1.

INTRODUCTION
1.1 OVERVIEW
With the rapid growth in population and industrialization is leading to the depletion of
natural resources and causing major environmental problems such water pollution, soil pollution
etc. The environmental problem which is of our concern is water pollution which is mainly
caused due to the discharge of heavy metals from steel, dairy, textile, paper and pulp and
fertilizer industries, etc. An excessive amount of the discharge may cause several health related
problems by causing eutrophication and acidification of water bodies. To overcome this process
there are various methods which have been used for decades but the question arises is which
process is more economical and beneficial over others.
Among all the secondary treatment process equipments, aerobic biofilm reactors are the
most recent systems. These are compact, capable of being installed in urban areas, highly
resistant to variations in temperature and to toxicity shock loads having better operational
stability to treat low concentration of organic pollutants. These systems also have the ability to
take higher (three times or more) organic load than other processes and have higher degradation
rates. These systems are less expensive to construct and conceptually simpler than any other
secondary treatment systems.
Based on the state of biomass fixation, aerobic biofilm reactors are classified into hybrid
reactors, reactors with suspended biomass and reactors with attached biomass. And among the
several types of aerobic biofilm reactors, the reactors with moving beds, particularly the two- and
three-phase inverse fluidized bed aerobic biofilm reactors have drawn much attention in recent
years which have the support medium in permanent movement (mainly hydraulically driven)
Among many conventional processes available for wastewater treatment, inverse
fluidization process which is a three phase fluidization process has been widely used for many
applications such as hydro-treating and conversion of heavy petroleum and synthetic,
crystallization, food processing, biomedical engineering, methanol production, treatment of
municipal sewage wastewater and similarly many processes. Some of the benefits which ones
process can gain if this unit is used are easy to handle, less consumption of power, low space
requirement, less chemical waste and eco-friendly as it does not produce any chemical as its
waste after the process. Indeed it is the most significant feature of it is high efficiency as
compared to the other conventional fluidization processes.

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1.2 INVERSE FLUIDIZATION
The name inverse fluidization comes from the direction of flow of liquid and gas which
depends upon the density of the particle. Here the liquid is fed continuously from the top using
pump if it is a continuous process and gas is released from using sparger form the bottom after it
has been compressed in a compressor, thus it makes the process counter current flow process. In
this counter current flow process the density of the particle is lesser than that of the liquid which
is in a continuous phase
Why inverse fluidization technique and not the conventional one?
The bio film thickness grows very fast on the surface of the solid particle, if provided
proper conditions .Sometimes it also happens that bio film thickness increases so much
that it causes bloom and proper mixing and growth of film is degraded. Thus some new
particles have to be added to provide new surface to the biomass from time to time. The
advantage of IFBR lies here that it controls bio film thickness in a very narrow range.
Due to power failure sometimes it needs to start the fluidization process from the
beginning itself but with the IFBR this problem is almost sorted out as we can re-fluidize
the process.
Carryover of particles is minimized due to low particle or solid attrition.
High volumetric efficiency, long term stability and applicability to treat low
concentration pollutants
1.3 Aerobic Process
The groups of bacteria typically found in dairy industries are lactic acid bacteria,
psychrotrophic Gram-negative bacteria, Gram-positive spore formers and Salmonella. Aerobic
digestion is the degradation of nutrients by bacteria in the presence of oxygen which produces an
end-product of carbon dioxide. Aerobic digestion occurs much more efficiently at high
temperatures compared to temperatures in the mesophilic range, however can still be achievable
at lower temperatures. The 2 types of aerobic treatment include: suspended growth processes,
and attached growth processes. In this study we chose the latter. There are many advantages to
aerobic digestion compared to anaerobic digestion, such as its ease of operation, lower
equipment costs, the lower safety hazard level when cleaning, and its production of gases which
are not explosive digester gases. Aerobic digestion is also much faster than anaerobic digestion

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and requires a shorter hydraulic retention time. These factors, along with the renewed interest in
aerobic systems for bioremediation, led to the choice of aerobic bioreactor in this study.
1.4 BOD & COD
COD test is used to measure the amount of organic compounds in water i.e. we can say
that it is the amount of oxygen required to chemical oxidize the pollutants. The applicable range
of COD is 3 900 mg/ml.
BOD test is used to determine the amount of oxygen required by the microorganism to
break the organic material present in the sample at a particular temperature over a specific period
of time. Generally the time taken for test is 5days at a temperature of 20 degree Centigrade. It is
also a principle test which predicts the biodegradability of any water or wastewater sample. The
efficiency of wastewater is measured by measuring the effluent BOD and influent BOD of the
sample taken. Any effluent to be discharged into the water should have BOD less than 30mg/ml.
COD value is always greater than BOD value .It is found from the research that the COD
values for domestic and industrial wastewater is about 2.5 times the BOD value. The ratio of
BOD to COD if greater than 0.8 then it is considered that the water is highly polluted and
amenable to biological treatment.
1.5 Objectives
Measurement of the COD and BOD content of the inlet and outlet streams from the
inverse fluidization unit.
Preparation of bacteria culture for synthetic medium.

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 2. LITERATURE SURVEY

Muhammad Nadeem et al (2011) studied the performance of dairy wastewater using

anaerobic fixed film reactors with foam cubes, bamboo rings, firebricks, PVC rings and
gravels as support material to immobilize the biomass for the reduction of COD and VSS.
From their study, they have concluded that the fixed film reactor with fire brick as
support material performed better when compared to other support materials. The
bamboo rings with fixed film reactors, have shown the least performance. They have also
concluded that the material used as packing medium in the reactor provided a large
surface/volume ratio that permitted the tremendous growth of microorganism in the pores
of the support materials and hence the amount of biofilm was high which helped the
reactor to operate at short HRT.
Abbasnezhad (2009) studied the HUASB performance in different situations to obtain
the optimal conditions of reactor performance for treating dairy wastewater. The reactor
had a biofilter of 70% and a total volume of 26.5 liters. The influent COD Concentration
was in the range of 500 to 3000 mg/l.
Vijay kale et al (2006) found that dairy wastewater is distinguished by the high BOD
and COD content, high level of dissolved or suspended solids, including fats, oil and
grease, nutrients such as ammonia or minerals and phosphates. Since, it generates huge
quantities of wastewater, it is essential to treat the dairy wastewater before it is disposed
in land or nearby water bodies.
Nadais et al (2005) assessed the possibility of using flocculent sludge in reactors for the
treatment of dairy wastewater. Through their study, they have stated that the performance
of flocculent sludge was similar to that of granular sludge. They have also found that the
residence time of 12 hours was necessary to attain both soluble COD and VFA removals
to near 80%.
Gavala et al (1999) treated dairy wastewater and studied the performance of the reactor
at different OLRs. COD removal efficiencies of 83%-92% and residence time in the
range of 26-40 days were reported at an influent COD concentration of 60 g/l.
Cordoba et al (1995) studied the treatment of dairy industry wastewater in a laboratory
scale AHR, using polyurethane foam (200 x 10 x 10 m size; tied together as a bundle,

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 void volume - 91%) as packing material. Through their study, they have concluded that
the AHR performed well for the removal of organic matter (92%) and gas production of
4.64 l/d, at the highest OLR.
Anderson et al (1994) investigated the performance of AHR treating wastewater from a
milk bottling factory, using two types of packed media (PVC media - non porous type
and a porous sintered glass media i.e., Raschig rings). Their results showed a heavy
biomass attachment in the porous media, whereas, mainly unattached biomass were
retained in the voids of the non-porous media.
Ozturk et al (1993) did a significant work on the treatment of dairy effluent from a large
integrated industry using a laboratory scale reactor under thermostatically controlled
temperature. The upper 60% of the reactor was filled with cylindrical plastic rings. The
reactor was operated for more than 270 days under mesophilic conditions. The residence
time ranged from 0.21 to 0.96 days after the start-up. At an OLR of 8.5 g COD/L-d, COD
removal efficiency was more than 87%. Through their study they have observed that the
HUASB reactor tolerated a high OLR of 17 g COD/L-d, with an average COD removal
efficiency of 75% for two weeks.

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 3. MATERIALS AND METHODS
3.1 Isolation technique:

Experimental Procedure:
Three samples of wastewater from a local dairy industry were collected:
S 1: Wastewater sample from drain,
S 2: Pure wastewater sample, and
S 3: Soil sample where it is dumped.

Step 1: Preparation of Media

At first 500 ml of distilled water was taken in a beaker.
Following chemicals were weighed carefully
Peptone powder - 5 gm
Yeast extract - 2.5 gm
Sodium chloride - 2.5 gm
These chemicals were added into the beaker containing distilled water.
The solution was stirred thoroughly to get uniform solution.
This solution was now transferred carefully into 3 conical flasks equally amounting to
100 ml each and numbered as 1, 2, & 3.
Then the mouths of these flasks were sealed with cotton buds and wrapped with paper
along with 5 petri dishes and 20 test tubes.
These instruments were then put inside an Autoclave at a temperature of 121.5 0C for
about 1.5 hours for sterilization in order to prevent growth of unwanted microbes.

Figure 3.1: Media Preparation

Step 2: Preparation of microbial culture

1 ml from Samples 1 and 2 were taken in two test tubes using a pipette.
Few grams of soil from Sample 3 were dissolved in a beaker containing water. Then 1 ml
of this solution was transferred into another test tube.

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 About 9 test tubes were filled with 9 ml distilled water (each).
Dilution of each sample was conducted. For Sample 1, 1 ml of Sample 1 was put into one
of the 9 test tubes containing 9 ml distilled water, subsequently 1 ml from the latter was
taken and put into second test tube and again 1 ml from previous test tube was taken and
put into third one. This test tube was marked as 1.
Similarly dilution of other two samples was done and test tubes were marked as 2 & 3
respectively.
These three test tubes were sealed thoroughly using cotton buds.
Now these diluted wastewater samples were transferred into the above prepared media
contained in respective conical flasks in Laminar Air Flow region carefully.
Then these conical flasks were put inside incubator shaker at room temperature of 25 0C
for about 2-3 days. The purpose of this step is to allow growth of bacteria.
Growth of bacteria was visually observed from the colour change.

Figure 3.2: Preparation of Microbial Culture

Precautions:
All glass apparatus must be washed thoroughly with water before use.
The Laminar Air Flow region should be cleaned using Ethanol before transferring
samples to media to prevent growth of unwanted microbes.
The mouth of the conical flask should be heat treated with methanol spirit lamp before
wastewater sample was transferred into it.

Step 3: Growth of microbe culture

Step 1 was repeated to prepare fresh nutrient media and 5ml from each of the wastewater
samples was transferred into three conical flasks marked as N1, N2, and N3 respectively.
These conical flasks were put inside incubator shaker at room temperature of 25 0C for
about 2-3 days.
After 3 days, new samples also showed decent growth

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 Figure 3.3: Growth of Microbial Culture
Step 4: Formation of Colonies

At first, the following solution was prepared in 500 ml distilled water containing the
chemicals mentioned below.
Peptone powder - 5 gm
Yeast extract - 2.5 gm
Sodium chloride - 2.5 gm
Agar Agar - 7.5 gm
The solution formed was transferred into a 1000ml conical flask.
Sterilization process:
All the glassware i.e.
1000ml conical flask containing agar solution (cotton wrapped)
12 petri-dishes (paper wrapped)
10 to 15 micropipette tips in a small beaker (paper wrapped)
18 test tubes each containing 9ml distilled water (cotton wrapped)
are put inside an Autoclave for about an hour operating at a temperature of 121 0C and 15
psi pressure.
Immediately, the solution after sterilization was transferred equally to 12 petri-dishes
inside the Laminar Air Flow region and left for about 15 minutes to solidify.
(Note: Since, solution consists of agar agar, it would get solidified easily and hence
cannot be transferred to petri-dish)

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 Figure 3.4: Transfer of sterilized solution to petri dishes
Now both the old samples (3 nos.) and new samples (3 nos. containing fresh nutrient
media) were brought from incubator shaker to Laminar Air Flow region.
A new sample (say N1) was taken and 1ml of it was drawn into micropipette and spread
over a petri-dish (containing solidified agar media) by the help of L-shaped rod which
was marked as N1-b.
(Note: For drawing 1 ml of each sample a new micropipette tip should be used)
Similarly, again 1ml of new sample N1 was taken and it underwent three stage dilution
i.e., 1 ml of sample was put into one of the 18 test tubes containing 9 ml distilled water,
subsequently 1 ml from the latter was taken and put into second test tube and again 1 ml
from previous test tube was taken and put into third one. This 1ml from the third test tube
was taken and spread over another petri-dish using L-shaped rod and was marked as N1-
a.

Figure 3.5: Dilution of the sample

Above 2 steps were repeated for remaining 5 samples and petri-dishes were marked
accordingly (i.e. N2-b, N2-a
N3-b, N3-a
O1-b, O1-a

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 O2-b, O2-a
O3-b, O3-a)
Next, all the petri-dishes were covered and made air tight by wrapping parafilm, all
around them.
Then these petri-dishes were put inside an incubator at a temperature of 29 0C to 300C.
After 2-3 days, in some of the petri-dishes some dotted structures appeared showing
formation of colonies.

Step 5: Streaking technique

All the old samples showed decent formation of colonies, except for the new ones which
may due to contamination of sample.
These three samples (i.e. O1-a, O2-a, O3-a) were taken into consideration.
Again, the following solution was prepared in 200ml distilled water containing the
chemicals mentioned below and contents were transferred to 500ml conical flask.
Peptone powder - 2 gm
Yeast extract - 1 gm
Sodium chloride - 1 gm
Agar Agar - 3 gm
Sterilization process:
All the glassware i.e.
500ml conical flask containing agar solution (cotton wrapped)
3 petri-dishes (paper wrapped)
3 to 5 micropipette tips in a small beaker (paper wrapped)
are put inside an Autoclave for about an hour operating at a temperature of 121 0C and 15
psi pressure.
Immediately, the solution after sterilization was transferred equally to 3 petri-dishes
inside the Laminar Air Flow region and left for about 15 minutes to solidify.
Now an Inoculation loop is taken and heat treated inside Laminar Air Flow region using
methanol spirit lamp before dipping inside every petri-dish

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 Figure 3.6: Sterilization of loop
This loop after dipping inside O1-a is spread in a horizontal manner for cultivation of
microbes on petri-dish.

Figure 3.7: Dipping of loop into sample

Similarly, above step is repeated for other two samples also and petri-dishes were marked
accordingly.

Figure 3.8: Streaking of media

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 Next, all the petri-dishes were covered and made air tight by wrapping parafilm, all
around them.
Then these petri-dishes were put inside an incubator at a temperature of 30 0C.
After 2-3 days, in some of the petri-dishes some long horizontal strands appeared
showing cultivation of microbes.

3.2 BOD ANALYSIS

Apparatus required:

BOD incubator
Burette and Burette stand
300ml glass stopper Bod bottles
500ml conical flask
Pipettes with elongated tips
Graduated cylinders

Chemicals required:

Calcium chloride
Magnesium sulphate
Ferric chloride
Di-Potassium Hydrogen phosphate
Potassium Di-Hydrogen phosphate
Di-sodium hydrogen phosphate
Ammonium chloride
Manganous sulphate
Potassium hydroxide
Potassium iodide
Sodium Azide
Sulphuric acid
Starch indicator
Sodium Thiosulphate

Procedure for preparation of solution:

Phosphate buffer solution

8.5 g of KH2PO4, 21.75 g of K2HPO4 , 33.4 g of Na2HPO4.7H2O and 1.7 g of NH4Cl was
dissolved in 500 ml distilled water and diluted it to 1000ml. The pH was adjusted to 7.2.
Magnesium Sulphate solution
22.5g MgSO4.7H2O was dissolved in distilled water and dilute it to 1 litre.

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 Calcium Chloride solution
27.5 g of CaCl2 was dissolved in 1000ml distilled water.
Ferric Chloride solution
0.25 g of Ferric chloride solution was dissolved in 1000ml of distilled water.
Sodium sulphite solution
1.575 g of sodium sulphite is dissolved in 1000ml of distilled water.

Procedure of dissolved oxygen analysis:

Four 300ml glass stoppered BOD bottles were taken (two for the sample and two for
blank).
10ml of the sample was added to each of the two BOD bottles and the remaining was
filled with the dilution water.
The remaining two BOD bottles are for blank, to these bottles dilution water was added.
After addition, immediately the glass stopper was placed over BOD bottles.
The two BOD bottles were taken and 2 ml each of managanous sulphate solution and of
alkali iodize azide was added to it below the liquid level. The bottle must completely air
tight so that no air should enter into it. The sample was mixed properly. The presence of
oxygen is indicated by the appearance brownish orange cloud of precipitate or floc.
This floc can be disappeared by turning the bottle upside down and allowing it to settle.
2 ml of sulphuric acid was added to it via a pipette holding it just above the surface of the
sample. Again the bottle is inverted after carefully plugging the stopper into it to dissolve
the floc. Then the sample is kept for 8 hr.
Filled the burette with sodium thiosulfate solution.
2 ml starch solution was added so that blue colour forms.
The sample was titrated slowly till the end point .And end point is determined when the
blue colour disappears.
The concentration of dissolved oxygen can be determined by the number of ml titrant
used. As each ml of sodium thiosulphate added equals 1 mg/l dissolved oxygen.

3.3 COD ANALYSIS

Apparatus required:
COD digester
Burette and Burette stand
COD vials with stand
Erlenemeyer flask
Pipettes
Chemicals required:
Potassium dichromate
Concentrated sulphuric acid

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 Ferroin indicator
Ferrous Ammonium Sulphate(FAS)
Mercuric sulphate
Distilled water
Procedure:
4.913gm of K2Cr2O7, 33.3g of HgSO4 and 167ml of H2SO4 were dissolved in 1000ml
distilled water.
39.2 gm of FAS is dissolved in distilled water and then 20ml of Conc. Sulphuric acid was
added and the solution is diluted to 1000ml
Now 20 ml of the sample was taken in a 500 ml flask
Then 10 ml of K2Cr2O7 was added to it.
30 ml of conc.H2SO4 was added slowly and cautiously.
0.4 gm of Mercuric sulphate was then added then the sample was heated at 120 C for
around 10 min.
The sample was cooled to room temperature
The solution was diluted to two times its volume with distilled water
Fill the burette with FAS solution and add 2-3 drops of Ferroin indicator to the diluted
solution and titrate it against FAS solution. The end point of the titration is determined by
sharp colour change from blue green to reddish brown which persisted for 1 min.

3.4 INVERSE FLUIDIZATION UNIT

Experimental Setup:
The experimental setup (Figure 2) consists of a cylindrical column (ID = 100 mm,
thickness = 3 mm, and height = 1240 mm) made of Perspex material. There are conical
liquid distribution at the top section and conical liquid discharge at the bottom section.
The pressure taps are evenly spaced at100 mm intervals on the wall of the column and
connected to manometers. Water is used as the manometric fluid. The liquid discharge
section connects a pipe to the reservoir to transfer the liquid into the tank, so that it is re-
circulated if needed. A control valve is also provided in the discharge line to adjust the
flow rate of fluid. A non-returning valve is attached for the one way entry of air only (not
water).
Materials and methods:
6 mm polypropylene spherical balls of density 930 kg/m3 have been used for the
hydrodynamic study. Initially the bed is operated under batch mode. Here, the column is
filled with water upto a certain level and loaded with polypropylene solid particles of a
particular size and density to the required bed height. With zero liquid flow, the gas flow
rate is increased gradually till the bed is completely fluidized. And the pressure drop is
measured by manometers. The bed heights were measured by visual observation.

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Figure3.9: Schematic diagram of Inverse fluidization unit

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 4. RESULT AND DISCUSSION
4.1 COD
Before treatment,
Initial value of COD = 2580 mg/l
pH = 6.7
After treatment,
Final value of COD = 215 mg/l
pH = 8.5

Table 4.1: Variation of COD with wastewater

Sl. No. Number of hours of operation COD(mg/l)

1 0 2580
2 6 2098
3 12 1456
4 24 987
5 48 564
6 72 215

3000

2500

2000
COD(mg/l)

1500

1000

500

0
0 10 20 30 40 50 60 70 80
Time(Hours)

Figure 4.1: Variation of COD with wastewater

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4.2 BOD
Before treatment,
Initial value of BOD = 1139 mg/l

After treatment,
Final value of BOD = 25 mg/l

Table 4.2: Variation of BOD with wastewater

Number of hours of
Sl No. operation BOD(mg/l)
1 0 1139
2 6 950
3 12 710
4 24 495
5 48 221
6 72 25

1200

1000

800
BOD(mg/l)

600

400

200

0
0 10 20 30 40 50 60 70 80
Time(Hours)

Figure 4.2: Variation of BOD with wastewater

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4.3 EFFICACY OF THE PROCESS

Table4.3: Percentage reduction

CPCB
CHARACTERISTICS INLET(mg/l) OUTLET(mg/l) EFFICIENCY(%) NORMS
pH 6.7 8.5 26.86 5.5-9.0
BOD 1139 25 97.8 <30
COD 2580 215 91.66 <250

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 5. CONCLUSION

COD and BOD analysis of wastewater is one of the basic step which is needed to set up
any wastewater treatment plant and to control losses to the sewer system. Many ways of
chemical treating wastewater has been proved to be very expensive and produces harmful end
product which is very necessary to be avoided in todays century. This study which includes
treatment of dairy wastewater with the most abundantly available resource i.e. bacteria shows a
new pathway to achieve two major goals of any wastewater treatment plant first being the
economy and second being the efficiency in reduction of harmful components present in
industrial, domestic or municipal wastewater. Treatment in inverse fluidization unit is very
economical as it very cheap to procure, easy to handle and require low power to operate and in
addition to these using aerobic bacteria in it for degradation of hazardous components sorts out
problems such as cost of oxygen supply needed for conversion of organic compounds.
Continuous mixing with the help of solid particles in fluidization unit helps aerobic bacteria to
grow on its surface. Thus, this type of study is necessary before setting up any wastewater
treatment plant.

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