Biochemical changes across a carbon saturation gradient: Lignin, cutin, and

suberin decomposition and stabilization in fractionated carbon pools

Elizabeth M. Carrington
a,
*
, Peter J. Hernes
b
, Rachael Y. Dyda
b
, Alain F. Plante
c
, Johan Six
a
a
Department of Plant Sciences, University of California, Davis, CA 95616, USA
b
Department of Land, Air, and Water Resources, University of California, Davis, CA 95616, USA
c
Department of Earth and Environmental Sciences, University of Pennsylvania, Philadelphia, PA 19104-6316, USA
a r t i c l e i n f o
Article history:
Received 25 January 2011
Received in revised form
13 December 2011
Accepted 16 December 2011
Available online 5 January 2012
Keywords:
Soil organic carbon
Carbon saturation
Carbon stabilization
Carbon biochemistry
Lignin
Cutin
Suberin
a b s t r a c t
Soils that exhibit soil organic carbon (SOC) saturationprovide an opportunity to examine mechanisms of C
storage in soils with increasingly limited C-stabilization potential. A manure rate experiment in Leth-
bridge, Alberta, in which SOC responded asymptotically to long-term manure C additions, allowed us to
assess changes in SOC biochemical composition in response to soil C saturation. By quantifying the cupric
oxide oxidation products of lignin, cutin, and suberin in fractionated SOC pools that are characterized
by chemical (i.e., mineral-associated), physical (i.e., microaggregate-associated), or no protection (i.e., free
particulate organic matter), we evaluated the interaction between C saturation and the biochemical
characteristics of SOC.
We tested the specic responses of soil fraction lignin, cutin, and suberin to C saturation level by using
the bulk soil to approximate C-input composition across manure input treatments. Carbon-normalized
lignin (lignin-VSC/OC) in the chemically protected fractions did not differ, while in the non-protected
and physically protected soil fractions, it decreased with C saturation level. Neither the stabilization of
cutin and suberin, nor the lignin:cutin suberin ratio, differed in any of the measured soil fractions in
response to C saturation level.
These results indicate that with C saturation and decreased C stabilization potential, lignin, cutin,
or suberin were not preferentially stabilized or depleted in mineral protected soil C pools. The lack of
evidence for biochemical preference in mineral associations with C saturation supports the existence of
an outer kinetic zone of organomineral associations, in which partitioning of organic compounds, rather
than sorption, controls mineral SOC accumulation at high SOC loadings. Furthermore, despite theories of
inherent lignin recalcitrance, depleted lignin concentrations with C saturation in the non-protected and
aggregate protected fractions indicate that lignin was, in this study, preferentially decomposed when not
protected by association with mineral phases in the soil. In conclusion, C-input quantity, and not quality,
combined with physical and chemical protection mechanisms that govern long-term C storage, appeared
to control C saturation and stabilization at this site.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
The maximum C stabilization potential of a soil limits the
effectiveness of soil organic carbon (SOC) storage (Six et al., 2002;
Stewart et al., 2007; West and Six, 2007). Long-term eld studies
demonstrate that increased C-input or C-input proxies, such as
bulk soil C content or elevated CO
2
, do not produce a concomitant
increase in SOC for whole soils or mineral and aggregate soil
fractions (Kool et al., 2007; Chung et al., 2008; Gulde et al., 2008;
Stewart et al., 2008). The observed asymptotic C response
under equilibrium conditions has been termed soil C saturation,
a premise that infers that inherent limits to soil C stabilization
affect the rate, duration, and permanence of SOC (Six et al., 2002;
Stewart et al., 2007; West and Six, 2007). Although process-dened
biogeochemical models, such as DayCENT, Century, and EPIC,
model linear equilibrium SOC responses to C-input (Parton et al.,
1987; Schimel et al., 1994; Izaurralde et al., 2006), C saturation
models better t soils with high SOC stocks or high input levels
(Stewart et al., 2007; West and Six, 2007). Inclusion of a saturation
parameter in biogeochemical models would, therefore, improve
estimates of the rate and duration, as well as the total potential of C
* Corresponding author. Department of Plant Sciences, 1210 Plant and Environ-
mental Sciences Building, University of California, Davis, CA 95616, USA. Tel.: 1
541 890 8458; fax: 1 530 752 5262.
E-mail address: carrington@ucdavis.edu (E.M. Carrington).
Contents lists available at SciVerse ScienceDirect
Soil Biology & Biochemistry
j ournal homepage: www. el sevi er. com/ l ocat e/ soi l bi o
0038-0717/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2011.12.024
Soil Biology & Biochemistry 47 (2012) 179e190
stabilization in terrestrial soils (Hassink and Whitmore, 1997; West
and Six, 2007).
The role of SOC biochemical composition in C saturation and
stabilization processes remains unclear, but could inform our
understanding of SOC models. It has been suggested that biochem-
ical composition of inputs, i.e., residue quality, can affect C accu-
mulation through the addition of recalcitrant macromolecules
coupled with the availability of N substrate (Melillo et al., 1982;
Vanlauwe et al., 1996; Trinsoutrot et al., 2000). Current SOC models
implicitly incorporate these theories of biochemical C protection
by correlating C stabilization in slower turning over pools to residue
quality (i.e., lignin:N ratios) (Parton et al., 1987; Palm et al., 2001).
The preferential sorption of specic compounds to soil minerals
offers another mechanism to explain how SOC biochemical
composition can affect SOC stabilization. Preferential sorption could
result from ligand exchange reactions that favor complexation of
carboxyl and phenolic groups at mineral surfaces (Guggenberger
and Kaiser, 2003). Furthermore, the organomineral association of
specic molecules can be affected by the dominance of hydrophilic
or hydrophobic interactions with distance from the mineral surface
(Kleber et al., 2007). Alternatively, SOC stabilization can be inde-
pendent of SOC biochemical composition, as common decomposi-
tion processes can override biochemical input controls (Grandy and
Neff, 2008; Crow et al., 2009; Fierer et al., 2009) or common reac-
tions with mineral surfaces can attenuate biochemical differences
(Grandy et al., 2008).
Lignin, a structural plant biopolymer dominated by aromatic
moieties, and cutin and suberin, waxy biopolymers in roots and
leaf cuticles that are dominated by aliphatic moieties, have been
considered important contributors to stable SOC pools due to innate
biochemical recalcitrance (Kgel-Knabner et al., 1992; Stevenson,
1994; Kgel-Knabner, 2002; Krull et al., 2003; Lorenz et al., 2007).
More recent studies contradict the innate biochemical recalcitrance
of lignininsoils andsuggest that its importance instable SOCpools is
overstated (Thevenot et al., 2010). Lignin was found to cycle faster
than total SOC in
13
C tracer experiments (Dignac et al., 2005; Rasse
et al., 2006; Heim and Schmidt, 2007a), had shorter residence
times than proteins based on pyrolysis isotope ratio mass spec-
trometry measurements (Gleixner et al., 1999), and did not accu-
mulate in the refractory SOC pool of C-depleted soils (Kiem and
Kgel-Knabner, 2003).
The enrichment of cutin and suberin in the free particulate
organic matter (POM) fraction of soils converted from grasslands
to woodlands suggests that the recalcitrance of cutin and suberin
contributes to the accumulation of refractory POM-C (Filley et al.,
2008). The preservation of cutin and suberin in soils has been
attributed to the biochemical recalcitrance of alkyl C in SOM(Lorenz
et al., 2007). Furthermore, cutin suberin-derived aliphatic waxes
exhibit evengreater inherent recalcitrance than lignin, as evidenced
by their relative enrichment with depth and decreasing particle
size (Riederer et al., 1993; Nierop and Verstraten, 2003; Rumpel
et al., 2004; Lorenz et al., 2007), and the greater turnover of lignin
relative to cutin suberin (Feng and Simpson, 2007).
The interaction of specic biomolecules with mineral protection
of SOC is also contradictory. Kaiser and Guggenberger (2000)
found that lignin in hydrophobic dissolved organic matter (DOM)
fractions sorbed to goethite in preference to alkyl and carbonyl C.
Mikutta et al. (2006) observed that most stable lignin occurred
in mineral fractions, but that lignin had a quantitatively small
contribution to mineral-associated SOC. NMR spectroscopy found
greater mineral protection of aliphatic structures in mineral frac-
tions, which suggests greater preservation of compounds such as
cutin and suberin relative to lignin (Feng et al., 2005). Other studies
suggest that favorable conformational changes and amphiphilicity
of proteinaceous compounds leads to preferential mineral
protection of microbially-derived, rather than plant-derived C, at
mineral surfaces (Omoike and Chorover, 2006; Sollins et al., 2006;
Kleber et al., 2007).
Studies on the selective preservation of SOC biochemical
components, such as lignin, cutin, and suberin, are often limited to
the bulk soil or particle size separates, and, therefore, do not address
the effects of specic C protection mechanisms on SOC biochemical
composition. To date, this study is the rst we know of to addi-
tionally look at lignin, cutin, and suberin in aggregated versus non-
aggregated mineral and POM fractions, as well as to observe the
effects of C saturation on the stabilization of these compounds.
To accomplish this, soil C pools were fractionated after the concep-
tual model of Six et al. (2002) to isolate measurable pools corre-
sponding to C protection mechanisms. We isolated primarily
chemical protection inthe silt clay fraction (SC); primarily physical
protection in the microaggregate-associated POM fraction (iPOM);
combined physical and chemical protection in the macroaggregated
silt clay fraction (Magg-SC); and innate biochemical protection
in the non-protected POMfraction (cPOM) (Fig. 1). By measuring the
cupric oxide oxidation products of lignin, cutin, and suberin in these
fractions, we aimed to better constrain the role of chemical, physical,
and innate biochemical protection mechanisms in the preservation
of SOC.
We utilized a C saturation gradient, established by long-term
manure additions in Lethbridge, Alberta (Gulde et al., 2008), to
address the overall hypothesis that SOC preservation in all fractions
is compound-specic and that this specicity will amplify with
increased C saturation. Specically, we expected the progressively
limited protective capacity for mineral adsorption with increasing
C saturation (Hassink, 1997; Six et al., 2002, 2004) to limit the
preservation of plant-derived lignin, cutin, and suberin in the
Fig. 1. The physical and density fractionation scheme modied fromGulde et al. (2008),
which isolated functional SOC pools utilized in this study. The easily dispersed silt clay
(SC) was directly utilized; whereas, the coarse particulate organic matter (cPOM), the
micro-within macroaggregate protected intra-aggregate particulate organic matter
(iPOM), and the macroaggregated silt clay (Magg-SC) were bulked by fractional
proportion of large and small macroaggregates. After Six et al. (2002), these functional
pools model chemically-protected SOC in the SC, physically chemically-protected SOC
in the Magg-SC, physically-protected SOC in the iPOM, and non-protected SOC in the
cPOM. Shaded fractions were not utilized in this study.
E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 180
chemically-protected SC fractions due to specic sorption mecha-
nisms that favor amphiphilic and microbial compounds (Kleber
et al., 2007). We hypothesized that the maximum potential for
aggregate formation (Puget et al., 2000; Six et al., 2002; Denef et al.,
2004) would lead to increased turnover of these compounds in the
physically-protected iPOM. Finally, we expected that these plant-
derived biomarkers would increase in the biochemically-protected
cPOM due to increased input of these components and reduced
stabilization in the other fractions with increased C saturation.
2. Materials and methods
2.1. Site description
The experimental eld is a long-term manure addition experi-
ment at the Lethbridge Research Center in Lethbridge, Alberta,
which was established on a well-drained Dark Brown Chernozem
clay loam (Typic Haplustoll) (Sommerfeldt and Chang, 1985). It has
received aged solid beef cattle manure treatments of 0, 60, 120, and
180 Mg manure ha
1
yr
1
(wet weight) since 1973. This addition
is 0e3 times Canadas recommended manure application rate
(Hao et al., 2003) and equates to additions of 0, 6.6, 12.6, and 18.2
Mg manure-C ha
1
yr
1
(Gulde et al., 2008). Treatments were
applied in a strip plot randomized complete block designwith three
replicates. The plots sampled were cultivated under conventional
tillage, were irrigated at a rate of approximately 148 mm yr
1
,
and have been cropped under barley, canola, and maize. Further
experimental details regarding the Lethbridge manure trial can be
found in Sommerfeldt and Chang (1985).
2.2. Soil fractionation and SOC measurement
The fractions analyzed in this study were a subset of those
collected by Gulde et al. (2008), which previously published
the sampling, soil characterization, soil fractionation, and SOC
measurement protocols. Briey, two cores were collected at 15 cm
for each manure treatment replicate and composited for further
analysis. Standard methods were used for bulk density and particle
size analysis. Soil fractionation procedures were carried out
according to Sixet al. (2000) andare presentedinFig. 1. This involved
using, as a rst step, the wet-sieving method of Elliott (1986) to
isolate water stable macroaggregates (large macroaggregates gt;
2000 mm and small macroaggregates 250e2000 mm), micro-
aggregates (53e250 mm), and the easily dispersed silt and clay
fraction (SC, <53 mm) from air dry soil that was previously sieved
at 8 mm. Dispersion and density otation in 1.85 g cm
3
sodium
polytungstate separated the light fraction (LF < 1.85 g cm
3
) from
the aggregate fractions. Macroaggregates were further separated
into microaggregates (53e250 mm), inter-microaggregate fractions
(i.e., coarse POM(cPOMsand >250 mm)) andmacroaggregatedsilt
and clay (Magg-SC < 53 mm) by using the microaggregate isolator
developed by Six et al. (2000). Sand was separated from cPOM
by otationin2.3 g cm
3
sodiumpolytungstate. Intra-micro-within-
macroaggregated POM (iPOM, 53e250 mm) was separated from the
silt and clay of microaggregates-within- macroaggregates (micro-
aggregate SC, <53 mm) by dispersion and sieving at 53 mm after the
ne POM was oated off by density separation (Fig. 1). All fraction
weights were calculated on a sand-free basis to account for potential
textural shifts between treatments, as applied manure contained
more clay than the native soil. Carbonate-free SOC concentrations
were used to evaluate the effects of increasing C-inputs on soil C
saturation in the bulk soil and soil fractions. For a more complete
description of soil fractionation, SOC measurement, and C saturation
evaluation see Gulde et al. (2008).
For this study, the bulk soil, cPOM, iPOM, Magg-SC, and SC
fractions isolated by Gulde et al. (2008) were selected for further
biomarker analysis. Fractions isolated from large (2000e8000 mm)
and small (250e2000 mm) macroaggregates were bulked to
represent fractions from total macroaggregates (250e8000 mm).
Fractions and bulk soils were oven-dried at 60

C for 24 h prior to
cupric oxide (CuO) oxidation.
2.3. Cupric oxide oxidation
Alkaline CuO oxidation coupled with gas chromatographyemass
spectroscopy was used to measure lignin-derived phenols and
cutin suberin- derived substituted fatty acids. Cupric oxide
oxidation was performed after Hedges and Ertel (1982), as modied
by Hernes and Benner (2002). Vanillyl (V), syringyl (S), and cinnamyl
(C) lignin-derived phenol monomers (lignin-VSC) were quantied
by gas chromatographyemass spectroscopy (GCeMS) as a proxy of
total soil lignin after Hernes and Benner, 2002 (Table 1). Selected C16
andC18 substituted fattyacidmonomers (cutinsuberin-SFA) were
identied according to Goi and Hedges (1990a,1990b) and were
measured by GCeMS as a proxy for total soil cutin suberin after
Filley et al. (2008) (Table 1).
Depending on the C content of the soil, 50e100 mg of sample
was used for CuO oxidation. Samples were added to Monel reaction
vessels with excess cupric oxide, ferrous ammonium sulfate, and
glucose (Louchouarn et al., 2000; Hernes and Benner, 2002). Reac-
tion vessels were lled with argon-sparged 8% sodium hydroxide,
and then purged with argon. Oven temperatures were ramped from
35

C to 155

C at 4

C per minute (30 min), and samples were then
oxidized at 155

C for 150 min. After transferring oxidized samples
to culture tubes and rinsing with 4% sodium hydroxide, appropriate
amounts of internal standard were added to each sample. Samples
were acidied to pH 1 with 12 N H
2
SO
4
, extracted in ethyl acetate,
and dried under N
2
gas. Samples were stored at <0

C until further
GCeMS analysis.
For GCeMS analysis, dried samples were dissolved in pyridine
and derivatized by heating at 60

C for 10 min in equal parts of N,
O Bis(trimethylsilyl)triuoroacetamide (BSTFA), a trimethylsilyl
donor. The trimethylsilyl derivatives of lignin-derived VSCs and
cutin suberin-derived SFAs were separated by gas chromatog-
raphy on an Agilent 6890 gas chromatograph, tted with a DB5-MS
capillary column (30 m, 0.25-mm inner diameter, J&W scientic).
Table 1
Chemical names and abbreviations of lignin-derived phenol monomers, by phenol
subunit grouping (V,S, or C), and cutin suberin-derived substituted fatty acids, by
C16 or C18 chain length, for the cupric oxide oxidation products measured in this
study by gas chromatographyemass spectroscopy.
Lignin-derived phenols (VSC)
Monomer group Abbreviation Phenol
Vanillyl (V) VAL vanillin
VAD vanillic acid
VON acetovanillone
Syringyl (S) SAL syringaldehyde
SAD syringic acid
SON acetosyringone
Cinnamyl (C) CAD p-coumaric acid
FAD ferulic acid
Cutin Suberin-derived Substituted Fatty Acids (SFA)
Monomer group Abbreviation SFA
C16 acids u-C16 16-hydroxyhexadecanoic acid
C16DA hexadecane-1,16-dioic acid
(x,16)DHPA 8,16-; 9,16-; &
10,16-dihydroxyhexadecanoic acid
(x)-C16DA 7 & 8-hydroxyhexadecane-1,16-dioic acid
C18 acids u-C18:1 18-hydroxyoctadec-9-enoic
C18DA:1 9-octadecene-1,18-dioic acid
9,10uC18 9,10,18-trihydroxyoctadecanoic acid
E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 181
This was coupled with an Agilent 5973 mass selective detector
to identify individual monomers based on mass fragmentation
behavior. For optimal separation of lignin-VSCs and cutin
suberin-SFAs, the GC oven was ramped from 100 to 200

C at
3

C min
1
, then ramped from 200 to 270

C at 5

C min
1
and held
at 270

C for 16 min.
Quantication and identication of lignin and cutin suberin
monomers was achieved using a selective ion monitoring scheme,
adaptedfromHernes andBenner (2002). Table 1 lists the eight lignin-
derived VSC phenols and seven cutin suberin-derived SFA mono-
mers quantied. Compounds were identied by mass fragmentation
patterns of individual monomers and published elution times
(Hedges and Ertel, 1982; Goi and Hedges, 1990a; Louchouarn et al.,
2000). For quantication, we utilized a quadratic calibration with
internal standards after Hernes and Benner (2002). Our calibrations
were tted to four points and an intercept, using standard concen-
trations of lignin-VSC phenols and cutin suberin-SFA monomers
(w5 ng mL
1
, w10 ng mL
1
, w25 ng mL
1
, and w50 ng mL
1
).
Compounds were quantied based on the relative response ratio of
each compound to the internal standard. Cinnamic acid was used as
the internal standard for lignin-VSC phenols after Hernes and Benner
(2002), and DL-12 Hydroxystearic acid was used as the internal
standard for cutin suberin-SFA monomers after Filley et al. (2008).
As commercial compound standards were not available for all SFA
monomers, proxy compound standards with similar mass fragmen-
tationbehavior (2,3 dihydroxyhexadecanoic acid; ricinoleic acid; and
9,10,16 trihydroxyhexadecanoic acid) were utilizedfor the calibration
of (x,16)DHPA & (x)-C16DA; C18DA:1 & u-C18:1; and 9,10uC18
(Table 1), respectively, after the method of Filley et al. (2008). As
target ions for proxy standards were unpublished, we veried our
quantication of these compounds by comparing cutin suberin-
SFA ratios for live oak litters, sequoia litters, loblolly pine litter, and
Dabob-Bay sediment samples to published values (Goi and Hedges,
1990a, b, c). All sample lignin-VSC and cutin suberin-SFA
measurements were corrected by the background concentrations in
the reagent blanks that were oxidized with each set of samples.
Blanks averaged w111 ng lignin-VSC and w141 ng cutin suberin-
SFA, and across samples, blanks were less than 1% of total lignin-VSC
and generally less than 3% of total cutin suberin-SFA, but never
higher than 10% in samples with the lowest SFA content.
2.4. Statistical analysis
All statistical analyses were performed after consultation with
the Department of Statistics at the University of California, Davis.
Changes in C-normalized and dry fraction weight lignin-VSC and
cutin suberin-SFA contents were tested using a mixed effects
model ANOVA for a repeated measures randomized complete block
design, where treatment was considered the main plot and fraction
considered the subplot. As soil fractions are spatially repeated
measures within the bulk soil, the bulk soil was considered the rst
measure and the soil fraction considered the second, repeated
measure. The PROC Mixed procedure in SAS 9.1.3 was employed for
the analysis, with conservative degrees of freedom for the repeated
measures, xed effects of fraction and treatment, and a random
effect of block. Signicance of simple treatment effects within each
fraction and the bulk soil were determined for mass concentrations
of SOC, lignin-VSC, and cutin suberin-SFA at the alpha 0.05 level.
Tukeys test for the least square adjusted means was performed to
group signicantly different treatments within each fraction.
As the unique biochemical C compositions of discrete soil C pools
is well documented (Guggenberger et al., 1994; Amelung et al., 1999;
Six et al., 2001), we did not focus on the differences in lignin-VSC
and cutin suberin-SFA between isolated fractions. Rather, we
assessed the differences in the stabilization of these plant-derived
compounds within each fraction with increased C saturation level
(i.e., manure treatment). Unfortunately, the nonlinear mixing of
manure and plant inputs with increasing manure input treatment
confounded estimates of the change in lignin-VSC and
cutin suberin-SFA C-input contents across treatments. For this
reason, we used the bulk soil to approximate C-input chemistry.
The rationale for using the bulk soil to approximate input
composition comes from the mathematical proof by Stewart et al.
(2008), which validates this proxy based on the convergent
behavior of discrete soil C pools (Fig. 2). In this way, we tested the
signicance of a C saturation level response within each fraction by
testing the statistical fraction treatment ANOVA interaction effect
on C-normalized lignin-VSC and cutin suberin-SFA from the bulk
soil to each measured fraction. As this analysis was performed
once per fraction, a maximum experiment-wise error rate was
established at a 0.012 for the fraction treatment interactions
using the Bonferroni correction for a 95% condence level. Further
details on statistical models used and the relevance, necessity, and
interpretation of the statistical fraction treatment interaction are
presented in the online supplementary materials.
3. Results
3.1. SOC, lignin-VSC, and cutin suberin-SFA per dry weight in bulk
soils and soil fractions across manure input treatments
Lignin-VSC and cutin suberin-SFA contents on a dry fraction
weight basis (g VSC or SFA kg
1
dry weight fraction) generally
covaried with SOC concentrations across treatments (Fig. 3). Soil
organic carbon, lignin-VSC, and cutin suberin-SFA in the bulk soil
increased signicantly (p < 0.001) and linearly with increased
manure input (Fig. 3a, f, k). In the Magg-SC and SC, these parame-
ters increased signicantly (p 0.016 and p 0.001) and asymp-
totically (Fig. 3d, e, i, j, n, o). Although both SOC and lignin-VSC per
fraction weight in the iPOM increased signicantly (p < 0.001 and
p 0.002) and asymptotically with manure input, the signicance
groupings by treatment varied for these two parameters (Fig. 3c, h).
Cutin suberin-SFA per fraction weight also increased
Fig. 2. Theorized increases in fraction C (C
f
) as a function of bulk soil organic carbon
(C
t
) content. Fraction one (C
f1
) exhibits asymptotic behavior, while fraction two (C
f2
)
exhibits saturating behavior. Fractions one and two converge to sum to the bulk soil
carbon content, which is represented by the 1:1 line. The dashed vertical line repre-
sents the theorized saturation limit for all fractions and the bulk soil. Adapted from
Stewart et al. (2008) with permission.
E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 182
asymptotically in the iPOM with manure input, yet this change was
not signicant (Fig. 3m).
Soil organic carbon concentrations in the non-protected cPOM
signicantly decreased (p 0.010) with the initial 60 Mg manure
ha
1
yr
1
treatment and then remained statistically unchanged at
higher manure inputs (Fig. 3b). Similarly, cutin suberin-SFA in the
cPOM decreased (non-signicantly) with the initial 60 Mg manure
ha
1
yr
1
treatment (Fig. 3l). In contrast, lignin-VSC cPOM concen-
trations initially increased from the 0e60 Mg manure ha
1
yr
1
treatments, then decreased signicantly (p 0.020) fromthe 60e180
Mg manure ha
1
yr
1
treatments (Fig. 3g).
Increasing manure inputs changed the proportional contribution
of each fraction to total soil mass, SOC, lignin-VSC, and cutin
suberin-SFA. The mass contribution of the cPOM increased with
manure addition from w1% of bulk soil mass to 34% (Fig. 4a). The
proportional contribution of cPOM lignin-VSC and cutin suberin-
SFA to the bulk soil increased from less than 20% in the no manure
treatment to more than 60% in the highest manure input treatment
(Fig. 4bed). The increased percent contribution of the cPOM to total
SOC, lignin-VSC, andcutinsuberin-SFAoccurreddespite decreasing
concentrations of these compounds in the cPOM with increased
manure input (Fig. 3b, g, l).
In contrast to the cPOM, the iPOM, Magg-SC, and SC all decreased
in mass percent contribution to the bulk soil with manure input
(i.e., from8 to 6%for the iPOM, 24e11%for the Magg-SC, and 9e4%for
the SC from the 0 to 180 Mg ha
1
yr
1
manure treatments (Fig. 4a)).
The SOC, lignin-VSC, and cutin suberin-SFA contribution of these
fractions to the bulk soil declined more rapidly than the total mass
contributions in physically and chemically protected soil fractions
(Fig. 4bed). This was most notable for the Magg-SC, in which SOC
declinedfrom33to 5%, lignin-VSCfrom23to 3%, andcutinsuberin-
SFA from27 to 4% fromthe 0 to 180 Mg manure ha
1
yr
1
treatments
(Fig. 4bed). The proportional contributions of the other, unmeasured
soil fractions, including the light fraction, the ne POM, and the
micro-within-macroaggregated silt and clay, were determined by
difference and exhibited non-consistent behavior across manure
treatments (Fig. 4).
3.2. Carbon-normalized lignin-VSC and cutin suberin-SFA in bulk
soils and soil fractions across manure input treatments
Carbon-normalized lignin-VSC (lignin-VSC/OC) increased
signicantly for the bulk soil from the 0 to the 60 Mg man-
ure ha
1
yr
1
treatments and for the SC from the 0 to the 120
manure Mg ha
1
yr
1
treatments (Table 2). Manure treatment had
a signicant effect on cPOM lignin-VSC/OC (p 0.039); although,
the direction of this effect was not consistent with increasing
manure input. Both the cPOM and the iPOM (although not signi-
cantly for iPOM) exhibited an initial enrichment fromthe 0 to 60 Mg
manure ha
1
yr
1
treatments and then continued depletion with
Fig. 3. Concentrations (on a dry fraction weight basis) of soil organic carbon (SOC), lignin-derived phenols (lignin-VSC), and cutin suberin-derived substituted fatty acids
(cutin suberin-SFA) for the bulk soil, coarse particulate organic matter (cPOM), micro-within macroaggregate intra-aggregate protected particulate organic matter (iPOM),
macroaggregated silt clay (Magg-SC), and the non-aggregated silt clay (SC) across manure treatments (0, 60, 120, and 180 Mg manure ha
1
yr
1
). Carbon contents are adapted
from data in Gulde et al. (2008). Error bars represent standard error of the mean, while letters indicate signicant differences between treatments within each fraction at the
a 0.05 level.
E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 183
increasing manure input to 180 Mg manure ha
1
yr
1
(Table 2).
Measured lignin-VSC/OC for manure applied between the years
2004e2008 ranged from 6.28 to 9.18 mg VSC (100 mg OC)
1
and averaged 7.89 0.64 mg VSC (100 mg OC)
1
(Table 2). Only the
cPOMfraction lignin-VSC/OC exceeded the manure input signatures
for every treatment, while bulk soil lignin-VSC/OC for the 120 and
180 Mg ha
1
yr
1
manure treatments exceeded the lower end of the
manure input range (Table 2).
Carbon-normalized cutin suberin-SFA/OC did not signicantly
differ with manure treatment for the bulk soil or any of the soil frac-
tions (Table 2). The cPOM fraction had the greatest cutin suberin-
SFA/OC concentration in each treatment, with cutin suberin-SFA/OC
concentrations greater than or equal to the average measured manure
content of 0.22 mg SFA(100 mg OC)
1
(Table 2) for all treatments. The
bulk soil had the next highest cutin suberin-SFA/OC concentration
in each treatment (Table 2), with concentrations comparable to the
Fig. 4. Percent contribution of coarse particulate organic matter (cPOM), micro-within macroaggregate-protected intra-aggregate particulate organic matter (iPOM), macroaggre-
gatedsilt clay (Magg-SC), and the non-aggregatedsilt clay (SC) to bulk soil mass (a), soil organic carbon (SOC, b), lignin-derived phenols (lignin-VSC, c), and cutinsuberin-derived
substituted fatty acids (cutin suberin-SFA, d) on a dry soil weight basis. Other refers to soil fractions not tested in this analysis, and determined by difference, namely the light
fraction, free microaggregates, and micro- within-macroaggregate protected silt clay.
Table 2
Soil organic carbon (SOC), C-normalized lignin (lignin-VSC/OC), C-normalized cutin suberin (cutin sub-SFA/OC), vanillyl and syringyl acid to aldehyde lignin phenol ratios,
root-derived SFA/OC (a, u-alkanedioic acids), shoot-derived SFA/OC (mid-chain hydroxy acids), and the ratio of root:shoot SFAs in the bulk soil, four soil fractions
(cPOM coarse particulate organic matter; iPOM intra-micro-within-macro particulate organic matter; Magg-SC macroaggregated silt clay; SC non-aggregated
silt clay), and the added manure. Manure treatments (0, 60, 120, and 180 Mg manure ha
1
yr
1
) constitute an increasing C saturation gradient. SED provides the standard
error of the mean for Tukey signicance tests for treatment differences within each fraction. Treatments with different letters were signicantly different at the a 0.05 level.
Soil Fraction Manure
(Mg ha
1
yr
1
)
SOC mg
(100 mg
1
soil)
Lignin-VSC/OC Cutin Sub-SFA/OC Phenol Acid:
Aldehyde
Root-SFA/OC Shoot-SFA/OC Root:Shoot SFAs
mg (100 mg
1
OC) Vanillyl Syringyl mg (100 mg OC)
1
Bulk Soil 0 2.08 a 3.60 a 0.14 0.37 0.57 0.02 0.08 0.27
60 5.07 b 5.43 b 0.19 0.34 0.54 0.02 0.14 0.17
120 7.36 c 6.69 b 0.20 0.34 0.53 0.02 0.15 0.14
180 9.69 d 6.88 b 0.22 0.35 0.55 0.02 0.16 0.16
SED 0.40 0.35 0.02 0.02 0.03 0.003 0.02 0.03
cPOM 0 20.59 a 9.75 ab 0.29 0.30 0.45 0.04 0.15 0.25
60 17.45 b 11.94 a 0.23 0.35 0.53 0.02 0.16 0.15
120 17.17 b 8.86 ab 0.21 0.34 0.53 0.02 0.15 0.13
180 16.63 b 7.87 b 0.25 0.30 0.53 0.02 0.18 0.13
SED 0.58 0.85 0.02 0.04 0.04 0.005 0.02 0.04
iPOM 0 2.73 a 5.37 0.13 0.36 0.59 0.02 0.06 0.34 b
60 6.50 b 6.17 0.13 0.31 0.53 0.02 0.08 0.23 ab
120 9.96 bc 5.13 0.14 0.31 0.48 0.02 0.09 0.18 a
180 9.26 c 4.80 0.12 0.34 0.53 0.02 0.08 0.20 ab
SED 0.70 0.58 0.03 0.03 0.04 0.003 0.02 0.03
Magg-SC 0 2.78 a 2.42 0.12 0.53 0.67 0.02 0.07 0.25
60 4.22 b 3.28 0.13 0.56 0.72 0.01 0.09 0.13
120 4.69 b 3.58 0.12 0.46 0.62 0.01 0.09 0.14
180 4.53 b 4.22 0.15 0.48 0.61 0.01 0.11 0.12
SED 0.21 0.42 0.01 0.05 0.05 0.002 0.01 0.03
SC 0 1.83 a 2.51 a 0.14 0.63 0.72 0.01 0.09 0.16
60 3.26 b 3.15 ab 0.13 0.57 0.76 0.01 0.09 0.14
120 3.48 b 4.21 b 0.16 0.52 0.67 0.01 0.12 0.12
180 4.36 b 4.44 b 0.12 0.48 0.63 0.01 0.09 0.13
SED 0.26 0.42 0.02 0.06 0.07 0.001 0.02 0.02
Manure Input average 2.16 7.89 0.22 0.30 0.44 0.02 0.14 0.09
SED 0.09 0.64 0.04 0.02 0.01 0.01 0.05 0.03
E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 184
manure input itself in the 120 and 180 Mg manure ha
1
yr
1
treatments.
3.3. Interactions of fraction lignin-VSC, and cutin suberin-SFA
with C saturation
Specic effects of C saturation on SOC biochemical composition
(i.e., lignin-VSC/OC and cutin suberin-SFA/OC) were determined
by a signicant statistical fraction treatment interaction between
the bulk soil and each fraction. The statistical fraction treatment
interaction effect on lignin-VSC/OC was not signicant for the Magg-
SC and SC fractions (Fig. 5eeh). In contrast, the fraction treatment
interaction effect on lignin-VSC/OC was highly signicant in the
cPOM (p 0.006) and iPOM (p 0.002). This effect is visualized by
the divergent and intersecting lines from the bulk soil to the POM
fractions (Fig. 5a, c). In the POM fractions, the direction of this
effect indicates a depletion of lignin-VSC per SOC with increasing C
saturation.
C saturation did not affect cutin suberin-SFA/OC concentra-
tions in any fraction, as indicated by the absence of a signicant
statistical fraction treatment interaction effect with increasing
manure input (Fig. 5b, d, f, h). Although marginally signicant
(p 0.090) due to the large standard errors of the measurements,
the cutin suberin-SFA/OC concentrations of the cPOM were
depleted in comparison to the bulk soil input proxy for the 60e180
manure Mg ha
1
yr
1
treatments relative to 0 Mg manure ha
1
yr
1
treatment (Fig. 5b).
In spite of the contrasting signicant results for lignin-VSC/OC
and cutin suberin-SFA/OC in the cPOM, no signicant fraction
treatment interaction was found for the ratio of VSC:SFA biomarkers
in any fractions (data not shown). This indicates that the relative
contribution of each class of compounds (dominantly aromatic
lignin-VSC versus dominantly aliphatic cutin suberin-SFA) did not
change with C saturation level. In the absence of an interactive
effect, the main effect of fraction was signicant. When averaged
across all manure treatments, the VSC:SFA ratio was signicantly
greater in the cPOM and iPOM than the bulk soil, Magg-SC, and SC
(Fig. 6a).
3.4. Ratios of lignin and cutin suberin monomers
The ratios of both vanillyl and syringyl acid:aldehyde lignin-
derived phenols, which increase with increasing side-chain alter-
ation of the lignin phenol precursors, did not signicantly change
with manure treatment in any SOC fraction (Table 2). Though not
signicant, these ratios tended to decrease in the Magg-SC and SC
with increasing manure input (Table 2). Furthermore, the C satura-
tion effect, tested by the statistical interaction of fraction treat-
ment, was not signicant for any fraction across manure treatments
(data not shown). Inthe absence of a signicant interaction, the main
effects of fraction on vanillyl and syringyl acid:aldehyde ratios were
signicant. When averaged across all treatments, acid:aldehyde
ratios were signicantly higher in the Magg-SC and SC than in the
bulk soil, cPOM, and iPOM (Fig. 6b).
Across all fractions and the bulk soil, the ratio of a, u-alkanedioic
acids to mid-chain hydroxy acids, a proxy for the contribution of
root to shoot cutin suberin (Mendez-Millan et al., 2010), tended
to decrease with increasing manure input (Table 2). This effect was
only signicant, however, for the iPOM (p 0.028), in which the
root:shoot ratio of SFAs decreased more rapidly than any other
Fig. 5. Change in the distribution of C-normalized lignin-derived phenols (lignin-VSC/OC, a, c, e, g) and C-normalized cutin suberin-derived substituted fatty acids
(cutin suberin-SFA/OC, b, d, f, h) from the bulk soil (approximating input composition) to each measured soil fraction (i.e., coarse particulate organic matter (cPOM, aeb),
micro-within macroaggregate intra-aggregate protected particulate organic matter (iPOM, ced), macroaggregated silt clay (Magg-SC, eef), and non-aggregated silt clay
(SC, geh)) with manure treatment (0, 60, 120, and 180 Mg manure ha
1
yr
1
). Signicant differences in treatment slopes from the bulk soil to each fraction illustrate the presence of
a signicant (at the Bonferroni corrected a 0.012) overall interaction between C saturation level (i.e., manure input treatment) and each soil fraction. Error bars represent standard
error of the mean.
E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 185
fraction from the 0 to the 120 manure Mg ha
1
yr
1
treatment
(Table 2). The trend in decreasing root:shoot ratios in all fractions
(non-signicant in cPOM, Magg, SC, and the bulk soil) resulted from
increased shoot-SFA/OC fraction contents with manure input, as
manure was enriched in shoot versus root SFAs relative to the soil
(Table 2). Neither root-SFA/OC and shoot-SFA/OC, nor the root:shoot
ratio exhibited a signicant fraction treatment interaction (data
not shown), indicating no signicant C saturation effect on the
concentration of root- versus shoot-derived SFAs. In the absence of
a signicant interaction, the main effect of fraction on root:shoot
ratios, when averaged across all treatments, determined signi-
cantly greater ratios in the iPOM than in the SC fraction (Fig. 6c).
4. Discussion
4.1. Carbon-normalized lignin-VSC and cutin suberin-SFA
Carbon-normalized lignin-VSC bulk soil concentrations of
3.60 mg VSC (100 mg OC)
1
inthe 0 Mg manure ha
1
yr
1
treatment
(Table 2) were comparable to results of previous studies in culti-
vated and grassland soils. Our results fell within the observed range
of 1.77e2.38 mg VSC-C (100 mg OC)
1
for an arable wheat- and
maize-cropped soil from a German eld trial (Heim and Schmidt,
2007b) and were greater than measurements of 1.03 and 1.13 mg
VSC-C (100 mg OC)
1
for a French wheat- and maize-cropped soil
(Dignac et al., 2005). The units in the above studies are normalized
for the amount of C in VSC, which ranges from 55 to 66% C per VSC,
depending on the monomer. Similarly, lignin-VSC/OC for the 0 Mg
manure ha
1
yr
1
treatment fell withinthe range of 2.5e4.9 mg VSC
(100 mg OC)
1
found across European arable and grassland
soils (Heim and Schmidt, 2007a), yet were larger than yields of
0.9e2.41 mg VSC (100 mg OC)
1
from grassland soils across
a North-American climate gradient (Amelung et al., 1999). Bulk soil
lignin-VSC/OC in the 60e180 Mg manure ha
1
yr
1
treatments
rangedfrom5.43 to 6.88 mg VSC (100 mg OC)
1
(Table 2), exceeding
the values published for most arable soils (Thevenot et al., 2010).
Higher bulk soil lignin-VSC/OC in the manure plots, as compared to
previous studies, likely resulted from the high application rates of
manure, whichis enriched in lignin-VSC relative tothe soil (Table 2).
Bulk soil cutin suberin-SFA measurements of 0.14 mg
SFA (100 mg OC)
1
in the 0 Mg manure ha
1
yr
1
treatment were
consistent with measurements for the same cutin suberin-SFA
monomers in a long-term agroecosystem experiment in France,
which determined 0.15 mg SFA (100 mg OC)
1
for a maize-cropped
soil and 0.11 mg SFA (100 mg OC)
1
for a wheat-cropped soil (data
calculated from individual monomers: Mendez-Millan et al., 2010).
Furthermore, cutinsuberin-SFA/OCinBrownChernozemandDark
Brown Chernozem grassland soils from Alberta, Canada, provided
comparable estimates of 0.14e0.15 mg SFA (100 mg OC)
1
(data
calculated fromindividual monomers: Otto andSimpson, 2006). The
enriched cutin suberin-SFA/OC bulk soil content in the 60e180 Mg
manure ha
1
yr
1
treatments exceeded published results for arable
and grassland soils. Since the Alberta grassland soil studied by
Otto and Simpson (2006) experienced climatic conditions and
high C-input levels similar to the soils in this study, comparable
cutin suberin-SFA/OC values across bulk soil samples were
expected. The higher SFA/OC contents of the Lethbridge soils under
manure input treatments compared to the Alberta grassland soils
indicates that enriched SFA/OC manure signatures, combined with
high manure application rates, control this enrichment.
4.2. Saturation of SOC, lignin-VSC, and cutin suberin-SFA
4.2.1. Saturation models and the C saturation gradient
According to the C saturation model (Stewart et al., 2007), the
asymptotic SOC response to increased manure C-inputs in the in
iPOM, Magg-SC, and SC (Fig. 3) illustrates the C saturation response
and the decreasing C stabilization potential of these soils at higher
levels of C-input (Gulde et al., 2008). These asymptotic SOC trends
and the reduced sequestration efciency from 19.9% to 16.9% of
C-input stabilized with increasing manure input treatments (Gulde
et al., 2008) validates the C saturation gradient at the Lethbridge
manure experiment.
The asymptotic increase in lignin-VSC and cutin suberin-SFA
per dry weight fraction in the Magg-SC and SC and of lignin-VSC in
the iPOM implies that these biochemical components also saturate
with increasing manure C-input (Fig. 3). This concomitant satura-
tion of plant-derived compounds per dry weight fraction and total
SOC was expected since lignin-VSC and cutin suberin-SFA are
components of the total SOC pool. The determination of the specic
C saturation effects on SOC biochemical composition thus required
an assessment of the relative saturation of lignin-VSC and
cutin suberin-SFA components compared to SOC.
WithSOCsaturation, carbon-normalized lignin-VSC/OCincreased
in the bulk soil, Magg-SC, and SC and decreased in the POMfractions
(Table 2). The increased carbon-normalized concentrations suggest
that lignin saturates more slowly than SOC in the bulk soil and the SC
fractions, while the decreased carbon-normalized concentrations
suggests ligninsaturates more rapidly thanSOC inthe POMfractions.
On the other hand, changes in C-normalized lignin, cutin, and
suberin concentrations with increased manure input could also
result from different input concentrations across treatments.
Although the lignin, cutin, and suberin input composition could not
be quantied, the nonlinear mixing of plant and manure inputs
Fig. 6. Main fraction effects on biomarker ratios of lignin-derived VSC phenols to cutin suberin-derived substituted fatty acids (VSC:SFA, a); phenolic acids to aldehydes for
vanillyl (AD:AL
V
) and syringyl (AD:AL
S
) lignin subunits (b); and a, u-alkanedioic acids to mid-chain hydroxy acids from cutin suberin-SFAs as indicators of root:shoot-SFA
contributions (root:shoot, c). Ratios compared the bulk soil, coarse particulate organic matter (cPOM), micro-within macroaggregate intra-aggregate protected particulate
organic matter (iPOM), macroaggregated silt clay (Magg-SC), and non-aggregated silt clay (SC) across all manure treatments. Tukey groupings were found signicant at the
alpha 0.05 level. Error bars represent standard error of the mean.
E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 186
across treatments likely leads to an increase in input lignin-VSC/OC
and cutin suberin-SFA/OC concentrations across treatments (See
Supplementary materials). To mitigate this confounding factor across
treatments, we tested C saturation specically by using the bulk
soil as a proxy of lignin-VSC and cutin suberin-SFA inputs and
statistically testing a fraction treatment interaction from the bulk
soil to each fraction.
4.2.2. POM fraction compositions with C saturation
Lignin-VSC/OCacross fractions at eachC saturationlevel generally
followed previously observed trends of decreasing lignin concen-
trations with decreasing particle size (Guggenberger et al., 1994;
Amelung et al., 1999; Six et al., 2001). In each treatment, concen-
trations of lignin-VSC/OC followed the order cPOM> iPOM> Magg-
SC z SC. With increased C saturation level, however, these differ-
ences became less pronounced, as lignin-VSC/OC continued to
increase in the SC fractions and decrease in the POM fractions
(Table 2).
The signicant statistical fraction treatment interaction for
lignin-VSC/OC in the cPOM and iPOM (Fig. 5a, c) indicated specic
C saturation effects on lignin contents. Decreased lignin-VSC/OC at
the 120 and 180 Mg manure ha
1
yr
1
treatments, i.e. higher C
saturation level, versus the 0 and 60 Mg manure ha
1
yr
1
treat-
ments, i.e. lower C saturation level, showed that lignin-VSC was
depleted relative to SOC when not protected (i.e., cPOM) or pro-
tected only by aggregates (i.e., iPOM) with increasing C saturation.
Carbon saturation level did not have a signicant effect on
cutin suberin-SFA/OC in the POM fractions (Fig. 5b, d). These
results are inconclusive, however, due to the large standard errors for
this measurement and the resulting lack of statistical signicance.
Trends in the cPOM fraction suggest that the C saturation effects on
these compounds in non-protected pools warrants further testing.
Non-protected cPOM composition is controlled by input and
decomposition. Depleted lignin-VSC/OC as soil C saturation was
approached implies that although the cPOM initially accumulated
the lignin added from the manure input, the decomposition of
these components was faster than the decomposition of total
cPOM-C. Similarly, although the iPOM initially stabilized available
input through physical aggregation, lignin stabilization was
progressively limited by increased decomposition. Gulde et al.
(2008) found increased aggregate turnover at this site with soil
C saturation, an effect that could also contribute to decreased
iPOM lignin stabilization through heightened interaction with the
lignin-depleted non-protected cPOM pool.
The reduced stabilization of lignin-VSC compared to SOC in non-
protected and aggregate-protected POM fractions corroborates
similar ndings of more rapid lignin than SOC turnover in
C-depleted soils (Kiemand Kgel-Knabner, 2003) and in
13
C natural
abundance tracer experiments (Dignac et al., 2005; Hofmann et al.,
2009). Increased cPOM and iPOM lignin-VSC decomposition with
C saturation could result from the effect of increased available
C substrate on microbial activity in the non-protected fraction.
Manure additions, in particular, could affect lignin decomposition
through increased addition of labile N, which can interact with
lignin-degrading enzymes (Grandy et al., 2008). The direction of
a hypothesized Neffect, however, is uncertain due to the high wood
chip content of the manure, which could immobilize the manure-N,
as well as the variable potential effects of N addition on SOM
decomposition across pools and biochemical components (Neff
et al., 2002; Grandy et al., 2008; Grandy and Neff, 2008).
Increased lignin decomposition in POM fractions with C satu-
ration could also reect the inuence of reduced C stabilization
capacity in the chemically protected SC fractions. Miltner and Zech
(1998) found decreased lignin decomposition with increased
mineral C stabilization potential and suggested that the reduced
availability of substrate for decomposer organisms drove this
response. The saturation of SC fractions with increased C-input
could, therefore, increase the substrate available for lignin degra-
dation, as lignin requires cometabolic microbial decomposition in
most environments (Miltner and Zech, 1999; Kgel-Knabner, 2002;
Thevenot et al., 2010). In this way, saturation of the SC fractions
could contribute to greater lignin decomposition in the non-
protected cPOM.
4.2.3. Mineral fraction compositions with C saturation
The lack of signicant statistical fraction treatment interac-
tions with C saturation level for lignin-VSC/OC and cutin suberin-
SFA/OC contents in the chemically-protected Magg-SC and SC
(Fig. 5e, g) indicated that lignin, cutin, and suberin stabilization
in organomineral associations is not affected by C saturation.
Although Magg-SC and SC lignin-VSC/OC increased with C satura-
tion level (Table 2), they did so in concert with the bulk soil, a proxy
for input composition. This suggests that, contrary to our initial
hypothesis, reduced capacity for chemical protection in the Magg-
SC and SC fraction did not progressively limit the chemical stabi-
lization of lignin-VSC in preference to other plant- or microbially-
derived carbon compounds. Furthermore, macroaggregate protec-
tion in the Magg-SC fraction did not affect the stabilization of
these compounds with C saturation, despite the addition of phys-
ical, combined with chemical, protection and the potential inter-
actions of aggregation on the mineral protection of SOC.
The nite potential of mineral surfaces to stabilize C, proposed
by Hassink (1997) and Mayer (1994), and elaborated by Six
et al. (2002), provides a mechanism for the observed saturation
of the chemically protected SC fractions (Kool et al., 2007; Chung
et al., 2008; Gulde et al., 2008; Stewart et al., 2008). However,
even with reduced mineral stabilization potential, we did not nd
evidence of diminished lignin, cutin, and suberin stabilization or of
preferential stabilization of aliphatic cutin and suberin compared
to aromatic lignin with C saturation. The consistent organomineral
stabilization of lignin, cutin, and suberin with decreased mineral
stabilization capacity supports the existence of an outer kinetic
zone, as outlined by the zonal model of organomineral associations
(Kleber et al., 2007), in which partitioning of hydrophobic moieties,
rather than sorption, is the dominant mechanism of mineral C
stabilization at high mineral SOC loadings.
4.3. Fraction effects on SOC biochemical composition
The absence of a C saturation effect on the VSC:SFA ratio for any
fraction indicates that the rates of decomposition and stabilization
of these compounds, with respect to each other, did not change.
This fails to support the hypothesis that greater recalcitrance of
cutin and suberin compared to lignin would amplify compositional
differences with C saturation level in the POM fractions. It also
further contradicts the idea that the preferential stabilization
of aliphatic to aromatic moieties would amplify compositional
differences in the SC fractions, even given the reduced stabilization
potential of these fractions with C saturation.
When averaged across treatments, the main effects of fraction
on VSC:SFA ratios showsignicantly greater lignin in the cPOMand
iPOMfractions and cutin suberin in the Magg-SC and SC fractions
(Fig. 6a). These fractional differences suggest that aliphatic
cutins and suberins are either preferentially protected on mineral
surfaces, as suggested by previous workers (Feng et al., 2005, Feng
and Simpson, 2007), or preferentially decomposed in the POM
fractions. The lack of a interaction effect on this ratio however,
indicates that the mechanism involved in fractional differences is
not affected by C saturation, i.e., the reduced stabilization potential
of mineral surfaces or increased turnover of POM fractions.
E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 187
4.3.1. Fraction effects on lignin acid:aldehyde ratios
Increased ratios of lignin-derived vanillyl or syringyl acids to
aldehydes in soils can indicate increased levels of lignin alteration
due to greater side-chain oxidation within the lignin polyphenol
(Ertel et al., 1984; Kgel, 1986). We hypothesized that acid:aldehyde
ratios of lignin would increase with C saturation in the chemically
protected Magg-SC and SC, due to decreased stabilization of fresh
input in the mineral fractions. Acid:aldehyde ratios, however, did
not change in any fraction with increased manure input. Further-
more, there was no evidence for a specic C saturation effect on the
acid:aldehyde ratio, as the fraction treatment interactions were
non-signicant for every fraction. If interpreted as indicative of
alteration status, this result suggests that lignin is not more or less
decomposed in any fraction with increased C saturation.
Averaged across treatments, acid:aldehyde ratios decreased
with decreasing particle size (Fig. 6b), in accordance with other
studies (Guggenberger et al., 1994; Amelung et al., 1999; Six et al.,
2001). Acid:aldehyde ratios in the cPOM and iPOM were signi-
cantly lower than in the Magg-SC and SC fractions (Fig. 6b).
Traditionally, these fraction differences were interpreted to reect
the presence of older, more altered lignin in mineral fractions.
Elevated acid:aldehyde ratios of colloidal bound SOC also result
frompreferential sorption of acid versus aldehyde lignin precursors
of fresh litter leachate (Hernes et al., 2007). This study exhibited no
change in mineral fraction acid:aldehyde ratios with increased C
saturation or manure input, despite the decreased stabilization
potential of these fractions with C saturation (Gulde et al., 2008).
This lends support that sorption, at least partially, controls the
increased acid:aldehyde ratios with decreasing particle size seen in
this study.
4.3.2. Contributions of root versus shoot carbon
Isotope studies have shown that root-derived C has a longer
mean residence time than shoot-derived C in soils, a result
primarily attributed to the preferential chemical and physico-
chemical protection of root-C (Puget and Drinkwater, 2001; Rasse
et al., 2005; Kong and Six, 2010; Mendez-Millan et al., 2010).
Along C saturation gradients established by increased plant input,
the hypothesized preferential stabilization of root versus shoot C
could play an important role in SOC biochemical composition
with reduced C stabilization potential. We hypothesized that with
increasing C saturation, the preferential protection and recalci-
trance of root-derived C would lead to increased root- versus
shoot-derived SFAs with decreasing C stabilization potential.
In this case, increasing C saturation level did not affect the
root:shoot ratios (i.e., the ratio of a, u-alkanedioic acids to mid-chain
hydroxy acids) of any fraction, as determined by no signicant
fraction treatment interactions. This implies that any preferential
stabilization of root versus shoot biomarkers did not increase with
increasingly limited stabilization potential. This result, however,
cannot be extrapolated to C saturation studies established by net
primary productivity, as the Lethbridge C saturation gradient was
established by above-ground manure additions, which affected the
composition and placement of new C-input.
The manure C-input, derived primarily from shoot-derived feed,
had lower root:shoot ratios than any measured soil fractions and
drove the observed reduction in fraction root:shoot ratios with
manure input (Table 2). In the iPOM, the root:shoot-SFA ratio for the
120 Mg manure ha
1
yr
1
treatment was signicantly lower than
the 0 Mg manure ha
1
yr
1
. The signicant root:shoot reduction in
the iPOM fraction is explainable by the observed increased aggre-
gate turnover with C saturation (Gulde et al., 2008). With increased
aggregate turnover, iPOM could become increasingly derived from
the fresh input (i.e., shoot-derived manure), leading to signicant
reductions in the root:shoot ratio.
Despite the greater reduction in root:shoot ratios in the iPOM
versus any other fraction, it still had the greatest fraction root:shoot-
SFA ratio at every manure treatment level (Table 2), and a signi-
cantly greater ratio than the SC fraction when averaged across treat-
ments (Fig. 6c). These results support the role of physical protection in
the stabilization of root versus shoot-C due to the formative role of
roots in aggregation (Denef and Six, 2006). If increased aggregate
turnover, however, is a feature common to C saturated soils, then the
contributions of root versus shoot-C to stable iPOM-C pools could
prove less important in soils close to C saturation.
5. Conclusions
The depletion of lignin in the non-protected (cPOM) and physi-
cally protected (iPOM) fractions indicates that inherent lignin
recalcitrance was not, in this study, a mechanism for SOC accumu-
lation with increased C saturation. In light of the greater fractional
distribution of C-input into the non-protected fraction with C
saturation (West and Six, 2007; Gulde et al., 2008; Stewart et al.,
2008), these results indicate that C saturation actually increased
lignin turnover through accelerated decomposition and increased
aggregate turnover. In the mineral protected Magg-SC and SC frac-
tions, C saturation did not apparently affect lignin, cutin, or suberin
biochemical composition. Fraction comparisons show greater
cutin suberin than lignin in mineral vs. POM fractions, suggesting
that cutin suberins contributed to the aliphatic C preferentially
found on mineral surfaces (Feng et al., 2005) and to alkyl C found in
stable SOC pools (Kgel-Knabner et al., 1992; Rumpel et al., 2004).
Likewise, greater root:shoot stabilization in aggregate-protected
iPOM versus mineral fractions points to the preferential contribu-
tion of root-derived C to aggregate C (Denef and Six, 2006). That
these fraction effects do not change with C saturation argues that
the mechanisms of these preferential associations are not affected
by the reduced stabilization potential of mineral surfaces or aggre-
gate structures that is hypothesized to control soil C saturation
(Six et al., 2002).
With C saturation, the observed increasing decomposition of
recalcitrant plant-derived compounds, the greater turnover of
aggregate fractions, and the lack of preferential biochemical protec-
tion in organomineral associations signies that biochemical SOC
composition did not inuence long-term C protection in this study.
This conclusion, therefore, contests the exibility of land manage-
ment practices to control long-term soil C stocks through manipu-
lation of SOC composition. The results of this study support the idea,
inherent to the denition of the C saturation model itself, that only C
quantity, not biochemical C composition, may control the saturation
of SOC and the mechanisms of SOC protection.
Acknowledgements
We thank Drs. Susan Crowand TimFilley for their help in setting
up the SFA methods and Drs. Cathy Stewart and Robert Spencer
for data analysis discussions. We thank Agriculture and Agri-Food
Canada for access to the Lethbridge manure experiment and are
indebted to the earlier work of Sabrina Gulde in sampling and
fractionating the Lethbridge soils. Finally, we sincerely thank the
reviewers for their helpful insights and suggestions during the
submission process.
Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.soilbio.2011.12.024.
E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 188
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