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IJMTES | International Journal of Modern Trends in Engineering and Science ISSN: 2348-3121


Priyadharshini.M1, R.Santhosh Kumar2
(Environmental Engineering, Anna University, Chennai, India,
(Department of Civil Engineering, The Kavery Engineering College, Salem, India)

Abstract—This work was planned to identify the microbes which are effective for the biological degradation of dairy wastewater. The
activated sludge process facilitates the removal of carbonaceous BOD and the stabilization of organic matter by a variety of microorganisms
(principally bacteria). Bacillus supergene is one of the predominant bacteria found in dairy waste water. The bacteria Bacillus .sp was
isolated from the dairy wastewater and inoculated into the samples taken from the dairy industry. Laboratory tests were carried out by
inoculating varied cell concentrations and incubating under aerobic conditions to determine the removal of COD and protein from the
samples. During the incubation, microbial growth was monitored by the measurement of optical density. The biodegradation ability of the
native bacteria was also compared with the commercial inoculum of the same isolate. The COD removal was higher for the commercial
inoculums and the protein removal was higher in the selected native strain.

Keywords—Biodegradation; Bacterial isolates; Chemical Oxygen Demand (COD); Dairy wastewater


1. INTRODUCTION environment. Large concentration of pollutants in terms of

quantity and quality of liquid, solid and gaseous pollutant
Dairy industry is most polluting food industry in terms of
shows harmful effects on flora and fauna as well as on many
large amount of water use and considering huge amount of
areas on environment. Effluent treatment plant in industry
water is used during production of milk and milk products.
installed for meet regulating norms governs by pollution
The wastewater contains dissolved sugars, proteins and fat
control board. Every industry have a wastewater treatment
which is organic in nature and bio-degradable. However,
plant which helps to lower down pollution level of
efficient multi products dairy plants are using one lit water
wastewater going to be discharge in to the environment from
per lit of milk processed. An efficient waste water system is
industrial premises. According to the pollution board norms
of primary importance to any commercial dairy plant. The
industry need to be follow land and stream wastewater
characteristics of these effluents also vary widely both in
discharge norms.
quantity and quality, depending on the type of system and
The economy has suffered because many developing
the methods of operation used.
countries have resorted to importation of fish to sustain local
Wastes from milk product manufacturing plants contain milk
demands. To save the natural bodies of the water from
constituents such as casein, lactose, fat, inorganic salt,
possible complete deterioration, stricter and more defined
besides detergents and sanitizers used for cleaning in a more
effluent standards should be imposed by the government on
or less dilute condition, but in varying concentration. So that
different industrial firms. This needs to employ efficient
dairy wastewater is considered as high concentration of
facilities for the various waste materials.
organic matter and high BOD. Such untreated wastewater
B. Objectives And Scope Of The Study
pollutes land and river system so that proper treatment of
The main objective of the study was planned to identify
dairy wastewater is necessary before disposal in
the microbes which are effective for the biological
degradation of dairy wastewater. The wastewater sample was
The disposal of large quantities of this wastewater with or
subjected to microbial analysis to isolate the specific bacteria
without treatment and continuous pile of solid waste from
Bacillus sp. by spread plate method on Nutrient agar media.
the industrial and domestic source rapidly causes
These selected microorganisms were inoculated to the
deterioration of the environment. The effects of presence of
wastewater with different concentrations of 1x106 CFU/ml
these wastes include contamination of drinking water, killing
to 5x106 CFU/ml at 30oC in the shaker incubator at 120 rpm
of aquatic life, increased danger in swimming and
to examine their ability in reducing the chemical oxygen
objectionable physical condition such as off odours and
demand and protein in the wastewater.
accumulation of debris. e.g. higher concentrations of organic
The removal of organic matter from wastewater using
matter have led to the depletion of dissolved oxygen supply
chemicals is used in many industrial wastes, and these
for fish due to the oxidative actions of microorganisms in the
methods are more expensive than the biological treatment
surrounding media.
methods. The chemical treatment also creates waste
A. Need For The Study
disposable problems, because neutralization and
Dairy industry contains high amount of organic
detoxification of the discharged wastewater is necessary
constituents. So that it is need to provide required treatment
which also causes further contaminations to the environment.
before discharge into the environment. The treatment mostly
To overcome the shortage of chemical treatments, studies
classified as aerobic and anaerobic treatment. Due to rapid
have been conducted using microorganisms or proteolytic
industrial growth world’s economy improve with rapid
enzymes for the biological degradation of industrial wastes.
growth but also that make impact in terms of pollution on

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C. Removal of COD and Protein treatment. The microorganisms of choices should have a
The chemical oxygen demand test (COD) determines, the strong degradative capacity and high toxic resistance. The
oxygen required for chemical oxidation of organic matter process of seeding inoculation of micro organisms for
with the help of strong chemical oxidant. The COD is a test degrading the waste materials on streams, rivers and
which is used to measure pollution of domestic and industrial treatment tanks has been rapidly increasing practice in many
waste. The waste is measure in terms of equality of oxygen countries because it is economical and the application is
required for oxidation of organic matter to produce CO2 and uncomplicated.
water. It is a fact that all organic compounds with a few Many researchers involved with activated sludge
exceptions can be oxidizing agents under the acidic treatments assume that simply adding additional bacteria to
condition. COD test is useful in pinpointing toxic condition the sludge in a digestion vessel wound improve the
and presence of biological resistant substances. performance of the system without understanding the ‘effect’
of the process condition in which the bacteria are expected to
2. REVIEW OF LITERATURE live and breed. Although industry has started to the provide
A. Problems in Biological Treatment pre-cultured, specific strain ‘packages’, the knowledge of
Activated sludge technology has becomes the favoured how to use them is still very limited and is restricted to
method for the biological treatment of organic wastewater existing systems, primarily the activated sludge process.
streams, it suffers from a large number of inherent problems. Hence, the ability to use specific strain cultivation is limited.
For example, the treatment process relies on the natural Measurement of Growth rate of Bacteria
growth of bacteria within the sewage and sludge, and hence Growth refers to increase in population size rather than
it requires the ‘correct’ bacteria to be cultivated, which are enlargement of the microorganism. Four recognizable phases
capable of effectively digesting the waste. However, the are seen when the increase in cell number is determined in
wrong species of bacteria can easily reproduce in the relation to time viz. Lag phase, log phase, Stationary phase
digestion vessel, and these ‘incorrect’ bacteria can out and Death phase.
compete the correct bacteria, and consequently inhibit the The curve shown in Figure.1 represents what occurs in a
efficiency of the system, or even prevent it from working at batch reactor in which, at time zero, the substrate and
all. Hence the process is incapable of tolerating large swings nutrients are present in excess and only a very small
in strength or type of microbial contamination. population of biomass exists.
In addition the process is highly susceptible to toxic
shocks and has a very slow recovery rate if a micro-organism
culture wipe out occurs. A further problem with the existing
activated sludge protocols is that a high volume of surplus
sludge is produced. This sludge is bulky and requires
disposal, which is expensive. The routes available for the
disposal of sludge derived from wastewater treatment are
becoming more tightly controlled due to environmental
pressures and costs of land-fill and incineration are rising
due to shortage of facilities and more complex policing.
Another problem with existing methods is that a high
level of operational expertise is required to operate
wastewater/sewage treatment plants efficiency, and the
operatives need a good grounding in biological techniques of
wastewater treatment. Hence, there is a requirement for
constant staff attention to ensure that the system operates Fig.1 Typical Growth of curve
efficiently. In addition, due to the large number of vessels i).The Lag Phase
required system tend to often time-consuming and expensive. The lag phase represents the time required for the
Because of its large footprint requirement, activated sludge organisms to acclimate to the new environment. There is no
treatment often proves impractical for industrial purposes increase in the number of viable cells. It is a period of active
where companies are short of space, even though the process growth without cell division and cells for preparation.
would be the most environmentally suitable for the purpose. ii).The Log Phase
The degradation activity of the indigenous During this period the cells divide at a rate determined
microorganisms present in these conventional systems may by their time and their ability to process food. Cell
not be sufficient to obtain efficient and reliable treatments. population increase logarithmically.
In activated sludge systems, bioaugmentation has been iii). The Stationary Phase
demonstrated to enhance the treatment system performance Here the population remains stationary. The cells have
by adding external microorganisms with high degradation exhausted the substrate or nutrients necessary for growth and
capacity of specific contaminant compounds. hence can no longer reproduce.
Seeding Inoculation of Microorganisms iv).The Death Phase
Due to capital draining expenses attention has been During this process, the bacteria death rate exceeds the
drawn to the use of microbial culture preparations for waste production of new cells. The death rate is usually a function
of the viable population and environmental characteristics.

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IJMTES | International Journal of Modern Trends in Engineering and Science ISSN: 2348-3121

3. METHODOLOGY Nutrient agar media was prepared for isolating bacteria

A. Sampling Of The Dairy Wastewater with the following ingredients dissolved in distilled water
The dairy wastewater used in this study was collected and sterilized for 20 minutes in an autoclave.(Composition:-
from Aavin, Salem District Milk Producers Association, Peptone- 5.0 g ,Beef extract- 3.0 ,Sodium chloride -5.0,Agar
Sithanur, Salem. They were stored at 4oC, until the study -15.0 g, Distilled water -1000 ml).
was carried out. The influent characteristics of the dairy Since bacteria plays an important role in the degradation
wastewater are shown in the Table 1. of wastewater, some of the aerobic bacteria were isolated
from the mixed microbial population by serial dilution and
TABLE.1 CHARACTERISTICS OF RAW DAIRY WASTEWATER was plated on Nutrient agar by spread plate method. The
results are shown in fig.3.
Parameters Unit Concentration

pH mg/l 7–8

BOD mg/l 1200 – 1400

COD mg/l 1800 – 2800

Suspended mg/l 750 – 800


Protein mg/l 0.28

B. Isolation Of Bacteria From The Mixed Microbial Fig. 3 Bacterial colonies obtained in the dilution of 10-9
Population In The Wastewater C. Isolation Of Bacillus sp. From The Bacterial Colonies
The sample collected was initially subjected to the serial Macconkey agar media is prepared for isolating
dilution (10 fold dilution) to isolate the bacteria from the Bacillus Sp. from mixed culture with the following
dairy wastewater and the procedure is as follows. ingredients dissolved in distilled water and sterilized for 20
Serial Dilution minutes in an autoclave.
One ml of the wastewater was initially diluted in the 9ml Preparation Of Macconkey Agar Media
of the sterile distilled water to get the dilution of 10-1 and Compositions are Peptone-1.7g, Protease peptone
again one ml of the dilution waste water was diluted in the -0.3 g, Lactose- 1.0 g, Bile salt- 0.15 g, Sodium chloride- 0.5
9ml of the sterile distilled water to get the dilution of 10-2. g, Neutral red - 0.003 g, Crystal violet- trace, Agar - 1.5 g,
Further it was diluted logarithmically up to the dilution of Distilled water- 100 ml.
10-9. From this 1ml of the diluted sample was plated on As a result lactose degrading bacteria were identified
Nutrient agar media by Spread plate method to obtain and was added to an enrichment cultural medium such as
Bacterial colonies. nutrient broth to enrich the growth of Bacillus sp. 1ml of the
Spread Plate Method suspended solution was inoculated to 250 ml of sterile
The serially diluted sample was placed on Nutrient agar nutrient broth and shaked for 24 – 48 hrs at 30oC. For
media and spreaded evenly over the surface by means of inoculum preparation, slant tubes are prepared with each
sterile L shaped rod and incubated at 37oC to obtain bacterial strain and incubated at 37oC for 48 hrs.
colonies. A loop full of the isolated organism was plated on
Macconkey agar media which clearly differentiates the
lactose and non lactose fermenting organisms
Enrichment cultural media
After isolating the lactose fermenting organisms, they
are added to an enrichment cultural medium such as Nutrient
broth to have a good growth of Bacillus sp. and transferred to
the prepared slant.
D. Growth curve of Microbial culture
During incubation, the optical density was measured by
taking absorbance at 620nm by Colorimeter as the
measurement of the microbial cell growth. The Optical
density (OD) against time are shown in table.2

Fig.2 Serial Dilution For Isolation Of Bacteria

Preparation Of Nutrient Agar Media

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IJMTES | International Journal of Modern Trends in Engineering and Science ISSN: 2348-3121


Time Optical
(days) Density
0 0.08
1 0.10
2 0.11
3 0.17
4 0.26
5 0.32
6 0.39
7 0.42
Fig.5 Standard graphs for working protein solution
8 0.46 From standard graph the corresponding amount of protein
9 0.46 for different cell concentrations of Bacillus sp. was measured
10 0.46 as the slope of optical density for the working standard
11 0.46 protein solutions and the results were tabulated in table.3
12 0.38
13 0.34
14 0.31
15 0.26 Time Influent S –I S –II S-III S-IV S –V S-V
16 0.23 mg/ml mg/ mg/ mg/ mg/ mg/ mg/
17 0.20 ml ml ml ml ml ml
2 0.28 0.27 0.25 0.24 0.22 0.19 0.17
4 0.28 0.25 0.22 0.20 0.17 0.14 0.11
6 0.28 0.22 0.18 0.15 0.11 0.11 0.09
8 0.28 0.18 0.14 0.11 0.09 0.07 0.05
10 0.28 0.16 0.08 0.06 0.05 0.04 0.04
12 0.28 0.15 0.06 0.04 0.04 0.03 0.02
14 0.28 0.12 0.06 0.03 0.03 0.02 0.02

S-I - Sample I Control solution

S-II - Sample II Addition of 1x106CFU/ml of Bacillus sp.
S-III - Sample III Addition of 2x106CFU/ml of Bacillus sp.
S-IV - Sample IV Addition of 3x106 CFU/ml of Bacillus sp.
S-V - Sample V Addition of 4x106 CFU/ml of Bacillus sp.
S-VI -Sample VI Addition of 5x106 CFU/ml of Bacillus sp.

Fig.4 Microbial growth curve

Fig.4 shows the microbial growth curve. The curve clearly
showed the four distinct phases, log phase, stationary phase
and death phase of the microorganisms.
E. Removal of Protein by Lowry’s method
To measure the protein content in the sample, the
optical density of working standard protein solutions were
observed at 600 nm in a colourimeter.
Standard graph is prepared for the working standard
protein solution by taking concentration of protein in X axis
and optical density in Y axis as shown in fig.5.

Fig.6 Protein degradation for different cell concentrations

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IJMTES | International Journal of Modern Trends in Engineering and Science ISSN: 2348-3121

The protein removal was comparatively higher in the 10 2190 854.4 711.9 698.4 679.3 345.6 258.5
sample VI with 5x106 CFU/ml of bacterial strain within 5 12 2190 735.5 682.5 587.7 380.9 334.0 192.2
days; hence sample inoculated with cell concentrations more
14 2190 735.0 680.2 586.8 362.2 332.2 191.5
than 5x106 CFU/ml was not taken for this study purpose. It
was also observed that in the 6 to 8 days of incubation, rapid 16 2190 732. 680.0 586.2 360.2 330.1 190.2
reduction of protein content was noted and after this the 2
changes became slow. 18 2190 732. 679.8 585.7 360.1 330.0 190.2
Comparison of protein degradation for different cell
concentration is shown in fig.7.

Fig.7 Percentage of Protein degradation for different cell concentrations Fig.8 COD removal for different cell concentrations
F. COD removal After 2 days of incubation, the sixth sample showed COD
The samples which are inoculated with different cell reduction up to 50% and after 4 days, the fourth and fifth
concentrations of Bacillus sp. were examined for their ability samples also showed more than 50% of reduction. The
to reduce the chemical oxygen demand. The initial sample VI showed the maximum COD removal efficiency as
concentration of the wastewater was found to be 2190 mg/l. 91.3% after 18 days.
The COD measurement was carried out for the samples for a
period of 18 days and the reading were tabulated in 4.7.
Then the results were plotted with COD against time on
a graph as shown in fig 4.8. From the graph it was observed
that due to the inoculation of additional bacteria in the
sample, there is an appreciable COD reduction after 2 days.
The COD reduction was comparatively higher in the samples
IV, V and VI with 3x106 CFU/ml, 4x106 CFU/ml and 5x106
CFU/ml of bacterial strain respectively. It was also observed
that in the 4 to 10 days of incubation, rapid reduction of
COD was observed and after this time, it remained
unchanged in all the samples.

Tim Influen S –I S –II S-III S-IV S –V S-V

e in t
days mg/ mg/ mg/ mg/ mg/ mg/
ml ml ml ml ml ml Fig.9 Percentage of COD removal for different cell concentration
2 2190 1994 1625. 1225. 1192. 1126. 1054.
4 4 2 2 4
Commercial inoculum and Selected strain of Bacillus sp.
4 2190 1421 1304. 1206. 1080. 1001. 869.0 Were inoculated with the native microorganisms obtained
1 4 9 6 from dairy wastewater and commercial inoculum of Bacillus
6 2190 1372 1250. 1175. 954.9 623.3 597.5 sp. for 5 days in an orbital shaker at 30°C with a shaking rate
6 3 of 120 rpm. The results of COD and protein removals for the
selected strains and a commercial inoculum after 5 days are
8 2190 969.0 731.8 711.5 700.2 610.9 533.3
presented in table.5 and Fig.10 shows the removal

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IJMTES | International Journal of Modern Trends in Engineering and Science ISSN: 2348-3121

efficiencies of selected strains and commercial inoculum of treatment system was increased with the addition of
Bacillus sp. bacterium to the microbial mixture of the activated sludge.
From the figure it has been observed that the The biodegradation ability of these native
biodegradation of the model effluent in terms of COD microorganisms was also compared with the commercial
removal was higher for the commercial inoculum than for inoculum of the same isolate. The COD removal was higher
the selected strains (63% for commercial inoculum and 57% for the commercial inoculum (63% for commercial inoculum
for the selected strains). But the protein removal was higher and 57% for the selected strains) and the protein reduction
in selected strains than the commercial inoculum of the same was higher in selected strains (56% for commercial inoculum
genus (56% for commercial inoculum and 71% for the and 71% for the selected strains). Therefore the native
selected strains). microorganisms are effective in removing milk proteins.


BACILLUS SP. [1] Adamse.A.D, (1966): “Some bacteriological aspects of Dairy
waste Activated sludge”, Laboratory of microbiology, Agri
University- Washington, pp 19-27.
Removal efficiency in % [2] Jen-Kuo Yanga, et al (1999): “Production and purification from
a Bacillus subtilis that can deproteinize crustacean wastes”,
Bacterial COD Proteins Elsevier, pp 406-413.
type [3] Kosseva M.R et al (2002): “Thermophilic bioremediation
strategies for a dairy waste”, Elsevier, pp 125-130.
Commercial 63% 56%
[4] Lyliam Loperena, et al (2008): “Isolation and selection of native
inoculum microorganisms for the aerobic treatment of simulated dairy
wastewater” Bioresource Technology, pp 1762-1766.
Selected 57% 71%
[5] Nadais H et al (2005): “Treatment of dairy wastewater in UASB
strains reactor inoculated with flocculent biomass”, pp 603-608.
[6] Schneider and Topalova (2008): “Amendment of the acitivity of
microbial preparation Laktazym to protein biodegradation for
dairy wastewater”, Biotechnol. & Biotechnol. EQ., pp 810-815.
[7] Vida Maghsoodi et al (2007); “Biodegradation of effluents from
dairy plant by bacterial isolates”, Vol. 26, pp 55-59.

Fig.10 Removal efficiencies of selected strains and commercial inoculums of

Bacillus sp.
Addition of effective biodegradable bacteria to the
microbial mixture of the activated sludge reduced the
bulking problems by preventing the load of organic matter
(by the removal of fat and protein) becoming too high and
thereby increased the overall efficiency of the treatment
Today most modern wastewater treatment processes rely
on the composition and activity of their microbial
communities. Recent developments in molecular methods for
analysis of the microbial communities have retriggered
public interest in the microbiology of activated sludge. This
work assesses the possibility of using lactose fermenting
bacteria isolated from activated sludge of dairy wastewater to
remove COD and protein from milk based medium and
compared to a commercial bio augmentation inoculums.
A protease producing strain that can be used to
deproteinize dairy wastewater was isolated and identified as
a strain Bacillus sp. Hence the overall efficiency of the

Volume: 03 Issue: 07 2016 138

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