Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 641-649
ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 641-649
http://www.ijcmas.com
Original Research Article
Effect of Cement Dust Pollution on Microbial Properties and Alkaline
Phosphatase Enzyme Activity in Soil
S.Kulandaivel*, S.Nagarajan, A.Priyanga, R.Saravanapandian and A.Thangarani
Department of Zoology and Microbiology, Thiagarajar college, Madurai-625 009, India
*Corresponding author
ABSTRACT
Keywords
Alkaline
phosphatase,
Cement dust
soil,
Bacteria,
Fungi
Totally 10 different soil samples were collected from two cement factory areas (5
from Sankar cement factory,Thalaiyuthu and 5 Ramco cement factory from
Thulukkapatti) of Tirunelveli. It was named as SC 1-5 and RC 1-5.Enumerate the
microbial population (bacteria and Fungai) in the soil. Maximum microbial count
was observed in the sample RC-5 (276X102 of bacteria and 20X102 of fungi).
Effect of phosphate source (K2PO4 and CaPo4) for alkaline phosphatase production
was analysed by batch fermentation. In our study, 0.03g/100ml of both substrates
have gien maximum enzyme activity (35.61 U/ml and 29.47 U/ml). Alkaline
phosphatase enzyme was optimized at different pH (6-10) and temperature (30-50).
The maximum activity was occurred at the pH9.0 and temperature 40oC.
Introduction
Air pollutants generated by the cement
manufacturing process consist primarily of
alkaline particulates from the raw and
finished materials. The direct effects of
cement dust pollution are the alkalization of
the ecosystem and the changing of the
chemical composition of soil [1]. The main
impacts of the cement activity on the
environment are the broadcasts of dusts and
gases. The pollutant particles can enter into
the soil as dry, humid or occult deposits and
can undermine it physicochemical properties
[2].
cement dust pollution. The most commonly
used microbial activity indicators for soil
health monitoring are microbial biomass,
soil respiration and soil enzyme activity
[3].Soil enzyme activities depend on
optimum condition of moisture, pH and
temperature and substrate concentration.
Soil pH can affect enzyme activity by
influencing the concentration of inhibitors or
activators in the soil solution and the
effective concentration of the substrate.
Enzyme may vary under stress when soil is
concentrated by heavy metals [4-5].
Thus cement dust pollution has a negative
effect on the soil. Soil microbial activity is
important for the nutrient biogeochemical
cycling and it is negatively affected by the
Acid phosphatase, alkaline phosphatase,
arylsulfatase, invertase, amidase and urease
activity in 0 to 7.5 cm surface soil were
significantly greater in soil [6].Alkaline
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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 641-649
phosphatase is a hydrolyzing enzyme
responsible for removal of phosphate group
from different types of organic molecules
like proteins, nucleic acids and alkaloids.
filed, Neem tree, Papaya tree, Mango tree,
Coconut tree, Chilly field, Penndy field and
Brinjal field. In this research, soil sample
were taken from 0-20 cm depth in sterile
poly bags and immediately brought to the
laboratory or stored at 4oc for further
analysis.
The process of removal the phosphate group
is called phosphorylation. Soil itself has no
any enzyme activity for solubilization as
well as mobilization of minerals. But the
huge number of microorganisms present in
soil makes it possible to recycle the nutrients
from both organic acid inorganic substances
nitrate reductase and alkaline phosphatase
activity of soil is the product of microbial
secretion of this enzyme to its nearest soil
particles. Activity of soil nitrate reductase is
useful for maintaining the nitrogen ratio in
the atmosphere as well as removal of
hazardous nitrate compounds of soil. Acid
as well as alkaline phosphatases are
responsible for quick regeneration of
organic phosphates and making them easily
available for plant.
Soil microbial population
Soil samples were subjected to serial
dilution and spread on nutrient agar plates
and potato agar plates, incubated for 48 hrs
to grow the microbial colonies proper.
Colony forming units were counted by
colony counter.
Measurement of microbial activity in soil
Soil contains a variety of microbes. The live
microorganisms respire and evolve CO2
from the soil. This can be measured and
assessed as an index of microbial activity of
soil. Weigh 100g of soil sample from field
and transfer into a sterile flask. Mix distilled
eater to adjust soil moisture to its 33% of
water holding capacity. Pour 10 ml of
freshly prepared N/10 NaOH solution into
two test tubes with a thread, and hung the
tubes in the two flasks. Close the mouth of
the flasks with rubber stopper and seal with
molten wax to make them airtight. Incubate
the flasks at 30o C. CO2 will evolve as the
result of microbial activity which will be
absorbed in alkali in the tubes and react as
below
In this study, we aimed to detect possible
impact of cement dust pollution, which is
generated by cement factory, on soil
nutrients (minerals), microbial population
(Bacteria and Fungi), microbial respiration
and alkaline phosphatase enzyme activity.
Materials and Methods
Experimental site and soil sampling
The study was conducted in the area of two
cement
industries
(Ramco
cement,
Thulukkapatti, near Sattur and Sankar
cement, Thalaiyudhu, near Thirunelveli).
Based on the area distribution, the soil
sampling sites were selected at 1 km away
from each site. Total 10 sampling sites were
selected
for
soil
collection.
The
experimental area soil nutrients were
determined. The major crops grown in this
area are Rice field, Sugarcane field, Cotton
2NaOH+CO2 Na2CO3+H2O
At weekly intervals take out NaOH solution
from each flask, transfer into a flask and add
2-3 drops of phenolphalein indicator. colour
of NaOH solution turns pink or red. Titrate
it against N/10 HCL solution pouring in a
burette and measure the residual amount of
NaOH unturned to Na2CO3
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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 641-649
NaOH + HCl Nacl + H2O
quantity of the enzyme required to release
1.0 micromol of p-nitrophenyl from pNPP in
1 minute.
Measure volume of Hcl through end point
colour turns to colourless. Calculate the
amount of CO2 by using the following
formula
Effect of pH on enzyme activity
After production of enzyme, activity of
enzyme was studied on different pH ranges
6-10. The pH of the substrate 2mM pnitrophenyphosphate was adjusted by 0.1 M
Tris-Hcl buffer and 0.2 M Nacl.
Mg CO2 = V X N X 22
Where, V= Volume of Hcl required to titrate
residual NaOH. N=Normality of Hcl.
Screening
microbes
of
phosphatase
producing
Effect of temperature on enzyme activity
Effect of temperature was studied on
enzyme activity by incubating the reaction
mixture on different temperature 30-50oC
for 10 minutes.
From the isolated microbes, to screening the
phosphatse producing microbes, Pikovskaya
medium was used. Which shows the clear
zone around the colony, was capacity to
isolate solubilize phosphate and producing
phosphatase enzyme. The isolate was
identified by staining and biochemical
techniques. Pure culture was maintained on
nutrient agar slants.
Results and Discussion
In our study, we have isolated different
bacterial and fungal species from soil in 10
different location in and around two cement
factory and it was named as RC-1,RC-2,RC3,RC-4 and RC-5 from RAMCO cement
factory, Sattur ; SC-1,SC-2,SC-3,SC-4 and
SC-5 from SANKAR cement factory,
Thirunelvelli (Table-2).
Effect of substrate concentration on
production of enzyme
Effect of substrate concentration was studied
on production and activity of ALPase
enzymes.
In
our
study,
different
concentration of K2HPO4 and Calcium
phosphate (0.02-0.04 g/100ml) was used.
The isolated bacteria and fungi were
screened for the production of alkaline
phosphatase enzyme. Most of the microbes
were produced the enzyme but we selected
only 2 microbes from each factory area (1
bacteria and 1 fungi) based on the clear zone
formation around the colonies in pikovasky
medium and it was named as RC-2,RC4,SC-3 and SC-5.
Assay of Alkaline phosphatase enzyme
The standard assay for ALPase activity was
carried out at 37oc for 30 minutes using
2mM p-nitrophenyphosphate as a substrate
in 0.1 M Tris-Hclbffer, pH9.5, containing
0.2 M Nacl. The reaction was terminated by
adding 2 ml of cold 0.5M NaOH, and the
absorbance of p-nitrophenol formed was
measured at 400nm as the difference
between the assay and control sample. One
unit of ALPase activity was defined as the
Further the microbes were identified by
staining and biochemical test and tentatively
the microbes were identified as RC2=Bacillus
cereus,RC-4=Aspergillus
niger,SC-3=Flavobacterium sp. And RC-5
A. niger.
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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 641-649
The quantitative analysis of
CO2-C
production in RC and SC soil are given in
table-1. According to the result, the CO2-C
production rate was depending on distance
from the cement factory. In increasing
distance, the highest CO2-C production was
356.4 mg in RC factory and 367.4 mg in SC
factory area. The CO2 production depends
on the numbers and types of microbes
present in the soil. From this observation,
we found that microbial load was lower in
nearest cement factory than the distanced
area. Higher deposition of phosphate and
other dusts are deposited in the cement
factory area will created higher pH and soil
hardness.
the quantity and quality of plant residues
placed in soil, their time and space
distribution, a ratio of above-ground input to
underground input of organic residues and
changes in nutrient inputs [7-10].The
amount of bacteria is indirectly measured by
the amount of CO2evolution in the soil. Soil
respiration technique was used for the
quality soil (Table-1). In our study the
amount of CO2 evolution and mineralization
were increased
when the increased the distance from the
cement industries. This indicated that the
microbial load was higher (276x102 bacteria
and 20x102 fungi) from cement plant area
compared to the nearest place (56x102
bacteria and 2x102 fungi). One of the
parameters which are most often studied for
the purposes of evaluating the biological
status of soil is the amount of microbial
biomass [11].
The quantitative analysis of bacteria and
fungi in soils are given in Table-2. Average
numbers of bacteria and fungi were
observed among different from the cement
factory. Effect of substrate concentration on
enzyme production and activity was shown
in Table 3.We used different concentration
(0.02, 0.03 and 0.04 g/100ml) of two
different substrates such as K2HPO4 and
CaPO4. Maximum enzyme activity was
occurred at 0.03g/100ml of both the
substrates by all the isolates.
According to Franken berger and Dick [12],
dehydrogenase activity is often correlated
with microbial respiration when exogenous
C sources are added to soil. Skujins [13] and
Casida [14] reported close correlations of
dehydrogenase activity with CO2 release and
O2 uptake, respectively. The highest activity
occurred in top 3 cm of an arid soil, but
there was no correlation with microbial
number because the dehydrogenase activity
depends on the total metabolic activity and
soil microorganisms.
Activity of alkaline phosphatase enzyme
was studied on different pH ranges 6.0-10.0.
In our study, the optimum enzyme activity
was found at pH 9.0 (Table 4).Effect of
temperature was studied on enzyme activity
by incubating the reaction mixture on
different temperatures 30, 40 and 500C
(Table 5). Maximum enzyme activity was
observed on 40oc incubation. It shows that
the higher temperature increase the kinetic
energy of molecules which break the bond
that holding the active amino group and
enzyme gets denatures.
The value of metabolic activity and soil
microorganisms in different soils, containing
different populations, does not always
reflect the total numbers of viable
microorganisms that are isolated on a
particular medium. Differences on average
number of bacteria and fungi were observed
among different distances from the cement
factory in CT and NT soils. The highest
number of bacteria were observed at
Soil
microorganisms
and
soil
microbiological processes are affected by
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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 641-649
distance of 15 km (387.2 x 106 CFU g-1
soil) from the cement plant in CT and the
lowest were observed at distance of 1 km
(296.3x106 CFU g-1 soil) from the cement
plant in CT. The highest number of bacteria
was obtained at a distance of 15 km (370.6 x
106 CFU g-1 soil) in NT and the lowest were
obtained at distance of 1 km (215.7 x 106
CFU g-1 soil) from the cement plant in NT
soils. Average numbers of bacteria were
higher in CT (332.5x106 CFU g-1 soil) soils
than in NT (282.8 x 106 CFU g-1) soils.
in NT soils. Beyer et al., [15]; Friedel et al.,
[9]; Kandeler et al., [15] reported that the
soil biological activity was change due to
different soil cultivation and fertilization
methods, as well as the whole system of soil
management. The carbon mineralization in
the soil was shown in Table-1. Maximum
carbon mineralization was observed in the
sample SC-4 (100.2 mg/100g). Beyer et al,
[7] reported that the values were 35.48 mg
C.kg-1 higher on average, but without
statistical significance, in the fields where
alfalfa, silage maize and pea (Pisum
sativum) were grown.
The highest number of fungi was obtained at
distance of 7 km and the lowest was
obtained at a distance of 1 km from the
cement plant in CT (37.2 x 104; 21.6 x 104
CFU g-1 soil) and NT (71.4 x 104; 42.6 x
104 CFU g-1 soil) soil. Average number of
fungi was determined 58.1 x 104 CFU g-1
soil in NT and 29.7 x 104 CFU g-1 soil in
CT soils. Increasing distance increased CO2C production in CT and NT soils. The
highest CO2-C production was observed at
distance of 15 km from cement factory in
CT (31.5 mg CO2-C m2 h-1) and NT (21.8
mg CO2-Cm2 h-1) soils. The lowest CO2-C
production was observed at distance of 1 km
from cement factory in CT (21.6 mg CO2-C
m2 h-1) and NT (16.0 mg CO2-C m2 h-1)
soils. The average CO2-C production was
determined 27.2 mg CO2-C m2 h-1 soil in
CT, and 19.3 mg CO2-C m2 h-1 soil in NT
soils.
An influence of the quality and amount of
plant residues ploughed in soil has been
confirmed for both systems. The crops
significantly affected the quantity of
biomass in either of the experimental years,
the greatest values being found for the soil
under alfalfa in the year 2000 (199.28 mg
C.kg-1 dry soil) and under bean with under
sown alfalfa in 1999 (187.3 mg C.kg-1 dry
soil). Effect of crops was observed in 1999,
with the highest value for the soil under
wheat (184.38 mg C.kg-1 dry soil). The
dynamics of microbial biomass in soil
within individual experimental years was
affected by the timing of soil sampling.
The characteristic feature of soil microbial
communities is their functional stability,
resulting from their large number, generic
diversity, high adaptation ability, as well as
from internal regulation. Soil microbes and
plant roots are sources of extracellular
enzymes, mainly through either secretion
from living cells or from lysed cells [17].
Once in the soil, enzymes may be protected
from denaturation by being absorbed onto
organic or inorganic surfaces. In this
absorbed state, extracellular enzymes
develop stability to desiccation and heat, and
can remain active for several years [18]. Soil
management during this period may allow
The highest AlkP enzyme activity was
observed at distance of 2 km (84.60 µg pNP
g-1 soil h-1), and the lowest AlkP enzyme
activity was observed at distance of 15 km
(58.40 µg pNP g-1 soil h-1) away from the
cement factory in CT soils. The highest
AlkP enzyme activity was observed at
distance of 1 km (68.10 µg pNP g-1 soil h1) and the lowest AlkP enzyme activity was
observed at distance of 10 km away from the
cement factory (47.50 µg pNP g-1 soil h-1)
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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 641-649
enzyme activity to gradually increase, and
contribute to an improvement in soil
fertility.
Soil enzymes contribute to the
mineralization of nutrients contained in
organic matter. One group comprises the
phosphatases, which breakdown organicphosphate compounds releasing the essential
plant nutrient.
and enzyme production respectively. The
growth increasing up to 2% sodium nitrate
the results are was extremely slow at pH 4.0
and 11.0 and reached maximum after 24
hours of incubation at pH 7.0.
The influence of initial pH on the production
of enzyme was maximum at pH 9.0 (1773
U/ml), production of alkaline phosphatase
by Escherichia coli beyond this production
rate was low. The pH dependent using 2.0%
molasses and 2.0% sodium nitrate in mineral
enzyme production might have been due to
pH control medium when incubated at 37°C
for 24 hours over the growth of bacteria or
pH dependent control the enzyme synthesis
gene expression [21].
Plants meet their phosphorus requirement
through the uptake of phosphate anions from
the soil. To be available to plants, organic
forms of soil phosphorus must be
mineralized by those processes, which are
mediated by phosphatase enzymes. A part of
the total phosphorus in soil occurs in organic
forms. The average content of organic
phosphorus in soils ranges from 5 to 50% of
total P [19] and forest soils have a higher
organic P content than arable or cultivated
soils.
Temperature is also a major factor for the
enzyme production. In our study maximum
enzyme activity was occurred in40oc (Table5).The maximum yield of alkaline
phosphatase was noted at 40°C. Similar
results are reported by Danielle and
Raymond [22] in the case of alkaline
phosphatase production by E.coli at 37°C
after 16 hours of incubation and Dahot et al
.,(1986) in the case of alkaline phosphatase
production by Penicillium expansum at
40°C. Temperature influence all the
physiological activities in a living cell and is
one of the important environmental factor to
control the growth, microbial activities,
normal functioning of enzyme and many
enzymes control the nutritional requirement
of the cell and subsequently its composition
[23].
Halstead & Mckercher [20] state that as
much as 5 10% of the organic phosphorus is
associated with living microbial tissue. So
we used two different phosphate compounds
for enzymes production (Table 3).
Maximum alkaline phosphatase enzyme
activity was observed in fungi culture (35.61
U/ml) in .03 g/100ml of potassium
phosphate medium. But in calcium
phosphate medium, 29.74 U/ml enzyme
activities were observed by bacteria. From
this result, we found that 0.03 g/100ml of
the both substrate were optimum
concentration for enzyme activity.
Table-4 Shows effect of pH on phosphatase
enzyme activity. The assay was carried out
using various buffers with specific pH. The
maximum of phosphatase activity was at pH
9.0. The enzyme activity above or below of
this pH showed less degradation of the
substrate used in these assays. The optimum
pH 7.0 and 9.0 were noted for production of
alkaline phosphatase the yield was growth
Nowadays soils are easily polluted by
various factors such as plastics, papers, oils,
factory wastes etc., indirectly the soil
fertility was affected that is, decreases the
microbial numbers and nutrients. We chose
cement factory area for microbial analysis
and the effect of phosphate on microbial
activity. In this area predominately Bacillus
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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 641-649
sp. and Aspergillus sp. were found for
adapted to the environment. These two kinds
of microbes mineralize and utilize the
phosphate to produce the alkaline
phosphatase enzyme. This enzyme has used
for molecular biology and genetic
engineering experiments. So we can use the
two microbes.
Table.1 Measurement of microbial activity in soil respiration
Sample
number
RC-1
RC-2
RC-3
RC-4
RC-5
SC-1
SC-2
SC-3
SC-4
SC-5
Amount of CO2
evolution (mg)
275
312.4
334.4
356.4
286
206.8
272.8
314.6
367.4
264
Amount of CO2
mineralization (mg)
75
85.2
91.2
97.4
78
56.4
74.4
85.4
100.2
72
RC-Ramco cement factory; SC-Sankar cement factory
Table.2 Microbial population of different soil samples
Sample
number
RC-1
RC-2
RC-3
RC-4
RC-5
SS-1
SS-2
SS-3
SS-4
SS-5
Bacterial X 102
(CFU/g)
78
134
167
212
276
56
149
189
220
266
Soil
Environment
Rice field
Sugarcane field
Cotton field
Neem tree
Papaya tree
Mango tree
Coconut tree
Chilly field
Penndy field
Brinjal field
Fungal X 102
(CFU/g)
3
8
12
17
20
2
2
10
11
15
Table.3 Effect of various concentration of substrates ( K2HPO4, CaPO4) on enzyme production
Name of the
Isolates
Bacillus cereus
Aspergillus niger
Flavobacterium
Aspergillus niger
Enzyme activity
(U/ml) in 0.02 %
K2HPO4 CaPO4
17.67
22.10
11.87
11.58
05.93
13.69
11.86
17.80
Enzyme activity
(U/ml) in 0.03%
K2HPO4 CaPO4
29.23
29.47
35.61
22.48
29.67
23.74
22.45
19.20
647
Enzyme activity
(U/ml) in 0.04%
K2HPO4 CaPO4
24.56
19.10
25.84
23.58
15.33
21.33
26.96
23.47
Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 641-649
Table.4 Effect of various pH (6, 7, 8, 9, 10) on enzyme activity of K2HPo4 and CaPO4 medium
Name of the
Isolates
Bacillus cereus
Aspergillus
niger
Flavobacterium
Aspergillus
niger
Enzyme activity (U/ml) in K2PO4
medium
pH 6 pH 7 pH 8 pH 9 pH 10
05.93 17.20 24.0 29.23 21.03
10.10 19.50 27.56 35.61 27.41
Enzyme activity (U/ml) in CaPO4
medium
pH 6 pH 7 pH 8 pH 9 pH 10
09.21 15.56 18.76 29.47 16.67
09.47 13.54 17.59 22.48 13.56
05.18
07.16
12.09
11.45
13.70
16.10
19.98
21.67
29.67
22.45
17.03
17.45
17.90
13.57
20.86
28.54
23.74
29.20
19.86
25.53
Table.5 Effect of various temperatures (30, 40, 50oC) on enzyme activity of
K2HPo4 and CaPO4 medium
Name of the Isolates
Bacillus cereus
Aspergillus niger
Flavobacterium
Aspergillus niger
Enzyme activity (U/ml)
in K2PO4 medium
30oC
40oC
50oC
22.90
34.56
31.46
24.42
37.20
33.52
19.97
31.54
29.32
21.43
27.50
25.75
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Acknowledgement
The authors are grateful thanks to
Management and Department of Zoology
and Microbiology, Thiagarajar College,
Madurai
625009, Tamilnadu, India for
providing the necessary facilities.
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