Journal of Environmental Protection, 2011, 2, 655-661
doi:10.4236/jep.2011.25075 Published Online July 2011 (http://www.scirp.org/journal/jep)
Implications of Secondary Treated Distillery
Effluent Irrigation on Soil Cellulase and Urease
Activities
Devendra Mani Tripathi, Smriti Tripathi, B. D. Tripathi*
Banaras Hindu University, Varanasi, India.
Received March 30th, 2011; revised May 3rd, 2011; accepted June 14th, 2011.
ABSTRACT
Currently distillery effluents have attracted worldwide attention for their application in agricultural land. The present
investigation deals with the effect of application of various dosages of distillery effluent irrigation on soil physico-
chemical, Cellulase and Urease activities in a tropical agricultural field. Experiment was designed in factorial model
by using randomized block design. Soil cores were sampled from the selected pits of both polluted and non polluted
(control) sites. Majority of soil physicochemical properties (e.g. silt, clay, electrical conductivity, organic matter, total
nitrogen contents, cellulase and urease activities) were significantly higher in the samples from polluted site than the
non polluted site just after 15 to 30 days of incubation. Although application of effluents at lower rate substantially in-
creased the enzyme activities, the same decreased at high effluent concentration. Prolonged incubation period resulted
in gradual suppression of enzyme activity in both polluted and nonpolluted soil samples. Thus, the present investigation
suggest that with the passage of time substrate for enzyme activity decreases which in association with residual toxicity
resulted in the reduced enzyme activity.
Keywords: Distillery Effluent, Cellulase, Urease, BOD, COD, Electrical Conductivity
1. Introduction and Methods
Application of distillery effluent on degraded soils is one
of the most economical resources for the soil fertility
amelioration through improvement in soil water-holding
capacity, texture, structure, nutrients retention, roots pe-
netration, and reduction in soil acidity (O’Brien et al.
2002; Aravena et al. 2007; Rato Nunes et al. 2008). Now
a day in our country due to the increasing number of
sugar mills and distillery units, application of distillery
effluent on soil nearly become mandatory.
However, its application in soil also results in environ-
mental problems (Cruz et al. 1991) because apart from
organic content and nutrients, sludge also includes heavy
metals, colored compounds, dissolved inorganic salts,
chlorinated lignin, and phenolic derivatives (Chandra et
al. 2004). These compounds may change soil physico-
chemical properties and soil enzyme activities. Soil enzy-
mes activities play an essential role in catalyzing reac-
tions which are necessary for the decomposition of or-
ganic matter and nutrient cycling in ecosystems, involv-
ing a range of plants, microorganisms, animals and their
debris (Johansson et al., 2000). Therefore, changes in en-
zymes activity could alter the availability of nutrients for
plant uptake and these changes are potentially sensitive
indicators of soil quality (Ajwa et al., 1999; Albiach et
al., 2000).
Dick and Tabatabai (1992) expressed that measure-
ments of several enzymatic activities have been used to
establish indices of soil biological activity. Cellulase and
Urease are the two important enzymes which play a sig-
nificant role in soil environment. Cellulase is a core en-
zyme which contains exo, endo and β-glucosidases. This
enzyme synergistically acts on cellulose, the most abun-
dant polysaccharide of plant cell walls and representing
significant input to soils (Richards, 1987). Urease cata-
lyzes the hydrolysis of urea and amides to carbon dioxide
and ammonia. It acts on carbon-nitrogen (C-N) bonds
other than peptide linkage (Bremner and Mulvaney, 1978;
Karaca et al., 1999). Urease is a constitutive intracellular
enzyme with three subunits of α, ß and γ and two nickel
ions. Furthermore, liberation of these enzymes by mi-
crobes during litter decomposition may be influenced by

Implications of Secondary Treated Distillery Effluent Irrigation on Soil Cellulase and Urease Activities 656
too many factors like temperature, pH and substrate con-
centration in the soil environment (Linkins et al., 1984).
Therefore, the main objectives of the present study
were to evaluate the effect of different application rates
of distillery effluent, on Urease and Cellulase activity in
the test and the control soil samples.
2. Materials and Methods
2.1. Soil Collection and Soil Physico Chemical
Property measurement
This study was performed at the agricultural farm at the
Narang distillery (26°52' N, 82°08' E and 98 m above the
mean sea level) in northern India. Figure 1, indicates the
sampling site. Experiment was designed in randomized
block design with four replicate plots of 5 m × 5 m size
for various doses of effluent amendment. Likewise four
un-amended plots were also established as control. Com-
posite soil samples were collected from A-horizon (0 - 20
cm soil depth) of the plot without any crop at different
time intervals from the experimental field. The experi-
mental soil is an inceptisol with a pale brown colour, and
sandy loam texture. Ten soil samples were collected and
composited into one sample then packed in sterile poly-
thene bags and were stored at 4˚C in the dark and main-
tained at 50% water holding capacity then it was sieved
through a 2 mm-pore size sieve before use. The physico-
chemical properties of amended and un-amended soil,
including organic matter, cations exchange capacity etc.
were estimated using standard methods (Kalra et al. 1988,
APHA 2005). Moisture content was determined by wet
oxidation method. Soil pH was determined using an
electrode and a 1:1 soil/water mixture (Thomas, 1996).
Electrical conductivity was estimated by the addition of
100 ml of water to 1 g of soil sample in the conductivity
meter. The method described by Johnson and Ulrich
(1960) was employed for estimating 70% water holding
capacity. Organic C and Total Nitrogen content was mea-
sured by using the Walkely and Black method (Nelson
and Sommers, 1996), and Microkjeldhal method (Jack-
son, 1973), respectively. The extractable heavy metal
concentrations in soil samples were measured by atomic
absorption spectrometry after extraction with aquaregia.
Figure 1. Sampling site.
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Implications of Secondary Treated Distillery Effluent Irrigation on Soil Cellulase and Urease Activities657
2.2. Enzyme Activity Measurement
Both the amended and control soils were moistened to
70% soil water holding capacity and incubated for 90
days, at 28 ± 4˚C in a large size vessel. Soil samples in
triplicate were taken after 0, 15, 30, 45, 60, 75 and 90
days of incubation. For Cellulase activity measurement, 5
g of soil samples were treated with 0.5 ml of toluene in
50 ml Erlenmeyer flasks, mixed thoroughly, and after 15
minutes treated with 10 ml of acetate buffer of 0.5 M (pH
5.9) and 10 ml of 1% carboxy methyl cellulose (CMC).
After 30 min of incubation, approximately 50 ml of dis-
tilled water was added, filtered by Whatman No.1 filter
paper and the volume of the filtrate was made upto 100
ml with distilled water (Pancholy and Rice 1973). The
resultant filtrate was used for the determination of re-
ducing sugar content by Nelson-Somagyi method (1944)
in a digital spectrophotometer. Cellulase activity was ex-
pressed in terms of micrograms of Glucose Equivalents
per g of soil per 30 min (µg GE g–1 30 m–1).
For Urease activity, determination citrate buffer (0.75
ml) of pH 6.7, 1 ml of 10% urea substrate solution and
0.25 ml toluene were added to 1 g of soil sample and
incubated for 3 h at 37˚C. The formation of ammonium
was determined spectrophotometrically and the results
were expressed as μg NH4+ g-1 dry soil (Hoffmann and
Teicher 1961).
3. Results and Discussions
3.1. Soil Physicochemical Characters
Effluent discharged soil samples underwent significant
changes (Table 1 and Table 2) in all measured parame-
ters in comparison to control. Soil texture in terms of
percentage of sand, silt and clay were 31.6, 56.4 and 12.0
in the control soils, respectively. The results indicated
that distillery effluent discharged soil had relatively higher
clay and silt contents than the control soil Other studies
have found the same, like long term application of sew-
age effluents (Abdelnainm et al., 1987) and cotton gin-
ning mill effluents (Narasimha et al., 1999). However,
increased water holding capacity and electrical conduc-
tivity in the test soil may be due to accumulation of or-
ganic wastes and salts in the distillery effluents. Likewise,
similar results were observed in soils discharged with
effluents from cotton ginning mills (Narasimha et al.,
1999), paper mills (Medhi et al., 2005) and sewage irri-
gated soils (Renukaprasanna et al., 2002). The pH of all
the test samples increased upto 8.2 from 6.8 upon the
release of effluents. Electrical conductivity, organic mat-
ter, Phosphate, Sulphate, Phenol and Total Nitrogen con-
tents were higher in the test samples over the control
samples and were 2.02 mS·cm–1, 3.72 %, 432 mg·kg–1,
321.36 mg·kg–1, 102.45 mg·kg–1 and 116.56 mg·kg–1 of
Table 1. Physico-chemical characteristics and metal contents in Distillery effluent.
Parameter Values for Distillery Effluent
pH 9.2 ± 0.45
COD (mg·L−1) 56800 ± 127.83
BOD (mg·L−1) 22500 ± 213.54
TSS (mg·L−1) 12560 ± 315.56
TDS (mg·L−1) 13760 ± 229.56
Sulphate (mg·L−1) 785.80 ± 34.67
Phosphate (mg·L−1) 626.65 ± 58.34
Potassium (mg·L−1) 369.88 ± 74.83
Chloride (mg·L−1) 278.67 ± 26.78
Total Nitrogen (mg·L−1) 412.78 ± 15.89
Electrical Conductivity (mS·cm−1) 15.84 ± 1.05
Copper (0.20)* (mg·L−1) 2.78 ± 0.63
Cadmium (0.01)* (mg·L−1) 3.56 ± 0.04
Chromium (0.10)* (mg·L−1) 5.88 ± 0.56
Zinc (2.0)* (mg·L−1) 34.27 ± 1.49
Iron (5.0)* (mg·L−1) 218.78 ± 7.98
Nickel (0.20)* (mg·L−1) 7.89 ± 0.85
Manganese (0.20)* (mg·L−1) 4.92 ± 0.43
Lead (5.0)* (mg·L−1) 2.56 ± 0.18
All the values are means of three replicates ±S.D; *Permissible limits for metals in agricultural land irrigation water (Rowe and Abdel-Magid,1995).

Implications of Secondary Treated Distillery Effluent Irrigation on Soil Cellulase and Urease Activities 658
Table 2. Physicochemical properties of experimental unamended soil and soil amended with different dosages of distillery
effluent (values represent mean n=3 ± SE). All values presented in mg·kg-1 exce pt elec tric al conductivity (mS·cm-1) and pH.
Parameter Soil 10% 50% 100%
pH 6.8 ± 0.27 7.3 ± 0.17 8.47 ± 0.35 8.2 ± 0.31
Sand (%) 31.6 ± 2.56 18.35 ± 0.54 13.00 ± 1.05 8.5 ± 1.9
Silt (%) 56.4 ± 3.56 69.06 ± 2.67 71.50 ± 4.76 73.87 ± 4.22
Clay (%) 12.0 ± 1.05 12.75 ± 0.81 15.46 ± 1.54 18.20 ± 2.50
Moisture (%) 70 ± 1.67 71 ± 1.62 72 ± 3.43 74 ± 3.22
Organic matter (%) 1.09 ± 0.03 1.89 ± 0.056 2.86 ± 0.86 3.72 ± 0.75
EC (mS·cm-1) 1.55 ± 0.024 1.85 ±0.12 1.93 ± 0.083 2.02 ± 0.12
Phosphate (mg·kg-1) 265 ± 2.88 316 ± 3.27 383 ± 6.92 432 ± 8.43
Sulphate (mg·kg-1) 55.68 ± 1.53 188.67 ± 1.82 278.50 ± 6.45 321.36 ± 8.77
Phenol (mg·kg-1) 3.09 ± 0.21 34.78 ± 1.47 85 .02 ± 8.82 102.45 ± 11.23
Total Nitrogen (mg·kg-1) 56.76 ± 2.34 73.34 ± 2.66 92.32 ± 7.33 116.56 ± 13.08
Sodium (mg·kg-1) 13.67 ± 0.82 29 .14 ± 0.85 48.45 ± 2.18 69.44 ± 2.07
Chloride (mg·kg-1) 96.72 ± 1.51 109.28 ± 3.57 168.45 ± 8.89 211.14 ± 9.19
Magnesium (mg·kg-1) 9.48± 0.32 9.88 ± 0.48 14.42 ± 0.77 17.22 ± 0.63
Calcium (mg·kg-1) 11.55 ± 0.21 19.33 ± 0.64 45.3 ± 2.21 62.12 ± 3.46
Aluminum (mg·kg-1) 3.94 ± 0.27 4.34 ± 0.47 8.12 ± 0.34 10.88 ± 0.64
Potassium (mg·kg-1) 44.39 ± 0.017 72.82 ± 0.13 108.55 ± 0.41 183.33 ± 0.88
Cadmium (mg kg-1) 0.06 ± 0.0021 0.32 ± 0.016 1.02 ± 0.042 1.38 ± 0.04
Chromium (mg·kg-1) 0.89 ± 0.014 2.88 ± 0.065 2.92 ± 0.12 3.08 ± 0.07
Copper (mg·kg-1) 2.25 ± 0.043 18.22 ± 0.78 62.88 ± 3.27 88.17 ± 4.77
Iron (mg·kg-1) 3.73 ± 0.069 79.12 ± 0.67 114.34 ± 2.11 173.74 ± 3.38
Manganese (mg·kg-1) 1.86 ± 0.047 32.66 ± 0.48 78.55 ± 1.94 112.55 ± 2.7
Nickel (mg·kg-1) 1.11 ± 0.021 23.66 ± 0.63 39.33 ± 0.87 75.6 ± 1.34
Zinc (mg·kg-1) 12.18 ± 0.15 38.65 ± 0.86 76.55 ± 2.24 91.61 ± 2.45
Lead (mg·kg-1) 4.31 ± 0.062 8.86 ± 0.32 23.72 ± 1.28 22.44 ± 1.55
the test against 1.55 mS·cm–1, 1.09%, 256 mg·kg–1, 55.68
mg·kg–1, 3.09 mg·kg–1 and 56.76 mg·kg–1 of the control,
respectively.
3.2. Enzyme Activity
Cellulase and Urease activity in the control and the soil
samples treated with various concentrations of effluents
such as 10%, 50% and 100% were observed with the
amendment of substrate and results were depicted in the
Figure 2. Little information is available on the effect of
industrial effluents on soil cellulase activity. In this di-
rection, cellulase activity was enhanced in soils treated
with the effluents of textile and sugar industry (Kannan
and Oblisami, 1990b), cotton ginning mills (Narasimha,
1997), paper mill effluent and amendment addition (Chi-
nnaiah et al., 2002), solid urban waste (Renukaprasanna
et al., 2002) and sodium based black liquor from fiber
pulping for paper making (Xiao et al., 2005) over un-
treated soils. The present results clearly indicate that the
activity of cellulase was greatly enhanced in the distillery
effluent amended soil over the control (Figure 2). By
increasing the soil incubation period, the cellulase activ-
ity was increased upto 30 days interval, and was declined
in all the soil samples. Cellulase activity of the 10 % ef-
fluent amended soil sample at 0 day was 29.45 mg GE
g–1 30 m–1, it was increased by 400 % to 119.22 mg GE
g–1 30 m–1 at 30 days, and later declined by to 48.8 mg
GE g–1 30 m–1 in 90 days. Similar results were also ob-
served in the rest of the concentrations. Control soil was
nearly static. The test sample of 100 percent effluent
treated soil exhibited 16 % more cellulase activity over
the control at 0 day interval, it was 37.88 mg·mg GE g–1
30 m–1 against 32.55 mg mg GE g–1 30 m–1 of the control
soil and same trend was continued at the rest of the in-
cubation periods (Figure 2). Soil treated with 50% ef-
fluent has shown higher activity over 10% and 100%
effluent treated soils. For instance, 50% soil sample
showed 37.88 mg GE g–1 30 m–1 activity at 0 day against
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Implications of Secondary Treated Distillery Effluent Irrigation on Soil Cellulase and Urease Activities659
Figure 2. Cellulase and Urease activity in soil (with sub-
strate) after incubation as influenced by different concen-
trations Distillery effluent (the mean values are presented, n
= 3).
32.55, 29.45 and 22.56 mg GE·g–1 30 m–1 activity of the
control, 10% and 100% samples respectively. In terms of
increasing percentage of activities, the 50% effluent
treated sample has shown 400%, 50% and 15% more
activities over the control, 10% and 100% effluent irri-
gated samples in 30 days, respectively. Similar trend was
also seen at the rest of the incubations. Urease activity
increased rapidly and reached the maximum level some-
what earlier in comparison to cellulase i.e. after 15 to 30
days in treated soils, although it remained constant in
control treatment. The highest activity was in soil treated
with the highest rate of effluent application nearly after
30 days of incubation. For other treatments its activity
decreased significantly in 15 days for lower rate of
treatment and after 30 days for higher rate of treatment.
But the amended soil activity was always higher than
control treatment. However, more interestingly at 75 and
90 days urease activity of distillery effluent amended soil
was lower than control treatment. The highest values of
enzyme activity were observed in high rates of effluent
application, therefore we can express that increases or
decreases of these enzymes is proportional to microbial
biomass and available substrate which would increase by
high organic matter (Nannipieri, 1994). In general, Cel-
lulase and Urease activity can be divided in 2 stages, in
the first stage its activity was dramatically upward and it
was as a result of adding organic matter to the soil and
the second stage lasting to the end of the incubation time
in which a notable reduction in enzyme activity in soil
treated with effluent was observed. For this stage we can
express the effect of toxic compounds and metals. En-
zymatic activity diminished with increasing available
concentration of metals (Tyler, 1974; Kizilkaya et al.,
2004). Increased levels of heavy metals will react of en-
zymes causing inhibition or inactivation of the enzymatic
activity (Nannipieri, 1994). Metals also indirectly affect
soil enzymatic activities by altering the microbial com-
munity which synthesizes enzymes (Kandeler et al.,
1996). The organic matter-heavy metal fractions which
are readily available for plant uptake, occur in organic
matter and soil solutions. This would prevent the heavy
metal from interacting directly with the active sites of
enzyme, thus affecting the enzyme,s activity (Doelman
and Haanstra, 1984). The rapid decomposition of organic
matter which occurs after the application of distillery
effluent to soil increases the proportion of available met-
als as a result of mineralization of organically complexed
metals (Dudley et al., 1986). Decreased activity of cellu-
lase at higher concentrations of effluents may be due to
the exposure of cell free enzyme to highly concentrated
effluent. But, inhibitory effect of organic matter (Gian-
freda 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 de-
creased activity. Similar observation was made by
Sreenivasulu (2005) that, at high concentration of fungi-
cide in soil, the cellulase activity was inhibited. Accord-
ing to Joshi et al. (1993), enzyme activity was greatly
increased in soils high amount of substrate and increased
enzyme activity was positively correlated with fungal,
bacterial number and moisture content of litter. Nonethe-
less, high significant correlation between cellulase activ-
ity and soil respiration was observed by Splading (1979)
and microbial biomass by Kanazawa and Miyashita
(1987) and Donnelly et al. (1990). Additionally, by in-
creasing the effluent concentration in the control sample,
the cellulase activity was increased, maximum at 50%,
there after decreased.
4. Conclusions
The results of the present investigation clearly indicated
that discharge of effluents from distillery has altered the
physicochemical properties and enhanced the cellulase
and urease activity of the soil, but it was declined with
the time. Furthermore, by increasing the effluents con-
centration, the enzyme activity was improved upto 50%
and later decreased. Also, a suitable treatment of distill-
ery effluent is essential to remove heavy metals and other

Implications of Secondary Treated Distillery Effluent Irrigation on Soil Cellulase and Urease Activities 660
toxic organic compounds. This observation, therefore,
greatly warrants a prior treatment of distillery effluents
before discharging on to agricultural land.
5. Acknowledgements
The authors are thankful to the University Grant Com-
mission, New Delhi for their financial assistance and the
Centre for Environmental Science & Technology, BHU,
Varanasi, for technical help.
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