Pedosphere 23(1): 111119, 2013

ISSN 1002-0160/CN 32-1315/P

c 2013 Soil Science Society of China

Published by Elsevier B.V. and Science Press

Enzyme Activity in Water-Stable Soil Aggregates as Aected by

Long-Term Application of Organic Manure and Chemical
Fertiliser1
LIU Yi-Ren1,2 , LI Xiang1 , SHEN Qi-Rong1 and XU Yang-Chun1,2
1 Jiangsu Provincial Key Laboratory for Solid Organic Waste Utilization, College of Resources and Environmental Sciences, Nanjing
Agricultural University, Nanjing 210095 (China)
2 Institute of Soil Fertilizer and Resource Environment, Jiangxi Academy of Agricultural Sciences, Nanchang 330200 (China)

(Received August 25, 2012; revised November 22, 2012)

ABSTRACT
The activities of invertase, protease, urease, acid phosphomonoesterase, dehydrogenase, and catalase in dierent fractions of waterstable aggregates (WSA) were examined in long-term (26 years) fertilised soils. The long-term application of organic manure (OM)
with chemical fertiliser (CF) signicantly increased macroaggregate and decreased microaggregate percentages, enhanced the mean
weight diameter, and signicantly increased soil total carbon (TC) and total nitrogen (TN) contents of WSA in dierent size fractions.
Combined fertilisation with OM and CF also increased invertase, protease, urease, acid phosphomonoesterase, dehydrogenase, and
catalase activities of WSA in dierent size fractions. Enzyme activities were higher in macroaggregates than in microaggregates.
The distribution of enzyme activities generally followed the distribution of TC and TN in WSA. The geometric mean of the enzyme
activities in dierent WSA of OM-treated soils was signicantly higher than that in soils treated with 100% CF or no fertiliser. The
results indicated that the long-term combined application of OM with CF increased the aggregate stability and enzyme activity of
dierent WSA sizes, and consequently, improved soil physical structure and increased soil microbial activity.
Key Words:

distribution, mean weight diameter, stability, total carbon, total nitrogen

Citation: Liu, Y. R., Li, X., Shen, Q. R. and Xu, Y. C. 2013. Enzyme activity in water-stable soil aggregates as aected by long-term
application of organic manure and chemical fertiliser . Pedosphere. 23(1): 111119.

INTRODUCTION
Fertilisation is a primary management practice
used to increase crop yields in agricultural production.
Dierent fertilisation practices have variable impacts
on soil quality. Mineral fertilisation provides readily
available nutrients for plant growth but does not contribute to the improvement of soil physical condition.
Soil amendment through the input of organic matter
(OM) can maintain the soil organic matter (SOM) status, increase plant nutrient levels, and improve the
physical, chemical, and biological soil properties that
directly or indirectly aect soil fertility (Doran and
Smith, 1987; Ferreras et al., 2006; Nayak et al., 2007).
Soil enzymes derived from plants, animals, and microbes are important components of soil. They participate in the natural material cycle involved in biochemical processes and play a key role in soil development
(Nannipieri et al., 2002). Soil enzymes catalyse the decomposition of organic substrates, release plant nutri1 Supported

ents, and inuence the depletion or sequestration of

soil organic carbon (SOC; Fansler et al., 2005). Soil enzyme activities have been used as a biological activity
parameter to study the eects of agricultural management on soil C, nitrogen (N), phosphorus (P), sulphur
reactions and soil nutrient supplies (Dick, 1994; Marinari et al., 2006).
Soil aggregation plays an important role in maintaining soil structure and sustaining soil fertility (Elliott, 1986; Six et al., 2000; Bronick and Lal, 2005). Differences in nutrient content, moisture, and aeration
status of an aggregate can aect microbial nutrient
turnover and enzyme activities, impacting soil nutrient
release and plant absorption (Ladd et al., 1996). Thus,
the study of enzyme activities in these microenvironments can provide further information about the microbial turnover of soil nutrients (Roldana et al., 2005).
Some reports have described the distribution of
major nutrients in dierent sizes of soil fractions and
aggregates. For example, Miller and Dick (1995) found

by the National Basic Research Program (973 program) of China (No. 2007CB109304) and the Special Fund for Agroscientic Research in the Public Interest of China (No. 200803031).
2 Corresponding author. E-mail: ycxu@njau.edu.cn.

112

that a soil management system that increased root

activity and C input signicantly improved soil aggregation and the maintenance of organic C pools.
Many similar studies have been published (Ladd et
al., 1996; Adesodun et al., 2005; Onweremadu et al.,
2007; Yang et al., 2007). However, few studies have examined enzyme activities in dierent soil fractions and
aggregates. Stemmer et al. (1998) and Poll et al. (2003)
studied invertase and xylanase activity distributions in
particle-sized fractions. Kandeler et al. (1999a) studied
protease, urease, alkaline phosphatase, xylanase, and
arginine deaminase activities in dierent particle-size
fractions. The activities of other enzymes, such as dehydrogenase, lipase, arylsulfatase, leucine aminopeptidase, -glucosidase, -cellobiohydrolase, N-acetyl-glucosaminidase, and -xylosidase, have also been examined in dierent particle-size fractions (Marx et al.,
2005; Saviozzi et al., 2007; Qin et al., 2010). These
studies concluded that enzyme activities were distributed unequally among particle-sized soil fractions.
Variations in the results of these studies are probably
due to dierences in experimental methods or soil matrices.
Long-term eld treatments may be the only means
of directly quantifying the inuences of management
practices, including organic amendments, on soil characteristics (Collins et al., 1992). However, the eects
of long-term fertilisation on the distribution of enzyme
activities among dierent soil particles remain unclear,
especially in paddy soil.
The objective of this study was to understand the
eects of long-term organic or inorganic fertilisers on
soil enzyme activities in dierent soil particle sizes. We
measured invertase, protease, urease, phosphate, dehydrogenase, and catalase activities in dierent waterstable aggregates (WSA) from long-term fertilised soil
and explored the relationship of total N (TN) and total C (TC) to enzyme activity in dierent aggregates,
so as to improve the understanding of nutrient-cycling
mechanisms in soil.
MATERIALS AND METHODS
Study site
This long-term fertilisation experiment was established in 1984 in Nanchang County (115 56 E, 28
34 N), Jiangxi Province, China. In this typical subtropical climate, mean annual rainfall is 1 632 mm and
mean annual temperature is 18.4 C. The cropping
regime was rice-rice-fallow. The eld soil was sandy
loam developed from Quaternary red clay. The physi-

Y. R. LIU et al.

cal and chemical properties of the ploughed layer were

pH 6.50, OM 25.6 g kg1 , TN 1.36 g kg1 , total P
0.49 g kg1 , slowly available potassium (K) 240 mg
kg1 , alkali-hydrolysable N 81.6 mg kg1 , available P
20.8 mg kg1 , available K 35.0 mg kg1 , and cation
exchange capacity (CEC) 7.54 cmol(+) kg1 .
The long-term fertilisation experiment included
ve treatments: 1) CK (no fertiliser), 2) 100% CF
(100% chemical fertiliser of N, P and K ), 3) 70% CF
+ 30% OM, 4) 50% CF + 50% OM, and 5) 30% CF +
70% OM. Each experimental plot was 33.3 m2 , with 3
replicates. During the early rice season, the application
rates of N, P (calculated as P2 O5 ), and K (calculated
as K2 O) were 150, 60, and 150 kg ha1 , respectively.
During the late rice season, these rates were 180, 60,
and 150 kg ha1 , respectively. Fresh vetch (3.03 g N
kg1 , 0.80 g P2 O5 kg1 , 2.30 g K2 O kg1 , and 60.5 g
C kg1 ) was applied during the early rice season and
fresh pig manure (4.50 g N kg1 , 1.90 g P2 O5 kg1 ,
6.00 g K2 O kg1 , and 87.0 g C kg1 ) was applied during the late rice season. N, P, and K were applied
as urea, superphosphate, and potassium chloride, respectively; OM, P, and 50% of N were applied as base
fertiliser, and K and remaining N were top-dressed in
equal amounts at the tilling and young panicle stages.
Separation of water-stable aggregates
Soil samples were collected from the surface (020
cm) after the late rice harvest in 2009. After removing surface litter, ve surface-soil cores were collected
randomly from each plot and combined into a single
sample. The moist samples were passed through an
8-mm sieve by gently breaking apart the soil. Subsamples (100 g) were subjected to WSA analysis using
a slight modication of the wet-sieving technique described by Elliott (1986). Briey, subsamples were wetsieved manually through a series of ve sieves to obtain six size fractions: 1) > 5.000 mm, 2) 2.0005.000
mm, 3) 1.0002.000 mm, 4) 0.2501.000 mm, 5) 0.053
0.250 mm, and 6) < 0.053 mm. Before wet-sieving,
the samples were submerged in deionised water for 10
min on the largest screen. Soils were sieved underwater by gently moving the sieve 3 cm vertically 50
times for 2 min through water contained in a shallow
pan. Material remaining on the sieve was transferred
to an aluminium container and freeze-dried. Soil that
passed through a particular sieve into the shallow pan
was transferred to the next (ner) sieve and the process was repeated. Material smaller than 0.053 mm
(silt and clay particle-size fraction) was allowed to settle before centrifugation at 2 500 g for 10 min and

FERTILISER EFFECT ON ENZYME ACTIVITY IN AGGREGATES

freeze-drying. Floating material was removed and discarded during the sieving process.
The percentage of each (> 5.000, 2.0005.000,
1.0002.000, 0.2501.000, 0.0530.250, or < 0.053 mm)
WSA was determined as reported by Kemper and
Chepil (1965):
MWD =

Xi Wi

(1)

i=1

where MWD is the mean weight diameter (mm) of

WSA, Xi is the mean diameter (mm) of each size fraction, Wi is the proportion of the total WSA sample in
the corresponding size fraction, and n is the number of
size fractions.
Measurement of soil enzyme activities
Invertase activity was measured as reported by
Ohshima et al. (2007) using 15 mL of 80 g kg1 sucrose
as a substrate. Protease activity was determined as reported by Ladd et al. (1976). Urease activity was analysed using citrate-acid buer solution (pH 6.7; Homann and Teicher, 1961). Acid phosphomonoesterase
activity was measured according to the method described by Tabatabai (1994). Catalase activity was
analysed using the permanganimetric method (Johnson and Temple, 1964). Dehydrogenase activities were
measured using the technique described by Tabatabai
(1994).
We used the geometric mean of the enzyme activities (GMea) as the comprehensive index of soil enzyme activities in WSA that had received dierent fertilisation treatments. For each soil sample, the GMea
was calculated as

GMea =

113

Inv Pro Ure AcP Deh Cat

(2)

where Inv, Pro, Ure, AcP, Deh, and Cat are invertase,
protease, urease, acid phosphomonoesterase, dehydrogenase, and catalase activities, respectively (GarcaRuiz et al., 2008).
Carbon and nitrogen analyses
Soil TC and TN were determined by dry combustion using an elemental analyser (Vario EL III; Elementar Analysensysteme GmbH, Hanau, Germany).
Statistical analyses
All analyses were replicated three times. Statistical analyses were performed using Excel 2003 (Microsoft Corporation, Redmond, WA, USA) and the
SPSS software package (version 13.0 for Windows;
SPSS, Inc., Chicago, IL, USA). Statistically signicant
dierences (P < 0.05) were identied using analysis of
variance (ANOVA) and Duncans multiple comparison
test. Data were compared using the least signicant
dierence (LSD) test with P < 0.05 considered to indicate statistical signicance. All values are presented
as means standard deviations.
RESULTS
Aggregate size distribution and stability
WSA content was highest in the 0.2501.000-mm
fraction and lowest in the 2.0005.000-mm fraction
(Fig. 1). Long-term fertilisation increased the contents
of 1.0002.000-mm and 2.0005.000-mm WSA and de-

Fig. 1 Contents of water-stable aggregates that received dierent treatments. Error bars represent standard errors of the means
(n = 3). Data designated by the same letter(s) do not dier signicantly at P < 0.05, as determined by Fishers least signicant
dierence test. CK = no fertiliser; 100% CF = 100% chemical fertiliser of N, P and K; OM = organic matter.

114

Y. R. LIU et al.

creased the contents of 0.2501.000-mm and < 0.053mm WSA. The contents of WSA that received OM
+ CF treatments were higher than those that received 100% CF treatments in the 1.0002.000-mm and
2.0005.000-mm fractions and lower in 0.2501.000mm and < 0.053-mm WSA.
Signicant dierences in MWD, which can indicate aggregate stability, were found among treatments.
MWD values were highest in samples that had received
combined applications of OM + CF and higher in samples that had received 100% CF treatments than in
those that had received CK treatments (Fig. 2). The
increase in OM rate did not aect the MWD value.
Thus, the long-term combined application of OM and
CF increased WSA stability.
Distribution of TC and TN in WSA of dierent soil
fractions
TC and TN in dierent fractions of WSA showed
similar trends (Fig. 3). For all treatments, the highest
TC (22.936.4 g kg1 ) and TN (2.203.60 g kg1 ) contents were found in the 1.0002.000-mm fraction and
the lowest TC (11.918.3 g kg1 ) and TN (1.302.00
g kg1 ) contents were found in the < 0.053-mm fraction. The TC and TN contents of WSA in all size
classes were higher in samples that had received OM

Fig. 2 Mean weight diameters (MWD) of water-stable aggregates that received dierent treatments. Error bars represent
standard errors of the means (n = 3). Data designated by the
same letter(s) do not dier signicantly at P < 0.05, as determined by Fishers least signicant dierence test. CK = no
fertiliser; 100% CF = 100% chemical fertiliser of N, P and K;
OM = organic matter.

+ CF treatments than in those that had received 100%

CF or CK treatments, and higher in samples that had
received 100% CF treatments than in those that had
received CK treatments. The TC and TN contents of

Fig. 3 Total carbon (C) and total nitrogen (N) contents of dierent water-stable aggregates. Error bars represent standard errors
of the means (n = 3). Data designated by the same letter(s) do not dier signicantly at P < 0.05, as determined by Fishers least
signicant dierence test. CK = no fertiliser; 100% CF = 100% chemical fertiliser of N, P and K; OM = organic matter.

FERTILISER EFFECT ON ENZYME ACTIVITY IN AGGREGATES

dierent WSA fractions generally increased with increasing rates of applied OM.

115

was signicantly higher than that in WSA of CKtreated soil, with increases ranging from 42.9%658.2%
(Fig. 4e). Generally, the application of OM increased
dehydrogenase activity in WSA compared with that of
WSA from 100% CF-treated soil. Increasing OM application rates did not always increase dehydrogenase
activity in WSA. Signicant dierences were observed
between 30% CF + 70% OM- and 70% CF + 30% OMtreated soils in the 2.0005.000-, 0.2501.000-, 0.053
0.250-, and < 0.053-mm aggregate sizes.
Signicantly lower catalase activity in CK-treated
soils than in 100% CK-treated soils occurred only in
2.0005.000- and < 0.053-mm aggregates (Fig. 4f). OM
fertilisation generally increased the catalase activity in
WSA, but OM application rates did not always have a
signicant eect.

Enzyme activities of dierent water-stable aggregates

Invertase activity in dierent WSA fractions was
higher for all fertilisation treatments than for CK treatment. Invertase activity in WSA of OM-treated soils
was generally higher than that in WSA of 100% CFtreated soils (Fig. 4a). Generally, higher OM application rates resulted in higher invertase activity.
Compared with CK treatment, fertilisation treatments increased protease activities in dierent WSA
aggregates by 41.9%177.2% (Fig. 4b). Protease activities in all WSA of OM-amended soils were higher
than those in 100% CF-treated soils. Protease activity
increased with increasing rates of OM application, except in the < 0.053-mm fraction, but dierences between treatments were not always signicant.
Urease activity in WSA for all fertilisation treatments was signicantly higher than that in CKtreated soils, with increases ranging from 20.9%79.7%
(Fig. 4c). Urease activities in WSA of soil fertilised
with OM and CF were generally higher than those
in 100% CF-treated soil. Urease activity was highest
in the 1.0002.000-mm size fraction. Generally, urease
activity in WSA did not dier signicantly with increasing OM application rates.
Acid phosphomonoesterase activities in WSA were
generally highest in the 0.2501.000-mm size fraction and lowest in the < 0.053-mm size fraction
(Fig. 4d). The activity of this enzyme in all WSA sizes
of fertilised soils was higher than that in CK soil, with
increases ranging from 18.0%134.3%. Acid phosphomonoesterase activity in WSA of soils treated with OM
and CF was not always signicantly higher than that
in WSA of 100% CF-treated soil. Generally, the enzyme activity in WSA did not increase with increasing
OM application rates.
Dehydrogenase activity in WSA of fertilised soils

Comprehensive index of soil enzyme activities in

water-stable aggregates
Fertilisation aected the comprehensive index of
soil enzyme activities in dierent WSA (Table I).
GMea was signicantly higher in dierent WSA of fertilised soils than in CK-treated soils, with increases
ranging from 37.8%153.6%. GMea in dierent WSA
of OM-treated soils was signicantly higher than that
in 100% CF-treated soils and increased with increasing
OM application rates. Furthermore, GMea was signicantly higher in 70% OM-treated soils than in 50%
and 30% OM-treated soils in all size fractions except
for the 1.0002.000-mm fraction. GMea for dierent
treatments was highest in 1.0002.000-mm WSA and
lowest in < 0.053-mm WSA.
DISCUSSION
Twenty-six years of combined OM and CF fertilisation increased the contents of 1.0002.000-mm and
2.0005.000-mm WSA, and decreased the contents of
0.2501.000-mm and < 0.053-mm WSA (Fig. 1). Our

TABLE I
Comprehensive indices of soil enzyme activities in water-stable aggregates that received dierent fertilisation treatments
Treatmenta)

CK
100% CF
70% CF + 30% OM
50% CF + 50% OM
30% CF + 70% OM
a)

Aggregate size (mm)

> 5.000

2.0005.000

1.0002.000

0.2501.000

0.0530.250

< 0.053

0.32db)
0.52c
0.61b
0.62b
0.67a

0.33d
0.58c
0.63b
0.66b
0.70a

0.46d
0.64c
0.73b
0.78a
0.80a

0.30d
0.48c
0.63b
0.63b
0.67a

0.37d
0.51c
0.63b
0.65b
0.72a

0.17d
0.32c
0.40b
0.41b
0.43a

CK = no fertiliser; 100% CF = 100% chemical fertiliser of N, P and K; OM = organic matter.

values followed by the same letter within each column do not dier signicantly at P < 0.05.

b) Mean

Fig. 4 Invertase (a), protease (b), urease (c), acid phosphomonoesterase (d), dehydrogenase (e), and catalase (f) activities in dierent water-stable aggregates. Error bars represent
standard errors of the means (n = 3). Data designated by the same letter(s) do not dier signicantly at P < 0.05, as determined by Fishers least signicant dierence test. CK =
no fertiliser; 100% CF = 100% chemical fertiliser of N, P and K; OM = organic matter.

116
Y. R. LIU et al.

FERTILISER EFFECT ON ENZYME ACTIVITY IN AGGREGATES

results showed that long-term combined OM + CF

fertilisation promoted the formation of water-stable
macroaggregates from microaggregates, improving the
fundamental composition of WSA and soil physical
structure. This eect was probably due to the microbial production of polysaccharides induced by the
addition of organic substrates, which can increase aggregate cohesion (Bandyopadhyay et al., 2010). Six et
al. (1999) and Yang et al. (2007) similarly found that
organic residues stimulated the formation of macroaggregates.
Aggregate stability, expressed as MWD, was increased by long-term fertilisation, and OM + CF treatment was more eective than 100% CF treatment
(Fig. 2). Several studies have reported similar results
(Tripathy and Singh, 2004; Hati et al., 2007; Singh et
al., 2007; Bandyopadhyay et al., 2010). This eect may
be related to the high SOM contents of OM-treated
soils. Thus, long-term combined application of OM
and CF increased the stability of WSA.
As anticipated, fertilisation with OM increased the
TC and TN contents of dierent WSA (Fig. 3). When
high C:N materials, such as vetch and pig manure,
were applied to the soil, microaggregates were bound
together by SOM into macroaggregates; C and N concentrations thus increased with increasing aggregate
size classes and macroaggregates had higher C and N
contents than microaggregates (Elliott, 1986). Similar results have been reported for other agricultural
soils (Whalen and Chang, 2002; Hao et al., 2003) and
in many long-term experiments (Christensen, 1996;
Smith et al., 1997). In the present study, the highest
TC and TN contents were found in the 1.0002.000mm fraction and the lowest were found in the < 0.053mm fraction. Aoyama and Kumakura (2001) found
that the application of animal manure increased SOM
and, consequently, the formation of macroaggregates
(0.2501.000 mm). They also found that manure application increased the accumulation of macroaggregateprotected C and thus sequestered more organic C
in soils. Other researchers (Mikha and Rice, 2004;
Kong et al., 2005; Marx et al., 2005; Lagomarsino
et al., 2009) have also reported higher concentrations
of C and N in macroaggregates than in microaggregates. However, Stemmer et al. (1998), Kandeler et
al. (1999b), and Poll et al. (2003) found that TC and
TN concentrations increased with diminishing particle
size. The use of dierent aggregate separation methods
and soil types may explain these conicting results.
Generally, the application of CF and OM increased
enzyme activities in dierent aggregate fractions, and
fertilisation with OM was more eective (Fig. 4). Poll

117

et al. (2003) reported that the addition of farmyard

manure enhanced invertase activity in dierent fractions in comparison with control samples. Nayak et al.
(2007) reported that dehydrogenase and urease activities could be ranked as compost + inorganic fertiliser
> compost > inorganic fertiliser > control. The application of fertiliser, particularly OM and fertiliser, has
been found to stimulate microbial growth (Bandyopadhyay et al., 2010) and thus enzyme production (Nannipieri et al., 2002). However, fertilisation also stimulated plant root development and possibly enzyme secretion by roots. Thus, both plant and microbial cells
could have contributed to the increase in enzyme activities.
Enzyme activities were generally higher in macroaggregates (acid phosphomonoesterase in 0.2501.000
mm, dehydrogenase in 2.0005.000 mm and the others
in 1.0002.000 mm) than in microaggregates (< 0.053
mm). The distribution of enzyme activities mainly followed the distribution of TC and TN. Allison and Jastrow (2006) observed that degradative enzyme activities were greatest in C-rich fractions. Marx et al.
(2005) found a positive and signicant relationship
between hydrolase activities and C concentrations in
size-classed grassland soil particles. This relationship
is reasonable for fractions with abundant labile C, such
as the > 200-m particle size class of Marx et al.
(2005), the particulate OM fractions of Allison and Jastrow (2006), and the macroaggregates in the present
study. Active microbes may use the newly introduced
and more labile C in growth and enzyme production,
thereby increasing enzyme activities in macroaggregates (Gregorich et al., 1996). The high TC and TN
contents of coarser sand fractions may explain the high
levels of enzyme activity observed in these fractions.
During degradation, the amounts of C and N available
for microbial growth and enzyme synthesis decrease
(Ladd et al., 1996), which may explain the lower levels
of enzyme activity in microaggregate than in macroaggregate fractions. The distribution pattern of acid
phosphomonoesterase that we observed was similar to
the ndings of Rojo et al. (1990), who observed that
phosphatase activity was associated mainly with large
soil fractions (1002 000 m). Marx et al. (2005) also
found that carbohydrase predominated in the coarser
fractions, whereas phosphatase and leucine aminopeptidase were predominant in the clay-sized fraction. Enzyme activities in macroaggregates were probably associated with microbial cell activity in microenvironments with available organic substrate, whereas enzyme activities in microaggregates may have been due
to the stabilisation of extracellular enzymes through

118

their association with surface reactive particles (Nannipieri et al., 2002).

Although we found obvious dierences in enzyme
activities in WSA that received dierent fertilisation
treatments, we observed no consistent trend in individual soil enzyme activity. Garca-Ruiz et al. (2008)
reported that the use of the GMea was an appropriate
means of condensing a set of soil enzyme values into
a single numerical value. We thus used the GMea as
a synthesising index of soil enzyme activity quality.
The GMea values of dierent WSA of OM-treated soil
were signicantly higher than those of WSA of 100%
CF- and CK-treated soils (Table I), probably due to
the increased SOM in soils that received OM + CF
treatments. These results indicate that the long-term
application of OM with CF can improve enzyme activity in dierent WSA.
CONCLUSIONS
The long-term combined application of OM and
CF signicantly increased the percentage of macroaggregates, decreased the percentage of microaggregates,
and enhanced the MWD of WSA, indicating that longterm additions of OM can improve soil physical structure. Invertase, protease, urease, phosphatase, dehydrogenase, and catalase activities were higher in
macroaggregates and lower in microaggregates, according to the distribution of TC and TN. OM improved
enzyme activities in WSA in comparison with 100%
CF treatment. The GMea values of dierent WSA of
OM-treated soils were signicantly higher than those
of 100% CF- and CK-treated soils, indicating that the
long-term application of OM with CF can improve enzyme activity in dierent WSA. Future research should
investigate the status of enzyme activity (associated
with active soil microbial cells or stabilised extracellular enzymes) and the origin (microbial, plant, or animal) of these soil enzymes.
ACKNOWLEDGEMENT
We would like to thank Prof. P. Nannipieri of the
University of Florence, Italy, for his careful revisions
of the manuscript.
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