC fractions ranged from 1.8 to
1.2 g kg-1 with no statistically significant (p \ 0.05) dif-
ference between the minesoils (Fig. 3a). Average FBHSOC
fractions ranged between 9.2 (Mylan Park) and 0.6 g kg-1
(WVO1), decreasing significantly (p \ 0.05) following the
order Mylan Park [ New Hill [ WVSK [ WVO1 along
the chronosequence (Fig. 3a). The RSC after the final base
hydrolysis (0.5M NaOH) step constituted the humin, the
most stable and resistant SOC fraction resulting from
extensive soil humification (Ussiri and Lal 2007).
Relative proportions (%) of SOC fractions
along the chronosequence
Expressing SOC fractions as percentages of TSC eliminated
the confounding effect arising from the differences in TSC
concentrations between the minesoils and thus offered a
better insight into relative contribution of each SOC fraction
to the overall soil-C budget and SOC sequestration. Inter-
estingly, contrasting trends were apparent when the per-
centages of the SOC fractions were compared to their
respective absolute concentrations (g kg-1). For example,
the average BHSOC (%) ranged from about 31 to 22 % of
TSOC and decreased significantly (p \ 0.05) following the
order WVSK & WVO1 [ Mylan Park & New Hill along
the chronosequence which was opposite to what was
observed for the absolute concentrations of the BHSOC
(g kg-1) fraction (Fig. 3b). Within the chronosequence, the
acid-hydrolysable fraction accounted for about 41 and 24 %
of TSOC and decreased significantly (p \ 0.05) following
the order WVO1 & New Hill [ Mylan Park & WVSK
along the chronosequence (Fig. 3b). Proteins and carbohy-
drates have their origins in microbial and plant exudates
(Spielvogel et al. 2007). With increasing time since recla-
mation vegetative development and soil biological activi-
ties were rejuvenated in the minesoils leading to increased
production of amino acids, proteins, and sugars (Mummey
et al. 2002). This accounted for higher absolute concentra-
tion (g kg-1) of the labile SOM species (e.g. proteins and
polysaccharides) in the older minesoils. However, higher
relative proportions (%) of these labile fractions in younger
minesoils (e.g. WVO1) indicated that with increasing age
the relative contribution of labile fractions to the overall soil
C budget had decreased.
Although no significant (p \ 0.05) differences were
observed in the absolute concentrations (g kg-1) of the
HFHSOC fractions among the minesoils, relative propor-
tions of HFHSOC (%) revealed a decreasing trend with
increasing time since reclamation following the order
WVO1 & WVSK [ New Hill & Mylan Park (Fig. 3b).
Hydrofluoric acid (HF) selectively dissolves alluminosili-
cate minerals (clays) thereby releasing the mineral-bound
C (Dick et al. 2006; Eusterhues et al. 2007; Mikutta et al.
2007). Organo-mineral associations reduce C degradation
(Jones and Edwards 1998; Kalbitz et al. 2005) by microbial
enzymatic reactions (Kaiser and Guggenberger 2007).
Highest HFSOC (%) found at WVO1and WVSK indicated
that organo-mineral complex formation was an important
mode of SOC stabilization at the younger minesites. Higher
Fig. 3 Comparison of the average ± standard deviation values for
a individual SOC (g kg-1) fractions and b relative abundance of each
individual SOC fractions (expressed as percentages of TSOC) for
each reclaimed minesoil along the chronosequence. Same letter for
each individual TSOC fraction indicates no significant difference
between the minesoils at a = 0.05. Years in the parenthesis indicate
time since reclamation. (BHSOC base hydrolysable SOC fraction,
AHSOC acid hydrolysable SOC fraction, HFHSOC 10 % hydrofluoric
acid hydrolysable SOC fraction and FBHSOC final base hydrolysable
SOC fraction, MP Mylan Park, NH New Hill)
Environ Earth Sci
123
mineral-bound SOC (%) at WVSK and WVO1 probably
resulted from significantly (p \ 0.05) higher clay contents
at WVO1 and WVSK compared to the older minesoils
(Mylan Park and New Hill) (Table 1).
Relative proportions (%) of the humin fraction (FBH-
SOC %) accounted for about 44 (Mylan Park) and 7 %
(WVO1) of TSOC and followed similar trend as that of
FBHSOC (g kg-1) (Fig. 3b). Humin represents a complex
biochemical assemblage, remaining after various phases of
C decomposition, constituting the most stable SOC frac-
tions (Almendros et al. 1996; Augris et al. 1998; Piccolo
et al. 2004). The presence of significantly (p \ 0.05) higher
humin fraction at Mylan Park indicated that with increasing
time since reclamation biochemical stabilization of SOC
became a key mode of soil C preservation and sequestra-
tion in this minesoil chronosequence. This was in agree-
ment with the previous molecular investigations performed
in this minesoil chronosequence which indicated signifi-
cantly (p \ 0.05) higher degree of SOC-humification at
Mylan Park, characterized by presence of more polycon-
densed and polysubstituted aromatic-C species probably
imparting additional biochemical stability to the organic
molecules (Chaudhuri et al. 2012b).
Distribution of coal-C along the chronosequence
The average concentration (g kg-1) of coal-C ranged from
1.76 to 0.6 g kg-1 along the chronosequence with no sta-
tistically significant (p \ 0.05) differences observed
between the minesoils (Table 3). Higher coal-C at WVSK
probably resulted from the recent soil-scraping activities
that exposed the subsoil having more coal fragments.
Although no significant (p \ 0.05) differences were
observed in the absolute coal-C concentrations (g kg-1)
among the minesoils, relative proportions of coal-C (%),
however, differed significantly (p \ 0.05) along the
chronosequence. The highest and lowest coal-C (%) were
observed at Mylan Park (2.5 %) and WVO1 (14 %),
respectively, which suggested negligible contribution of
coal-C to overall soil C dynamics in this chronosequence
except at the youngest site (Table 2). In accordance with
this finding, no significant (p \ 0.05) differences were
observed between TSC (g kg-1) and TSOC (g kg-1) at any
minesite, except for WVO1, which indicated that TSC can
be considered a suitable surrogate for TSOC in this
chronosequence. This was in agreement with Ussiri and Lal
(2007) who noted that contribution of coal-C is negligible
in the surface horizons (*10 cm depth) where appropriate
reclamation strategies, such as topsoil application (similar
to this chronosequence), have been implemented. Differ-
ences in TSC and TSOC at WVOI probably resulted due to
more recent disturbances and shorter time since reclama-
tion as compared to the older minesites, inadequate to
initiate pedological and biological processes required for
soil profile development and SOC accumulation.
Interrelationship between TSC and SOC fractions
Significant (p \ 0.05) positive relationships were found
between TSC and the humin fractions (FBHSOC, g kg-1)
in all minesoils. However, the r2 values decreased from the
oldest to the youngest minesoil underscoring the varying
degree of influence of humin on overall soil-C budget
(Table 4). The highest r2 at Mylan Park indicated that as
minesoils aged, humification processes began to set in,
imparting biochemical resistance to the SOC mole-
cules from degradation leading to soil C stabilization and
long-term sequestration. In contrast, highest r2 values at
WVO1 were found between TSC and acid-hydrolysable
and mineral-bound SOC fractions, which indicated that soil
C-stabilization mechanisms in younger minesoils differed
substantially from the older minesoils (e.g. Mylan Park). At
younger minesites SOC stabilization mainly resulted from
formation of organo-mineral complexes. Lack of any sig-
nificant (p \ 0.05) relationship (r2) between coal-C and
TSC or any SOC fraction at any minesite indicated that the
overall soil C dynamics in this minesoil chronosequence
was unaffected by coal-C.
Interrelationship between SOC fractions and soil
quality parameters
Our previous study revealed increasing CEC and decreas-
ing EC with increasing time since reclamation along the
chronosequence (Table 1) (Chaudhuri et al. 2012a). Higher
CEC and coupled with lower EC at Mylan Park had
favorable impacts on soil biological processes, such as root
network development and microbial activities, which led to
increased SOC accumulation. Higher SOC contents at
Mylan Park, and New Hill, had positively influenced CEC
Table 3 Descriptive summary for coal-C along the chronosequence
Minesoil Mean Min Max
Coal-C (g kg-1)
Mylan Park (22 year) 0.59a 0.01 1.93
New Hill (5 year) 1.76a 0.01 3.94
WVSK (4 year) 1.68a 0.36 3.19
WVO1 (2 year) 1.46a 0.02 3.05
Coal-C (%)
Mylan Park (22 year) 2.69c 0.06 6.47
New Hill (5 year) 9.22b 0.09 9.22
WVSK (4 year) 14.34a 4.80 18.94
WVO1 (2 year) 13.45a 0.01 15.85
Same letters indicate no significant difference at a = 0.05
Environ Earth Sci
123
and EC which were more influenced by the clays at WVO1
(Chaudhuri et al. 2012a). At Mylan Park and New Hill,
both CEC and EC were strongly influenced by the stable
humin fraction (Table 5). In contrast, at WVO1 soil CEC
was more related with the labile acid-hydrolysable and the
mineral-bound SOC fraction while no significant
(p \ 0.05) effects of any SOC fraction found on EC
(Table 5). The above observations indicated that different
SOC fractions had varying effects on soil quality indicators
and were time-dependent phenomenon.
Conclusion
Due to well-defined ages minesoil chronosequences are
ideal for identifying spatio-temporal changes in soil prop-
erties including SOC sequestration. Intricate mixing of
SOC with coal-C in reclaimed minesoils, however, hinders
accurate estimation of soil carbon stocks and thus carbon
sequestration potential. Methods to distinguish between
coal-C and SOC in minesoils are often expensive and
resource-intensive. In the present context, we used a
relatively inexpensive, rapid, easy-to-use yet accurate
chemi-thermal fractionation scheme to isolate coal-C in a
chronosequence comprising four minesoils which differed
only by their respective times since reclamation. The
results indicated that contribution of coal-C to overall soil
C budget was negligible. A relatively higher proportion of
coal-C, however, was observed in the younger minesoils
which probably was due to lack of adequate time to initiate
pedologic processes at younger minesites that lead to SOC
accumulation.
The fractionation scheme effectively isolated a number
of chemically stabilized SOC fractions, each having a
distinct temporal trend and varying effect on soil quality
indicators. At the oldest site (Mylan Park), the soil-C
budget was dominated by the humin fraction underscoring
the importance of humification processes on carbon sta-
bilization and overall soil-C dynamics. In contrast, at the
younger minesites the labile and mineral-bound SOC
fractions controlled the SOC dynamics. Soil CEC and EC
were significantly (p \ 0.05) influenced by the SOC frac-
tions with differing strengths (r2) and were a function of
time since reclamation. Soil CEC and EC were influenced
Table 4 Coefficient of determination (r2) between TSC, SOC frac-
tions and coal-C along the minesoil chronosequence
Minesoil TSC
(g kg-1)
BHSOC
(g kg-1)
AHSOC
(g kg-1)
HFSOC
(g kg-1)
Humin
(g kg-1)
BHSOC (g kg-1)
MP 0.66*
NH 0.57*
WVSK 0.73*
WV01 0.24
AHSOC (g kg-1)
MP 0.57* 0.44
NH 0.60* 0.31
WVSK 0.57* 0.54*
WVO1 0.76** 0.15
HFSOC (g kg-1)
MP 0.12 0.03 0.01
NH 0.46* 0.44 0.07
WVSK 0.30 0.06 0.13
WV01 0.61* 0.15 0.30
Humin (g kg-1)
MP 0.88** 0.70** 0.38 0.08
NH 0.76** 0.35 0.26 0.24
WVSK 0.66* 0.31 0.29 0.12
WVO1 0.57* 0.11 0.40 0.29
Coal-C (g kg-1)
MP 0.00 0.03 0.05 0.16 0.00
NH 0.34 0.03 0.07 0.13 0.14
WVSK 0.30 0.09 0.06 0.00 0.12
WVO1 0.01 0.34 0.00 0.07 0.04
MP Mylan Park, NH New Hill
** Significant at p \ 0.01, * p \ 0.05
Table 5 Coefficient of determination (r2) between different SOC fractions and soil properties including CEC and EC
Soil property Minesoil name BHSOC (g kg-1) AHSOC (g kg-1) HFSOC (g kg-1) Humin (g kg-1)
CEC (cmolc kg-1) Mylan Park 0.43 0.41 0.08 0.82**
New Hill 0.44* 0.39 0.21 0.73*
WVSK 0.39 0.28 0.50* 0.33
WVO1 0.20 0.54* 0.51* 0.26
EC (dS m-1) Mylan Park 0.39 0.38 0.03 0.75*
New Hill 0.41 0.32 0.09 0.53*
WVSK 0.20 0.32 0.26 0.37
WV01 0.09 0.21 0.18 0.38
** Significant at p \ 0.01, * p \ 0.05
Environ Earth Sci
123
more by the stable humin fraction at the oldest minesite,
while more so by the acid-hydrolysable (labile) and min-
eral-bound SOC fractions at the youngest site. Overall,
appreciable soil-C recovery indicated that this fractionation
procedure can aptly be used to characterize SOC dynamics
in coal-C-contaminated soils.
With emerging environmental issues emanating from
progressive accumulation of CO2 and other greenhouse
gases in the atmosphere, the approach outlined in this study
will become increasingly relevant to assess and understand
SOC dynamics in reclaimed minesoils. However, some
effort to identifying the molecular properties and turnover
times of different SOC fractions isolated at different steps
of this sequential fractionation method will provide more
insight into the overall SOC dynamics in these disturbed
terrains.
Acknowledgments The authors are thankful to the scientific con-
tribution no. 3153 from the West Virginia Agricultural and Forestry
Experiment Station, Morgantown, WV for supporting this research.
References
Akala VA, Lal R (2000) Potential for mine land reclamation for soil
organic carbon sequestration in Ohio. Land Degrad Dev
11:289–297
Akala VA, Lal R (2001) Soil organic fractions and sequestration rates
in reclaimed minesoils in Ohio. J Environ Qual 30:2090–2104
Almendros G, Guadalix ME, Gonz0alez-Vila FJ, Martin F (1996)
Preservation of aliphatic macromolecules in soil humins. Org
Geochem 24:651–659
Augris N, Balesdent J, Mariotti A, Derenne S, Largeau C (1998)
Structure and origin of insoluble and non-hydrolyzable, aliphatic
organic matter in a forest soil. Org Geochem 28:119–124
Barriuso E, Portal JM, Andreux F (1987) Kinetics and acid hydrolysis
of organic matter in humic–rich mountain soil. Can J Soil Sci
67:647–658
Bruun S, Six J, Jense LS, Paustian K (2005) Estimating turnover of
soil organic carbon fractions based on radiocarbon measure-
ments. Radiocarbon 47:99–113
Chatterjee A, Lal R, Shrestha RK, Ussiri DAN (2009) Soil carbon
pools of reclaimed minesoils under grass and forest land uses.
Land Degrad Dev 20:300–307
Chaudhuri S, Pena-Yewtukhiw EM, McDonald LM, Skousen J,
Sperow M (2011) Land use effects on sample size requirements
for soil organic carbon stock estimations. Soil Sci 176(2):110–114
Chaudhuri S, Pena-Yewtukhiw EM, McDonald LM, Skousen J,
Sperow M (2012a) Early C sequestration rate changes for
reclaimed minesoils. Soil Sci 177:443–450
Chaudhuri S, McDonald LM, Pena-Yewtukhiw EM, Roy M (2012b)
Soil organic carbon molecular properties: effects of time since
reclamation in a reclaimed mineland chronosequence. Land
Degrad Dev (accepted)
Collins HP, Elliott ET, Paustian K, Bundy LG, Dick WA, Huggins
DR, Smucker AJM, Paul EA (2000) Soil carbon fractions and
fluxes in long-term Corn Belt agroecosystems. Soil Biol
Biochem 32:157–168
Dick DC, Knicker H, Avila LG, Inda AV Jr, Giasson E, Bissani CA
(2006) Organic matter in constructed soils from a coal mining
area in southern Brazil. Org Geochem 37:1537–1545
Eusterhues K, Rumpel C, Kleber M, Kogel-Knabner I (2003)
Stabilization of soil organic matter by interactions with minerals
as revealed by mineral dissolution and oxidative degradation.
Org Geochem 34:1591–1600
Eusterhues K, Rumpel C, Kogel-Knabner I (2007) Composition and
radiocarbon age of HF-resistant soil organic matter in Podzol
and a Cambisol. Org Geochem 38:1356–1372
Fontain S, Barot S, Barre P, Bdioui N, Mary B, Rumpel C (2007)
Stability of organic carbon in deep soil layers controlled by fresh
carbon supply. Nature 450:277–281
Guggenberger G, Kaiser K (2003) Dissolved organic matter in soils.
Challenging the paradigm of sorptive preservation. Geoderma
113:293–310
Haile-Mariam S, Cheng W, Johnson DW, Ball JT, Paul EA (2000)
Use of carbon-13 and carbon-14 to measure the effects of carbon
dioxide and nitrogen fertilization on carbon dynamics in
ponderosa pine. Soil Sci Soc Am J 64:1984–1993
Harris D, Horwath WR, van Kessel C (2000) Acid fumigation of
soils to remove carbonates prior to total organic carbon or
CARBON-13 isotopic analysis. Soil Sci Soc Am J 65:1853–
1856
Helfrich M, Flessa H, Mikutta R, Dreves A, Ludwig B (2007)
Comparison of chemical fractionation methods for isolating
stable soil organic carbon fractions. European J Sol Sci 58:
1316–1329
Herbert BE, Bertsch PM (1995) Characterization of dissolved and
colloidal organic matter in soil solutions: a review. In: McFee
WW, Kelly JM (eds) Carbon forms and functions in forest soils.
Soil Sci So. Am, Madison, pp 63–88
Jenkinson DS, Rayner JH (1977) The turnover of soil organic matter
in some of the Rothamsted classic experiments. Soil Sci
123:298–305
Johnson CD, Skousen JG (1995) Minesoil properties of abandoned
mine land sites in West Virginia. J Environ Qual 24:635–643
Jones DL, Edwards AC (1998) Influence of sorption on the biological
utilization of two simple carbon structures. Soil Biol Biochem
30:1895–1902
Kaiser K, Guggenberger G (2007) Sorptive stabilization of organic
matter by microporous goethite: sorption into small pores vs.
surface complexation. Eur J Soil Sci 58:45–59
Kalbitz K, Schwesig D, Rethemeyer J, Matzner E (2005) Stabilization
of dissolved organic matter by sorption to the mineral soil. Soil
Biol Biochem 37:1319–1331
Kiem R, Knicker H, Korschens M, Kogel-Knabner I (2000)
Refractory organic carbon in C-depleted arable soils, as studied
by 13C NMR spectroscopy and carbohydrate analysis. Org
Geochem 31:655–668
Kogel-Knabner I (2000) 13C and 15N NMR spectroscopy as a tool in
soil organic matter studies. Geoderma 80:243–270
Komada T, Anderson MR, Dorfmeir CL (2008) Carbonate removal
from coastal sediments for the determination of organic carbon
and its isotopic signatures, d13C and D14C: comparison of
fumigation and direct acidification by hydrochloric acid. Limnol
Oceanogr Methods 6:242–262
Lal R (2004) Agricultural activities and global carbon cycle. Nutr
Cycl Agro-Ecosyst 70:103–116
Lal R, Ussiri D (2005) Carbon sequestration in reclaimed minesoils.
Plant Sciences Critical Review. http://www.highbeam.com/doc/
1P3-890304481.html. Accessed 10 January 2012
Leavitt SW, Follett RF, Paul EA (1996) Estimation of the slow and
fast cycling soil organic carbon fractions from 6 N HCl
hydrolysis. Radiocarbon 38:230–231
Lee SB, Lee CH, Jung KY, Park KD, Lee D, Kim PJ (2009) Changes of
soil organic carbon and its fractions in relation to soil physical
properties in a long-term fertilized paddy. Soil Till Res
104:227–232
Environ Earth Sci
123
Lorenz K, Lal R (2007) Stabilization of organic carbon in chemically
separated fractions in reclaimed coal mine soils in Ohio.
Geoderma 141:294–301
Maharaj S, Barton CD, Karathanasis TAD, Rowe HD, Rimmer SM
(2007a) Distinguishing ‘‘New’’ from ‘‘Old’’ organic carbon in
reclaimed coal mine sites using thermogravimetry: I. Methods
development. Soil Sci 172:30–292
Maharaj S, Barton CD, Karathanasis TAD, Rowe HD, Rimmer SM
(2007b) Distinguishing ‘‘New’’ from ‘‘Old’’ organic carbon in
reclaimed coal mine sites using thermogravimetry: II. Field
validation. Soil Sci 172:302–312
Midwood AJ, Boutton TW (1998) Soil carbonate decomposition by
acid has little effect on d13C of organic matter. Soil Biol
Biochem 10–11:1301–1307
Mikutta R, Mikutta C, Kalbitz K, Scheel T, Kaiser K, Jahn R (2007)
Biodegradation of forest floor organic matter bound to minerals
via different binding mechanisms. Geochim Cosmochim Act
71:2569–2590
Mummey DL, Stahl PD, Buyer JS (2002) Soil microbiological
properties 20 years after surface mine reclamation: spatial
analysis of reclaimed and undisturbed sites. Soil Biol Biochem
34:1717–1725
Nierop GJK, Naafs DFW, Verstraten JM (2003) Occurance and
distribution of ester-bound lipids in Dutch coastal dune soils
along a pH gradient. Org Geochem 34:719–729
Oades JM (1984) Soil organic matter and structural stability: mecha-
nisms and implications for management. Plant Soil 76:319–337
Ostle NJ, Bol R, Petzke KJ, Jarvis SC (1999) Compound specific
15 N% values: amino acids in grassland and arable soils. Soil
Biol Biochem 31:1751–1755
Paul EA, Follett RF, Leavitt SW, Halvorson A, Peterson GA, Lyon
DJ (1997) Radiocarbon dating for determination of soil organic
matter fraction sizes and dynamics. Soil Sci Soc Am J
61:1058–1067
Paul EA, Morris SJ, Bohm S (2000) The determination of soil C
fraction sizes and turnover rates: biophysical fractionation and
tracers. In: Lal R, Kimble JM, Follett RF, Stewart BA (eds)
Assessment Methods for Soil Carbon. CRC Press, Boca Raton,
pp 193–206
Paul EA, Morris SJ, Bohm S (2001) The determination of soil C
fraction sizes and turnover rates: Biophysical fractionation and
tracers. In: Lal R et al (eds) Assessment Methods for Soil
Carbon. Lewis Publ, Boca Raton, pp 193–205
Paul EA, Morris SJ, Conant RT, Plante AF (2006) Does the acid
hydrolysis–incubation method measure meaningful soil organic
carbon fractions. Soil Sci Soc Am J 70:1023–1035
Piccolo A (2002) The supramolecular structure of humic substances: a
novel understanding of humus chemistry and implications in soil
science. Adv Agron 75:57–134
Piccolo A, Spaccini R, Neider R, Ritcher J (2004) Sequestration of
biologically labile organic carbon in soils by humified organic
matter. Climatic Change 67:327–343
Rumpel C, Knicker H, Kogel-Knabner I, Skjemstad JO, Huttl RF
(1998) Types and chemical composition of organic matter in
reforested lignite-rich mine soils. Geoderma 86:123–142
Rumpel C, Seraphin A, Dignac M, Michaelis W, Eusterhues K,
Kogel-Knabner I (2005) Effect of base hydrolysis on the
chemical composition of organic matter of an acid forest soil.
Org Geochem 36:239–249
SAS (1990) SAS user’s guide: Statistics, vol 2, 6th edn. SAS Inst,
Cary
Schnitzer M, Preston CM (1983) Effects of acid hydrolysis on the
C-13 NMR spectra of humic substances. Plant Soil Sci
75:201–211
Shrestha RK, Lal R (2006) Ecosystem carbon budgeting and soil
carbon sequestration in reclaimed minesoils. Environ Int
32:781–796
Shrestha RK, Lal R (2007) Soil carbon and nitrogen in 28-year-old
land uses in 10 reclaimed coal mine soils of Ohio. J Environ
Qual 36:1775–1783
Shukla MK, Lal R, Underwood J, Ebinger M (2004) Physical and
hydrological characteristics of reclaimed minesoils in southeast-
ern Ohio. Soil Sci Soc Am J 68:1352–1359
Shukla MK, Lal R, Ebinger MH (2005) Physical and chemical
properties of a minespoil eight years after reclamation in
northeastern Ohio. Soil Sci Soc Am J 69:1288–1297
Siewert C (2004) Rapid screening of soil properties using thermo-
gravimetry. Soil Sci Soc Am J 68:1656–1661
Siregar A, Kleber M, Mikutta R, Jahn R (2005) Sodium hypochlorite
oxidation reduces soil organic matter concentrations without
affecting inorganic soil constituents. European J Soil Sci
56:481–490
Six J, Bossuyt B, Degryze S, Denef K (2004) A history of research on
the link between (micro) aggregates, soil biota, and soil organic
matter dynamics. Soil Till Res 79:7–31
Solomon D, Lehmann J, Kinyangi J, Liang B, Schafer T (2005)
Carbon K-edge NEXAFS and FTIR-ATR spectroscopic inves-
tigation of organic carbon speciation in soils. Soil Sci Soc Am J
69:107–119
Solomon D, Lehmann J, Thies J, Schafer T, Liang B, Kinyangi J,
Neves E, Petersen J, Luizao F, Skjemstad J (2007) Molecular
signature and biochemical recalcitrance of organic C in Ama-
zonian dark earths. Geochim Cosmochim Ac 71:2285–2298
Spielvogel S, Prietzel J, Kogel-Knabner I (2007) Changes of lignin
phenols and neutral sugars in different soil types of a high-
elevation forest ecosystem 25 years after forest dieback. Soil
Biol Biochem 39:655–668
Stevenson FJ (1994) Humus Chemistry, 2nd edn. Wiley and Sons,
New York
Tan ZX, Lal R, Izaurralde RC, Post WM (2004) Biochemically
protected soil organic carbon at the North Appalachian exper-
imental watershed. Soil Sci 196:423–433
Trumbore S (1997) Potential response of soil organic carbon to global
climate change. Proc Natl Acad Sci 94:8284–8291
Trumbore S (2000) Age of soil organic matter and soil respiration:
radiocarbon constraints on belowground dynamics. Ecol Appl
10:399–411
Ussiri DAN, Lal R (2007) Method for determination of coal carbon in
the reclaimed minesoils contaminated with coal. Soil Sci Soc
Am J 72:231–237
Ussiri DAN, Lal R, Jacinthe PA (2006) Soil properties and carbon
sequestration in afforested pastures in reclaimed minesoils of
Ohio. Soil Sci Soc Am J 70:1797–1806
White AF, Schultz MS, Vivit DV, Blum AE, Stonestrom DA, Harden
JW (2005) Chemical weathering rates of a soil chronosequence
on granitic alluvium: III. 19 Hydrochemical evolution and
contemporary solute fluxes and rates. Geochim Cosmochim Ac
69:1975–1996
Zegouagh Y, Derene S, Dignac MF, Baruiso E, Mariotti A, Largeau C
(2004) Demineralization of a crop soil by mild hydrofluoric acid
treatment: influence on organic matter composition and pyroly-
sis. J Anal Appl Pyrol 71:119–135
Zimmerman M, Leifield J, Abiven A, Schimdt MWI, Fuhrer J (2007)
Sodium hypochlorite separates an older soil organic matter
fraction than acid hydrolysis. Geoderma 139:171–179
Environ Earth Sci
123
