Pedosphere 24(5): 605–612, 2014

ISSN 1002-0160/CN 32-1315/P

c© 2014 Soil Science Society of China

Published by Elsevier B.V. and Science Press

Dynamics of Soil Organic Carbon Fractions and Aggregates in

Vegetable Cropping Systems∗1

LIANG Cheng-Hua∗2, YIN Yan and CHEN Qian

Department of Soil and Environment, Shenyang Agricultural University, Shenyang 110866 (China)

(Received January 24, 2014; revised July 22, 2014)

ABSTRACTFertilisers significantly affect crop production and crop biomass inputs to soil organic carbon (SOC). However, the long-term effects

of fertilisers on C associated with aggregates are not yet fully understood. Based on soil aggregate and SOC fractionation analysis, this

study investigated the long-term effects of organic manure and inorganic fertilisers on the accumulation and change in SOC and its

fractions, including the C concentrations of free light fraction, intra-aggregate particulate organic matter (POM) and intra-aggregate

mineral-associated organic matter (MOM). Long-term manure applications improved SOC and increased the concentrations of some C

fractions. Manure also accelerated the decomposition of coarse POM (cPOM) into fine POM (fPOM) and facilitated the transformation

of fPOM encrustation into intra-microaggregate POM within macroaggregates. However, the application of inorganic fertilisers was

detrimental to the formation of fPOM and to the subsequent encrustation of fPOM with clay particles, thus inhibiting the formation of

stable microaggregates within macroaggregates. No significant differences were observed among the inorganic fertiliser treatments in

terms of C concentrations of MOM, intra-microaggregate MOM within macroaggregate (imMMOM) and intra-microaggregate MOM

(imMOM). However, the long-term application of manure resulted in large increases in C concentrations of MOM (36.35%), imMMOM

(456.31%) and imMOM (19.33%) compared with control treatment.

Key Words: long-term fertilization, physical fractionation, soil aggregates, soil organic matter

Citation: Liang, C. H., Yin, Y. and Chen, Q. 2014. Dynamics of soil organic carbon fractions and aggregates in vegetable cropping

systems. Pedosphere. 24(5): 605–612.

INTRODUCTION

Soil organic carbon (SOC) has a profound effecton soil quality. It encourages aggregation, increaseswater retention, nutrient supply and soil organismactivity and improves soil fertility and productivity(Karlen et al., 1997), thereby, ensuring the long-termsustainability of an agroecosystem. Soil can act as asink for atmospheric CO2, and the increased sequestra-tion of C in agricultural soils can potentially mitigatethe global increase in atmospheric greenhouse gases(Young, 2003).

Increasing C sequestration in agricultural soils andmaking soil a net sink for atmospheric C can beachieved by adopting the best management practices,such as conservation tillage, application of fertilisersand bio-solids or organic amendments, crop rotationand improved residue management (Lal, 2004). In par-ticular, the benefits of a balanced application of inor-ganic fertilisers (i.e., the combined application of N, P

and K fertilisers) and organic manure in maintainingand increasing SOC levels in agricultural soils havebeen well documented (Rudrappa et al., 2006). Manylong-term fertiliser experiments worldwide have provedthat a balanced fertilisation using inorganic fertilisersand organic manure improves the nutrient status of thesoil and maintains high crop yields and high levels ofresidues that can be returned to the soil to increaseSOC concentration (Holeplass et al., 2004). In an ex-periment in north China, Meng et al. (2005) showedthat a balanced application of N, P and K fertilisersand organic manure significantly increases SOC accu-mulation to averages of 0.1 and 1.01 Mg ha−1, respec-tively, over a 13-year period. In studying organic Cfractions, Yang et al. (2005) suggested that the con-tinuous application of organic manure and inorganicfertilisers increases soil light fraction (LF) and C con-centration of particulate organic matter (POM). Li etal. (2004) suggested that the long-term use of fertili-sers enhances LF concentration and C concentration of

∗1Supported by the National Natural Science Foundation of China (No. 31171997), the Fifth Session of Geping Green Action—123

Project of Liaoning Environmental Research and Education (No.CEPF2012-123-1-4), and the Innovative Graduate Training Program

of Shenyang Agricultural University of China.∗2Corresponding author. E-mail: liang110161@163.com.

606 C. H. LIANG et al.

POM in paddy soil in this order: NPK (inorganicfertilisers) + OM (organic manure) > 2NPK (dou-ble the NPK application rates) > NPK > control (nofertiliser). In addition, Zotarelli et al. (2007) reportedthat the combination of no-tillage and green manureapplication promotes the stabilisation of aggregate-associated C.

Six et al. (2002) revealed a physical fractionationmethod in SOC. In this method, SOC is divided intorelatively homogeneous and differently functional frac-tions according to the protection extent of the soilaggregate. For example, SOC may be divided intoLF, POM, and inter- and outer-aggregates of mineral-associated organic matter (MOM). The physical frac-tionation technique also elucidates soil processes andmechanisms involved in the storage of SOC. Aggre-gation is known to increase in less disturbed systems,and organic materials within soil aggregates (especiallymicroaggregates) have lower decomposition rates thanthose located outside aggregates (Oades, 1984; Elliottand Coleman, 1988; Six et al., 2000). Therefore, thepresent study aimed to determine the accumulation ofSOC and the C concentration of different SOC frac-tions using the physical fractionation technique and toinvestigate the effect of inorganic fertilisers and farm-yard manure on C changes in SOC fractions as appliedin northeastern China in the long run.

MATERIALS AND METHODS

Site description and experimental design

A long-term fertiliser experiment was conducted atthe College of Horticulture, Shenyang Agriculture Uni-versity, Liaoning Province, China (latitude 41◦ 31′ N,longitude 123◦24′ E). The soil was classified as TypicFimi-Orthic Anthrosol (CRG-CST, 2001) and sandyloam (Soil Survey Staff, 1998). The experiment wasinitiated in 1988. Before this period, the field was anopen vegetable plot. From 1988 to 1995, two field veg-etable rotation cycles were completed. The crops se-quentially cultivated in a two-crops-per-year mannerconsisted of Chinese cabbage, bean, carrot, onion, cu-cumber, potato, mustard leaf and pimento. The tillagedepth was 20 cm. In 1996, the soil was moved fromthe study plot to the greenhouses for microfield exper-iments. The fertiliser treatments in the greenhouse ex-periment were identical to those in the long-term fieldstudy. Between 1997 and 2007, vegetable rotation wasimplemented in a one-crop-per-year manner. Specifi-cally, eggplant was cultivated for two years, tomatofor five years, cucumber for one year, and pimento forthree years. After the pimento harvest in October 2007,

soil samples were collected in the cultivated horizon(0–20 cm) of five random locations in each plot. Thesamples were mixed prior to analysis. The air-driedsamples were passed through an 8 mm sieve, and thevisible crop residue and roots were removed.

In 1997, the treatments were organised into a splitplot within a randomised complete block design. Farm-yard manure treatments were assigned to the mainplots and inorganic fertiliser treatments to the sub-plots. The treatments consisted of 1) control (no fer-tiliser and manure), 2) IN (inorganic N fertiliser), 3)INPK (inorganic N, P and K fertilisers), 4) M (farm-yard manure), 5) MN (farmyard manure with inorganicN fertiliser), and 6) MNPK (farmyard manure com-bined with inorganic N, P and K fertilisers). The farm-yard manure used was horse dung. N, P and K wereapplied to the crops annually as urea (22.5 g N plot−1),acid phosphate (720 g P plot−1) and potassium sul-phate (63.79 g K plot−1) fertilisers. Farmyard manurewas applied at a level of approximately 11.25 kg plot−1

each year. Each treatment was repeated thrice, and thesize of each plot was 1.5 m2. The plots were separatedby ridges (0.3 m × 0.8 m). Farmyard manure, alongwith acid phosphate, potassium sulphate and urea, wasapplied annually prior to the cropping season.

Aggregate separation

The aggregate separation was conducted followingthe method of Elliott (1986), i.e., the wet sieving of soilthrough a series of three sieves (2, 0.25, and 0.053 mm)to obtain four aggregate size classes (Fig. 1). A subsam-ple of 100 g air-dried soil was submerged in deionisedwater on top of a 2 mm sieve for 5 min prior to sie-ving. The sieving was performed manually by movingthe sieve up and down at a 3 cm level for 50 times in2 min. Organic material floating on the water in the 2mm sieve was removed after the 2 min cycle becausethis material is by definition not considered SOM. Thefraction that remained on the 2 mm sieve was collectedin an aluminium pan and oven dried. Water and soilsifted through the 2 mm sieve were poured onto thenext sieve, and the sieving was repeated. All fractionswere gently back-washed into an aluminium pan anddried overnight (50 ◦C). The next day, all fractionswere weighed.

Free LF separation

The method of separating the free LF was adoptedfrom the method of Dalal and Mayer (1986) (Fig. 1).Briefly, the free LF associated with different aggregatesize classes was isolated through the density flotationmethod by bromoform of 2.0 g cm−3. After the isola-

SOIL ORGANIC C AND AGGREGATES IN VEGETABLE SYSTEMS 607

Fig. 1 Fraction scheme to isolate aggregate and aggregate-associated soil organic carbon (SOC) fractions. LF = light fraction; HF =

heavy fraction; MOM = mineral-associated organic matter; cPOM = coarse particulate organic matter (POM); fPOM = fine POM;

HMP = hexametaphosphate; imMPOM = intra-microaggregate POM within macroaggregate; imMMOM = intra-microaggregates

MOM within macroaggregate; imPOM = intra-microaggregate POM; imMOM = intra-microaggregate MOM.

tion of the LF, residues were washed with 95% ethanolthrice to eliminate the bromoform.

Isolation of microaggregate out of macroaggregate

The method of isolating the coarse POM (cPOM,> 0.25 mm), fine POM (fPOM, 0.053–0.25 mm), intra-microaggregate POM within macroaggregate (imM-POM) and MOM was adopted from Six et al. (2000). Adevice was used to allow the complete breakup of themacroaggregates while minimising the breakdown ofthe released microaggregates. Exactly 10 g of macroag-gregates was immersed in deionised water on top of a0.25 mm mesh screen and shaken with 50 glass beads(diameter = 4 mm). A continuous and steady waterflow through the device flushed all the released mi-croaggregates immediately onto a 0.053 mm sieve toprevent further disruption from the beads. After thecomplete breakup of the macroaggregates, only thecPOM remained on the 0.25 mm mesh screen. Underthe 0.053 mm sieve was the MOM. The fPOM, imM-POM and imMMOM were isolated by density flotationand dispersion as described above (Fig. 1).

Carbon analysis

SOC and the C concentrations of SOC fractions

were measured using an element analyser (ElementarII, Germany). Based on Dumas combustion, SOC wascompletely combusted, and the CO2 released was de-termined through a thermal conductivity detector tomeasure C concentrations.

Statistical analysis

The data were analysed with SPSS (version 18.0)statistical package for analysis of variance, and theleast significant difference (LSD) was used to comparethe means of the significant treatments and interac-tions. Statistical significance was assessed at P < 0.05.

RESULTS

Total SOC increased with treatment: no fertiliser <

inorganic fertilisers alone < farmyard manure (Fig. 2).SOC increased by 19% in the inorganic fertiliser treat-ments relative to the control. However, the SOC con-centration in the farmyard manure treatment was al-most twice of that in the no-fertiliser treatment. SOCconcentration was the highest in the MNPK treatmentand lowest in the IN treatment (Fig. 2). No significantdifferences were observed in the SOC concentrations inthe control, IN and INPK treatments. This result indi-cated that the long-term application of inorganic ferti-

608 C. H. LIANG et al.

lisers had no significant effect on SOC accumulationin tilled cropping systems, in which crop residue androots were removed. By contrast, the SOC concentra-tion in the manure treatments increased significantly.The SOC concentration in the MNPK treatment washigher than those in the M and MN treatments.

Fig. 2 Total soil organic carbon (SOC) concentration at 0–20

cm soil depth under different fertiliser treatments. Vertical bars

represent standard errors of the means (n = 3). Bars with the

same letter(s) are not significantly different among treatments

at P < 0.05. Control = no fertiliser and manure; IN = inor-

ganic N fertiliser; INPK = inorganic N, P and K fertilisers; M

= farmyard manure; MN = farmyard manure with inorganic N

fertiliser; MNPK = farmyard manure combined with inorganic

N, P and K fertilisers.

The concentrations of free LF carbon in the con-trol and IN treatments did not significantly different(Fig. 3). However, the amount of free LF carbon in theMN treatment increased significantly compared withthose in the control and IN treatments. The amount

of LF carbon in the long-term manure application, es-pecially with manure combined with inorganic fertili-sers, was higher than that in the other treatments. In-organic N fertiliser had no effect on the increase ofLF carbon. In addition, the amount of LF carbon in-creased with LF size classes (Fig. 3).

Fig. 3 Carbon concentrations of free light fraction (LF) as-

sociated with 0.053–0.25 mm (LF53) and 0.25–2 mm (LF250)

aggregate sizes at 0–20 cm soil depth under different fertiliser

treatments. Vertical bars represent standard of the means (n

= 3). Bars with the same letter(s) for the same aggregate size

are not significantly different among treatments at P < 0.05.

See Fig. 2 for description of fertiliser treatments.

The C concentrations of imPOM were the high-est among all the SOC fractions in the different fer-tiliser treatments because of the large proportion ofthe 0.053–0.25 mm microaggregates. Furthermore, theSOC fractions varied across the fertiliser treatments(Fig. 4). The long-term application of inorganic fertili-

Fig. 4 Carbon concentrations of intra-aggregate particulate organic matter (POM) associated with aggregate size classes (a) and

POM sites in aggregates (b) at 0–20 cm soil depth under different fertiliser treatments. Vertical bars represent standard errors of

the means (n = 3). Bars with the same letter(s) for the same POM fraction are not significantly different among treatments at

P < 0.05. cPOM = coarse POM; fPOM = fine POM; imMPOM= intra-microaggregate POM within macroaggregate; imPOM =

intra-microaggregate POM. See Fig. 2 for description of fertiliser treatments.

SOIL ORGANIC C AND AGGREGATES IN VEGETABLE SYSTEMS 609

sers alone or farmyard manure increased C concentra-tions of cPOM and fPOM. C concentrations of cPOMand fPOM increased from 0.16 g kg−1 (control) to 1.14(INPK treatment) and 1.71 g kg−1 (M treatment) andfrom 0.08 g kg−1 (control) to 0.09 (INPK treatment)and 0.61 g kg−1 (M treatment), respectively. In thelong-term manure treatments, the C concentrations ofimMPOM and imPOM increased significantly, exceptfor that in the MN treatment. The application of in-organic fertilisers did not significantly increase the Cconcentrations of imMPOM and imPOM. The imM-POM and imPOM constitute the stabilised POM frac-tions within the microaggregates. In the treatments ofno fertiliser, inorganic fertilisers and farmyard manure,the stabilised POM were 4.44, 2.04 and 5.21 g kg−1,respectively.

The C concentrations of intra-aggregate MOM be-tween the manure and inorganic fertiliser treatmentsdiffered significantly (Fig. 5). Inorganic fertilisers hadno effect on the C concentration of intra-aggregateMOM relative to the no fertiliser treatment. However,the long-term application of farmyard manure alone orin combination with inorganic fertilisers significantlyincreased C concentrations of MOM, imMMOM andimMOM (36.35%, 456.31% and 19.33%, respectively)relative to the control.

Fig. 5 Carbon concentrations of intra-aggregate mineral-asso-

ciated organic matter (MOM) at 0–20 cm soil depth in different

fertiliser treatments. Vertical bars represent standard errors

of the means (n = 3). Bars with the same letter(s) for the same

MOM fraction are not significantly different among treatments

at P < 0.05. imMMOM = intra-microaggregates MOM within

macroaggregate; imMOM = intra-microaggregate MOM. See

Fig. 2 for description of different fertiliser treatments.

DISCUSSION

SOC and SOC fractions

In this experiment, the application of inorganic fer-

tilisers did not significantly affect SOC relative to theno-fertiliser treatment. However, it decreased the Cconcentration in some SOC fractions (e.g., LF, imM-POM and imPOM) relative to the control (Figs. 6 and7). This result was consistent with that of a long-termfertilisation experiment in northeast China (Yang et al.2003), in which inorganic N and NPK fertiliser did notmaintain SOC levels, and aboveground crop residueswere not returned to the soil. In general, the long-term application of inorganic fertilisers can increaseSOC content relative to no-fertiliser treatments whencrop yields increase and residue is returned to the soil(Haynes and Naidu, 1998). Other factors, such as therate of application of fertilisers, rotation regime andmanagement practices, affect the SOC concentration.In the USA, Raun et al. (1998) reported that SOC in-creased when N was applied at rates in excess of thatrequired for maximum yield; however, when N was ap-plied in their experiment at rates ≥ 90 kg ha−1, thesurface SOC was equal to or slightly greater than thatof the control (no N applied). Some long-term experi-ments in the northern Great Plains have shown thatN fertiliser increases crop residue returns but does notusually increase SOC sequestration (Halvorson et al.,2002), whereas SOC increases in others. Crop responseto N fertiliser and a low initial C pool increased SOC atthe Breton plots in Alberta, Canada, whereas 5 mg Cha−1 was sequestered over 28 years in fertilised treat-ments at Indian Head, Saskatchewan (Janzen et al.,1998). By contrast, Russell et al. (2005) reported thatN fertilization usually has a net negative effect on Csequestration. In our experiment, the application of ei-

Fig. 6 Carbon (C) concentrations in free light fraction (LF) as-

sociated with 0.053–0.25 mm (LF53) and 0.25–2 mm (LF250)

aggregate sizes at 0–20 cm soil depth under different fertiliser

treatments. Vertical bars represent standard errors of the means

(n = 3). Bars with the same letter(s) for the same aggregate size

are not significantly different among treatments at P < 0.05.

See Fig. 2 for description of different fertiliser treatments.

610 C. H. LIANG et al.

Fig. 7 Carbon (C) concentrations in intra-aggregate particulate organic matter (POM) associated with aggregate size classes (a) and

POM sites in aggregates (b) at 0–20 cm soil depth in different fertiliser treatments. Vertical bars represent standard errors of the

means (n = 3). Bars with the same letter(s) for the same POM fraction are not significantly different among treatments at P < 0.05.

imMPOM = intra-microaggregates POM within macroaggregate; imPOM = intra-microaggregate POM; cPOM = coarse POM; fPOM

= fine POM. See Fig. 2 for description of different fertiliser treatments.

ther N fertiliser alone or farmyard manure combinedwith N fertiliser did not increase POM. However, ap-plication of farmyard manure alone or in combinationwith inorganic fertilisers significantly increased SOCand C concentrations in SOC fractions. This findingsuggested that the application of manure strongly af-fected SOC levels and accumulation in cropping sys-tems and was thus consistent with the results obtainedfrom long-term experiments elsewhere (Kanchikeri-math and Singh, 2001; Rudrappa et al., 2006).

Stocks of SOC are believed to be a function of thenet input of organic residues by the cropping system(Gregorich et al., 1996). Soil and crop managementpractices, such as crop rotation and residue and fer-tiliser management, have a substantial effect on SOClevels over time. Lal (2004) reported that the appli-cation of inorganic fertiliser is important in obtaininghigh yields but may have little impact on SOC concen-tration, except when no-till and residue managementare combined. In this study, SOC levels decreased be-cause of the combination of conventional tillage and theremoval of aboveground crop residue and roots. Conse-quently, the application of chemical fertilisers withoutfarmyard manure did not maintain SOC levels in thelong term. These results are similar to those reportedby Nardi et al. (2004), who concluded that the incorpo-ration of limited crop residues has little effect on totalSOC level.

Change in SOC fractions

When fresh residues enter the soil, they becomesites of microbial activity and nucleation centres ofaggregation (Puget et al., 1995; Jastrow, 1996). Theenhanced microbial activity in these sites induces the

binding of residue and soil particles into macroaggre-gates. During this process, LF is incorporated intomacroaggregates and becomes cPOM. As the cPOM isformed and decomposed, microbial and decompositionproducts become associated with minerals, and cPOMsubsequently decomposes into fPOM. Within macroag-gregates, new microaggregates form during the last en-crustation of crop-derived fPOM with clay particlesand microbial by-products. The new microaggregatescontain a high proportion of crop-derived C and arepropitious for C accumulation.

Our results showed that the application of farm-yard manure alone and in combination with inorganicfertilisers accelerated the decomposition of cPOM intofPOM and improved encrustation from fPOM to imM-POM (Fig. 7). Nevertheless, inorganic fertilisers slowdown the formation of fPOM, and the subsequent en-crustation of fPOM with clay particles leads to theformation of stable microaggregates within macroag-gregates being inhibited. In this study, fertilisers didpromote C accumulation.

The role of intra-macroaggregate POM in the for-mation and stability of microaggregates, and the func-tions of microaggregates in C sequestration and stabi-lity have been described extensive in the literature (Sixet al., 1998; Six et al., 2000; Del Galdo et al., 2003;Denef et al., 2004). Six et al. (2002) reported that thePOM protected by microaggregates forms an SOC poolthat is sensitive to ecosystem changes. This SOC poolis stable in the long term and accounts for a substan-tial amount of total SOC stocks. It also varies acrossecosystems. In this study, the application of farmyardmanure did not increase imPOM but significantly in-creased imMPOM. The long-term application of far-

SOIL ORGANIC C AND AGGREGATES IN VEGETABLE SYSTEMS 611

myard manure promoted the formation of intra-agg-regate fPOM with clay particles and microbial by-products, and the formation of new microaggregateswithin macroaggregates. The new microaggregates inmacroaggregates were more stable than those not as-sociated with large aggregates. In addition, the im-POM had the greatest C concentration in all SOCfractions because the 0.053 to 0.25 mm microaggre-gates had a large percentage in the aggregate distribu-tion. Thus, both imMPOM and imPOM constitutedthe total POM fractions that stabilised within themicroaggregates. Organic C protected by microaggre-gates is a significant factor in C stability and stocksover the long term because of the slow turnover rate ofC in these aggregates (Besnard et al., 1996; Jastrow,1996; Six et al., 2002). However, the differences be-tween the microaggregates in the soil matrix and thosein complexes of microaggregates within macroaggre-gates are still unclear. Although the complexes of mi-croaggregates within macroaggregates protect a largeamount organic C in POM, mineralisation is high rela-tive to that in imPOM C. Once microaggregates are nolonger protected within macroaggregates, they becomesusceptible to disrupting factors and are broken downinto small microaggregates with low POM concentra-tion. Microaggregates released from macroaggregatesare probably incorporated into new macroaggregatesin the next macroaggregate formation cycle (Tisdalland Oades, 1982).

In the present study, the response of intra-aggre-gate MOM to the fertiliser was significant, particularlyin the sand-sized particles. Recent research suggeststhat the formation of macroaggregate sand increasesthe proportion of macroaggregates and organic C con-centrations. Jastrow (1996) found that 20% of the ac-crual of C in macroaggregates is in the form of POMand that the majority of accumulated C occurs in themineral-associated fraction of macroaggregates. Six etal. (1999) reported that POM is the nucleus for the for-mation of micro and macroaggregates, but C seques-tration within micro and macroaggregates is associatedwith minerals and POM C. Recent studies suggest thatMOM responds significantly to tillage and soil manage-ment (Six et al., 1999; Freixo et al., 2002; Del Galdo etal., 2003; Denef et al., 2004). Our study showed thatdifferent fertiliser treatments, especially farmyard ma-nure treatments, significantly increased MOM.

CONCLUSIONS

The long-term application of inorganic fertiliseralone was found to be insufficient to maintain SOClevels in the cropping systems with tillage, especially

when the aboveground crop residues were not returnedto the soil. The application of farmyard manure with orwithout inorganic fertiliser increased SOC concentra-tion and transformed fractions of intra-macroaggregatePOM into stable POM fractions in the microaggre-gates. In addition, different fertiliser treatments, es-pecially farmyard manure treatments, increased thecontent of imMMOM. Application of farmyard manurealone or in combination with inorganic fertilisers wouldsignificantly increase SOC.

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