1. [1]
ON
QUALITY ASSESSMENT OF INDUSTRIAL WASTE WATER AND ITS
BIOREMEDIATION WITH Pseudomonas aeroginose
CT Institute of Pharmaceutical Sciences
Partappura road, Shahpur (near lambra), Nakodar road, Jalandhar
Submitted to PUNJAB TECHNICAL UNIVERSITY, JALANDHAR
For the partial fulfilment of the requirement for the award of degree of
BACHELORS OF BIOTECHNOLOGY
(Session 2011-2014)
SUBMITTED BY:
Amrit
B.Sc Biotechnology
1111781
SUPERVISED BY :
Ms Navdeep Sidhu
HOD Biotechnology

2. [2]
SHAHPUR, JALANDHAR
TO WHOM IT MAY CONCERN
This is to certify that the project report entitled “QUALITY ASSESSMENT OF
INDUSTRIAL WASTE WATER AND ITS BIOREMEDIATION WITH Pseudomonas
aeruginose” has been completed by Amrit Univ. Roll No. 1111781 under my supervision
and guidance. To the best of my knowledge, this is an original work under taken by the
candidate and has not been submitted elsewhere in full or in parts for the award of any other
degree.
I approve it for submission the partial fulfilment of the requirement for the degree of
Bachelor of Biotechnology.
Place- CTIPS, SHAHPUR, JALANDHAR Ms. Navdeep Sidhu
HOD Biotechnology

3. [3]
ACKNOWLEDGEMENT
Hereby I would like to express my humble thanks to GOD for HIS bless, strength,
inspiration, and knowledge given to me throughout the completion of this dissertation report.
I am extremely thankful to Dr. Anil Sharma (Director CTIPS), to allow me to complete this
project. I wish my million grateful to my supervisor Ms. Navdeep Sidhu through her
support, motivation, encouragement and guidance that finally bring me to the completion of
this dissertation report. Her kindness and her gentleness have encouraged me in finishing this
report.
In spite of, the most important persons in my life not to be forget my father and my mother
for their inspiration and moral support throughout my study in CTIPS that has brought me to
this level. Without them I would be nowhere. This appreciation would not be complete
without my friends and classmates Pooja, Alka, Navneet, Subreena and not to be forget my
project partner Manpreet Kaur who has always perform equal work as me.
The last but not least lecturers in CTIPS and all teachers who have give full co-operation
during my study, their supportive ideas and critics had developed my knowledge and I’m
truly appreciating all their restless effort throughout my whole years study in this beloved CT
Institute.

4. [4]
ABSTRACT
The volume of waste in water is increasing each year due to the urbanization and industrial
development all around the world. It is concerned that the increase of waste in the water
could cause severe impact to the environment and to human health. Bioremediation of waste
from industrial waste water using Pseudomonas culture is being studied to overcome this
problem. The ability of the microorganism to degrade the waste is observed by applying
some parameters on waste water after bioremediation of 15 days. Results obtained before and
after microbial bioremediation are determined which shows that this bacterial species can be
used to decrease the amount of waste present in the water.

5. [5]
TABLE OF CONTENT
CHAPTER NO. TITLE PAGE NO.
(A) List Of Abbreviations 10
CHAPTER 1 INTRODUCTION
1.1 Background Of Study 12-13
1.2 Problem Statement 14-15
1.3 Bioremediation 16
1.4 Micro-Organism Used For
Bioremediation
17
1.5 Objectives 18
1.6 Parameters Used For Quality
Assessment Of Industrial Waste
Water
19-21
CHAPTER 2 REVIEW OF LITERATURE 22-26
CHAPTER 3 MATERIAL AND METHODS
3.1 Apparatus Used 28
3.2 Equipments Used 28
3.3 Preparation Of Reagents 29
3.4 Preparation Of Media 30
3.5 Protocols 31-38
CHAPTER 4 RESULT AND DISCUSSION
4.1 Areas Of Sampling 40
4.2 Observations 41-49
4.3 Observation Table 50-51

6. [6]
4.4 Discussion 52
CHAPTER 5 CONCLUSION 53-54
CHAPTER 6 REFERENCES 55-58

7. [7]
LIST OF FIGURES
FIGURE NO. TITLE PAGE
1 Addition of effluent by industries in drains 13
2 Intermixing of industrial effluent and drain
water.
15
3 Capping of bottle inside water to prevent any
traces of air.
31
4,5,6 BOD observations 41-42
7,8,9 COD observations 43
10,11,12 pH observations 44
13,14,15 TSS observations 45-46
16,17 TS observations 47
18,19 Alkalinity observations 48
20,21 Before and after bioremediation 49

8. [8]
LIST OF TABLES
TABLE NO. TITLE PAGE
1. Areas from where sample has
been collected for analysis
40
2. Table showing readings
before bioremediation
50
3. Table showing readings after
bioremediation
51

9. [9]
LIST OF ABBREVIATIONS
BOD- biochemical oxygen demand
COD - chemical oxygen demand
oC - degree Celsius
g - Grams
L - Litre
Mg - milligrams
HCl - hydrochloric acid
NaCl - sodium chloride
TS - Total solids
TSS - total suspended solids

10. [10]
1. INTRODUCTION

11. [11]
1.1 BACKGROUND OF STUDY
It is needless to emphasize the importance of water in our life. Without water, there is no life
on our planet. Water for different purposes has its own requirements for composition and
purity. Each body of water needs to be analysed on a regular basis to confirm to suitability.
The types of analysis could vary from simple field testing for a single analyte to laboratory
based multi-component instrumental analysis. The measurement of water quality is a very
exacting and time consuming process, and a large number of quantitative analytical methods
are used for this purpose.
Wastewater is generally divided into two broad classifications:
1. Domestic wastewater
2. Industrial wastewater.
Domestic wastewater comes from communities of homes, businesses, and institutions.
Domestic wastewater is 99.9% water and only 0.1% solids. The solids in domestic
wastewater are both dissolved and suspended solids. Suspended solids can be settled out or
filtered but dissolved solids will have to be converted to suspended solids during the
treatment process.
Industrial wastewater is one of the important pollution sources in the pollution of the water
environment. Wastewaters obtained from industries are generally much more polluted than
the domestic or even commercial wastewaters. The industrial waste water may comprise of
pathogens such as bacteria, viruses, worms; organic particles such as hair, food, plant
material; soluble organic material such as urea, sugars, soluble proteins, drugs,
pharmaceuticals; inorganic material such as ammonia, cyanide, hydrogen sulphide,
thiocyanates, thiosulphates; emulsions such as paints, adhesives, hair colourants, oils; toxins
such as pesticides, poisons etc. These wastes present in water are considered as serious types
of hazardous pollutants in aquatic organisms.

12. [12]
Fig 1: Addition of effluent by industries in drains

13. [13]
1.2 PROBLEM STATEMENT
During the last century a huge amount of industrial wastewater was discharged into rivers,
lakes and coastal areas. Still, however, several industrialists try to discharge their effluents
into natural river streams, through unauthorized direct discharges. Such a tendency, on the
part of industries may pollute the entire river water to a large extent, thereby making its
purification almost an impossible task. In modern societies proper management of waste
water is a necessity, not an option. Sometimes, the industries discharge their polluted
wastewaters into municipal sewers, thereby making the task of treating that municipal
sewage, a very difficult and costly exercise. The industries are, therefore, generally prevented
by laws, from discharging their untreated effluents. It, therefore, becomes, necessary, for the
industry to treat their wastewaters in their individual treatment plants, before discharging
their effluents either on land or lakes or rivers, or in municipal sewers. The characteristics of
the produced wastewater will usually vary from industry to industry, and also vary from
process to process even in the same industry. Many industrial wastewaters require pre-
treatment to remove non-compatible substances prior to discharge into the water bodies. Such
industrial wastewaters cannot always be treated easily by the normal methods of treating
domestic wastewaters, and certain specially designed methods or sequence of methods may
be necessary. In order to achieve this aim, it is generally always necessary, and advantageous
to isolate and remove the troubling pollutants from the wastewaters, before subjecting them
to usual treatment processes. The sequence of treatment processes adopted should also be
such as to help generate useful bi-products. This will help economize the pollution control
measures, and will encourage the industries to develop treatment plants.

14. [14]
Many types of treatment technologies are in use to remove contaminants from the waste
water like air sparging, bioremediation, thermal desorption, soil washing, chemical
dehalogenation but out of all the technologies used bioremediation is considered to be most
efficient among all. Biological treatment by bioremediation has been found to be the most
efficient method for removing fat, oil and grease by degrading them into miscible molecules.
Fig 2: Intermixing of industrial effluent and drain water.

15. [15]
1.3 BIOREMEDIATION
Bioremediation can be defined as a natural or managed biological degradation of
environmental pollution. Bio-remediation in the wastewater is the biological breakdown of
organic compounds in the wastewater system. Bio-remediation is proving to be very
successful in wastewater systems in a range of climates and operating conditions. It is an
environmentally friendly process that is resulting in significant savings for councils and
factories, particularly in reduction of odour and acid attack, reduced cleaning costs and
significantly improved performance of existing treatment plant infrastructure.
Bioremediation can be done in two ways:-
a) Using plants; known as phytoremediation and
b) Using micro-organisms; known as microbial bioremediation.
Phytoremediation is the direct use of living green plants for the removal, degradation, or
containment of contaminants in soil, sludge, sediment, surface water and groundwater.
Microbial bioremediation is basically the addition of microbes specially chosen to more
rapidly and completely biologically degrade the various types of organic waste. Micro-
organisms can be added to the various components of treatment plants and the different types
of lagoon systems. As various combinations of microbes and methods are trialled and proven,
the benefits are becoming obvious throughout sewerage systems, from the collection network
to final effluent quality and also for effluent re-use.
Bioremediation uses naturally occurring microorganisms and other aspects of the natural
environment to treat wastewater. Bioremediation can prove less expensive than other
technologies that are used for cleanup of hazardous waste. Industrial and wastewater
treatment systems are inoculated with micro-organisms for a wide variety of reasons, from
pre-treatment of sewage, to final product quality. Bio-remediation can lead to better oxygen
uptake, solids removal and overall efficiency of removing organic material such as BOD and
suspended solids. Higher operating temperatures, pH and alkalinity in digesters can lead to
greater operating efficiency and stability. Micro-organisms are used to digest a larger
proportion of the accumulated organic solids and improves final effluent quality to opens the
opportunities for reuse.

16. [16]
1.4 MICRO-ORGANISM USED FOR BIOREMEDIATION
Pseudomonas aeruginosa (ATCC NO. 2453)
Pseudomonas aeruginosa is classified as gram-
negative bacterium. They are aerobic bacterium
with unipolar motility. P. Aeruginosa is a free-
living bacterium, commonly found in soil and
water. However, it occurs regularly on the
surfaces of plants and occasionally on the
surfaces of animals. It can survive under
conditions that few other organisms can tolerate,
it produces a slime layer that resists phagocytosis (engulfment), and it is resistant to most
antibiotics.
Kingdom: Bacteria
Phylum : Proteobacteria
Class: Gamma proteobacteria
Order: Pseudomonadales
Family: Pseudomonadaceae
Genus: Pseudomonas
Species: aeruginosa

17. [17]
1.5 OBJECTIVES
The aim of the project is
 Waste water study and analysis
 Physical analysis of waste water collected from various areas.
 Chemical analysis of waste water collected from various areas.
 Bioremediation of waste water using micro-organism Pseudomonas aeruginosa.

18. [18]
1.6 PARAMETERS USED FOR QUALITY ASSESSMENT OF
INDUSTRIAL WASTE WATER
1.6.1 pH:
pH is a measure of the acidity or basicity of solution or substance. It is determined by the
number of free hydrogen ions (H+) in a substance. Solutions with a pH less than 7 are said to
be acidic and solutions with a pH greater than 7 are basic or alkaline. Pure water has a pH
very close to 7. The pH is a logarithmic factor; when a solution becomes ten times more
acidic, the pH will fall by one unit. As a chemical component of the wastewater, pH has
direct influence on wastewater treatability regardless of whether treatment is
physical/chemical or biological. Because it is such a critical component of the makeup of the
wastewater, it is therefore critically important to treatment.
1.6.2 Biochemical oxygen demand:
Biochemical oxygen demand represents the amount of oxygen consumed by bacteria and
other microorganisms while they decompose organic matter under aerobic conditions at a
specified temperature. The biochemical oxygen demand (BOD) is an empirical test, in which
standardised laboratory procedures are used to estimate the relative oxygen requirements of
wastewaters, effluents and polluted waters. Micro- organisms use the atmospheric oxygen
dissolved in the water for biochemical oxidation of organic matter, which is their source of
carbon. The BOD is used as an approximate measure of the amount of biochemically
degradable organic matter present in a sample. The 5-day incubation period has been
accepted as the standard for this test.
The BOD test was originally devised by the United Kingdom Royal Commission on Sewage
Disposal as a means of assessing the rate of biochemical oxidation that would occur in a
natural water body to which a polluting effluent was discharged.
1.6.3 Chemical oxygen demand:

19. [19]
The chemical oxygen demand (COD) is the amount of oxygen consumed by organic matter
from boiling acid potassium dichromate solution. It provides a measure of the oxygen
equivalent of that portion of the organic matter in a water sample that is susceptible to
oxidation under the conditions of the test. It is an important and rapidly measured variable for
characterising water bodies, sewage, industrial wastes and treatment plant effluents. The
dichromate method has been selected as a reference method for the COD determination
because it has advantages over other oxidants owing to its oxidising power, its applicability
to a wide variety of samples and its ease of manipulation.
1.6.4 Total Suspended Solids:
Total suspended solids (TSS) give a measure of the turbidity of the water. Total suspended
solids include all particles suspended in water which will not pass through a filter. Suspended
solids are present in sanitary wastewater and many types of industrial wastewater. As levels
of TSS increase, a water body begins to lose its ability to support a diversity of aquatic life.
Suspended solids absorb heat from sunlight, which increases water temperature and
subsequently decreases levels of dissolved oxygen. Photosynthesis also decreases, since less
light penetrates the water. As less oxygen is produced by plants and algae, there is a further
drop in dissolved oxygen levels. TSS can also destroy fish habitat because suspended solids
settle to the bottom and can eventually blanket the river bed. Suspended solids can smother
the eggs of fish and aquatic insects, and can suffocate newly-hatched insect larvae.
Suspended solids can also harm fish directly by clogging gills, reducing growth rates, and
lowering resistance to disease. Changes to the aquatic environment may result in a
diminished food sources, and increased difficulties in finding food. Natural movements of
aquatic populations may be disrupted.
1.6.5 Total Solids:
Total solids (TS) are a measure of all the suspended, colloidal, and dissolved solids in a
sample of water. This includes dissolved salts such as sodium chloride, calcium, chloride,
bicarbonate, nitrates, phosphates, iron and NaCl, and solid particles such as silt and plankton.
An excess of total solids in rivers and streams is a very common problem. Elevated levels of
total solids, however, can lead to eutrophication of the stream. Eutrophication result in a

20. [20]
decrease in stream water quality. If the levels of total solids are too high or too low, it can
impact the health of the stream and the organisms that live there. High levels of total solids
will reduce the clarity of the water. This decreases the amount of sunlight able to penetrate
the water, thereby decreasing the photosynthetic rate. Reduced clarity also makes the water
less aesthetically pleasing. While this may not be harmful directly, it is certainly undesirable
for many water uses. When the water is cloudy, sunlight will warm it more efficiently. This
occurs because the suspended particles in the water absorb the sunlight which, in turn, warm
the surrounding water. This leads to other problems associated with increased temperature
levels.
1.6.6 Alkalinity:
Alkalinity is a measure of the capacity of water to neutralize acids. Alkaline compounds in
the water such as bicarbonates, carbonates, and hydroxides remove H+ ions and lower the
acidity of the water. They usually do this by combining with the H+ ions to make new
compounds. Without this acid-neutralizing capacity, any acid added to a stream would cause
an immediate change in the pH. Measuring alkalinity is important in determining a stream's
ability to neutralize acidic pollution from rainfall or wastewater. It's one of the best measures
of the sensitivity of the stream to acid inputs. Alkalinity in streams is influenced by rocks and
soils, salts, certain plant activities, and certain industrial wastewater discharges.

21. [21]
2. REVIEW OF
LITERATURE

22. [22]
According to Yusufu Luka, Haruna Mavakumba Kefas, Jackson Robinson Genza, Bukhari
Auwal Abdul-hamid in 2014 the use of Bacillus subtilis microorganism to study the kinetics
of bioremediation of Shinko drainage wastewater used for agriculture and channelled into
River Benue was carried out. The bioremediation was observed for a period of twenty six
days at an interval of two days. A bioreactor was used under aerobic condition to achieve the
degradation of contaminant. Bacillus subtilis was isolated from Shinko drainage wastewater
and effective in reducing the substrate concentration from65.35 mg/l to 15.59 mg/l for
bioaugmented sample.
Usman D. Hamza, Ibrahum A. Mohammed and Abdulahhi Salem in 2012 studied that
industrial wastewaters containing petroleum hydrocarbon is highly toxic and posed a great
danger especially to refinery nearby communities. Therefore, there is need for effective
treatment before discharge. Natural process employing microorganisms is considered to be
very effective and environmentally friendly method of decontamination. This research is
mainly focused on the use of indigenous microbes in biodegradation of petroleum
hydrocarbons in contaminated water. The wastewater was characterized for physicochemical,
organics, inorganics and metallic parameters. The ability of Serratia mercescens, Bacillus
subtilis, Micrococcus luteus, Bacillus cereus and biostimulated samples were studied for the
hydrocarbon reduction from petroleum refinery wastewater. The performance of the system
was measured by monitoring chemical oxygen demand (COD) reduction and increase in
optical density (OD) of the flask cultured samples in Bushnell-Haas medium. Bacillus subtilis
demonstrated high COD reduction of 56.19% while Micrococcus luteus reduced COD by
52.43%. Micrococcus showed higher growth in the Bushnell-Haas medium with the refinery
wastewater as a sole carbon source.
According to Zhao S , Hu N, Chen Z, Zhao B, Liang Y in 2009 organic matter and nitrogen
removal from reclaimed wastewater used as landscape water was carried out by in situ
bioremediation. A denitrifying bacterium Bacillus cereus DNF409 was introduced for this
purpose, and the amount of B. cereus used was optimized. The total nitrogen (TN) content
and chemical oxygen demand (COD) of the landscape water decreased from 9.86 to 3.1 mg/L
(removal rate, 68.6%) and from 127 to 36 mg/L (removal rate, 71.7%). The transparency of
water increased from 0.2 to 0.55 m.

23. [23]
Emmanuelle Gratia, Frederic Weekers, Rosa Margesin, Salvino D’Amico, Philippe Thonart,
Georges Feller in 2009 invented that amongst more than 1000 isolates collected in various
cold environments, the strain Arthrobacter psychrolactophilus Sp 31.3 has been selected for
its ability to grow and to produce exoenzymes at low temperatures, its inability to grow at
37°C, its non-halophilic character and its growth versatility on various media. This non-
pathogenic strain displays a strong resistance to desiccation and storage at room temperature
and is suitable for the production of freeze-dried bacterial starters. When grown in a synthetic
wastewater at 10°C, the strain induces a complete clarification of the turbid medium and
efficiently hydrolyses proteins, starch and lipids in the broth. Furthermore, this strain has a
remarkable capacity to improve the biodegradability of organic compounds in wastewater as
indicated by a BOD5/COD.
According to Shuyan Zang, Bin Lian, in 2008, 2-Naphthol, which originates widely from
various industrial activities, is toxic and thus harmful to human liver and kidney. A new
compound biodegradation system was adopted to degrade 2-naphthol-contaminated
wastewater. As a co-metabolic substrate, salicylic acid could induce the two microorganisms
(Fusarium proliferatum and Bacillus subtilis) to produce a large amount of degradation
enzymes for 2-naphthol. The key enzymes were confirmed as polyphenol oxidase (PPO) and
catechol 2, 3-dioxygenase (C23O). The degradation extent of 2-naphthol, determined by high
performance liquid chromatography (HPLC), was enhanced by nearly 15% on the 6th day
after the addition of the co-metabolic substrate. The results obtained thus clearly indicated
that the co-metabolic process was the most important factor affecting the degradation of the
target contaminant. The optimal concentration of 2-naphthol was 150 mg L−1
, and the optimal
pH value was 7.0. The degradation extent of 2-naphthol was further enhanced by nearly 10%
after the addition of Tween 80, which increased the bioavailability of 2-naphthol. In a
practical treatment of industrial wastewater from medical manufacture, the synergistic
degradation system resulted in a high degradation efficiency of 2-naphthol although its lag
time was a little long in the initial stage.

24. [24]
Rajbir Singh, Debarati Paul, Rakesh K. Jain in 2006 invented that biofilms are assemblages
of single or multiple populations (like Bacillus Subtilis) that are attached to abiotic or biotic
surfaces through extracellular polymeric substances. Gene expression in biofilm cells differs
from planktonic stage expression and these differentially expressed genes regulate biofilm
formation and development. Biofilm systems are especially suitable for the treatment of
recalcitrant compounds because of their high microbial biomass and ability to immobilize
compounds. Bioremediation is also facilitated by enhanced gene transfer among biofilm
organisms and by the increased bioavailability of pollutants for degradation as a result of
bacterial chemotaxis. Strategies for improving bioremediation efficiency include genetic
engineering to improve strains and chemotactic ability, the use of mixed population biofilms
and optimization of physico–chemical conditions.
According to Dr. Ayodhya Dattatray Kshirsagar in 2004 i) Wastewater was treated with
Aspergillus terreus, Aspergillus niger, Rhizopus nigricans and Cunninghamella sp. and ii)
Wastewater was treated without these organisms (Control). The sample was analyzed for pH,
BOD, COD nitrate and phosphate before and after wastewater treatment using fungi at
various time intervals for 5th, 10th, 15th and 20th days wastewater samples were analysis
using standard methods. Result reveled that A. terreus and A. niger which showed excellent
pollutant removal capabilities. A. terreus were showed the best removal of nitrate and BOD
while A. niger showed best removal of phosphate and COD capacity from wastewater.
Present investigation focuses on the bioremediation of wastewater by using aquatic fungi.
According to Wiyada Mongkolthanaruk, Saovanee Dharmsthiti in 2002, a mixed bacterial
culture comprising Pseudomonas aeruginosa LP602, Bacillus sp. B304 and Acinetobacter
calcoaceticus LP009 for use in treatment of lipid-rich wastewater. The intended role of B304
was that of a protease and amylase producer while those of LP602 and LP009 were those of
lipase producers. The BOD value and lipid content were reduced within 12 days under
aerobic conditions.
According to Rahul S. Kulkarni, Pradnya P. Kanekar in 1998, the degradation of ε-
caprolactam in waste water was found to be optimal over a wide range of pH from 5.0 to 9.0,

25. [25]
temperature of 30°C, and under shake or aerated conditions, with an inoculum density of 105
cells/ml and with an incubation period of 24 – 48 h. Thus, Pseudomonas aeruginosa MCM B-
407 isolated from the activated sludge exposed to ε-caprolactam may play an important role
in the bioremediation of ε-caprolactam from the waste waters of industries manufacturing
nylon-6.

26. [26]
3. MATERIALS AND
METHODS

27. [27]
3.1 APPARATUS USED
i. Beaker
ii. BOD bottles
iii. Burette
iv. Burette stand
v. Dropper
vi. Erlenmeyer flask
vii. Funnel
viii. Glass rod
ix. Measuring cylinder
x. pH strips
xi. Pipettes
xii. Porcelain dish
xiii. Reagent bottles
xiv. Titration flask
xv. tripod stand
3.2 EQUIPMENTS USED
i. Autoclave
ii. BOD incubator
iii. Hot air oven
iv. Laminar air flow
v. Water bath

28. [28]
3.3 PREPARATION OF REAGENTS
1. Alkaline iodide sodium azide solution: Dissolve 700g of potassium hydroxide and
150g potassium iodide in fresh distilled water. Prepare 10g sodium azide in 40ml
water. Slowly add, with stirring, the azide solution to the alkaline iodide solution
bringing the total volume to 1000mL. Store the solution in clean reagent bottle.
2. Hydrochloric acid solution: Add 8.9mL of conc. Hydrochloric acid in 991mL
distilled water and store it in clean reagent bottle.
3. Manganese sulphate: Dissolve 168.4g of manganese sulphate in distilled water and
make the volume upto 1000mL. Store the solution in clean reagent bottle.
4. Methyl orange: Dissolve 1g of methyl orange in 100 mL distilled water. Freshly
prepare this solution before use.
5. Potassium dichromate solution: Dissolve 12.259 g of K2Cr2O7 in distilled water and
prepare the volume to 1000mL. Store the solution in clean reagent bottle.
6. Potassium iodide solution: Dissolve 10g of potassium iodide in distilled water and
make the volume upto 100mL. Store the solution in clean reagent bottle.
7. Starch indicator: Dissolve 2g of starch in hot distilled water and make the volume
upto 100mL. Add 0.2g salicylic acid as a preservative. Store the solution in clean
reagent bottle.
8. Sulphuric acid (concentrated)
9. Sodium thiosulphate stock solution: Weight approx. 25g of sodium thiosulphate and
dissolve it in boiled distilled water. Make the volume upto 1000mL. Add 1g sodium
hydroxide to preserve it. Store the solution in clean reagent bottle.

29. [29]
3.4 PREPARATION OF MEDIA
3.4.1 NUTRIENT BROTH
3.4.2 NUTRIENT AGAR

30. [30]
3.5 PROTOCOLS
3.5.1 COLLECTION OF WATER SAMPLE:
1. A labelled sterile bottle was taken with closed lid.
2. The sterile bottle was held in one hand near the base, and then the screw cap was
carefully removed and held with the other hand.
3. Bottle was held by its base and plunged into the water source with the neck facing
down.
4. The bottle was turned until the neck was pointing slightly upward and the mouth
was directed towards flow of stream.
5. Bottle was allowed to fill upto the brim.
6. The bottle was capped inside the water to prevent any traces of air.
Fig 3: capping of bottle inside water to prevent any traces of air.

31. [31]
3.5.2 BOD (Biochemical oxygen demand)
3.5.3 COD (Chemical Oxygen Demand)
3.5.4 pH
3.5.5 TOTAL SUSPENDED SOLIDS
3.5.6 TOTAL SOLIDS

32. [32]
3.5.7 ALKALINITY

33. [33]
4. RESULTS AND
DISCUSSION

34. [34]
4.1 AREAS OF SAMPLING
SAMPLES AREAS
Sample 1 Nijhran
Sample 2 Basti bawa
Sample 3 Nakodar
Sample 4 Maqsudan
Sample 5 Urban estate phase 2
Table 1: Areas from where sample has been collected for analysis

35. [35]
4.2 OBSERVATIONS
4.2.1 BOD
Fig 5: Colour after adding starch
Fig 4: Initial colour

36. [36]
Fig 6: End point

37. [37]
4.2.2 COD
Fig 7: Initial colour Fig 8: Colour after adding starch
Fig 9: End point

38. [38]
4.2.3 pH
Fig 10: Dip pH strip in sample water
Fig 11: Place the pH strip
Fig 12: Compare the colour of strip

39. [39]
4.2.4 TOTAL SUSPENDED SOLIDS (TSS)
Fig 13: Weighing the filter paper
Fig 14: Pouring sample through filter paper

40. [40]
Fig 15: Dry the sample before weighing

41. [41]
4.2.5 TOTAL SOLIDS (TS)
Fig 16: Evaporation of Sample on Water Bath
Fig 17: Residue Left after Evaporation

42. [42]
4.2.6 ALKALINITY
Fig 18: Colour after adding methyl orange
Fig 19: End point after titration

43. [43]
4.2.7 BIOREMEDIATION
Fig 20: Sample before Bioremediation
Fig 21: Sample after Bioremediation

44. [44]
4.3 OBSERVATION TABLE
4.3.1 BEFORE BIOREMEDIATION
PARAMETERS Ph BOD COD TSS TS ALKALINITY
SAMPLES
NIJHRAN
BASTI BAWA
NAKODAR
MAQSUDAN
URBAN ESTATE
PHASE-2
Table 2: Table showing readings before bioremediation

45. [45]
4.3.2 AFTER BIOREMEDIATION
PARAMETERS pH BOD COD TSS TS ALKALINITY
SAMPLES
NIJHRAN
BASTI BAWA
NAKODAR
MAQSUDAN
UBAN ESTATE
PHASE-2
Table 3: Table showing readings after bioremediation

46. [46]
4.4 DISCUSSION
In this project we had analyzed the quality and purity of water by applying certain physical
and chemical parameters. Samples were collected from the five different drains and analyzed
for the estimation of BOD, COD, TSS, TS and Alkalinity of water. When these tests were
performed, results were calculated which shows a significant decrease in the quality of water
of different drains. The quality of water varies from drain to drain. The results obtained
before and after bioremediation are stated underneath with discussion.
4.4.1 BEFORE BIOREMEDIATION:
When certain parameters were applied before bioremediation in order to check the quality of
water, the results shows a large amount of wastes present in the water bodies. The BOD,
COD, TSS, TS and Alkalinity values before bioremediation were 8.56mg/L, 43.2mg/L,
0.838mg/L, 2.09mg/L and 214.8mg/L respectively. A higher level of BOD and COD
represents the large amount of organic waste present in the water which indicates the
depletion of oxygen present in water gradually. A higher TSS and TS value indicates the
turbidity and cloudiness of water which adversely effect the aquatic life inside the water
bodies. These values have increased due to the presence of large amount of effluent from the
industries.
4.4.2 AFTER BIOREMEDIATION:
The values of BOD, COD, TSS, TS and Alkalinity after bioremediation of 15 days using
Pseudomonas aeruginosa have reduced to 5.06mg/L, 18.34mg/L, 0.238mg/L, 1.116mg/L and
113.4mg/L respectively. The differences between the results obtained from before and after
bioremediation shows a great decrease in the level of pollution in water by using the bacterial
strain P. aeruginosa that was increased by the addition of industrial effluent in water.

47. [47]
5. CONCLUSION

48. [48]
It is concluded from the present study that water bodies are highly polluted by the addition of
industrial waste without its prior treatment. To decrease the level of pollution in water
microbial bioremediation is necessary and also easy and cost effective technique than the
existing technologies. Results calculated shows that and Pseudomonas aeruginosa can be
used as the successful removal tool for the decrease in physical (TSS and TS) and chemical
(BOD, COD and Alkalinity) parameters in order to increase the quality of water. This
bacterial strain has shown a great decrease in the BOD and COD requirement. It has also
helped to decrease the content of TSS and TS in water and also decreases the high alkalinity
of water.

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