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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.
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.
pH mg/l 7–8
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
Time Optical
(days) Density
(OD)
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
TABLE.3 PROTEIN ESTIMATION AGAINST
13 0.34
TIME
14 0.31
15 0.26 Time Influent S –I S –II S-III S-IV S –V S-V
in
16 0.23 mg/ml mg/ mg/ mg/ mg/ mg/ mg/
days
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
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
0
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.
TABLE.4 COD REMOVAL AGAINST TIME
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.