Impact of sugar industry efuents on soil cellulase activity

M. Nagaraju
a,
*
, G. Narasimha
b
, V. Rangaswamy
c
a
Department of Microbiology, National P.G. College, N.G.Os colony road, Nandyal-518502, India
b
Department of Virology, Sri Venkateswara University, Tirupathi-517502, India
c
Department of Microbiology, Sri Krishnadevaraya University, Anantapur-515003, India
a r t i c l e i n f o
Article history:
Received 6 May 2009
Received in revised form
26 August 2009
Accepted 9 September 2009
Available online 9 October 2009
Keywords:
Efuents
Biological
Physicochemical parameters
Cellulase
a b s t r a c t
An assessment on the impact of sugar industry wastes on soil physicochemical and cellulase activity in
waste dump sites, carried out in urban Nandyal, Southern India. Some core samples were collected from
the selected prole pits at different soil horizons of dump (test) and non-dump sites (control). The
experimental results indicated that, most of the physicochemical properties such as silt, clay, electrical
conductivity, water holding capacity, organic matter and total nitrogen contents, microbial population
and cellulase activities were signicantly higher in the test sample than in the control. Furthermore,
though the application of efuents substantially increased the cellulase activity, but was declined at high
efuent concentration. Nevertheless, enzyme activity was gradually dropped upon prolonged incubation
period in all three samples, such as control, test and efuent amended samples.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Soil is one of the most vital natural resources. It produces food
for teeming millions and supplies raw materials for a large number
of industries on which the world economy is sustained. In fact, on
the other hand, progress of civilization and rapid industrialization
brought with it danger of soil pollution. A perusal of the literature
on the discharge of efuents on to the soil (Monanmani et al., 1990;
Kannan and Oblisami, 1990a; Narasimha et al., 1999), strongly
indicates that, they cause marked changes in physicochemical,
biological and enzymatic properties. Thus, the determination of
specic enzyme activity and microbial biomass, together with the
use of chemical soil parameters, seems to be the best approach for
evaluating the state of microbial activity and understanding its
response to cultivation practices and environmental factors.
In this review, Sugar industry is an agro based industry and
discharging of its efuents into surrounding terrestrial and aquatic
systems has become general practice. In fact, efuents majorly
contain considerable amounts of organic and inorganic pollutants
including sugar cane baggage, molasses, carbonates, bicarbonates
etc,. Though other industries have made considerable progress in
their process technology, but sugar industry has been operating
practically the same processes for the last 50 years. Realizing this
fact, besides, the sugar sector has not capitalized the newhigh-tech
system for global economy, no studies have been performed to
evaluate the inuence of sugar industry efuents in relation to
environmental pollution.
An attempt has, therefore, been made to determine the effects of
sugar industry efuents on soil physicochemical, biological prop-
erties and cellulase activity. The specic objectives of the study
were to (i) quantify the activities of cellulase in the test and the
control samples, and (ii) assess the activity in the soil by applying
the efuents of various concentrations.
2. Materials and methods
2.1. Soil collection
Two soils used in the experiments were collected from (top
10 cm) near by surrounding areas of Sri. Rayalaseema Sugar and
Energy Limited, at Ayyaluru Metta village, Nandyal mandal, Kur-
nool district of Andhra Pradesh, India. Prior to testing, the soils
were air-dried, passed through a 2 mm (millimeters) sieve and
stored at 4

C.
2.2. Physicochemical characters
The percentage of coarse fragments (>2 mm) was quantied
from the weight of material retained after sieving the initial soil
cores through a 2 mm sieve (Alexander, 1977). Soil pH was deter-
mined using an electrode and a 1:1 soil/water mixture (Thomas,
1996). Electrical conductivity was estimated by the addition of
* Corresponding author. Tel.: 91 9392227027; fax: 91 8514241158.
E-mail address: raju8875@yahoo.co.in (M. Nagaraju).
Contents lists available at ScienceDirect
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j ournal homepage: www. el sevi er. com/ l ocat e/ i bi od
0964-8305/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2009.09.006
International Biodeterioration & Biodegradation 63 (2009) 10881092
100 ml (milliliter) of water to 1 g (gram) of soil sample in Elico
conductivitymeter. The method described by Johnson and Ulrich
(1960) was employed for estimating 60% water holding capacity.
We quantied organic C and total nitrogen content using the
Walkely and Black method (Nelson and Sommers, 1996), and
Microkjeldhal method (Jackson, 1973), respectively.
2.3. Biological characters
Microbial cultures such as bacteria and fungi, both isolated and
enumerated from two soil samples by taking 1 g of soil sample
separately and serially diluted upto 10
10
. Diluted suspensions of
0.1 ml samples were plated and spread with sterile spreader on
nutrient agar (pH 7.4) and Martin Rose Bengal agar (pH 5.5)
medium for bacteria and fungi, respectively. Nutrient agar plates
were incubated at 37

C (centigrade) for 24 h, where as Martin Rose

Bengal agar medium plates were at 28

C for 7 days. After the
incubation period, colonies appeared on the agar surface and were
counted by colony counter.
2.4. Cellulase activity
Cellulase activity of two soil samples was determined by placing
5 g of each soil sample with 60% water holding capacity in separate
boiling test tubes (25 200 mm) at 28 4

C. The activity of
cellulase was assayed as described by Pancholy and Rice (1973).
Triplicate samples (5 g) of each soil were with-drawn after 0, 10, 20,
30 and 40 days of incubation, placed in 50 ml Erlenmeyer asks and
0.5 ml of toluene was added. Contents in the asks were mixed
thoroughly, after 15 min, 10 ml of acetate buffer of 0.5 M (pH 5.9)
was added and followed by 10 ml of 1% carboxy methyl cellulose
(CMC). After 30 min of incubation, approximately 50 ml of distilled
water was added. Then the suspension was ltered by Whatman
No.1 lter paper and the volume of the ltrate was made upto
100 ml with distilled water. The resultant ltrate was used for the
determination of reducing sugar content by Nelson-Somagyi
method (1944) in Elico digital spectrophotometer. Finally, cellulase
activity was expressed in terms of micrograms of Glucose Equiva-
lents per g of soil per 30 min (mg GE g
1
30 m
1
).
Similarly, cellulase activities of undischarged soils treated with
10, 50 and 100% efuents were also assayed.
3. Results
3.1. Physicochemical characters
Efuent discharged soil samples underwent signicant changes
(Table 1) in all measured parameters in comparison to control. Soil
texture in terms of percentage of sand, silt and clay were 51, 29 and
20 in the test; 64, 24 and 14 in the control soils, respectively. The
above results indicated that test samples had relatively lower sand
and higher silt and clay contents than control samples. The pH of
the test sample was decreased to 7.62 from8.30 upon the release of
efuents. Water holding capacity, electrical conductivity, organic
matter and total nitrogen contents were higher in the test samples
over the control samples and the were 0.34 ml/g, 1.71 mS/cm (micro
siemens cm
1
), 6.432 g/kg (grams kilograms
1
) and 0.22 g/kg of the
test against 0.28 ml/g, 0.24 mS/cm, 3.602 g/kg and 0.14 g/kg of the
control, respectively.
3.2. Biological characters
The microora of both soil samples were enumerated and listed
in Table 2. Three fold higher bacterial and two fold higher fungal
populations were observed in the test soil over the control soil.
3.3. Cellulase activity
The present results clearly indicate that the activity of cellulase
was greatly enhanced in test soil over the control (Fig. 1). By
increasing the soil incubation period, the cellulase activity was
increased upto 30 days interval, and was declined in both samples.
For example, cellulase activity of the test sample at 0 day was
36.66 mg GE g
1
30 m
1
, it was increased by 300% to 110.0 mg GE g
1
30 m
1
at 30 days, and later declined by 30.3% to 76.66 mg GE g
1
30 m
1
at 40 days. Same trend was also noticed in the control soil.
Furthermore, higher activity was recorded in test sample than in
control sample at all incubation periods. For instance, the test
sample exhibited 57% more cellulase activity over the control at
0 day interval, it was 36.66 mg GE g
1
30 m
1
against
23.33 mg GE g
1
30 m
1
of the control soil and same trend was
continued at the rest of the incubation periods (Fig. 1).
Cellulase activity in the control sample treated with various
concentrations of efuents such as 10, 50 and 100% were observed
with the amendment of substrate and results were depicted in
Fig. 2. By increasing the soil incubation periods, cellulase activity
was also increased upto 30 days then the activities were decreased
in all concentrations of the efuents. For example, cellulase activity
of 10% efuent treated soil sample at 0 day was 16.66 mg GE g
1
30 m
1
, this was increased by 500% to 83.33 mg GE g
1
30 m
1
at 30
days and later decreased by 8% to 76.66 mg GE g
1
30 m
1
at 40 days
interval. Similar results were also observed in the rest of the
concentrations (Fig. 2). Moreover, by increasing the concentration
of efuent upto 50% in the soil, cellulase activity was signicantly
increased and then declined. Soil treated with 50% efuent has
shown higher activity over 10 and 100% efuent treated soils. For
instance, 50% soil sample showed 33.33 mg GE g
1
30 m
1
activity at
0 day against 23.33, 16.66 and 26.66 mg GE g
1
30 m
1
activities of
control, 10% and 100% samples respectively. In terms of increasing
percentage of activities, the 50% efuent treated sample has shown
142%, 200% and 125% more activities over the control, 10% and 100%
samples at 0 day, respectively. Similar trend was also seen at the
rest of the incubations.
Table 1
Physicochemical characters of soil as affected by sugar industry efuents.
Characters Control
a
Test
b
Colour Gray Thick black
Odor Normal Bad
pH 8.30 7.62
Texture (%)
Sand 64 51
Silt 22 29
Clay 14 20
Electrical conductivity (mS/cm) 0.24 1.71
Water holding capacity (ml/g) 0.28 0.34
Organic carbon (%) 3.602 6.432
Total nitrogen (g/Kg) 0.14 0.22
a
Soil without sugar industry efuents.
b
Soil polluted with sugar industry efuents.
Table 2
Biological characters
c
of soil as affected by sugar industry efuents.
Microora Control
a
Test
b
Bacteria 64 10
4
192 10
4
Fungi 7 10
4
15 10
4
a
Soil without sugar industry efuents.
b
Soil polluted with sugar industry efuents.
c
Microbial population in terms of colony forming units g
1
of soil.
M. Nagaraju et al. / International Biodeterioration & Biodegradation 63 (2009) 10881092 1089
4. Discussion
In general, organic amendments such as crop residues, animal
manures, logging and wood manufacturing residues, various
industrial organic wastes, sewage wastes, food processing and ber
harvesting wastes, are naturally occurring compounds that are
used as additives to improve soil physical conditions and/or plant
nutrition. One of the possible reasons for improving the soil
properties could be due to organic waste that may contribute to
maintain or increase the organic matter and nutrient content in the
soil (Bollag et al., 2002). Wealth of information is available on
industrial efuents and their inuence on soils. Invariably, with
most studies showing that most efuent application is benecial,
such a role may be limited to situations where the efuent is to be
applied at an amount, or has a composition that has not been
previously tested (Speir, 2002).
In this study, sugar industry efuent discharged soil had rela-
tively higher clay and silt contents than the control soil. Other
studies have found the same, like long term application of sewage
efuents (Abdelnainmet al., 1987) and cotton ginning mill efuents
(Narasimha et al., 1999). However, increased water holding capacity
and electrical conductivity in the test soil may be due to accumu-
lation of organic wastes and salts in the sugar industry efuents.
Likewise, similar results were observed in soils discharged with
efuents from cotton ginning mills (Narasimha et al., 1999), paper
mills (Medhi et al., 2005) and sewage irrigated soils (Renukapra-
sanna et al., 2002). High electrical conductivity was also observed
in soils treated with distillery efuents (Devarajan et al., 2002) and
sodium based black liquor from ber pulping for paper making
(Xiao et al., 2005). In contrast, soils polluted with cement dust from
cement industries had low water holding capacity and high elec-
trical conductivity (Shanthi, 1993; Sivakumar and John De Brito,
1995). The slight drop in the pHof the test soil is explained in terms
of release of efuents with acidic in nature, containing agro based
chemicals from sugar industry. Same was noticed in the discharges
of sugar cane residues from sugar industry (Zende, 1996), applica-
tion of sewage efuents (Bhogal et al., 2002) to soils decreased the
pH. The higher organic matter of the test soil may be due to the
discharge of efuents in an organic nature. Similarly, disposal of
municipal organic compost (Chuasavathi and Trelo-ges, 2001), long
term municipal waste (Anikwe and Nwobodo, 2002), and the
efuents from cotton ginning mills (Narasimha et al., 1999) into
soils, signicantly increased the soil organic matter and total
nitrogen content.
Higher microbial population in the test soil possibly due to the
presence of high organic matter in acidic efuents. Similarly,
Monanmani et al. (1990) and Narasimha et al. (1999) reported that
microbial populations were profusely increased in soils polluted
with alcohol and cotton ginning mills efuents respectively.
Cellulase is a core enzyme, it consists of exo, endo and
b-glucosidases. This enzyme synergistically acts on cellulose poly-
mer substrate, are abundantly available on earth surface in the form
of wood, chips, rocks, municipal wastes. On the other hand, cellu-
lose is the most abundant polysaccharide of plant cell walls and
represents signicant input to soils (Richards, 1987). Cellulose
hydrolysis into glucose is mainly achieved by complex enzyme
cellulase, produced by fungi (Maile and Linkins, 1978). However,
these enzymes are extensively studied in plant litter (Sinsabaugh
and Linkins, 1987; Wood and Bat, 1988; Linkins et al., 1990).
Furthermore, liberation of these enzymes by microbes during litter
decomposition may be inuenced by too many factors like
temperature, pH and substrate concentration (Linkins et al., 1984).
The activity of cellulase was indicated by the substrates like cellu-
lose polymer of cellophane (Markus, 1955; Kiss and Peter, 1959),
cellulose powder (Rawald et al., 1968) and carboxy methyl cellulose
(Kong and Dommergues, 1972). Nevertheless, cellulase activity was
potentially correlated with fungal and bacterial populations in soil
(Joshi et al., 1993).
Little information is available on the effect of industrial efuents
on soil cellulase activity. In this direction, cellulase activity was
enhanced in soils treated with the efuents of textile and sugar
industry (Kannan and Oblisami, 1990b), cotton ginning mills
(Narasimha, 1997), paper mill efuent and amendment addition
(Chinnaiah et al., 2002), solid urban waste (Renukaprasanna et al.,
2002) and sodium based black liquor from ber pulping for paper
making (Xiao et al., 2005) over untreated soils. Similarly, urban
expansion into wild lands signicantly increased the cellulase
activity (Douglas and Oleksyszyn, 2002). Contrary to this, soil
contaminated with cement dust, the cellulase activity was ceased
(Shanthi, 1993).
In this assessment, results showed that the cellulase activity in
the test sample was relatively higher than in the control sample at
all incubations. The increased percentage of cellulase activity of the
test sample range was in between 22 and 57 over the control. Thus,
activity was increased gradually but not signicantly. However,
0
20
40
60
80
100
120
140
0 10 20 30 40
Incubation period in days

g

o
f

g
l
u
c
o
s
e

/

g

o
f

s
o
i
l

/

3
0

m
i
n
Control
10%
50%
100%
* g of glucose g
-1
of soil 30 min
-1
.
** incubation, in minutes, of soil with carboxy methyl cellulose (1% w/w)
Control: Soil without sugar industry effluents.
10%, 50% and 100%: Lab contaminated soils with various concentrations of effluents.
Fig. 2. Cellulase activity* in soil (with substrate) after 30 min** incubation as inu-
enced by different concentrations sugar industry efuents.
0
20
40
60
80
100
120
0 10 20 30 40
Incubation period in days

g

o
f

g
l
u
c
o
s
e

/

g

o
f

s
o
i
l
/

3
0

m
i
n
Control
Test
* g of glucose g
-1
of soil 30 min
-1
.
** incubation, in minutes, of soil with carboxy methyl cellulose (1% w/w).
Control. Soil without sugar industry effluents.
Test. Soil polluted with sugar industry effluents.
Fig. 1. Cellulase activity* in soil (with substrate) after 30 min** incubation as inu-
enced by sugar industry efuents.
M. Nagaraju et al. / International Biodeterioration & Biodegradation 63 (2009) 10881092 1090
increased cellulase activity in soils with efuent discharges may be
due to high availability of substrate, and increased cellulolytic
microorganisms. But the activity was declined with time,
maximumat 30 days, it is probably because of the exhaustion of the
readily available substrate. It has been very well established that
the discharge of efuents from tomato processing unit (Sarade and
Joseph, 1994), cotton ginning mill (Narasimha, 1997), paper mill and
pressmud addition (Chinnaiah et al., 2002), and potassium based
black liquor from straw pulping (Xiao et al., 2005) increased the
cellulase activity in the test over the control sample. Ramakrishna
Parama et al. (2002) reported that the soil treated with urban
wastes along with additives such as cow dung, rock phosphate,
green leaves and coir dust increased the cellulase activity in the
early incubations, later it was stabilized. Similarly, by increasing the
incubation period, cellulase activity in soils treated with and
without fungicide were increased upto 20 days, later were
decreased (Sreenivasulu, 2005). According to Joshi et al. (1993),
cellulase activity was greatly increased in soils treated with cellu-
lose and increased cellulase activity was positively correlated with
fungal, bacterial number and moisture content of litter. Nonethe-
less, high signicant correlation between cellulase activity and soil
respirationwas observed by Splading (1979) and microbial biomass
by Kanazawa and Miyashita (1987) and Donnelly et al. (1990).
Additionally, by increasing the efuent concentration in the
control sample, the cellulase activity was increased, maximum at
50%, there after decreased. Decreased activity of cellulase at higher
concentrations of efuents may be due to the exposure of cell free
enzyme to highly concentrated efuent. But, inhibitory effect of
organic matter (Gianfreda and Bollag, 1994, 1996), high acidity
(Ruggiero et al., 1996) and short living enzymes in the soil environ-
ment (Ahn et al., 2002) are also the reasons for the decreased activity.
Similar observation was made by Sreenivasulu (2005) that, at high
concentration of fungicide in soil, the cellulase activity was inhibited.
5. Conclusion
The results of the present investigation clearly indicated that
discharge of efuents from sugar industry has altered the physi-
cochemical properties, affected the microora and enhanced the
cellulase activity of the soil, but it was declined with the time.
Furthermore, by increasing the efuents concentration, the enzyme
activity was improved upto 50% and later decreased. This obser-
vation, therefore, greatly warrants a prior treatment of sugar
industry efuents before discharging into a water body or on to
agricultural land and additional research will be necessary to
discriminate the type of cellulase producing microorganisms
(genera and species).
Acknowledgements
We wish to thank Dr. S. Imthiyaz Ahamed, Principal, National
P.G. College, Nandyal for providing lab facilities and encouraging us
through out this study.
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