Gradiente de Saturacion Lignina Cutina

8/12/2019 Gradiente de Saturacion Lignina Cutina

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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, USAb Department of Land, Air, and Water Resources, University of California, Davis, CA 95616, USAc 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) saturation provide an opportunityto 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 CO2, 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 responseunder 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: [email protected](E.M. Carrington).

Contents lists available atSciVerse ScienceDirect

Soil Biology & Biochemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . co m / l o c a t e / s o i l b i 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
mailto:[email protected]://www.sciencedirect.com/science/journal/00380717http://www.elsevier.com/locate/soilbiohttp://dx.doi.org/10.1016/j.soilbio.2011.12.024http://dx.doi.org/10.1016/j.soilbio.2011.12.024http://dx.doi.org/10.1016/j.soilbio.2011.12.024http://dx.doi.org/10.1016/j.soilbio.2011.12.024http://dx.doi.org/10.1016/j.soilbio.2011.12.024http://dx.doi.org/10.1016/j.soilbio.2011.12.024http://www.elsevier.com/locate/soilbiohttp://www.sciencedirect.com/science/journal/00380717mailto:[email protected]


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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 andNeff, 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 ligninin soilsand suggest that itsimportance in stableSOC poolsis

overstated (Thevenot et al., 2010). Lignin was found to cycle faster

than total SOC in 13C tracer experiments (Dignac et al., 2005;Rasse

et al., 2006; Heim and Schmidt, 2007a), had shorter residencetimes 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 thebiochemical recalcitrance of alkyl C in SOM(Lorenz

et al., 2007). Furthermore, cutin suberin-derived aliphatic waxes

exhibit even greater inherent recalcitrance than lignin,as evidenced

by their relative enrichment with depth and decreasing particle

size (Riederer et al., 1993; Nierop and Verstraten, 2003; Rumpelet 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 in the silt clayfraction (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 POM fraction (cPOM) (Fig.1). By measuring the

cupric oxide oxidation products of lignin, cutin, and suberin in these

fractions, we aimedto better constrain the role of chemical, physical,

and innate biochemical protection mechanisms in the preservation

ofSOC.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. Thephysicaland density fractionationscheme modiedfrom Gulde et al.(2008),

which isolated functional SOCpools utilized in thisstudy.The easilydispersedsilt 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) 179e190180


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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 ha1 yr1 (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 18.2Mg manure-C ha1 yr1 (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 yr1,

and have been cropped under barley, canola, and maize. Further

experimental details regarding the Lethbridge manure trial can be

found inSommerfeldt 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 cmfor 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) and arepresented in Fig.1. Thisinvolved

using, as a rst step, the wet-sieving method ofElliott (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, 250mm)) and macroaggregatedsiltand clay (Magg-SC


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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 min1, then ramped from 200 to 270 C at 5 C min1 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,

adapted fromHernesand Benner(2002). Table1 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 afterHernes 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 mL1, w10 ng mL1, w25 ng mL1, and w50 ng mL1).

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 phenolsafter 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-

tation behavior (2,3 dihydroxyhexadecanoic acid; ricinoleic acid; and

9,10,16 trihydroxyhexadecanoic acid) wereutilized for the calibration

of (x,16)DHPA & (x)-C16DA; C18DA:1 & u-C18:1; and 9,10uC18

(Table 1), respectively, after the method ofFilley 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 rstmeasure 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

signicanceof 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 onlinesupplementary 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 kg1 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 (Cf) as a function of bulk soil organic carbon

(Ct) content. Fraction one (Cf1) 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.



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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

ha1 yr1 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

ha1 yr1 treatment (Fig. 3l). In contrast, lignin-VSC cPOM concen-

trations initially increased from the 0e60 Mg manure ha1 yr1

treatments, then decreased signicantly (p 0.020) from the 60e180

Mg manure ha1

yr1

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, and cutin suberin-SFA occurred despite 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 to6% for the iPOM, 24e

11% for the Magg-SC,and 9e

4%for

the SC from the 0 to 180 Mg ha1 yr1 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

declined from 33 to 5%,lignin-VSCfrom 23 to 3%,and cutin suberin-

SFA from27 to4% fromthe 0 to180 Mgmanure ha1 yr1 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 manuretreatments (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 ha1 yr1 treatments and for the SC from the 0 to the 120

manure Mg ha1 yr1 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-

cantlyfor iPOM) exhibited an initial enrichment from the 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 ha1

yr1

). Carbon contents are adaptedfrom data inGulde et al. (2008). Error bars represent standard error of the mean, while letters indicate signi cant differences between treatments within each fraction at the

a 0.05 level.

E.M. Carrington et al. / Soil Biology & Biochemistry 47 (2012) 179e190 183


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increasing manure input to 180 Mg manure ha1 yr1 (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/OCexceeded the manure input signatures

for every treatment, while bulk soil lignin-VSC/OC for the 120 and

180 Mgha1 yr1 manure treatments exceededthe lower endof 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) forall 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), andthe non-aggregatedsilt clay(SC) to bulksoil mass(a), soilorganiccarbon(SOC, b),lignin-derivedphenols(lignin-VSC, c),and cutin suberin-derived

substituted fatty acids (cutin suberin-SFA, d) on a dry soil weight basis. Otherrefers 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 ha1 yr1) constitute an increasing C saturation gradient. SED provides the standard

error of the mean for Tukey signicance tests for treatmentdifferences within each fraction. Treatments with different letters were signicantly different at the a 0.05 level.

Soil Fraction Manure

(Mg ha1 yr1)

SOC mg

(100 mg1 soil)

Lignin-VSC/OC Cutin Sub-SFA/OC Phenol Acid:

Aldehyde

Root-SFA/OC Shoot-SFA/OC Root:Shoot SFAs

mg (100 mg1 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 ab120 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



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manure input itself in the 120 and 180 Mg manure ha1 yr1

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 forthe 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 ha1 yr1 treatments relative to 0 Mg manure ha1 yr1

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

(datanot shown).In theabsence of a signicantinteraction,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 thebulk soil, cPOM, and iPOM (Fig. 6b).

Across all fractionsand the bulk soil, the ratio ofa, 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 ha1 yr1). 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.



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fraction from the 0 to the 120 manure Mg ha1 yr1 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(Table2). 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 mgVSC(100 mgOC)1 in the 0 Mgmanureha1 yr1 treatment(Table 2) were comparable to results of previous studies in culti-

vated and grasslandsoils. 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 ha1 yr1 treatmentfell withinthe range of 2.5e4.9mgVSC

(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 soillignin-VSC/OC in the 60e180 Mg manure ha1 yr1 treatments

rangedfrom 5.43to 6.88mg VSC (100 mgOC)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, which is enriched in lignin-VSC relativeto thesoil (Table2).

Bulk soil cutin suberin-SFA measurements of 0.14 mg

SFA (100 mg OC)1 in the 0 Mg manure ha1 yr1 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, cutin

suberin-SFA/OC in Brown Chernozem and Dark

Brown Chernozem grassland soils from Alberta, Canada, provided

comparable estimates of 0.14e0.15 mg SFA (100 mg OC)1 (data

calculated from individual monomers: Otto and Simpson,2006).The

enriched cutin suberin-SFA/OC bulk soil content in the 60e180 Mg

manure ha1 yr1 treatments exceeded published results for arableand 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 iniPOM, 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 specicC 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.

With SOC saturation, carbon-normalized lignin-VSC/OCincreased

in the bulk soil, Magg-SC, and SC and decreased in the POM fractions

(Table 2). The increased carbon-normalized concentrations suggest

that ligninsaturates more slowly than SOC in thebulk soil and the SC

fractions, while the decreased carbon-normalized concentrations

suggests lignin saturates more rapidlythan SOC in the POM fractions.

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 quanti

ed, 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:ALV) and syringyl (AD:ALS) 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.



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across treatments likely leads to an increase in input lignin-VSC/OC

and cutin suberin-SFA/OC concentrations across treatments (See

Supplementary materials). Tomitigate thisconfounding 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 saturation level 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 ha1 yr1 treatments, i.e. higher C

saturation level, versus the 0 and 60 Mg manure ha1 yr1 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

resultsare 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 totalcPOM-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 (Kiem and Kgel-Knabner, 2003) and in 13C 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 availableC 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 N effect, 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 ofthese 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 kineticzone, 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 stabilizationof 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 show signicantly greater lignin in the cPOM and

iPOM fractions 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.



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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

from preferential sorption of acid versus aldehyde lignin precursorsof 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 ofa, 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 netprimary 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 ha1 yr1 treatment was signicantly lower than

the 0 Mg manure ha1 yr1. 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 Tim Filley 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

submissionprocess.

Appendix. Supplementary material

Supplementary material associated with this article can be

found, in the online version, atdoi:10.1016/j.soilbio.2011.12.024.

http://dx.doi.org/10.1016/j.soilbio.2011.12.024http://dx.doi.org/10.1016/j.soilbio.2011.12.024


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Gradiente de Saturacion Lignina Cutina