Biol Fertil Soils (2012) 48:325336

DOI 10.1007/s00374-011-0629-2

ORIGINAL PAPER

Effects of long-term compost and fertilizer application

on stability of aggregate-associated organic carbon
in an intensively cultivated sandy loam soil
Hongyan Yu & Weixin Ding & Jiafa Luo & Ruilin Geng &
Anwar Ghani & Zucong Cai

Received: 6 July 2011 / Revised: 20 September 2011 / Accepted: 30 September 2011 / Published online: 18 October 2011
# Springer-Verlag 2011

Abstract The study examined the influence of compost

and mineral fertilizer application on the content and
stability of soil organic carbon (SOC). Soil samples
collected from a long-term field experiment were separated
into macroaggregate, microaggregate, and silt + clay
fractions by wet-sieving. The experiment involved seven
treatments: compost, half-compost N plus half-fertilizer N,
fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and
control. The 18-year application of compost increased SOC
by 70.7121.7%, and mineral fertilizer increased by 5.4
25.5%, with no significant difference between control soil
and initial soil. The C mineralization rate (rate per unit dry
mass) in microaggregates was 1.522.87 mg C kg1 day1,
significantly lower than in macroaggregate and silt + clay
fractions (P<0.05). Specific C mineralization rate (rate per
unit SOC) in silt + clay fraction amounted to 0.48
0.87 mg C g1 SOC day1 and was higher than in
macroaggregates and microaggregates. Our data indicate
that SOC in microaggregates is more stable than in
macroaggregate and silt + clay fractions. Compost and
mineral fertilizer application increased C mineralization rate
in all aggregates compared with control. However, compost
application significantly decreased specific C mineralization rate in microaggregate and silt + clay fractions by 2.6
28.2% and 21.925.0%, respectively (P<0.05). By contrast, fertilizer NPK application did not affect specific C
H. Yu : W. Ding (*) : R. Geng : Z. Cai
State Key Laboratory of Soil and Sustainable Agriculture,
Institute of Soil Science, Chinese Academy of Sciences,
Nanjing 210008, China
e-mail: wxding@issas.ac.cn
J. Luo : A. Ghani
Climate, Land and Environment, AgResearch,
Hamilton 3240, New Zealand

mineralization rate in microaggregates but significantly

increased that in silt + clay fractions. Carbon sequestration
in compost-amended soil was therefore due to improving
SOC stability in microaggregate and silt + clay fractions. In
contrast, fertilizer NPK application enhanced SOC with low
stability in macroaggregate and silt + clay fractions.
Keywords Mineral fertilizer . Compost . Mineralization .
Carbohydrate . Wet-sieving . Enzyme

Introduction
The stability of soil organic C (SOC) is defined as its
resistance to microbial decomposition and is correlated to soil
C sequestration potential and soil fertility (Leinweber et al.
2008). The aggregate fractionation technique is used to
distinguish SOC into different pools, including more easily
decomposed and less easily decomposed. A wealth of studies
have shown that SOC stability in aggregate generally
increases with smaller aggregate size (Puget et al. 2000;
Ashman et al. 2003) primarily due to physical protection in
microaggregates and/or physico-chemical stabilization in the
silt + clay fraction (Hassink 1997; Six et al. 2000;
Jagadamma and Lal 2010). However, Razafimbelo et al.
(2008) observed a significantly higher accumulation of the
mineralized C in mesoaggregates (20200 m) than from
macroaggregates (2002,000 m) during a 28-day incubation period in 11-year share-plowed soil with no residues and
in a no-tilled soil with residue mulching. Liao et al. (2006)
also reported a 1517% organic C loss in the silt + clay
fraction 10130 years after grassland was converted into
forestland. Gleixner et al. (2002), Dignac et al. (2005),
and Bol et al. (2009) identified carbohydrate and protein,
which are defined as labile organic C, in small aggregates,

326

e.g., silt + clay fraction. Bayer et al. (2006) reported that

within the silt + clay fraction, the strongest organo-mineral
interactions were found in the clay fraction (<2 m), but
the most recalcitrant organic C was in the fine silt fraction
(202 m). Their finding suggests that in some cases the
SOC in small size aggregate may not be as inert as
expected. Carbon sequestration may also be variable due to
physical protection in microaggregates or physico-chemical
stabilization in the silt + clay fraction, depending on both soil
type and management (Hassink 1997; Six et al. 2002).
Organic waste and residue management are recommended to increase SOC by improving stabilization of organic
C (Fonte et al. 2009; Liang et al. 2011). For example,
Bidisha et al. (2010) and Majumder and Kuzyakov (2010)
observed that applying manure for 20 years lowered the
decomposability of SOC in soil, which facilitated C
retention in cultivated soil. Six et al. (2000) and Chivenge
et al. (2007) suggested that an observed increase in organic
C in cultivated soils following manure or residue application could be attributed to physical protection of the newly
added organic C by occlusion in microaggregates. In
contrast, Razafimbelo et al. (2008) found that the increment
of SOC in soil after an 11-year period of no-tillage, together
with residue mulching, was due to increased organic C
associated within the clay and silt fractions via physicochemical protection in aggregates. These inconsistent
reports in the literature suggest that further work is needed
to understand the organic C stability in aggregates and to
accurately explain the accumulation process of organic C in
soil.
Potentially mineralizable C through incubation experiment is frequently reported as a measure of SOC stability.
Since SOC labile pools are exhausted after 6 months to a
year, depending on the soil material, some researchers
suggested that short-term soil incubation studies are less
reliable (Chen and Stark 2000). However, Chevallier et al.
(2004) point out that if the protected SOC in aggregate was
labile (refer to quality of SOC), long-term incubation would
smoothen the importance of this SOC pool. In this study,
therefore, we used short-term incubation to evaluate SOC
stability in aggregates. In general, carbohydrate represents a
main source of nutrients and energy for soil microorganisms and is easily mineralized (Jolivet et al. 2006). SOC
decomposition has been determined by the activities of the
C-cycle enzymes, including dehydrogenase, invertase,
xylanases, cellulases, ligninases, and so on (Stemmer et
al. 1999; Allison and Jastrow 2006). We also measured
carbohydrate content and the activities of cellobiohydrolase
(CBH), -glucosidase (BG), xylosidase, invertase, and
polyphenol-oxidase (PPO) in aggregates. The objectives
of this study were to (1) evaluate SOC stability in soil and
aggregates and (2) understand the accumulation process of
SOC in the compost-amended and fertilized soil.

Biol Fertil Soils (2012) 48:325336

Materials and methods

Field experiment and soil sampling
A long-term field experiment was established to evaluate
the effect of compost and mineral fertilizer application
on soil fertility and crop yield where two crops per year,
winter wheat (Triticum aestivum L.) and summer maize
(Zea mays L.), were cultivated. The experiment described
in this paper started in September 1989 on a well-drained
field at the Fengqiu State Key Agro-Ecological Experimental Station, Fengqiu County, Henan Province, China
(3500 N, 11424 E). The 30-year mean annual temperature
was 13.9C, with the lowest mean monthly value of 1.0C in
January and highest mean monthly value of 27.2C in July.
The mean precipitation was 615 mm. The soil, derived
from alluvial sediments of the Yellow River, is classified as
Aquic Inceptisol with 52% sand, 33% silt, and 15% clay
(Ding et al. 2007).
The experiment included seven treatments: compost
(CM), half-compost N plus half-fertilizer N (HCM),
fertilizer NPK (NPK), fertilizer NP (NP), fertilizer NK
(NK), fertilizer PK (PK), and control (CK). The treatments
were arranged in a randomized block design with four
replicates. Each plot size was 9.55 m. The detailed
experimental design and application amount of mineral
fertilizers and compost were summarized in Table 1. The
basal fertilizers (mineral fertilizers and compost) were
evenly broadcast into the plowed soil (020 cm in depth)
by tillage before sowing. The supplementary fertilizer urea
was surface-applied by hand then brought into the plowed
layer with irrigation water.
For the experiments, compost was prepared by mixing
wheat straw, oil cake, and cotton cake in a ratio of
100:40:45 and fermenting for 2 months at the experimental
farm. This proportion was calculated on the basis of
component C and N contents, with the goal of applying a
total amount of organic C in composted manures (per
hectare per season) equal to the amount in harvested wheat
straw and organic N equivalent to 150 kg N ha1. These
materials were machine-ground to ~5 mm in length before
composting. The oil cakes and cotton cakes were the
machine-dried residues of oil-harvested rapeseeds and
cottonseeds, obtained from a commercial cooking oil
business. The amounts of phosphorus and potassium were
generally lower than the prescribed doses, so the compost
was supplemented with calcium superphosphate and potassium sulfate prior to application. The compost had an
average C/N ratio of approximately 8. After maize harvest,
on 28 September 2007, eight soil samples were taken from
the 020-cm layer at different positions in each plot using a
5-cm-diameter stainless steel soil sampler and were carefully mixed to form a composite. All samples were

Biol Fertil Soils (2012) 48:325336

327

Table 1 Experimental design and application amount of mineral fertilizers and compost
Treatment Basal fertilizer
N (kg Nha1)

P (kg P2O5 ha1)

Supplementary fertilizer
urea (kg Nha1)

K (kg K2O ha1)

Total Compost Urea Total Compost Calcium superphosphate Total Compost Potassium sulfate
Maize
CK

HCM

75

75

75

25.5

49.5

150

32.5

117.5

75

CM

150

150

75

51

24

150

65

85

NPK

60

60

75

75

160

150

90

NP
NK

60
60

0
0

60
60

75
0

0
0

75
0

0
150

0
0

0
150

90
90

PK
Wheat

75

75

150

150

CK

HCM
CM
NPK

90
150
90

75
150
0

15
0
0

75
75
75

22.5
45
0

52.5
30
75

150
150
160

31.5
63
0

118.5
87
150

60
0
60

NP
NK
PK

90
90
0

0
0
0

0
0
0

75
0
75

0
0
0

75
0
75

0
150
150

0
0
0

0
150
150

60
60
0

The supplement amount of mineral fertilizers added in the HCM and CM treatments was calculated according to the average N, P, and K contents
in the compost for 18 years
CK control, HCM half-compost N plus half-fertilizer N, CM compost, NPK fertilizer NPK, NP fertilizer NP, NK fertilizer NK, PK fertilizer PK

immediately stored in glass vials and kept on ice in coolers,

directly transported to the laboratory, and stored at 4C
before analysis.
Soil fractionation
Moist soil samples were gently broken apart along natural
break points and passed through an 8-mm sieve. Plant and
organic debris in the sieved soil were carefully removed
with forceps. After thorough mixing, a subsample was dried
at 105C to measure soil moisture. Another subsample was
air-dried for analysis of the soil properties. The remaining
moist soil was used for wet-sieving.
The water-stable aggregates of moist soils were separated
using the wet-sieving protocol (Elliott 1986). One hundred
grams of oven-dried soil samples was submerged in
deionized water on top of a 250-m sieve (aggregates with
size >2,000 m were not found in the tested soil) for 5 min
at room temperature. The sieve was manually moved up and
down by 3 cm, and this process was repeated 50 times over a
2-min period. Floating organic material was discarded. The
fraction remaining on the 250-m sieve was collected in a
pre-weighed aluminum pan to obtain macroaggregates
(>250 m). Water plus soil with particle size <250 m was
poured through a 53-m sieve, and the sieving procedure
was repeated. The fraction remaining on the 53-m sieve

was collected in another pre-weighed aluminum pan to

obtain microaggregates (53250 m). All materials
through the 53-m sieve were transferred into 250-ml
centrifuge tubes and centrifuged at 3,750g for 30 min at
4C. The pellets were resuspended in deionized water and
recentrifuged under the same conditions three times to
exclude the possibly temporary adsorption of dissolved
organic C by the mineral particles in silt + clay fraction.
Finally, the pellets were collected to obtain the silt + clay
fraction (<53 m), and centrifugal supernatants were
decanted. An aliquot of each fraction was used to
determine the moisture content. Another subsample of
each fraction was dried at 50C and used to analyze the
soil properties. The remaining moist sample was used for
incubation and measurement of enzyme activities.
Laboratory incubation
Ten grams of bulk soil through 2-mm sieve or aggregates
from each plot was placed in a 150-ml glass jar. All bulk
soil samples were adjusted to 60% of water holding
capacity, and the same amount of water was added to the
aggregates. The water holding capacity of bulk soils was
measured according to Cai et al. (2001). The jars were
covered with a plastic wrap for air exchange and incubated
in the dark at 25C. During incubation, soil moisture was

328

adjusted every second day by adding deionized water (to

maintain the initial weight). Carbon dioxide (CO2) emission
rate was measured at 0.5, 1, 2, 3, 5, 7, 9, 11, 13, 15, 18, 21,
24, 28, and 32 days after the beginning of incubation. To
measure CO2 emission, each bottle was sealed using an
airtight butyl rubber stopper perforated by centered Perspex
tubes for sampling. Immediately after and again 12 h after
closure, 1 ml headspace gas of the jar was sampled using an
airtight syringe for the measurement of CO2 concentration.
The CO2 concentration was measured using a gas chromatograph equipped with a thermal conductivity detector
operated at 60C (Agilent 7890, Santa Clara, CA, USA).
Separation was done using a 177/149-m (80/100 mesh)
Chromosorb 102 column (Advanced Minerals, Santa
Barbara, CA, USA) at 40C. The temperature of the
injecting port was 100C. The carrier gas (H2) flow rate
was 80 ml min1. Carbon dioxide gas standards were
supplied by the National Research Center for Certified
Reference Materials, Beijing, China. The rates of CO2
emission were calculated by the linear model from the
change of CO2 concentration in the jar over a 12-h period
with an average chamber temperature (25C).
Soil analysis
Moist and air-dried soil was dried at 105C to determine the
soil moisture content. Soil organic C contents in soil and
aggregates were determined by the wet oxidationredox
titration method (Carter 1993). The carbohydrate contents
in soil and aggregates were determined according to Puget
et al. (1999). Briefly, 1 g of finely ground soil or aggregate
was hydrolyzed with a concentrated sulfuric acid solution.
The supernatant was filtrated, and the filtrate was analyzed
with the phenol-sulfuric acid method to determine the total
sugar content (Doutre et al. 1978).
Cellobiohydrolase (EC 3.2.1.91) activity was measured
using the method of Desphaude et al. (1984). One gram (on
an oven-dried basis) of soils or aggregates was incubated
with p-nitrophenyl--D-cellobioside and acetate buffer (pH
5.0) in the dark at 50C for 30 min. The reaction was then
stopped by adding 1.0 M Na2CO3. The color intensity of
the filtered supernatants (obtained due to the production of
p-nitrophenol) was measured at 410 nm on an ultravioletvisible light spectrophotometer (UV-1800, Mapada Instruments Co., Shanghai, China). The procedures of Tabatabai
(1994) and Lama et al. (2004) were used to determine the
activities of -glucosidase (EC 3.2.1.21) and xylosidase
(EC 3.2.1.37), using p-nitrophenyl--D-glucoside and pnitrophenyl--D-xyloside as substrates, respectively. Briefly, 1 g (on an oven-dried basis) of soils or aggregates plus
maleate buffer (pH 6.0) and substrate were incubated for
1 h in the dark at 37C. Then, 0.5 M CaCl2 and 0.2 M trishydroxymethylaminomethanesodium hydroxide (pH 12)

Biol Fertil Soils (2012) 48:325336

for BG and 1.0 M Na2CO3 for xylosidase were added,

respectively. The amount of p-nitrophenol produced was
determined by spectrophotometry at 410 nm. Invertase (EC
3.2.1.26) activity was determined as suggested by Schinner
and von Mersi (1990). One gram (on an oven-dried basis)
of soils or aggregates was incubated with 1.2% (w/v)
sucrose solution and acetate buffer (pH 5.5) in the dark at
50C for 3 h. After incubation, the contents were filtered,
and 1 ml of the filtrate was used to estimate the amount of
reducing sugars (i.e., as glucose) by using the 3,5dinitrosalicylic acid method (Miller 1972). Polyphenoloxidase (EC 1.14.18.1) activity was measured spectrophotometrically using pyrogallol as the substrate (Huang et al.
2009). One gram (on an oven-dried basis) of soils or
aggregates and 1% pyrogallol was incubated for 2 h in the
dark at 30C. After incubation, citrate buffer (pH 4.5) and
ethyl ether were added to extract the reaction product, and
the absorbance of the organic phase was measured at
430 nm. Enzyme activities were measured in soil samples
of each treatment using controls made by mixing buffer
with either soil fractions or substrate solution. The values
were corrected by subtracting the combined absorption
values for the sample and substrate controls from those for
the analytical samples. The activity of CBH, BG, and
xylosidase was expressed in mg p-nitrophenol (pNP)
released kg1 dry soil 1. The activity of invertase and
PPO was expressed in g glucose (GE) released kg1 dry soil
h1 and g purple gallic (PG) released kg1 dry soil 1,
respectively.
Calculation and statistical analyses
The SOC content in aggregates was not corrected for sand
content as there was no obvious difference in particle size
distribution between macroaggregates and microaggregates
in all treatments. Soil organic C content in soil and
aggregates was expressed as g Ckg1 soil and g Ckg1
aggregate, respectively.
The sum of the mineralized C in macroaggregate, microaggregate, and silt + clay fraction was counted as follows:
X
Sum
total mineralized C in aggregate 
mass ratio of aggregate in soil

Specific C mineralization rate and specific activity of

enzymes in soil and aggregates were calculated as follows:
Specific C mineralization rate
C mineralization rate = SOC content or
Specific enzyme activity
enzyme activity=SOC content

Biol Fertil Soils (2012) 48:325336

329

Statistical analyses were performed using the SPSS

11.0 software package for Windows. The differences in
SOC, carbohydrate, C mineralization rate, specific C
mineralization rate, and enzyme activities in soil or
aggregates among treatments and in different aggregates
of the same treatment were examined using repeatedmeasures ANOVA. Regression analyses were used to test
relationships between the C mineralization rate and SOC
or carbohydrate content and between the specific C
mineralization rate and specific enzyme activity in soil
and aggregates.

The lowest carbohydrate content occurred in microaggregates (0.280.54 gC kg1 aggregate), and the highest was
observed in macroaggregates (0.731.28 gC kg1 aggregate);
the latter was significantly higher than the silt + clay fraction
in the PK, NK, and CK treatments, but not in the CM, HCM,
NPK, and NP treatments (Table 3). Fertilization increased
carbohydrate content in bulk soil and microaggregates
compared with the CK treatment, while compost application
simultaneously substantially enhanced carbohydrate content
in bulk soil and all aggregates.
Mineralization of organic C in soil and aggregates

Results
Soil organic C and carbohydrate content in soil
and aggregates
The recovery of the soil aggregate mass after wet-sieving
ranged between 97.3% and 101.3%. The 18-year application of compost and mineral fertilizer increased SOC
content by 70.7121.7% and 5.425.5%, respectively, and
there was no significant difference between unfertilized soil
(CK) and initial soil (Table 2). The SOC content in
macroaggregates varied from 6.63 to 12.17 gC kg1
aggregate, which was significantly higher than those in
microaggregate and silt + clay fractions. The SOC content
in microaggregates was significantly lower than in bulk soil
in all treatments and close to the value in the silt + clay
fraction in the HCM and NP treatments. The SOC content
in the silt + clay fraction was higher than those in
microaggregates in treatments CM, NPK, NK, and PK.
Fertilization significantly increased the SOC contents in
bulk soil, macroaggregate, and silt + clay fractions, and
compost application also elevated SOC content in microaggregates compared to the control treatment, CK.

The sum of the mineralized C in macroaggregate, microaggregate, and silt + clay fraction during the 32-day
incubation period was equivalent to 100.8124.6% of the
total mineralized C in bulk soil in all treatments. The C
mineralization rates (per unit dry mass) in bulk soil in the
HCM and CM treatment were 3.15 and 3.19 mg C kg1 soil
day1, respectively. These were significantly higher than
those in the other treatments and approximately 2.1 times
higher than that in the CK treatment (Fig. 1). Fertilization
also significantly elevated the C mineralization rate in all
treatments except for the NK treatment. The mineralization
rate of organic C over a 32-day experimental period in
microaggregates was 1.522.87 mg C kg1 soil day1,
significantly lower than the 3.536.26 mg C kg1 soil day1
in macroaggregates and the 2.025.24 mg C kg1 soil day1
in the silt + clay fraction. In comparison with the control
treatment CK, the C mineralization rate in the HCM, CM,
and NPK treatments was increased by 75%, 159%, and
139% in the silt + clay fraction, respectively, but only by
20%, 71%, and 36% in macroaggregates, respectively
(Fig. 1). Compost application and fertilization had less
effect on C mineralization rate in microaggregates, compared with macroaggregate and silt + clay fractions.

Table 2 Organic C content in bulk soil (g Ckg1 soil) and aggregates (g Ckg1 aggregate) as affected by the 18-year application of compost and
mineral fertilizers
Macroaggregate (>250 m)

Microaggregate (53250 m)

Silt + clay fraction (<53 m)

d
d
b
a
c

6.630.06 eA
11.800.04 bA
12.170.13 aA
11.680.27 bA

4.220.06
7.591.60
8.800.25
5.040.12

3.150.03 gC
7.320.11 bB
10.510.22 aB
5.770.07 cB

5.430.03 c
4.710.05 d
5.110.11 c

10.970.07 cA
8.660.32 dA
8.920.11 dA

4.810.06 bB
3.660.05 bC
4.820.02 bC

Treatment

Bulk soil

Pre-soil
CK
HCM
CM
NPK

4.470.27
4.430.06
7.630.07
9.910.06
5.610.14

NP
NK
PK

bB
aB
aC
bC

4.020.02 eB
3.430.03 fB
5.060.02 dB

Values are means (n=4) with standard error. Different lowercase letters within the column indicate significant differences between treatments at P<0.05
and different capital letters within the row indicate significant differences between aggregates for the same treatment at P<0.05
CK control, HCM half-compost N plus half-fertilizer N, CM compost, NPK fertilizer NPK, NP fertilizer NP, NK fertilizer NK, PK fertilizer PK

330

Biol Fertil Soils (2012) 48:325336

Table 3 Carbohydrate content in bulk soil (g Ckg1 soil) and aggregates (g Ckg1 aggregate) as affected by the 18-year application of compost
and mineral fertilizers
Treatment

Bulk soil

Macroaggregate (>250 m)

Microaggregate (53250 m)

Silt + clay fraction (<53 m)

CK

0.490.04 eC

0.730.02 eA

0.290.04 dD

0.590.03 dB

HCM

0.780.06 bB

0.910.03 cA

0.540.05 aC

1.020.11 abA

CM
NPK

0.970.03 aB
0.600.02 cB

1.280.10 aA
0.780.01 dA

0.530.09 abC
0.460.01 bC

1.210.02 aA
0.770.05 cA

NP

0.580.04 cdB

0.870.06 cA

0.390.06 cC

0.910.03 bA

NK
PK

0.550.03 deB
0.510.07 eC

1.060.04 bA
1.110.07 bA

0.320.03 dcD
0.280.18 dD

0.450.06 Ec
0.860.12 bcB

Values are means (n=4) with standard error. Different lowercase letters within the column indicate significant differences between treatments at P<0.05
and different capital letters within the row indicate significant differences between aggregates for the same treatment at P<0.05
CK control, HCM half-compost N plus half-fertilizer N, CM compost, NPK fertilizer NPK, NP fertilizer NP, NK fertilizer NK, PK fertilizer PK

The specific mineralization rate of organic C, which was

expressed as the mineralization rate per unit SOC, ranged
from 0.48 to 0.87 mg C g1 SOC day1 in the silt + clay
fraction and was significantly higher than that in macro-

(0.320.55 mg C g1 SOC day1), bulk soil

mg C g1 SOC day1), and microaggregates
mg C g1 SOC day1) (Fig. 1). Long-term
of compost significantly reduced the specific C

C mineralization rate (mg C kg-1 soil d-1)

a.
C
CD

A
BC

8.0

A
ABC
A
a

A
C

6.0

AB
B

CD
A

BC
D
BCD
B

C
CD
D

AB
B

AB
B

C
a

a
a

4.0

bb

b
b

b b

CK

HOM
Soil

c
b

2.0

0.0

OM

NPK

Macroaggregate

NP

b
b

NK

Microaggregate

PK

Silt+clay fraction

b.
Specific C mineralization rate (mg C g-1 SOC d-1)

Fig. 1 The C mineralization

rate (a) and specific C mineralization rate (b) in bulk soil and
aggregates during the 32-day
incubation period. Treatments
CK, HCM, CM, NPK, NP, NK,
and PK represent control,
compost, half-compost N plus
half-fertilizer N, fertilizer NPK,
fertilizer NP, fertilizer NK, and
fertilizer PK, respectively.
Vertical bars denote standard
errors of the means (n=4).
Different lowercase and capital
letters indicate significant
difference between aggregates for
the same treatment and between
treatments for the same aggregate
at P<0.05, respectively. C
mineralization rate=(cumulative
mineralized C during the 32-day
incubation period/32). Specific C
mineralization rate=(C mineralization rate/SOC content)

aggregates
(0.320.52
(0.280.57
application

1.2

A
BC

AB
B

B
BC
C

A
C

AB
AB
BC

B
B

A
a

A
a

B
AB
B

A
A

0.8

b b

a
0.4

0.0

a
b

CK

a
b bb

b
b

HOM
Soil

b
b

bb
b

OM

Macroaggregate

NPK

NP

Microaggregate

NK
Silt+clay fraction

PK

Biol Fertil Soils (2012) 48:325336

331

mineralization rate in microaggregate and silt + clay

fractions by 2.628.2% and 25.021.9%, respectively,
compared with treatment CK. Although amending fertilizer
NPK slightly lowered the specific C mineralization rate in
macroaggregates and microaggregates, it significantly increased the specific C mineralization rate in the silt + clay
fraction (Fig. 1).

at 79.4%. Except PPO, enzyme activities in microaggregates were lower than that in macroaggregates, silt + clay
fraction, and bulk soil in all treatments (Fig. 2). The highest
PPO activity was observed in microaggregates in all
treatments, except NPK and PK.
Long-term compost amendment significantly increased the activities of invertase in the bulk soil,
microaggregates, and silt + clay fraction, and CBH, BG,
and xylosidase in bulk soil and all aggregates. The
increase of enzyme activities from compost application
in macroaggregates or in the silt + clay fraction was
more pronounced than that in microaggregates. Adding
mineral fertilizers NPK increased the activities of
invertase, CBH, BG, and xylosidase in bulk soil and

Enzyme activities in soil and aggregates

The mean recoveries of the activities of all enzymes except
BG, in macroaggregate, microaggregate, and silt + clay
fraction with the seven treatments, were 90.8102.2%
compared with those in bulk soils. BG was the exception

Xylosidase activity
-1 -1
(g pNP kg h )

BG activity
-1 -1
(mg pNP kg h )

CBH activitiy
-1 -1
(mg pNP kg h )

Invertase activity
-1 -1
(g GE kg h )

3.0
2.5

BC B C C C B BC

2.0
1.5

1.0
0.5

a
c cd cd
d

A B B A AA B
a
b
c
d
d
e
f

CC D C CBC

A A AAAAA
a
b

C DBB BCA

B A A B BB A
a

bc

a a

b c
c c

a
b

bc
d

0.0
80

C CABB B B A
60

B B A B BB A
a

40
20
0
200

a ab a
bc bc
c
c
B C C B BBB

150

100

50

e e

b
d

A A A AA A A
a
b
c
d
e
f ef

BDDC CBA

B B B C BC B B

A A A A A A AB
a
a
b
c
c
c
c

BCCABBB

0
60
50

B B B B AB BAB

40

a a

30

ab

20
10

bcbc bc

a
a
b ab ab b b

d d d

a
a
e

bc

a
b

b
b cd
e de

ab ab b b ab

c cd ef de

B A A A ABB A
a
a
bc b c b

0
1.0

PPO activity
-1 -1
(g PG kg h )

Fig. 2 Activities of invertase,

cellobiohydrolase (CBH), glucosidase (BG), xylosidase,
and polyphenol-oxidase (PPO)
in bulk soil and aggregates as
affected by the 18-year application of compost and mineral
fertilizes. Treatments CK, HCM,
CM, NPK, NP, NK, and PK
represent control, compost, halfcompost N plus half-fertilizer N,
fertilizer NPK, fertilizer NP,
fertilizer NK, and fertilizer PK,
respectively. Vertical bars
denote standard errors of the
means (n=4). Different capital
and lowercase letters indicate
significant difference between
aggregates for the same treatment and between treatments for
the same aggregate at P<0.05,
respectively

0.8

AABA A B ABB
a

0.6
0.4

D B B AB BA
a

c b b

d d d

c c

b
d d

B A A AA A A

C C A ABC A

a
b b b b

c c

b b

0.2
0.0

Bulk soil
CK

Macroaggregate
HCM

CM

Microaggregate
NPK

NP

Silt+clay fraction
NK

PK

332

Biol Fertil Soils (2012) 48:325336

all aggregates. The application of unbalanced fertilizers

had no obvious effect on the activities of CBH, BG,
xylosidase in bulk soil and all aggregates, and invertase
in bulk soil, microaggregates, and the silt + clay
fraction. In macroaggregates, compost or unbalanced
fertilizers application significantly decreased invertase
activity compared with CK. In contrast, PPO activities
in all aggregates were reduced by fertilization, regardless of
the fertilizer type.
Relationship between C mineralization rate and SOC,
carbohydrate, or enzyme activity
Correlation analysis showed that the C mineralization rate
was significantly logarithmically correlated to SOC content
in bulk soil (R2 =0.760, P=0.010) and the silt + clay fraction
(R2 =0.735, P=0.014) and marginally significantly correlated
with SOC content in microaggregates (R2 =0.546, P=0.058).
The C mineralization rate was also significantly correlated
with the carbohydrate content in the bulk soil (R2 =0.633, P=
0.32) and silt + clay fraction (R2 =0.604, P=0.040). The
specific C mineralization rate was significantly correlated
with the specific activities of -glucosidase in macroaggregates and with -glucosidase, polyphenol-oxidase,
and invertase activities in the silt + clay fraction (Table 4).
No significant relationship was observed between specific C
mineralization rate and the specific activities of any of the
tested enzymes in microaggregates.

Table 4 Correlation between

specific C mineralization rate
and specific activities of invertase, cellobiohydrolase, glucosidase, xylosidase, and
polyphenol-oxidase in soil and
aggregates affected by long-term
application of compost and
mineral fertilizers

Discussion
Stability of aggregate-associated organic C
The specific C mineralization rate in microaggregates was
lower than the rates in the macroaggregate and silt + clay
fractions (Fig. 1), which suggested that organic C in
microaggregates was more stable than it was in macroaggregates and also in the silt + clay fraction. Lisboa et al.
(2009) postulated that for a forest-to-pasture chronosequence, the turnover time of organic C in the slow-cycling
C pool of microaggregates (53250 m) amounted to
498 years, which is longer than the predicted time of
210 years for the silt fraction (253 m). It has been
proposed that excluding microbes and enzymes from pores
is the key protection mechanism for occluded organic C in
microaggregates (Sollins et al. 1996). Zimmerman et al.
(2004) and McCarthy et al. (2008) reported that mesopores
of less than 0.1 m in diameter, which could account for up
to 21% of the total pore volume in microaggregates, were
inaccessible to exoenzymes. The organic C in mesopores is
therefore protected from decomposition. McCarthy et al.
(2008) further pointed out that even if some pores in
microaggregates were suitable habitats for small bacteria
and accessible to exoenzymes, the enzymes produced might
not be physiologically capable of decomposing organic C
due to the tortuosity of the pore network. In the present
study, we found lower invertase, CBH, BG, and xylosidase

Aggregate

Enzyme type

Equation

R2

Bulk soil

Invertase

y 1:451lnx 3:233
y 0:104x 1:548
y 0:796lnx  0:282
y 0:122x 0:827

0.694
0.509
0.705
0.879
0.098
0.448
0.300

0.020
0.072
0.018
0.002
0.495
0.100
0.203

0.741
0.365
0.007
0.166
0.261
0.125
0.128
0.049
0.525
0.288
0.618
0.170
0.862

0.013
0.151
0.861
0.364
0.241
0.436
0.432
0.633
0.065
0.214
0.036
0.358
0.003

Macroaggregate (>250 m)

Microaggregate (53250 m)

Silt + clay fraction (<53 m)

Cellobiohydrolase
-Glucosidase
Xylosidase
Polyphenol-oxidase
Invertase
Cellobiohydrolase
-Glucosidase
Xylosidase
Polyphenol-oxidase
Invertase
Cellobiohydrolase
-Glucosidase
Xylosidase
Polyphenol-oxidase
Invertase
Cellobiohydrolase
-Glucosidase
Xylosidase
Polyphenol-oxidase

y 0:059x 3:316

y 2:002lnx 4:044

y 1:303lnx  0:834

y 1:883e2:632x

Biol Fertil Soils (2012) 48:325336

activities in microaggregate than that in macroaggregates

and silt + clay fractions. However, the activities of PPOdegrading macromolecular compounds like lignin were
higher in microaggregate (Fig. 2). Therefore, we argue that
the micro-environment in microaggregates may not be
favorable for microbial life and enzyme production, which
results in slow rates of organic C decomposition.
In macroaggregates, the specific C mineralization rate
was higher than that in microaggregates (Fig. 1), indicating
that organic C associated with macroaggregates was more
easily decomposed than that in microaggregates. Correlation analysis showed that the specific C mineralization rate
in macroaggregates was significantly correlated with the
specific activity of -glucosidase, an enzyme driving the
hydrolysis of cellobiose (Turner et al. 2002), and marginally significantly with that of invertase (Table 4). In general,
enzymes have high specificity for their substrate, and they
can even distinguish isomers of the same substrate
molecule. The mineralization rate of organic C in the
macroaggregate fraction might therefore be primarily
controlled by the decomposition of cellulose and sucrose.
Lignin and hemicellulose might be rich in macroaggregate, resulting in a temporary increase in SOC (Klbl
and Kgel-Knabner 2004).
We found that the specific C mineralization rate in the
silt + clay fraction was higher than that in microaggregates
(Fig. 1). This disagrees with the results of Six et al. (2000)
and Allison and Jastrow (2006), who reported that the
stability of organic C in aggregates increased with a
reduction in aggregate size. It has also been reported that
the organic C associated with silt and clay particles is very
recalcitrant, with turnover times ranging from 400 to
1,000 years (Buyanovsky et al. 1994). However, Baldock
et al. (1987) and Puget et al. (1999) reported a number of
carbohydrates and high activities for carbohydrate hydrolytic enzymes in the silt + clay fraction. Zimmerman et al.
(2004) and Allison and Jastrow (2006) suggested that the
low degradation capacities of these enzymes were probably
due to their adsorption onto mineral surfaces, resulting in a
loss of functional activity. In contrast, Mikha and Rice
(2004) observed that the percentage of total organic C
mineralized to C-CO2 in the 2053-m fraction over a 28day incubation period was 3.33%, higher than the 2.77% in
microaggregates (53250 m) and just slightly lower than
the 3.704.35% in small (2502,000 m) and large
(>2,000 m) macroaggregates. This was in spite of the
fact that the labile organic C content in the 2053-m
fraction was significantly lower than those in other
fractions. Mutuo et al. (2006) also monitored higher C
mineralization rate in the 2053-m fraction than in the 53
212-m fraction in an arenosol of western Kenya. John
(2003) reported that parts of organic C in the clay fraction
had a high turnover rate, which was believed to be caused

333

by a relatively high enrichment of organic C from fresh

residues and/or microbial biomass. In the present study,
we found that the carbohydrate content in the silt + clay
fraction was significantly higher than that in the bulk soil
and in microaggregates (Table 3). The C mineralization
rate was also significantly correlated with carbohydrate
content, and the specific C mineralization rate was
significantly correlated with the specific activities of
invertase and -glucosidase (Table 4). We suggested that
carbohydrates in the silt + clay fraction are not as stable as
previously reported (Zimmerman et al. 2004; Allison and
Jastrow 2006) and are more easily decomposed than in
microaggregates. This may be due to the enzymes
responsible for degradation being incompletely adsorbed
onto mineral surfaces in our studied soil and therefore
capable of retaining some level of activity in the silt + clay
fraction. Another possibility is that the increase of
carbohydrates may stimulate the growth and activity of
microorganisms, which in turn increased the synthesis of
enzymes in the silt + clay fraction.
Effect of fertilizer on stability of aggregate-associated
organic C
A large number of studies have shown that C mineralization
rates are significantly linearly correlated with SOC content
in bulk soil, regardless of soil management practices
(Cochran et al. 2007; Zhao et al. 2008), and in aggregates,
independent of aggregate size (Chevallier et al. 2004;
Barreto et al. 2009). However, in our study, we found that
there was a significantly logarithmic, rather than linear,
correlation between C mineralization rate and SOC content
in bulk soil. We also found that the specific C mineralization rate in compost alone-amended soil was lower than in
CK (control) and mineral fertilizer-amended soils (Fig. 1).
Our findings suggest that the long-term application of
compost more efficiently improved the stability of SOC
than did the mineral fertilizer. This finding agrees with the
results of Bidisha et al. (2010), who observed that a 20-year
addition of farmyard manure at 60 t ha1 year1 decreased
the specific C mineralization rate in soil compared to soil
with and without the addition of mineral fertilizer.
As discussed above, the organic C associated with
microaggregates was the most stable that was found in
any of the aggregate fractions. The rapid increase of SOC in
compost-amended soil was primarily attributed to an
accumulation of organic C in microaggregates (Table 2).
Said-Pullicino et al. (2007) noted that the increased organic
C derived from added compost in microaggregates included
lignin-derived phenols, microbial-derived carbohydrates,
and so on. In contrast, the water-extractable organic C in
compost, especially microbial-derived carbohydrates, was
generally stabilized in the silt + clay fraction (Zimmerman

334

et al. 2004; Allison and Jastrow 2006), or it was combined

with insoluble organic matter and organic components in
compost (which were well matured and more resistant as a
result of the fermenting process) to form microaggregates
(Tisdall and Oades 1982; Helfrich et al. 2008). One
possibility is that the reduced organic C biodegradability in
microaggregates could be attributed to the accumulation of
recalcitrant organic C derived from compost. We also found
that long-term application of compost somewhat increased
the population of bacteria but significantly reduced the
abundance of actinomycetes in microaggregates compared to
treatment with mineral fertilizer (data not shown). By
measuring phospholipids fatty acid, Poll et al. (2003) also
measured a shift towards a more bacteria-dominated community in the coarse sand fraction of a manured Luvic
Phaeozem. Vikman et al. (2002) demonstrated that the ability
of bacteria to degrade recalcitrant organic C is weaker than
that of fungi and actinomycetes. Therefore, another possible
explanation for the observed increase in SOC stability in our
CM treatment was the microbial community shift to bacterial
domination in microaggregates.
The application of compost also improved the stability
of organic C, even labile organic C such as carbohydrate, in
the silt + clay fraction compared to mineral fertilizer
(Fig. 1). Among carbohydrates, polysaccharides produced
by microorganisms are more enriched in the silt + clay
fraction in cultivated soils compared with plant-sourced
polysaccharides (Puget et al. 1999; Kiem et al. 2003).
Martin et al. (1966) and Greenland and Oades (1975) found
that a microorganism-derived polysaccharide, like Azotobacter polysaccharide, was relatively resistant to decomposition. As a consequence, the polysaccharides formed by
microorganisms became the main source of the recalcitrant
organic compounds in soil (Bottner et al. 1998; Spaccini et
al. 2000). In the present study, the compost had been
fermented for 2 months before use. During composting, the
proportion of the labile, hydrophilic, plant-derived organic
compounds gradually reduced. In contrast, that of more
stable, hydrophobic moieties, including lignin-derived
phenols and microbial-derived carbohydrates, increased in
water-extractable organic matter (Said-Pullicino et al.
2007). When these microbial-derived carbohydrates are
incorporated into the soil, they are not usually utilized by
soil microorganisms and can become stabilized by mineral
particles. In contrast, as previously described, applying
mineral fertilizer NPK accelerated the decomposition of
native organic C in our soil (Ding et al. 2010). This was
especially obvious in the silt + clay fraction (Fig. 1), of
which the newly increased organic C was mainly derived
from root residues and exudates, as no other exogenous
organic materials were amended in mineral fertilizer Nadded soils. Manna et al. (2007) demonstrated that fertilizer
NPK application significantly promoted the growth and

Biol Fertil Soils (2012) 48:325336

activities of microorganisms, which in turn accelerated the

mineralization of organic C in the silt + clay fraction.
Therefore, we argue that the application of fertilizer NPK
temporarily increased organic C in the silt + clay fraction,
such as carbohydrates. In this fraction, organic C, which is
mainly sourced from root residues and exudates, may not
be as stable as it is in compost-amended soil where
increased organic C also stemmed from recalcitrant organic
C in compost. This had resulted in the low level of
accumulation of organic C that we observed in the silt +
clay fraction in the NPK treatment.

Conclusion
The lowest C mineralization rate and specific C mineralization rate was in microaggregates, indicating that the most
stable organic C in aggregate fractions was associated with
microaggregates. The specific C mineralization rate in the
silt + clay fraction was significantly higher than in other
two aggregates in all treatments except CM and CK.
Therefore, we conclude that organic C in the silt + clay
fraction in our soil was not stable as expected.
Compared to mineral fertilizer, long-term compost application significantly increased the stability of organic C in the
microaggregate and silt + clay fractions, but not in the
macroaggregate fraction. Carbon sequestration in compostamended soil therefore may mainly correlate the accumulation
of organic C in the microaggregate and silt + clay fractions. In
contrast, fertilizer NPK application temporarily increased
organic C (with low stability) in the macroaggregate and silt
+ clay fractions compared to that in the control treatment, CK.
Further study is required to measure the structure of soil
organic matter and microbial community in macroaggregate,
microaggregate, and silt + clay fraction in soil amended with
compost and mineral fertilizer to understand C sequestration
mechanism.
Acknowledgements We would like to thank Dr. Andrea Donnison
for reviewing this paper. This study was financially supported by the
Chinese Academy of Sciences (KZCX2-YW-439), the Natural
Science Foundation of China (40725003, 41001173), and the Natural
Science Foundation of Jiangsu province (SBK200922477,
BK2008057, BK2009338).

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