Open Journal of Soil Science, 2013, 3, 235-243 Published Online September 2013 (
Physicochemical and Biochemical Reclamation of Soil
through Secondary Succession
Kamala Haripal, Sunanda Sahoo
Ecology Section, School of Life Sciences, Sambalpur University, Burla, India.
Received June 21st, 2013; revised July 21st, 2013; accepted July 29th, 2013
Copyright © 2013 Kamala Haripal, Sunanda Sahoo. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Conversion of forest to agricultural fields has become a common practice in India. Very often these fields have been
abandoned due to lack of sustainable production. In course of time these fallow lands undergo natural secondary suc-
cession. The present study was carried out to find out the restoration of soil physicochemical and biochemical properties
in a chronosequence of 2 yr, 4 yr, 6 yr, 11 yr, and 15 yr fallow lands. Soil enzyme activities play key roles in the bio-
chemical functioning of soils, including soil organic matter formation and degradation, nutrient cycling, and reflect the
change in soil management and land use. There was gradual improvement in the physical condition and nutrient status
along with increase in soil amylase, cellulase, dehydrogenase, phophatase, and urease activity in the present study with
the progress of fallow age which indicates the importance of natural secondary succession in soil restoration. However
the PCA analysis indicated that natural vegetational succession could reclaim the soil quality and promote ecosystem
restoration but it required a long time under the present local climatic condition.
Keywords: Sustainable Production; Secondary Succession; Soil Restoration; Land Use
1. Introduction
India has 329 million ha of total land area of which 43%
is under cropping and 23% is under forest [1]. From the
beginning human has exploited the natural resources for
agricultural activities. As a result most of the forests have
been converted into agricultural lands. Over recent years
there have been intensive agricultural practice basically
aimed to enhance the productivity to meet the food de-
mands of huge population throughout the world. In spite
of increased population, the productive potential has been
decreased in many areas of tropical countries. Intensive
cultivation leads to reduced soil fertility and increased
soil erosion in many areas of tropics [2]. Specific detri-
mental effects on biological and biochemical characters
of soil quality have also been noted due to changes in mi-
croclimate at the soil surface by tillage and on the rate
and quality of organic matter input to the soil. Therefore,
most of the agro-ecosystems are often abandoned due to
unsustainable agricultural production. Maintenance of
sustainable agricultural production has become a matter
of great concern for the scientists all over the world. In
many instances, the disturbed areas undergo natural re-
covery of vegetation within 5 to 20 years depending on
population pressure and land availability [3].
Soil enzymes (microbial exoenzyme) are recognized
as sensitive indicators of soil health and quality [4] due to
the rapid response to changes in soil management. In par-
ticular, enzyme activities are especially significant in soil
quality assessments because of their major contribution
to degrading organic matter [5]. In fact they have been re-
lated to soil physico-chemical characters, microbial com-
munity structure and disturbance [6]. It has been reported
that any change in soil management and land use is re-
flected in the soil enzyme activities, and that they can
anticipate changes in soil quality before they are detected
by other soil analyses [7]. The studies of soils from dif-
ferent regions indicated that enzyme activities are sensi-
tive to soil changes due to tillage [8], cropping systems
[9], and land use [10]. Most of the studies on soil enzyme
activities in different land use systems have focused on
temperate regions. Few literatures are available regarding
the enzyme activities due to land use changes in tropics
The present study of enzyme activity in soil along a
chronosequence of vegetation regrowth in the fallow
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Physicochemical and Biochemical Reclamation of Soil through Secondary Succession
lands can give insight into the role of soil enzymes in
restoring soil fertility during secondary succession. Cur-
rently, no information is available on microbial biomass
and enzyme activity that affected by conversion of forest
into agricultural land, and then into fallow land in India
except the work done by Maithani et al. [3] on microbial
biomass in 7, 13, and 16 year regrowth of a disturbed
subtropical humid forest in Meghalaya, India, and by
Ralte et al. [13] on microbial biomass and activity in
relation to shifting cultivation and horticultural practices
in subtropical evergreen forest ecosystem of North-East
India. Thus the present investigation was aimed to assess
some key enzyme activities along with some of the se-
lected physico-chemical characters involved in soil res-
toration in a chronosequence of abandoned rice fields in
Western Odisha, India.
2. Materials and Methods
2.1. Study Sites and Climate
The present study was confined to the revenue district
Sambalpur, located in the Western part of Odisha in In-
dia. Prior to 1950s the study sites were dense forest
forming a part of Barapahar forest range. After the estab-
lishment of multipurpose hydroelectric Hirakud Dam
project and subsequent industrialization, urbanization
and increase in population pressure, the district forest
coverage has been reduced to 30% [14]. Most of the for-
est lands were cleared and used for production of agri-
cultural crops (rice, maize, beans and sugarcane). The
climate is tropical monsoonal. The annual average rain-
fall during the study period (August, 2010-July 2011)
was 1597 mm out of which about 80% fell during rainy
season (July-October). The mean air temperature varied
from 5˚C (during December) to 44˚C (during May). The
soils of this district belong to mixed red and black soil,
Red sandy soil, mixed red and yellow lateritic soil [15].
The climatological data during the study period are
shown in Figure 1. In many places of the study area the
rice fields were derived by clearing natural forest which
had been subjected to abandonment by farmers due to
lack satisfactory agricultural production. All the sam-
pling sites were located on the same topographic situa-
tion and with the area of nearly 900 sq meters except 11
yr fallow land which covered 12000 sq meters. The age
of the fallow period was ascertained by asking the land
owners. By interviewing the elderly persons of the local-
ity it was known that the peoples of these localities were
practicing rain fed paddy cultivation since 1975. The
selected study sites were paddy fields abandoned since
1995 (15 yr·F), 1999 (11 yr·F), 2004 (6 yr·F), 2006 (4
yr·F), and since 2008 (2 yr·F).
The 4-year-old field was adjacent to the 6-year-old
field, while the 11-year-old field was adjacent to the
Figure 1. Climate pattern of study sites.
15-year-old field. All these sites were located at the
bottom of hill, Chandili Dunguri, 12 Km towards
south west from Sambalpur University campus at Burla
(20˚43N-20˚11N and 82˚39E-85˚13E longitude, lati-
tude at 263 m above mean sea level) in Sambalpur dis-
trict of Odisha, India.
2.2. Soil Physicochemical Analysis
The soils were sampled from the experimental plots
bimonthly during August 2010- June 2011. The soil
samples were taken by using a cylindrical soil sampler
having a diameter of 20 cm. and five random samples
were taken from 0 - 10 cm, 10 - 20 cm, and 20 - 30 cm
depths. The soil samples were packed in plastic zipper
bag and brought into the laboratory and stored at 4˚C
before analysis. The enzyme analyses were made within
four weeks after sampling because storage beyond two
weeks can cause decline in enzyme activities [16]. The
soil samples were air dried, gently crushed and sieved
through 2 mm sieve and then used for different physi-
cochemical analysis.
The laboratory analyses were conducted for bulk den-
sity and water holding capacity following the method
prescribed in TSBF hand book [17], soil pH by pH meter
using 1:5 soil water suspension, the organic carbon con-
tent by Walkely and Black’s titration method [18], total
nitrogen (TN) by Kjeldhal method [19], the total phos-
phorus by Bray and Kurtz [20] .Soil microbial biomass
carbon was calculated by chloroform fumigation-extrac-
tion method [21].
2.3. Soil Enzyme Activity
Soil amylase & cellulase activity was determined fol-
lowing Mishra et al. [22]. 3 g of soil sample was incu-
bated with 0.2 ml tolune, 6 ml substrate solution (starch
for amylase and carboxymethyl cellulose for cellulase)
and 6 ml Sorenson’s buffer (pH = 5.9) for 24 hr at 34˚C.
After incubation the reducing sugar test was performed
by adding 2 ml of 3, 5 dinitrosalicylic acid to 1 ml of
supernatant of incubated mixture in a test tube and al-
lowed to stand for 5 minutes for colour development.
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Physicochemical and Biochemical Reclamation of Soil through Secondary Succession
Copyright © 2013 SciRes. OJSS
The enzyme activity was quantified by spectrophotome-
ter at 540 nm and expressed in µg glucose g1 dry soil
Soil dehydrogenase was measured according to Casida
et al. [23]. 5 g of soil was incubated with 2 ml of 1%
soluble 2, 3, 5-triphenyl tetrazolium chloride (TTC) in a
sealed screw cap test tube at 32˚C for 24 hr. The
triphenyl formazon (TPF) formed in the incubated sam-
ple was extracted by methanol. The extrantant triphenyl
formazon (TPF) was determined by spectrophotometer at
485 nm, using methanol as control.
Acid phosphatase was estimated following the method
of Tabatabai and Bremnar [24] using paranitrophenyl
phosphate (p-NPP) as a substrate. A mixture of 1 g fresh
soil (<2 mm), 4 ml of modified universal buffer (pH 6.5)
and 1 ml of 100 mM p-NPP were incubated in a sealed
100 ml Erlenmeyer flask at 30˚C for 30 min. The sample
was adjusted to 100 ml with deionised water. After incu-
bation the para- nitrophenol (p-NP) was filtered through
Whatman-42 and quantified by spectrophotometer at 400
nm by taking p-NP as control.
Urease activity was determined as described by Dash
et al. [25]. 0.2 ml toluene was added to 5 g soil and 9 ml
of Tris-HCl buffer (pH = 9, 0.2 M) followed by 1 ml of
0.2 M urea (substrate solution) to the sample. The sam-
ples were subsequently incubated at 37˚C for 2 hrs. After
incubation the volume was made up to the mark (50 ml)
by adding KCl-AgSO4. The suspension was centrifuged.
To 1 ml of supernatant, 1 ml of phenol and 1 ml of alka-
line hypo chlorite solution were added. After 5 min, the
released ammonium was measured spectrophotometri-
cally at 625 nm.
2.4. Statistical Analysis
The data were statically analyzed using three way
analysis of variance (ANOVA) with SPSS 10 statistical
software. Pearson’s correlation analysis was performed
to determine the significant level of correlation. The PCA
was done using SX software to discriminate different
sites on the basis of the principal components Z1 and Z2.
3. Results
3.1. Soil Physico-Chemical Properties
Soil physico-chemical properties of the present study
sites have been presented in Table 1. Soil moisture con-
tent increased gradually along the chronosequence of
abandoned field (Table 1). ANOVA revealed a signifi-
cant impact of fallow period on soil moisture content.
Table 1. Soil physico-chemical properties of different abandoned fields at different depths.
Parameters Depth (cm) 2 yr 4 yr 6 yr 11 yr 15 yr
0 - 10 5.17 ± 2.39 6.55 ± 3.05 7.96 ± 3.43 9.44 ± 4.32 14 ± 4.91
10 - 20 4.70 ± 1.64 5.9 ± 2.34 7.24 ± 2.63 8.76 ± 3.62 12.12 ± 3.95
Moisture (%)
20 - 30 3.57 ± 1.18 4.76 ± 2.02 5.94 ± 2.56 7.84 ± 3.15 10.39 ± 3.52
0 - 10 1.53 ± 0.065 1.48 ± 0.061 1.42 ± 0.06 1.35 ± 0.067 1.25 ± 0.08
10 - 20 1.57 ± 0.051 1.52 ± 0.054 1.48 ± 0.063 1.41 ± 0.079 1.33 ± 0.074 Bulk density g·cm3
20 - 30 1.60 ± 0.054 1.56 ± 0.049 1.52 ± 0.054 1.48 ± 0.048 1.39 ± 0.098
0 - 10 24.84 ± 4.03 27.10 ± 3.92 30.16 ± 4.19 32.47 ± 4.50 36.72 ± 4.52
10 - 20 21.34 ± 2.87 24.00 ± 3.00 27.09 ± 3.25 29.52 ± 3.75 34.61 ± 4.03 WHC (%)
20 - 30 19.72 ± 2.54 22.32 ± 2.16 23.68 ± 2.83 28.16 ± 3.23 33.17 ± 3.72
0 - 10 5.56 ± 0.08 5.75 ± 0.12 6.00 ± 0.27 6.45 ± 0.11 6.50 ± 0.12
10 - 20 5.35 ± 0.39 5.80 ± 0.07 5.50 ± 0.14 5.64 ± 0.04 5.74 ± 0.03 pH
20 - 30 5.40 ± 0.18 5.45 ± 0.15 6.07 ± 0.16 6.17 ± 0.12 6.25 ± 0.14
0 - 10 6.17 ± 1.55 6.34 ± 1.61 8.7 ± 2.11 11.49 ± 2.82 13.43 ± 2.9
10 - 20 5.42 ± 1.23 5.02 ± 1.3 7.61 ± 1.68 9.78 ± 2.55 11.25 ± 2.89 SOC mg·g1
20 - 30 4.64 ± 0.96 4.24 ± 1.43 6.02 ± 1.81 8.18 ± 2.37 9.57 ± 2.62
0 - 10 0.66 ± 0.17 0.74 ± 0.18 0.91 ± 0.17 1.09 ± 0.23 1.19 ± 0.22
10 - 20 0.06 ± 0.13 0.64 ± 0.12 0.86 ± 0.13 0.98 ± 0.22 1.07 ± 0.23 TN mg·g1
20 - 30 0.53 ± 0.1 0.57 ± 0.13 0.72 ± 0.17 0.89 ± 0.2 0.93 ± 0.18
0 - 10 105.5 ± 32.12 155.36 ± 65.47 274.15 ± 95.26 398.97 ± 114.55 513.78 ± 109.57
10 - 20 81.04 ± 24.49 115.40 ± 42.45 220.35 ± 82.01 290.47 ± 85.44 364.24 ± 92.64 MBC µg·g1
20 - 30 58.93 ± 17.91 84.97 ± 31.11 158 ± 71 214.39 ± 71.75 280.03 ± 100.19
Physicochemical and Biochemical Reclamation of Soil through Secondary Succession
Figure 2. Amylase activity in soil of different abandoned
fields during different months and at different depths.
Decreasing level of soil moisture was found with in-
crease in depth irrespective of fallow period. The bulk
density ranged from 1.25 g·cm3 in the surface soil layer
(0 - 10 cm) of 15 yr·F land to 1.6 g·cm3 in the subsur-
face layer (20 - 30 cm) of 2 yr·F land. Lowest water
holding capacity (WHC) was recorded at 20 - 30 cm
depth of 2 yr abandoned field (19.72%) and highest
WHC was recorded in 0 - 10 cm depth of 15 yr aban-
doned field (36.7%). ANOVA revealed significant dif-
ference of WHC between different fallow lands and be-
tween depths at p < 0.001.
The soil pH was acidic in nature and varied from 5.35
to 6.28. The pH value increased over time since aban-
donment and highest value was seen in the upper soil
layer (0 - 10 cm) of 15 yr·F land. The soil organic matter
increased significantly with increasing year of abandon-
ment being highest in 0 - 10 cm depth of 15 yr aban-
doned field ( 13.43 mg·g1 ) and lowest in 20 - 30 cm
depth of 2 yr·F lands (6.17 mg·g1). Similar trend was
Figure 3. Cellulase activity in soil of different abandoned
fields during different months and at different depths.
observed for total nitrogen in the soil. The Soil microbial
biomass carbon ranged from 104.50 to 513 µg·g1 in top
soil (0 - 10 cm), 81.04 to 364 µg·g1 of soil in 20 - 30 cm
depth and 58.93 to 280 µg·g1 in 20 - 30 cm soil depth.
3.2. Soil Enzyme Activity
Figures 2-6 show amylase, cellulase, dehydrogenase,
phosphatase and urease activities respectively in relation
to sites, depth and season. Highest activities of all the
enzymes were observed in top soil (0 - 10 cm), and were
found to be decreasing with increasing depths. Seasonal
variations in the enzyme activities in different sites indi-
cated a peak during August (wet season) and lowest en-
zyme activities was observed in dry season i.e. during
April. All the enzyme activities increased with the in-
crease in years of abandonment. ANOVA revealed sig-
nificant difference between sites, depths, and between
different seasons at p < 0.001. Pearson’s correlation
analysis indicated significant correlation between en-
Copyright © 2013 SciRes. OJSS
Physicochemical and Biochemical Reclamation of Soil through Secondary Succession 239
Figure 4. Dehydrogenase activity in soil of different aban-
doned fields during different months and at different
zyme activities and soil physico-chemical parameters
(Table 2).
4. Discussion
4.1. Soil Physico-Chemical Properties
In the present study, soil was slightly acidic ranging
from 5.58 to 6.58. Leaching process tends to acidify
soils and partially offset by plant growth [26]. Gradual
establishment of plants with increasing year of aban-
donment is supposed to prevent much of the leaching in
WHC and decrease the bulk density. As the fallow pe-
riod gets extended, the inputs of organic residues from
colonizing plants increased the carbon and nitrogen
contents of soil. During the natural restoration in 3, 7,
10 year abandoned paddy fields an increase in pools of
organic matter, total nitrogen, exchangeable K, Ca, Mg
and pH was observed with increase of field age [27].
This supports the present findings. Microbial biomass
Figure 5. Phosphatase activity in soil of different abandoned
fields during different months and at different depths.
reflects the degree of immobilization of C and N in soil.
Gradual increase in microbial biomass carbon (MBC)
was recorded in the present study with a maximum
amount in the top soil of 15 yr·F land (513.78 µg·g1).
This may be attributed to greater input of plant detritus
that is ultimately incorporated into the soil and improves
the nutrient pool thereby increases the MBC [28]. The
MBC dynamic was significantly correlated with soil or-
ganic carbon content during the secondary succession as
reported by Arunachalam and Pandey [29]. The present
study supports the above findings. This result indicated
that organic matter played a pivotal role in the building
up and development of soil microbial biomass [30].
4.2. Soil Enzyme Activity
Soils enzymes are major indcators of microbial activity, i
Copyright © 2013 SciRes. OJSS
Physicochemical and Biochemical Reclamation of Soil through Secondary Succession
Copyright © 2013 SciRes. OJSS
Table 2. Pearson’s Correlation coefficients between soil properties.
SM 1
WHC 0.45** 1
BD 0.34** 0.71** 1
pH 0.41** 0.54** 0.51** 1
OC 0.85** 0.56** 0.51** 0.51** 1
MBC 0.85** 0.6** 0.54** 0.61** 0.90** 1
TN 0.86** 0.55** 0.47** 0.49** 0.97** 0.89** 1
AML 0.86** 0.55** 0.47** 0.51** 0.86** 0.88** 0.85** 1
CEL 0.83** 0.59** 0.6** 0.48** 0.81** 0.83** 0.81** 0.89** 1
DEH 0.82** 0.54** 0.44** 0.4** 0.81** 0.76** 0.81** 0.86** 0.86** 1
PHO 0.85** 0.64** 0.59** 0.53** 0.92** 0.88** 0.91** 0.87** 0.86** 0.87** 1
URE 0.89** 0.57** 0.52** 0.49** 0.85** 0.88** 0.84** 0.92** 0.93** 0.87** 0.89** 1
**Significant at p < 0.01; SMSoil moisture, WHC Water holding capacity, BDBulk density, OCOrganic carbon, TNTotal nitrogen, AMLAmylase activity, CELCellulase
activity, DEHDehydrogenase activity, PHOPhosphatase activity, UREUrease activity.
and their activities always depend upon soil types, vege-
tation cover, microbial biomass, and microbial diversity
during vegetation succession [31]. Dehydrogenase is an
intracellular enzyme that exists only in viable microbial
cells and it is considered as an index of microbial activity
[32]. In the present study the enzyme activities showed
peak value in rainy season (August) and lowest in sum-
mer (April). Some hydrolases have a tendency to in-
crease in the rainy season [33]. During summer, due to
moisture stress, the microbes are in dormant state and
their activities become low, but the entry of rainy season
caused spurt in flora and microbial population, and thus
increased the enzyme activities. Prolonged precipitation
along with enhanced plant growth and rhizoid position
result in increased extracellular activities [34,35]. The
activity of dehydrogenase, phosphatase and urease was
found to decrease in summer [36]. Thus the present find-
ing is in agreement with the above observations.
There was significant increase in extracellular en-
zymes like amylase, cellulase, phosphatase, urease, and
intracellular enzyme i.e. dehydrogenase with increasing
year of fallow period and significant decrease with in-
crease in soil depth. Significantly positive correlation
was found between all the enzyme activities with organic
carbon, total nitrogen and microbial biomass carbon (Ta-
ble 2). Increasing age of the land with respect to time
caused gradual addition of organic matter to the soil
through decomposition of shoot and root biomass of dif-
ferent vegetation developed on the surface layer. Higher
organic matter content provides higher substrate which
acts as energy source for microorganisms and supports
high amount of microorganism, hence higher enzyme
activities [30]. Later succession exhibited higher enzyme
activity than the early period and upper soil layer showed
higher enzyme activity than lower layer of soil. The de-
crease in these enzyme activities with depth might be
attributed to the diminution of biological activity down
the soil profile [37].
The data on physico-chemical parameters and enzyme
activities of present study and the data of natural forest,
natural grassland and crop field obtained in the same
topography [38] were subjected to principal component
analysis. Figure 7 illustrates the discrimination of differ-
ent sites on the basis of Z1 and Z2 whose cumulative per-
cent variance was 85.9%. The PC separated the 2 yr, 4yr,
6 yr, 11 yr, 15 yr fallow far away from grassland and
crop fields. However the 15 yr F is quite nearer to natural
forest which indicates that the chronosequence of aban-
doned fields showed a general trend of reclamation in
terms of some of the soil Physico-chemical and bio-
chemical parameters but still required more time to
achieve the soil status of natural forest, crop fields, and
grasslands. Self regeneration of vegetation on a degraded
land in tropics has relatively better reclamation potential
Physicochemical and Biochemical Reclamation of Soil through Secondary Succession 241
Figure 6. Urease activity in soil of differe nt abandoned fields
during different months and at different depths.
Figure 7. PCA analysis of different study sites.
than that of plantation species [39]. Thus the study justi-
fied that the degraded ecosystems derived from natural
forest when subjected to abandonment showed the symp-
toms of reclamation during the course of natural second-
dary succession.
5. Acknowledgements
The authors are grateful to Prof N. Behera, Head, School
of Life Sciences, Sambalpur University for providing ne-
cessary facilities to carry out the present work. Also the
authors are thankful to Dr. S K Pattanayak, P G De-
partment of Environmental Science, Sambalpur Univer-
sity for some of his important suggestions and comments
during the preparation of the manuscript.
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