BIOCHEMICAL REMOVAL OF METALS BY ALGAE (CHLORELLA …

ELECTROCHEMICAL AND

BIOCHEMICAL TREATMENT OF

INDUSTRIAL WASTE WATER

AA TThheessiiss ssuubbmmiitttteedd ttoo TThhee UUnniivveerrssiittyy ooff tthhee PPuunnjjaabb FFoorr tthhee AAwwaarrdd ooff DDeeggrreeee ooff

DDOOCCTTOORR OOFF PPHHIILLOOSSOOPPHHYY

IINN CCHHEEMMIISSTTRRYY

22001122

SUBMITTED BY

ZAMIR AHMAD ANSARI

SUPERVISED BY

PROF. DR. ZAID MAHMOOD

INSTITUTE OF CHEMISTRY

UNIVERSITY OF THE PUNJAB, LAHORE

2012

i

DEDICATION

This work is dedicated to

MY LATE PARENTS,

My Wife & My Daughter

ii

ACKNOWLEDGEMENT

All praises to the ALMIGHTY ALLAH who induced the man with

intelligence, knowledge, sight to observe, mind to think and judge. Peace

and blessings of Allah be upon the Hazrat Muhammad (S.A.W.W.) and

his pure and pious progeny who exhorted his followers to seek

knowledge from cradle to grave.

I am greatly obliged to my worthy upervisor Prof. Dr. Zaid Mehmood,

Institute of Chemistry, University of the Punjab, Lahore, whose

knowledge, skillful guidance, encouragement and kindness have helped

me in each and every stage of my research work. Indeed it is an honor

and leasure for me to work with him. I have been fortunate to learn a

great deal of Chemistry from him.

I am also grateful to Dr. Saeed Ahmad Nagra, Director, Institute of

Chemistry, University of the Punjab Lahore, for providing me research

facilities during my research work. I am indeed grateful to all teachers of

Chemistry section.

I am also grateful to Chairman, Depatment of Chemsitry, UET, Lahore

for providing analytical and micrological facilities for the completion of

this research work.

Zamir Ahmad Ansari

iii

CONTENTS

INTRODUCTION 2

Waste Water 2

Waste Water Constituents 2

Types of Waste Water 3

Sewage Water 3

Impurities in Sewage Water 3

Suspended Particles 3

Dissolved Inorganics 4

Dissolved Organic Impurities 4

Micro-organisms 4

Dissolved Gases 5

Heavy Metals 5

Commonly Announced Toxic Heavy Metals 5

Cadmium 5

Antimony 5

Arsenic 5

Mercury 5

Lead 6

Manganese 6

Chromium 6

Copper 6

Nickel 6

Selenium 6

LITERATURE SURVEY 10

BIOCHEMICAL TREATMENT 20

Collection of the Samples 20

iv

Purification of Algal Species 20

Repeated Washing Method 21

In the Breakers 21

In the Flask 21

Inoculum Development 21

Composition of Chu No. 10 Medium 22

Composition of Bristol media (stock solution) 22

Shaking Flask Technique 22

Preparation of Slants 23

Analysis of the Water Samples 23

Cobalt (Co) 24

Preparation of Standard Solutions 24

Solution Technique 24

Recommended Instrument Parameters 24

Atomic Absorption 24

Flame Emission 25

Copper (Cu) 25





Flame Emission 26

Europium (Eu) 26





Flame Emission 27

Potassium (K) 27

v





Flame Emission 28

Manganese (Mn) 28





Flame Emission 29

Sodium (Na) 29





Flame Emission 30

Nickel (Ni) 30





Flame Emission 31

Zinc (Zn) 31





Flame Emission 32

vi

Kinetic Parameters 32

pH 32

Temperature 32

Determination of Growth Rate 32

Methodology of Haemocytometer 33

Illumination 33

Conductivity 33

Determination of COD 33

Regeant Used 33

Procedure 34

Observations 35

Calculations 35

Determination of Total Phosphate 35

Reagents 35

Procedure 36

a) Sample pH adjustment 36

b) Color removalfrom sample 36

c) Color development in sample 36

d) Preparation of calibration curve 37

Calculation 37

Determination of Total Nitrogen 37

Reagents 37

Procedure 37

a) Digestion 37

b) Distillation 38

c) Titration 38

Calculations 38

vii

Determination of Sulphates 38

Solutions 38

Calculations 39

Determination of Chlorides 39

Calculations: 39

Determination of Total Solids 39

Calculations 40

Growth of Chlorella Vulgaris, Oocystus Pusilla, Maugetai

Genoflexa and Ulothrix in Waste Water 40

ELECTRO CHEMICAL TREATMENT OF WASTE WATER 43

Deposition v/s Surface Area 46

Deposition vs Rate of Rotation 47

RESULTS AND DISCUSSION 49

TABLES OF RESULTS 69

GRAPHS OF RESULTS 100

CONCLUSION 142

REFERENCES 146

LIST OF PUBLICATIONS 161

viii

ABBREVIATIONS

C.V. Chlorella Vulgaris

O.P. Oocystis Pusilla

M.G. Maugetia Genoflexa

G.R. Growth Rate

N. Total Nitrigen

P Total Phosphate

COD Chemical Oxygen Demand

TDS Total Dissolved Solids

Condt. Conductivity

Cu Copper

K Potasium

Na Sodium

Ca Calcium

Mg Megnasium

Mn Megnanese

Fe Iron

Zn Zinc

Mg/ml Milli Grams/Litre

P.A.M. Parts Per Million

Mill/ml Millions/ml

ix

SUMMARY

Industrial waste water disposal and its treatment is becoming a global

hazard nowadays. Present research work was conducted to suggest some

means to solve this problem.

Industrial waste water sample were collected from 3 different points of

Lahore canal, where the industrial and domestic waste water is being

disposed off. One sample from the river Ravi Disposal, one from

industrial area from Shahdra and one from industrial area of Sheikhupura.

For biochemical processes, Algal samples were collected from natural

habitate at different sites mostly from suburban areas of Lahore e.g.

Nandipur, Kalasha Kaku, Gujranwala, Mehmood Bhuti, and Jallo etc.

The samples were studied microscopically and were subjected to a series

of subculturings on various solid and liquid media following the standard

methods of pure cultures of Algae, the following Algal species were

screened out from the mixed field samples.

1) Chlorella Vulgaris

2) Oocystis Pusilla

3) Maugetia Genuflexa

4) Ulothrix

Species were collected in 1 liter conical flask with tap water as medium.

The species were first purified by repeated washing method in 800 ml of

water and then separated by identification under the microscope. The

species were further grown for inoculums development in Bristol medium

and Chu No. 10 medium. The luxurious growth occurred in Bristol

x

medium rather than in Chu No. 10. The fermentation was carried out at

27o – 35

oC temperature and 5000 Lux light intensity at a controlled pH of

7. And then transferred to the slant using czapecdox media and stored at

10oC in the incubator.

Waste water samples were analysed for the determination of metal ions,

COD, pH, conductivity, phosphates, total nitrogen, chlorides, sulphates

and total solids, before further studies.

Waste Water samples were subjected to the Algal fermentation using

Chlorella Vulgaris, Oocsystis Pusilla, Maugetia Genoflexa, Ulothrix and

mix species. Growth rate was studied at different parameters like, pH,

temperature, light intensity. Optimal microbial growth in all the algal

species occurred at pH-7, temperature 27o – 35

oC, illumination 5000 Lux

between 6 and 7 days of growth period. Maximum uptake of the

impurities occurred at these conditions. Water samples were analyzed

again for the determination of metal ions, COD, Total N, P, Cl, SO4,

TDS, pH, conductivity and a remarkable decrease in all impurities was

observed.In second phase, Electrochemical treatment with three

component system was adopted. Rectangular glass made take was used as

working cell. Cathodic and anodic parts were separated by polymeric

membrane.

Water samples were added in the central part one by one. Vitreous carbon

electrode was used as base for electro deposition of Pt to form doped c/pt

anode. Pure carbon rode was used as cathodes, ordinary power supply

was used as current source with DC rectifier. Voltmeter and ammeter

were used to determine voltage and current. C/pt and pb/sb electrodes

with surface area of 2cm and 3cm were used for the removal of the

xi

impurities from water. Average DC current of 20 volts and 3 amp was

applied. Potential was enhanced in anodic direction by 2 volt/10m and

potential curve was drawn. Studies were carried out at different potential

by varying rotation from 15 Rpm to 50 Rpm with and without using

H2SO4 as oxidizing agent. Maximum deposition occurred by using N/50

H2SO4 as oxidizing agent at a rotation of 15 Rpm and potential of 9V at

surface area 3 cm, and 11V 2 cm surface area with C/pt electrode.

Without using H2SO4, the maximum amount of impurities deposited at

11.V both with 3 cm and 2 cm surface area when C/Pt electrode was

used. With the increase in surface area of the disc the amount deposited

at electrode increases. Water left in the central part of the cell was again

analyzed for the determination of metal ions. Total N, P, COD,

conductivity, chlorides, sulphates, TDS, pH and conductivity.

Remarkable decrease in all these parameter was observed with c/pt

electrode of 3cm surface area as compared to pb/sb electrode.

The results were compared with the results obtained from Biochemical

process and it was observed that electrochemical process is a better

option than biochemical process but a combination of the two processes

can be a ideal one. Combination of mix species (Chlorella Vulgaris +

Oocystus Pucilla) followed by electrochemical process using c/pt

electrode gave excellent results for the removal of impurities as compared

to individual one and their combination of other species.

2

INTRODUCTION

Waste Water

Wastewater is the water that has been adversely affected in quality by

impurities and not fit for consumption.

Waste Water Constituents

The composition of wastewater differs widely. It contains both Pathogens

and Non-pathogenic bacteria viruses and parasitic worms, protozoa,

insects, arthropods, small fish etc. Impurities are of two types, i. e.

organic and inorganic which may be soluble or insoluble.

Wastewater quality depends on type of impurities both inorganic and

organic and biological also. These impurities affect the water in different

ways. Inorganic impurities such as different types of salts, gases and

ammonia compounds influence the quality of water by varying the pH

and providing a nutrient media for the growth of micro organism which

are the biological impurities normally present in waste water. Similarly

organic impurities also show an impact on the water quality as they are

both soluble and insoluble. Soluble organic impurities are sugar fruits,

pesticides and fungicides. They also provide a good media for biological

impurities. The biological impurities are bacteria, viruses, worm, E coli,

B coli and other micro organism responsible of causing the diseases like

cholera daiaria, dysentery. Waste water normally includes sewerage water

or industrial water and water coming from fields. All these three types of

water contain vary in concentration and are treated in different ways. The

waste water is given different types of treatment for the removal of

impurities.

3

Types of Waste Water

1) Sewage water or municipal waste water

2) Industrial waste water

3) Agricultural waste water

Sewage Water

Municipal waste water or the sewage water is the most impure form of

waste water. Sewage is created by residences, institutions, hospitals and

commercial and industrial places. Raw influent (sewage) includes

household waste, water from toilets, baths, showers, kitchens, sinks, and

so forth that is disposed of via sewers. In many areas, sewage also

includes liquid waste from industry.

Impurities in Sewage Water

The major categories of impurities found in it include:

Suspended particles, including colloids

Dissolved inorganic impurities

Dissolved organic compounds

Micro-organisms

Pyrogens

Dissolved gases

Suspended Particles

Suspended matter includes pipework debris, salt and colloids. Colloidal

particles (which can be organic or inorganic) are not truly in solution or

suspension and give rise to haze or pollution to water. The degree of

colloidal contamination can be determined by a fouling index test or by a

turbidometrer.

4

Dissolved Inorganics

Inorganic substances in solution include hardness salts derived from rock

strata. The bicarbonates of Ca++

and Mg++

give rise to „temporary

hardness‟, while the sulfates and chlorides cause “permanent hardness”.

Dissolved Organic Impurities

Organic impurities arise from the decay of vegetable matter - principally

humic and fulvic acids - and from farming, paper making and domestic

and industrial waste. These include detergents, fats, oils, solvents and

residues from pesticides and herbicides. In addition, water-borne organics

may include compounds leached from pipework, tanks and purification

media.

Micro-organisms

It normally contains more than 5000 different types of microorganisms

like bacteria, methogenic bacteria, flavobacteria E and B coli and viruses

which cause hepatic diseases and intestinal disturbance. It also contains

animal and human wastes. [86]

The microorganisms can grow at the pH close to neutral and temperature

also remains below 30°C as the pipes are covered. The only negative

factor is the non availability of sunlight in closed pipe lines. Anaerobic

fermentation of methogenic bacteria can occur. They produce methane

and H2S. These two gasses produce smell in gutters. In standing water,

growth occurs and CO2, CO, CH4 are produced by fermentation colour is

grey. Colour intensity depends on the percentage of organic substances.

[85]

5

Dissolved Gases

Oxygen and carbon dioxide are the two gases most commonly found in

natural waters. [87][88]

Heavy Metals

It contains heavy toxic metals which comes from mixing of the industrial

waste water, [wj 62] streat run off and ground sewage infiltration system

from the soil with domestic waste water. Concentration of these trace

elements depends on the living habits, cultural and socioeconomical

aspects, so it cannot be fixed. [85][89]

Commonly Announced Toxic Heavy Metals

Cadmium

It is a micronutrient excess leads to increased blood pressure, renal

disinfection, lung‟s diseases ultimately leading to lungs cancer.[117]

Antimony

It causes cancer and also nausea, vomiting and diarrohea.

Arsenic

It causes skin disease of eyelid, gastrointestinal irritation, kidneys and

liver is also damaged.[117]

Mercury

High intake often leads to psychotic stste resulting in hyper excitability

i.e madness. It damages growth tissues because of mucosal degradation,

toxicity develops in liver and kidney.[120][115][116]

6

Lead

It destroys the blood forming system causing anemia.[117] [112]

Manganese

Central nervous system is damaged due to high intake.

Chromium

Skin irritation ulceration along with kidney and liver is caused by access

intake. Circulatory and nervous tissues are also damaged.

Copper

Higher dosage causes anemia.lt produces intestinal irritation damages

stomach liver and kidney. Coppery normally adds to drinking water from

copper pipe lines as well as from additives designed to control algal

growth.

Nickel

It is needed for production of red blood cells but high doze leads to

decreased body weight, heart and liver diseases along with skin irritation.

Selenium

It causes fatigue and irritation. Nervous system is also damaged [118].

Algae being a very good source of proteins needs certain trace elements

for its growth.[64] Waste water of all the types contains a large number

elements e.g., they heavy metals serve as trace elements. The most

important trace element in this concern is copper [65]. However a definite

amount of metals present in the growth media is taken up by algal cells

[66]. If an excessive amount of copper is present in the growth media, it

causes toxicity, damaging the algal cell wall [67]. It also disturbs the

7

vitamine and nutrients [73]. There are conflicting reports whether the

copper is taken up in the form of copper ions or complex [67]. However

if copper is present in equimolar concentration, its up take is much higher

than that of their complexes like E.D.T.A or T.D.P.A [70].

Copper also takes part in oxidation reduction system of algal cells,

however its activity is affected by the concentration of other trace

elements in the working medium [71]. It also takes part in the

photosystems.

These substances initiate the rapid growth, a series of experiments were

carried out to demonstrate the effect of Co, Mn and Mb on growth of

Ulva lactuca. Sea water enriched with MgSO47H2O, K2HPO4 and

NH3NO3 was used as growth media. Two flasks were used, one

containing only water and other with trace elements. Then the algal

species was introduced the algae cell in the flask containing trace

elements showed more growth. [74] In a typical media, the concentration

of concentration of trace elements is in the order of 10-9

M. When

E.D.T.A is added to sea water cations present in it compete for it.

Because of great stability of E.D.T.A chellates such a culture media is

produced which have very low concentration of metal ions. So addition of

trace elements could compensate this effect these ions are metabolized by

the algal species and growth rate increases upto a certain limit under

controlled conditions of temperature and pH. Similar effects were

observed for Zn, Co, Mn, Mb.[75]

Alga, Chlorella vulgaris, was acclimated in wastewater for a period of 14

days before employed in treating primary settled wastewater. The

acclimated cells had significantly higher cellular chlorophyll content than

8

the unacclimated ones. The rate of chlorophyll synthesis in the acclimated

cells was double that of the unacclimated cells during the initial 3 days in

the course of wastewater treatment. This showed that the acclimated cells

were physiologically more active and used more nutrients from the

wastewater for their growth and metabolism, resulting in a nutrient

removal efficiency of 86% inorganic N and 70% inorganic P for a

retention time of 2 days. Such rates were much higher than those obtained

in the unacclimated system with similar algal density, where removal

rates for total inorganic N and P were 54% and 50%, respectively.

Methods showed a good way of the removal of ions from water by the

microalgal species which were not indigenous [9].

In biochemical treatment two Algae species C.V, O.P,M,G,Ulothrix were

grown in waste water and their growth rate per day was observed against

the change in pH. Temperature and illumination effect of Algae growth

on the metal ion, total nitrogen, phosphates, COD, Chlorides contents,

Sulphate and Total Solid contents were also observe. Combination of the

species was also used to observe the changes in above parameters of

waste water and optimal conditions were observed. Six water samples

(Ravi Disposal, Jallo, Ferozpur Road, Thokar, Sheikhupura and Shahdra)

were used for the studies. Two sets of waste water (Sheikhupura and Ravi

Disposal) were run after maintaining the observed optimal condition and

the effect on growth rate, metal ions, total nitrogen, phosphate, COD,

Chloride and Sulphate contents and on TOS were determined.

10

LITERATURE SURVEY

“An overview was given for current analytical methodologies, arsenic

speciation in environmental samples [1]. They are conventional

instrumental methods - mainly chromatographic techniques (HPLC, GC,

etc.) coupled with many detectors. However, methods using micro-

organisms were increasingly being applied for metal ions removal and

other metal species from water solutions. The use of the alga Chlorella

Vulgaris is proposed for the separation of arsenic (III) from the other

species. The As concentration is measured by hydride generation atomic

absorption spectrometry”.

“Hee-Mock Oh et al [12] Microbial flocculants for harvesting mass

cultured Chlorella Vulgaris were screened and that from Paenibacillussp.

AM49 was identified as the best. The efficiency of this bioflocculant

increased with the pH within a range of pH 5-11 and was 83%, which

was higher than the 72% and 78% produced by aluminum sulfate and

polyacrylamide, respectively. The highest flocculation efficiency was

with 6.8 mm CaCI2 as co-flocculant. Paenibacillussp. AM49 can be use d

effectively to harvest C. Vulgaris from large-scale cultures”.

“Luz E. Gonzalez et al [13] Coimmobilization of the freshwater

microalga and the plant-growth-promoting bacterium Azospirittum

brasilense in smalt alginate beads resulted in the increased growth of the

microalga. Dry and fresh weight, total number of cells, size of the

microalgal clusters (colonies) within the bead, number of microalgal cells

per cluster, and the levels of colored pigments significantly increased.

Light microscopy revealed that both microbial colonized the same

cavities inside the beads, though the tended to concentrate in the more

11

aerated outer circle while the bacteria colonized the entire bead. The

effect of indole-3-acetic acid addition to this culture prior to

immobilization of microorganisms in alginate beads partially imitated the

effect of A. brasilense. So coimmobilization of microalgae and plant-

growth-promoting bacteria is an effective means of increasing microalgal

populations within confined environments”.

“Anton F. Post et al [14] Research was carried out within the framework

of the Center for Reservoir Research at the Hebrew University and was

sponsored by the Mekoroth Water Company, Ltd., Tel Aviv. Thanks to

Dr. I. Dor for help in the identification of the Chlorella strains. They

reported on the characterization and isolation of two ecotypes of

Chlorella Vulgaris Beyerinck that coexist in wastewater reservoirs. One

ecotype (C1) contains high amounts of chlorophyll b, can autotrophically

growth, and can utilize only a few solutes for growth. The second ecotype

(C2) contains low amounts of chlorophyll b, vitamin B12 is required to

support its growth a large variety of organic compounds. Of the two

ecotypes, the latter showed slower growth rates when light was the sole

source of energy. Cells of C2-type C. attained higher photosynthetic

activities than C1-type cells at saturating irradiances. However, their low

chlorophyll b. content and lower light utilization efficiency suggest that

C2-type Chlorella contains relatively low amounts of light-harvesting

antennae, a disadvantage in severely light-limited ecosystems like

wastewater reservoirs. We can say C. types coexist by adopting different

lifestyles: C1-type cells rely largely on their photosynthetic potential for

energy conservation and growth, whereas C2-type cells may exploit their

heterotrophic properties for this purpose”.

“Amany El-Sikaily et al [15]

12

“Tang E.P.Y et al [18] 49 strains of filamentous, mat-forming

cyanobacteria isolated from the Arctic, subarctic and Antarctic

environments were isolated for their use in outdoor water treatment

systems designed for cold north-temperate climates. The most promising

isolate (strain E18, Phormidium sp. from a high Arctic lake) grew well at

low temperatures and formed aggregates (flocs) that could be readily

separated by sedimentation. The growth and nutrient uptake abilities of

E181 relative to the green algae (a Chlorococcalean assemblage, denoted

Vc) sampled from a tertiary treatment system in Valcartier, Canada. E182

had superior growth rates below 15°C Canada. (µ = 0.20 d-1

at 10oC

under continuous irradiance of 225 µmol photon m-2

s-1

) and higher

phosphate uptake rates below 10°C (k = 0.050 µmol at 5°C) relative to

Vc0 (µ=0.087 d

-1 at 10°C and k = 0.020 d

-1 at 5°C, respectively). The

assemblage generally performed good than E181 at high temperatures (at

25°C, µ = 0.39 d-1

and k = 0.34 d-1

for Vc; µ = 0.28 d-1

and k = 0.33 d-1

for E18). However, E182 removed nitrate more efficiently than Vc

1 at

most temperatures including 25°C. Polar cyanobacteria such as strain

E183 are appropriate species for water treatment in cold climates during

spring and autumn. Under warmer summer conditions, fast-growing Alga

such as the Vc2 are likely to colonize and dominate, but warm-water

Phormidium isolates could be used at that time”.

“Shan, Huifenqet al [26] Intensively cultured fish stock when fed

protein-rich feeds caused excrete high concentrations of total

ammoniacal-nitrogen (TAN) into the water column which can have

negative effects on productivity, and upon the environment when

aquaculture waste is discharged. A culture of nitrifying bacteria isolated

from prawn pond water and known to effectively remove TAN from

saline water was tested for its ability to remove TAN2 from freshwater.

13

The culture was readily adaptable to non-saline conditions and

maintained TAN3 at less than 0.25 mg/l(-1), even with a daily addition of

3.2 to 4.2 mg TAN l(-1) per d. The use of the culture of nitrifying bacteria

represents an innovative and economical in situ treatment technology for

removal of TAN4 in both saline and freshwater”.

“Yoshihiro Suzuki et al [29] The removal of microplutants from rearing

water and washing water in aquaculture systems, aquariums and fishing

port facilities is the most important means of lowering the risk of fish

diseases, improving public health and ensuring high food quality.

However, there are few methods of elimination, e.g., disinfection. Thus, it

is necessary to develop a technology for bacterial removal from coastal

seawater. In this study, the removal efficiency for several groups by foam

separation using dispersed bubbles and surface-active substances was

determined using both batch equipment and a continuous-flow unit. In

batch processing with only 1 mg/l milk casein added as a surface-active

substance and by supplying bubbles, enterococci, Vibrio, and Salmonella

were removed effectively at removal efficiencies of 80% or greater. In

addition, suspended solids were also remove. However, fecal conforms

were difficult to remove by foam separation. The removal efficiency for

viable bacteria was greater than 70% using a continuous system. Mass

were concentrated in a very small amount of generated foam and

removed from the water. The foam separation using dispersed bubbles

and surface-active substances is a feasible convenient technology for

seawater purification as a treatment prior to membrane filtration or

ultraviolet irradiation”.

“Marcel M. M. Kuypers et al [31] The availability of fixed inorganic

nitrogen (nitrate, nitrite and ammonium) limits primary productivity in

14

many oceanic regions. The conversion of NO3 to N2 by heterotrophic

bacteria (denitrification) is to be the only important reason for fixed

inorganic nitrogen in the ocean. Here we provide evidence for

anaerobicalty oxidize NH4+

ammonium with NO3 to N2 in the world‟s

largest anoxic basin, the Black Sea. Phylogenetic analysis of 16S

ribosomal RNA gene sequences shows that these are related to members

of the order Planctomycetales performing the anammox (anaerobic

ammonium oxidation) process in ammonium-removing bioreactors.

Micro nutrient profiles, fluorescentiy labelled RNA probes, 15

N tracer

experiments and the distribution of specific „ladderane‟ membrane lipids

indicate that ammonium diffusing upwards from the anoxic deep water is

consumed by anammox bacteria below the oxic zone. Anammox have

been identified and directly linked to the removal of fixed inorganic

nitrogen in the environment for the first time. The widespread occurrence

of ammonium consumption in suboxic marine setting indicates that

anammox might be important in the oceanic nitrogen cycle”.

“Ebrahim Vasheghani-Farahani et al [32] A combined physicochernical

and biological process for treatment of oil contaminated sea water. In this

process, a new polymeric surfactant is successfully applied with a dosage

of 0.0015 g/g of crude oil to accumulate oil spots on the sea water in a

microcosm. Microbial degradation of accumulated oil spots using isolated

bacteria from oil contaminated Caspian Sea water was studied. The

results of a proposed process for treatment of contaminated sea water in a

pilot scale, using a 1500-1 microcosm with several basins at different

conditions are presented”.

“Fabio Sergio et al [33] The production of inoculum is one of the

hindrances in the large scale application of arbuscuiar mycorrhizal fungi

15

(AMF). The objective of this work was to study the effect of nutrient

solutions with or without Tris-HCl buffer, on speculation of AMF. The

experiment was carried out in a greenhouse, using a substrate with sand

and vermiculite (1:1 v/v). Fifty spores of Gigaspora margarita,

Scutellospora heterogama, and Glomus etunicatum were inoculated in

Sorghum vulgare (sorghum) or Panicum miliaceum (fodder millet). The

substrate received the following micro nutrient solutions: Hoagland with

3 DM P (S1); Long Ashton II with 15.9 DM P (S2) and Hoagland with 20

M P (S3), with or without 50 mM of Tris-HCl buffer (pH 6.5); the control

treatment, consisting of a soil + sand + vermiculite (2:1:1 v/v) substrate

was irrigated with deionized water. Ten weeks after the beginning of the

experiment sporulation did not differ in treatments with sorghum.

Panicum miliaceum promoted higher sporulation of the AMF than

sorghum, and differences among treatments with nutrient solutions were

observed. Production of spores of G. margarita and S. heterogama

increased significantly after addition of buffer in 81 and 82, while that of

G, etunicatum was improved when the substrate was irrigated with S1 +

buffer and S3 solutions. Solution S1 + buffer benefited sporulation of the

three fungi. However, as observed, each AMF, host, and substrate system

should be studied separately for establishment of the most favorable

conditions for inoculum production”.

“Peter L. Sguros et al [34] The filamentous ascomycete Halosphaeria

mediosetigera and the deuteromycete Culcttalna achraspora were

gravimetricaily used by growth curves in natural and artificial seawater.

Response to growth media was very similar, and stationary phases of both

cultures showed autolysis. The organisms grew at all concentrations of

seawater, but growth maxima in dilutes. Neither seawater was able to

reduce the requirement for yeast extract. Glass-distilled water was

16

slightly better than artificial seawater for the survival of homogenized,

washed, resting mycelia over long periods at 4 C. Regardless of

suspending medium, this treatment did not appreciably alter the

regenerative vigor of inocula. Qualitative and quantitative variations of

artificial seawater, without rigid exclusion of inorganic traces from

experimental media, demonstrated separate growth requirements for Na+,

K+, Mg

++, Ca

+2, and SO4. Only K

++ and Ca

++ appeared nontoxic at

seawater concentrations, however. Some evidence of Na+ antagonism by

Mg++

was obtained. Tests were done on ions to observed the absence of

seawater amounts of NaCl. Reactions of these generically distinct fungi

to most experimental manipulations were markedly alike. Both fungi

appeared to be appropriately labeled „marine‟, judging by criteria

currently in general use”.

“E. Metcalf et al [35] The effect of 3-O-methyl glucose on the

carbohydrate metabolism of, and loss of 14

C-labelled compounds from,

mycelium of Dendryphiella salina pretreated with [U-14

CJmannitol was

investigated. Though concentrations of mycelial glycerol were higher

than previously reported, results for other internal carbohydrates were

generally similar. The sugar reduced the concentration of mannitol. Loss

of radioactivity into the medium was increased by 3-O-methyl glucose.

The pattern of loss cannot yet be explained but since there was no loss of

labelled mannitol or arabitol, unspecific membrane damage seems

unlikely”.

“Hung-Soo Joo Mitsuyo Hirai et al [36] To improve NH4+

removal

efficiency in treatment, a mixed culture of Alcaligenes faecalis no. 4 and

its mutant L1, both of which have heterotrophic nitrification and aerobic

denitrification abilities, was performed. In a batch culture, no. 4 has a

17

higher N24

removal ability than L1, but its ammonium removal rate was

lower. In a mixed continuous culture in the ammonium loading range of

750 to 3500 mg-N/l/d, the average ammonium removal rate and the

average denitrification ratio were 61 mg-N/l/h and 31%, respectively. In

the mixed culture, the ammonium removal rate was twofold higher than

that in a single culture of no. 4, the rate was similar to that in a single

culture of L1, and the removal ratio was very high compared with that in

the single culture of L1”.

“Diane Fournier et al [37] The stimulating effect of heterotrophic

microorganisms was investigated on the growth and on the iron oxidation

of Thiobacillus ferrooxidans in synthetic media and in wastewater sludge.

The addition of a sediment Rhodotorula rubra isolate or a strain of T.

acidophilus on two-layer agarose-geiled medium doubled the plating

efficiency of T. ferrooxidans. In liquid cultures, R. rubra had a slight but

significant effect on the growth rate of T. ferrooxidans. Moreover, the

yeast allowed a faster initiation of the ferrous iron oxidation and

acidification by T. ferrooxidans. In the bioleaching process, the co-culture

of T. ferrooxidans with R. rubra or with the indigenous microbial

assemblage from sludge was shown to be essential since the pure culture

of T. ferrooxidans failed to oxidize ferrous iron and to acidify wastewater

sludge. These results emphasize the importance of active heterotrophic

microorganisms in the metal bioleaching activity of T. ferrooxidans in

sludge”.

JieYing Jing et al [38] The studies were carried out to investigate COD8

removal efficiency of the coking-plant disposal by applying the moving-

bed technique batch reactor (MBBSBR). The operation is simple and

30%

18

WD-F10-4 BioM™ were packed as carrier materials. It was found that

the coking-plant wastewater could be effectively treated with 92.9% of

COD9 removal efficiency at a low organic loading rate (OLR) of 0.449 kg

COD.m-3

.d-1

. The removal efficiency decreased gradually down to 70.9%

when OLR increased to 2.628kg COD.m-3

.d-1

. The system has strong

tolerance to organic shock loading in this experiment. The COD10

removal results in the blank experiments of biofilm and sludge showed

that the attached biofilm has higher activity than suspended sludge and

contributes about 60%”.

20

BIOCHEMICAL TREATMENT

Collection of the Samples:

Water samples were collected in one liter sterilized conical flasks from

four points of Lahore Canal (Jallo, Icchra and Thokar), Ravi disposal and

from industrial area of Shahdra and Sheikhupura. The flasks were

immediately pluged with cotton and were moved to the Laboratory where

they were stored at 10oC in refrigerator. 03 samples from each source

were selected for further studies.

A number of Algal samples were collected from natural habitate at

different sites mostly from suburban areas of Lahore e.g. Nandipur,

Kalasha Kaku, Gujranwala, Mehmood Booti, and Jallo etc. The samples

were studied microscopically and were subjected to a series of sub

culturing on various solid and liquid media following the standard

methods of pure cultures of Algae, the following Algal species were

screened out from the mixed field samples.

5) Chlorella Vulgaris

6) Oocystis Pusilla

7) Maugetia Genuflexa

8) Ulothrix

Purification of Algal Species

The species were purified by repeated washing methods with 400 ml

water in one Litre Beaker and then identified under the microscope

(xS2 107BN NOIF). Their morphological characteristics were studied

and recorded.

21

Repeated Washing Method

It was done in two steps:

1) In the breakers

2) In the flasks

In the Breakers

Placed the samples in the breaker. One of the breaker was placed exactly

under the tape and opened the tape at full pressure. Due to high pressure

of falling water, cells of different size and weight settled down. Different

layers of cells were separated according to gravity. The heavier cells

settled down at the bottom of flask while the lighter ones were whirling

above. Above layer of smaller cells was poured in another flask while it

was whirling.

In the Flask

The flask was also placed under the tape. The tape was opened at its full

pressure. Again separation of cell took place into layers according to

gravity. The species were introduced in Bristol medium. The numbers of

cells were determined by Haemocytometer on 1st day.

Inoculum Development

5 litre capacity aspirators were used for inoculum development the pure

and indentified algal species were grown using Bristol media and Chu

No. 10 (modified medium). The growth was luxurious in Bristol medium

as compare to Chu No. 10 modified.

22

Composition of Chu No. 10 Medium

Nutrients Amount/litre

Ca(NO3)2 40 g

MgSO47H2O 25 g

K2HPO4 10 g

Na2CO3 20 g

Na2SiO3 (water glass) 25 g

FeK EDTA Solution 10 ml/litre

Composition of Bristol media (stock solution)

NaNo3 3grm/400ml

K2HPO4 3grm/400ml

KH2PO4 7grm/400ml

NaCl 1grm/400ml

mgSO4 3grm/400ml

CaCl2 1grm/400ml

10 ml of each of the stock solution was added to 940 ml of distill water

and 2 litre volume was made 20 flasks were prepared, they were plugged

with cotton and sterilized in incubator at 80oC with 15lbs/inch

2 pressure

for 30 minutes separately, 5 flasks were incubated with Chlorella

Vulgaris, 5 with Oocystus Pucilla, 5 with Maugetia Genoflexa and 5 with

Ulothrix.

Shaking Flask Technique

The broth culture was exposed to sun light for two hours daily under the

green house effect produced by cotton cloth along with occasional

23

shaking. This continued for 14 days approximately. Growth rate, pH and

temperature were noted after every 24 hours.

Preparation of Slants

For the preservation and further use the species were preserved on

Czapek-Dox media in 20ml test tubes. The tubes were sterilized and 10ml

of the Czapek-Dox medium was added in each test tube. Algal species

were incubated with sterilized spatula in each slant. 5 slants of CV and 5

of O.P., M.G. and Ulothrix were prepared and stored at 10oC in incubator

(Gallen kamp) plugged with sterilized cotton.

Composition of Czapek-Dox Medium modified CM 0097

KH2PO4 1 gm/liter of water

KCl 0.5 gm/liter of water

NaNO3 0.5 gm/liter of water

MgSO4 0.5 gm/liter of water

FeSO4 0.01 gm/liter of water

Sucrose 0.01 gm/liter of water

Agar 12 gm/liter of water

All the ingredients were weight on electric balance.

Analysis of the Water Samples

One liter waste water sample from each source was selected. All the

water samples were allowed to stand for 48 hours so as to settle the

suspended impurities. 300mg of supernated layers was analyzed for the

determination of metals ions. pH, conductivity, phosphate, COD, total

nitrogen, chlorides, sulphates and total solid as per following procedure.

Traces of oil and grease in industrial waste water and detergents in canal

24

water and Ravi Disposal water samples were present but no effort was

made for their removal as it was not economically feasible. Metal ions

were determined by atomic absorption spectrometer (varian).

The solutions for the determination of metal ions were prepared as per

following procedures.

Cobalt (Co)

Preparation of Standard Solutions

Recommended Standard Materials

Cobalt metal strip or wire 99.99%

Solution Technique

Dissolve 1000g of metal in a minimum volume of 1:1 nitric acid and

dilute to 1 litre to give 1000 µg/mL Co.

Recommended Instrument Parameters

Atomic Absorption

Working Conditions (Fixed)

Lamp Current 7 mA

Fuel Acetylene

Support Air

Flame stoichiometry oxidizing

Working Conditions (Variables)

Wavelength

(nm)

Slit width

(nm)

Optimum Working Range

(µg/mL)

240.7 0.2 0.05-15

304.4 0.5 1-200

346.6 0.2 2-500

347.4 0.2 4-1000

391.0 0.2 150-30000

25

Flame Emission

Wavelength 345.4 nm

Slit Width 0.1 nm

Fuel Acetylene

Support Nitrous oxide

Copper (Cu)



Copper metal strip or wire 99.99%

Solution Technique

Dissolve 1000g of copper metal in a minimum volume of 1:1 nitric acid

and dilute to 1 litre to give 1000 µg/mL Cu.


Atomic Absorption


Lamp Current 4 mA

Fuel Acetylene

Support Air

Flame stoichiometry oxidizing

Working Conditions (Variables)

Wavelength

(nm)

Slit width

(nm)


(µg/mL)

324.7 0.5 0.03-10

327.4 0.2 0.1-24

217.9 0.2 0.2-60

218.2 0.2 0.3-80

222.6 0.2 1-280

249.2 0.5 4-800

244.2 1.0 10-2000

26

Flame Emission

Wavelength 327.4 nm

Slit Width 0.1 nm

Fuel Acetylene


Europium (Eu)



Europium oxide Eu2O3 99.99%

Solution Technique

Dissolve 1.1579g of Eu2O3 in a minimum volume of 1:1 hydrochloric

acid and dilute quantitatively to 1 litre with water to give 1000 µg/mL Eu.


Atomic Absorption


Lamp Current 10 mA

Fuel Acetylene


Flame stoichiometry Reducing; red

cone 1-2 cm

Working Conditions (Variable)

Wavelength

(nm)

Slit width

(nm)


(µg/mL)

459.4 1.0 15-60

333.4 0.5 5000-20000

27

Flame Emission

Wavelength 459.4 nm

Slit Width 0.1 nm

Fuel Acetylene


Below 10 µg/mL, determination by flame emission is preferred, although

the wavelength required must be accurately isolated to avoid spectral

interference from other rare earth elements. At higher concentrations,

atomic absorption is normally used.

Potassium (K)



Potassium chloride KCl A.R. Grade

Solution Technique

Dissolve 1.907 g of dried potassium chloride in water and dilute to 1 litre

to give 1000 µg/mL K.


Atomic Absorption


Lamp Current 5 mA

Fuel (Note 1) Acetylene

Support Air

Flame stoichiometry Oxidizing

Note 1: An air-acetylene flame is normally used because interferences are

reduced and the signal/ noise ratio is improved.

28


Wavelength

(nm)

Slit width

(nm)


(µg/mL)

766.5 1.0 0.03-2.0

769.9 1.0 1-6.0

404.4 0.5 15-800

Flame Emission

Wavelength 766.5 nm

Slit Width 0.1 nm

Fuel Acetylene

Support Air

The flam emission determination of potassium is limited by flame

stability and by „pick up‟ of potassium from the air and storage vessels.

Air-acetylene can be used if an ionization suppressant is added.

Manganese (Mn)



Manganese metal strip or wire 99.99%

Solution Technique

Dissolve 1000g of manganese in a minimum volume of 1:1 nitric acid

and dilute to 1 litre to give 1000µg/mL Mn.


Atomic Absorption


Lamp Current 5 mA

Fuel Acetylene

Support Air


A nitrous oxide-acetylene flame can also be used but sensitivity is poorer.

29


Wavelength

(nm)

Slit width

(nm)


(µg/mL)

279.5 0.2 0.02-5

403.1 0.2 0.5-60

321.7 0.2 100-14000

Flame Emission

Wavelength 403.1 nm

Slit Width 0.1 nm

Fuel Acetylene


Sodium (Na)



Sodium chloride NaCl 99.99%

Sodium carbonate Na2CO3 99.99%

Solution Technique

Dissolve 2.542g of dried NaCl in water and dilute to 1 litre to give

1000µg/mL Na.


Atomic Absorption


Lamp Current 5 mA

Fuel Acetylene

Support Air


A nitrous oxide-acetylene flame can also be used but sensitivity is poorer.

30


Wavelength

(nm)

Slit width

(nm)


(µg/mL)

589.0 0.5 0.002-1.0

589.6 1.0 0.01-2.0

330.2 0.5 2-400

330.3

Flame Emission

Wavelength 589.0 nm

Slit Width 0.1 nm

Fuel Acetylene

Support Air

Nickel (Ni)



Nickel metal strip or wire 99.99%

Solution Technique

Dissolve 1000g of nickel in 1:1 nitric acid and dilute to 1 litre to give

1000µg/mL Ni.


Atomic Absorption


Lamp Current 4 mA

Fuel Acetylene

Support Air


A nitrous oxide-acetylene flame can also be used with poorer sensitivity.

31


Wavelength

(nm)

Slit width

(nm)


(µg/mL)

232.0 0.2 0.1-20

341.5 0.2 1-100

352.4 0.5 1-100

351.5 0.5 3-180

362.5 0.5 100-8000

Note: The 352.4nm line is preferred because the calibration is less curved

over the working range and the signal is less susceptible to non-atom

absorbance than at the more sensitive 232.0nm line.

Zinc (Zn)



Zinc metal or wire 99.99%

Solution Technique

Dissolve 1000g of zinc in 40mL 1:1 hydrochloric acid and dilute to 1 litre

to give 1000µg/mL Zn.


Atomic Absorption


Lamp Current 5 mA

Fuel Acetylene

Support Air


All other conventional flames can be used.

32


Wavelength

(nm)

Slit width

(nm)


(µg/mL)

213.9 1.0 0.01-2

307.6 1.0 100-14000

Flame Emission

Wavelength 213.9 nm

Slit Width 0.1 nm

Fuel Acetylene


Kinetic Parameters

These are pH and temperature and illumination:

pH

The maintenance of favourable pH is one of the most consequent steps

for the progression of growth rate of algal cells. It was controlled by

adding buffer solution of pH-7 initially. It was determined by pH meter of

(AD-1030 HANNA made) after every 24 hours.

Temperature

Temperature was determined by laboratory thermometer after every 24

hours and was controlled by illumination.

Determination of Growth Rate

Growth rate was determined by Haemocytometer (Superier Germany).

33

Methodology of Haemocytometer

Firstly the objective lens was washed out. The haemocytometer was

thicker than a normal microscope slide. Then objective lens was focused

away from the contact of the instrument and grid was used to determine

the proportion of the chamber to estimate correct number of cells during

counting.

After the addition of suspension of inoculums to the Haemocytometer it

was pressed with cover slide. The algal cells were examined

microscopically using 10x or 40x lens. Counted the algal cells on 5

random inner square within the central big square.

Illumination

Light intensity was controlled by electrical bulbs of 100 watts each at

night. The light intensity was varied by increasing or decreasing the

number of electric bulbs. It also helps in maintaining the temperature

between 27o to 30

oC.

Light intensity was determined by lux meter (Phillips made) with 0 to

10,000 lux intensity measure.

Conductivity

Conductivity was determine by conductivity meter (EC-215 HANNA).

Determination of COD

Reagents Used:

1) Potassium dichromate (Standard solution): K2Cr2O7 – 0.004167 M

(0.0250 N)

2) Mohr‟s Salt: Ferrous ammonium sulphate (Standard solution):

FeSO4 (NH4)2SO4 (0.025 M)

34

3) Mercuric Sulphate: Powdered HgSO4

4) Silver Sulphate: Powdered Ag2SO4

5) Phenanthroline ferrous sulphate indicator solution

6) Concentrated Sulphuric acid: H2SO4 18 M

3. Procedure:

50 ml of sample was taken into a refluxing flask and several boiling

stones were added. 0.1g HgSO4 was added to the solution. 5ml of

concentrated H2SO4 was also added to the solution. To ensure that HgSO4

dissolved completely, the solution was swirled slowly while adding

Sulphuric acid. 0.1 g of Ag2SO4 was added to this solution. Finally

Potassium dichromate was added. Thorough mixing of the solution was

ensured by swirling the flask in a water bath to recover any volatile

substances that may have escaped from the liquid state. The flask was

then attached to the condenser and further cooling was done. 20ml of

Sulphuric acid was added to the solution in the flask continuing cooling

and swirling to mix the solution. The solution was refluxed for 1 hour.

A blank run (using 50ml distilled water instead of sample) was

simultaneously conducted with the same procedure after cooling; the

solution was transferred to an Erlenmeyer flask. The reflux flask was

rinsed thrice, pouring the rinsing water to the Erlenmeyer flask. The

solution was diluted to about 300 ml and about 8 drops of Phenanthroline

ferrous sulphate was added to the solution as an indicator.

The solution was titrated against the Mohr‟s salt and the titer volume

required for the color change from blue-green to reddish blue was noted.

The procedure was repeated for the blank run.

35

4. Observations:

Sample Blank

Initial Reading (ml) Final Reading (ml) Titer volume (ml)

Solution Initial Reading

(ml)

Final Reading

(ml)

Titer Volume

(ml)

Sample 0.00 25.40 25.4 – Vs

Blank 0.00 29.20 29.2 – Vbl

5. Calculations:

COD = 8000 (Vbl – Vs) M / original volume of sample taken mg/1

Where,

Vbl = Titer volume for the blank

Vs = Titer volume for the sample

M = Molarity of Mohr‟s solution

COD = 8000 (29.2 – 25.4) 0.025 / 50

= 15.4 mg/L

Determination of Total Phosphate

3. Reagents

a. Phenolphthalein indicator aqueous solution.

b. Hydrochloric acid, HCl, 1 + 1. H2SO4, HC1O4, or HNO3 may be

substituted for HCl. The acid concentration in the determination is not

critical but a final sample concentration of 0.5N is recommended.

c. Activated carbon. Remove fine particles by rinsing with distilled

water.

d. Vanadate-molybdate reagent:

1) Solution A: Dissolved 25g ammonium molybdate, (Np4)6Mo7O24

4H2O, in 300mL distilled water.

36

2) Solution B: Dissolved 1.25g ammonium metavanadate, NH4VO3, by

heating to boiling in 300mL distilled water. Cool and add 330mL conc

HCl. Cool Solution B to room temperature, poured Solution A into

Solution B, mix, and diluted to 1 L.

e. Standard phosphate solution: Dissolved in distilled water 219.5 mg

anhydrous KH2PO4 and dilute to 1000mL; 1.00mL = 50.0 g P043-

-P.

4. Procedure

a. Sample pH adjustment:

If sample pH is greater than 10, add 0.05mL (1 drop) phenolphthalein

indicator to 50.0mL sample and discharge the red color with 1 + 1 HC1

before diluting to 100mL (pH was 7.8).

b. Color removalfrom sample:

Removed excessive color in sample by shaking about 50mL with 200mg

activated carbon in an erlenmeyer flask for 5 min and filtered to removed

carbon. Checked each batch of carbon for phosphate because some

batches produce high reagent blanks.

c. Color development in sample:

Placed 35mL or less of sample, containing 0.05 to 1.0mg P, in a 50mL

volumetric flask: Added 10mL vanadate-molybdate reagent and diluted to

the mark with distilled water. Prepared a blank in which 35 ml distilled

water was substituted for the sample. After 10 min or more, measured

absorbance of sample versus a blank at a wavelength of 400 to 490 nm,

depending on sensitivity desired. The color was stable for days and its

intensity was unaffected by variation in room temperature.

37

d. Preparation of calibration curve:

Prepared a calibration curve by using suitable volumes of standard

phosphate solution and proceeded as in 4c. When ferric ion is low enough

not to interfere, plot a family of calibration curves of one series of

standard solutions for various wavelengths. This permits a wide latitude

of concentrations in one series of determinations. Analyzed at least one

standard with each set of samples.

5. Calculation

mg P/L = sample mL

1000 volume)final 50mLP(in mg

Determination of Total Nitrogen

Reagents:

1) Digestion reagent (dissolve 134g K2SO4 and in about 800ml water

carefully and 134ml Conc H2SO4. Cool to room temperature, dilute

to 1 lit with water, mix well keep at temp 20oC to prevent

vaporization).

2) Phenolphthalein indicator

3) Sodium hydroxide

4) Mixed indicator solution

5) Indicating boric acid solution

6) Standard sulfuric acid titrant

7) Hydroxide thiosulfate reagent

Procedure:

a) Digestion

1) Took 280 mL of sample in a kjeldhal flask.

2) Added few glass beads to it then add 5mL digestion reagent.

38

3) Heated and continue boiling until solution remained 25-50 mL.

4) Cool it and added distilled water to it to make the volume 300 mL.

5) Added 0.5 mL phenolphthalein indicator.

6) Add 50ml thiosulfate hydroxide reagent solution.

b) Distillation

1) Collected the distillate in a flask containing boric acid solution.

2) Collected 200ml distillate into 50 ml boric acid solution.

(c) Titration

1) Titrated it against 0.02N H2SO4 solution until colour changes from

purple to green.

2) Carried the blank titration, following all steps of procedure.

Calculations:

Total Nitrogen (mg/L) = sample of mL

280B)-(A

A = volume of H2SO4 used for sample =

B = Volume of H2SO4 used for blank =

Total nitrogen (mg/L) =

Determination of Sulphates

Solutions:

Conc HCl, 2% solution of BaCl2. 50 mL of water sample was filtered

through Watman filter paper (42). It was boiled after adding 15 mL of

conc HCl and BaCl2 solution was added in portions during boiling till the

precipitation is completed. Filtered the precipitates through pre-weighed

Watman filter paper. Dried in the oven at 110oC and noted the difference

in weight which was the weight of BaSO4 precipitates.

39

Calculations:

233

96 esprecipetat theofWeight

Determination of Chlorides

Solutions: 0.1M AgNO3

K2CrO4 = Indicator

50 ml of water was titrated against 0.1M AgNO3 using K2CrO4 indicator

till the appearance of the brick red color. Burette reading was noted.

Calculations:

Sample: AgNO3

M1V1 = M2V2

readingBuritten0.150M1

in water Cl ofMobarity y50

n1.0M1

Mg/Liter of Cl 1000y5.35

Determination of Total Solids

50 ml water sample was filtered through pre-weighed Watman filter

paper dried in oven at 110oC and weighed again the difference in weight

was the amount of suspended solids in water. In a pre-weighed breaker,

filtrate was evaporated to dryness on a flam. Cold in a dessicator and

weighed again, the difference in weighed was the amount of dissolved

solids (B).

Total solid = A + B =C

50 ml of water contains total solids = C

40

Calculations:

solids totalofmg/liter 1000100050

C

Growth of Chlorella Vulgaris, Oocystus Pusilla,

Maugetai Genoflexa and Ulothrix in Waste Water

3 sets with Chlorella Vulgaris, 3 with Oocystms Pursulla, 3 with

Maugetia Genoflexa and 3 with Ulothrix were inoculated in the already

analyzed water from each source under batch system.

200 ml of water sample was taken in 5 liter sterilized aspirators and

diluted 10 times to make 2000 ml of sample with distilled water.

Chlorella Vulgaris was introduced with sterilized, spetula in aspirator No.

1, 2 and 3, in the sample from Ravi disposal. 4, 5, 6 in the sample from

Jallo, 7, 8, 9 in the sample from Ferozpur road, 10, 11, 12 from Thokar,

13, 14, 15 from Shahdra and 16, 17, 18 from Sheikhupura. Oocystms

Pursulla was added in the same order of the sample sources in aspirator

No. 19 to 36, M.G. in aspirator No. 37 to 54 and Ulothrix in aspirator No.

55 to 72 and subjected to fermentation species were taken from the slants

already prepared and preserve. 100 watts electrical bulbs were used for

illumination of the species. Initial pH was adjusted to 7.0 by adding

buffer solution of pH-7, 27o-30

oC and illumination at 5000 Lux.

Temperature was noted with laboratory thermometer. Growth rate was

determined after every 24 hours, batches were run under batch system for

fifteen days to establish optimal conditions of effect of pH, temperature

and illumination for further studies. on growth rate was determines in the

selected flask after every 24 hours.

Samples were finally run with Chlorella Vulgaris, Oocystms Pucilla,

M.G., Ulothrix, and mixed species of C.V.+O.P., C.V.+M.G.,

41

C.V.+Ulothrix, O.P.+M.G., O.P.+Ulothrix, M.G.+Ulothrix , after

maintaining the optimal condition of pH=7, Illuminates=3500 Lux and

Temperature=27o–35

oC as the mean growth rate was also observed at

these values. After 15 days the culture was analyzed for metal ions, COD,

conductivity, total N, phosphate, chlorides, sulphates and total solids.

43

ELECTROCHEMICAL TREATMENT OF

WASTE WATER

Electro chemical treatment was done in three component system:

a) Anodic part

b) Cathodic part

c) Central part

Rectangular glass made tank having 15cm height and 28cm length and

26cm width was used as the working cell. The cathodic and anodic part

was separated by polymeric membranes while the central part contains

water sample to be treated (Fig.-7)Rotating disc technique was employed

for the treatment and a rotator with variable speed was used for this

purpose. Ordinary power supply was used as current source with DC

rectifier (Philips) voltmeter and ammeter were used to measure the

current and voltage rectifier was connected with multimeter (DT803-B)

and voltmeter (DT-9205-A). Vitreous carbon was used as base for

electro-deposition of pt to form anode. Soft carbon rod was first pressed

to give disc shape of 2cm and3cm diameter with the help of spatula

before electro-deposition of Pt. For the contact of electrode copper wire

was used which was already fused in the electrode. Simple graphite rod

of 0.5cm thickness and length of 6cm and width of 3cm was used as

cathode. pb/sb electrode prepared in the same way, with the same

specification and diameter was also used for electrochemical treatment

with the same conditions of current (DC 20 volts) 3 ampere and rotation

15-50 Rpm.) The results were compared with c/pt electrode, finally the

water sample after the growth of C.V.+O.P. optimal conditions was

followed by electrochemical treatment with c/pt electrode. The tank was

44

filled with 800ml of water sample from each source every time and

connections were made. The anode was rotated initially at the rate of 15

rpm which was increased gradually upto 50 rpm to observe the effect of

deposition and rate of oxidation at different rotations. Average DC

current of 20 volts and 3 amp was applied potential was enhanced in

anodic direction manually by 2 volts/10min and potential curve was

recorded on point to point bases using voltmeter.

All the experiments were carried out at laboratory temperature both pt

and pb electrodes were first polished by buffering and then activated by

dipping in N/50 H2SO2.

Studies were carried out both by using H2SO2 as oxidizing agent and

without H2SO2. Amount of metals deposited was determined by weighing

the electrode before and after the experiment.

Oxidation rate of ions with respect to deposition was also observed on

point to point bases by drawing a graph between current and potential and

the peak value was observed at which maximum oxidation occurs causing

metal deposition on anode. Rate of deposition of metals with respect to

rotation and surface area with both the electrodes was observed for all the

water samples.

The diluted water was left in the central compartment after the treatment,

which was analyzed for the determination of conductivity, pH, COD.

Total N and phosphate, chlorides, sulphate, total solids for the

comparison with the results obtained from bio chemical treatment.

Finally the water samples Electrochemically treated with C/Pt electrode,

surface area 3 cm were treated Biochemically with a mix combination of

C.V.+ O.P.as the maximum removal of the impurities in Elecrochemical

45

treatment took place with C/Pt electrode of 3 cm surface area and with

C.V.+O.P.in case of Biochemical treatment.

Optimal conditions of pH, Temperature and illumination, already

observed were maintained during the Biochemical process.

46

Deposition v/s Surface Area

As two electrodes of surface area 2cm and 3 cm were used. The

deposition was determined by the initial and final difference in weight

before and after the process for all the water samples.

47

Deposition vs Rate of Rotation

The deposition was determined by the difference in weight of the

electrode for all the water samples. The Electrode was cleaned and wash

every time before the operation.

49

RESULTS AND DISCUSSION

Algal species were collected from local habitats after screening and

repeatedly cell culturing. Following four species were selected for further

studies:

1. Chlorella vulgaris

2. Oocystis pusilla

3. Maugetia genoflexa

4. Ulothrix

Two species Chlorella vulgaris and Oosystis pusilla are unicellular while

the other two namely Maugestia genoflexa and Ulothrix are filamentous

(Fig. 1-6, Table No. 1). Their growth rate was studied in Bristol and Chu-

No-10 modified media. Luxurious growth occurred in Bristol media

(Table No.2) as compared to Chu-No-10 (Table No. 3), which was not so

encouraging due to easy availability of nutrients. Because in Bristol

medium the ionization of NaCl and NaNO3 is greater than Ca(NO3) and

Na2SiO3. pH was kept between 6.5- 7.5 during the growth by adding a

buffer periodically as both acidic and basic pH decreases the growth due

to bacteriostate action. Temperature was kept at 27-30˚C by varying the

number of bulbs. Illumination was maintained at 5000 Lux, below and

above this intensity not only the temperature increases but the process of

photosynthesis is also affected. Growth rate was studied after every 24 hr.

The maximum growth rate was observed on the 6th and 7

th day of the

inoculation without depending on mean doubling time (Table 2 and 3).

The growth rate decreases in both media after 7th

day. This can be due to

the growth competition as the processes were carried out under batch

system. However the growth rate of Mangetia genoflexa and Ulothrix

50

was less than Chlorella vulgaris and Oosystis pusilla due to being

multicellular in nature.

Due to the decrease in nutrients, a sigmoid curved could be obtained at

the end. For the preservation of these purified algal species the slants

were prepared in Czapek-Dox medium. Modified CM0097 which a

internationally recommended media for slants preparation. The slants

were preserved at 10˚C. in incubator.

Waste water samples were collected from different points of Lahore canal

namely, Ravi disposal, Jallomore, Ferozpur Road bridge and Thokar Niaz

Baig as the main disposal points are located at these places. Industrial

waste water samples were collected from two main dispoal points

Shahdra Rohi Nala and Sheikhupura where maximum industries are

located. As the main emphasis was on the minimization of metals present

and the parameters like nitrogen, phosphates, COD, TDS, chlorides,

sulphates and conductivity.

These parameters were studies in waste water. The waste water was

allowed to stand for 72 hrs and the upper layer was used for the

determination of metal ions, nitrogen, phosphates, pH, COD, total solids,

chlorides ions, sulphate contents and conductivity (Table 4 and 5).

Common metal ions present in all six sources were Cu, K, Na, Mg, Ca,

Mn, Fe and Zn. However their concentrations varied in these sources. The

water samples from Lahore canal contains less amount of Cu, Mn, Fe and

Zn and more amount of Na and K as it contains more domestic waste

water as compared to industrial waste. The amount of Ca and Mg is

almost equal as they are used as soaps for cleaning purpose and as

runners in the industries.

51

Both industrial waste water from Shahdra and Sheikhupura contain more

amount of Cu, Mn, Fe and Zn as most of Foundries and Chemical

industries are located in these areas like Rustam Suhrab, Millat Tractors,

Shazoo Pharma, Alpha Farma and DH Urea Plant.

The amount of total nitrogen, phosphates, COD, TDS, chlorides and

sulphates is greater in Ravi disposal, Jallu and Ferozpur bridge samples as

most of the domestic waste water is added at these points. Due to the

same reason, TDS and conductivity is also high in these samples. The pH

is between 7.2-7.8 due to the presence of CO3, HCO3 and OH ions which

were not determined. The amount of nitrogen, phosphates, COD,

chlorides and sulphates, TDS and conductivity is less in the water

samples from Thokar as this is almost the end of canal water and not

much polluted water is added, the water is quite diluted here due to the

addition of rain drains. That is why the pH of this water is 7.2-7.5

Waste water sample from Shahdra and Sheikhupura drains contain more

nitrogen, phosphate and sulphate and chlorides as it receives waste water

from the industries. The values of COD, total solids and conductivity are

also less due to the same reasons. pH is acidic as most of the industries

dispose of acidic waste water from their plants.

The effect of pH on the growth of algal species was studied at different

pH ranges (Table No. 6-13). In analyzed waste water pH was varied by

adding buffer solutions, temperature was kept between 27-30 ˚C and at

Illumination S 5000 Lux.

The maximum algal growth was observed in analysed water sample at a

pH between 6.5 to 7 in case of all the four species while it decreases both

52

in highly acidic and basic pH. This is because nutireint remain dissiocited

in ionic form in this pH range and an easy uptake by the algal cells

occurs. Moreover the algal cell wall is partially damaged in highly acid

and basic pH causing a bacteriostatic action.

However Maugetia genoflexa and Ulotroix showed some stability in

growth rate at slightly basic pH due to being multicellular and filamentus

in nature as compared to the Chlorella vulgaris and Oocystis pusilla

which are unicellular algae. pH was maintained between 6.5 to 7 as the

optimal value for further studies.

Effect of temperature on the growth rate of algae in analysed waste water

was also observed at different ranges (Table No. 14-21). The temperature

was controlled by increasing and decreasing no of bulbs. pH was

maintained between 6.5 – 7 by buffer solution and at 5000 Lux light

intensity. Maximum growth occurred at a temperature range between 27-

30 ˚C above which the growth rate is decreased gradually. This range was

considered as optimal temperature range for further studies.

Growth rate vs illumination was also observed between 2000 to 6000 Lux

light intensity after .inoculating the algal stains in waste water (Table No.

22-29). Again the maximum algal growth occurred at a illumination

range between 4000 to 5000 Lux in all the algal species. This is due to the

optimal photosynthetic process in this range .below and above this

intensity photosynthesis is affected. Moreover, the temperature also

increases above 5000 Lux intensity as the number of bulbs are to

increased for increasing the light intensity which also decreases the

growth rate. Hence for rest of the studies the illumination was kept at

5000 Lux as an optimal value.

53

All the four Algal species and their mix combination was grown in all the

six water samples (Table No. A-F). Optimal conditions of pH,

temperature and illumination already established were maintained. Their

growth rate were studies after every 24 hours. Maximum growth occurred

between 7-8 days after which the growth started decreasing. Hence 8 days

old culture was selected for further studies.

In all the six samples, the amount of metal ions decreases reasonably after

the growth of CV and Oocystis pusilla as the growth rate was high in

these species being unicellular algae. The removal rate was less in

Maugetia genoflexaand Ullotrhix as these are the multicellular species

and the cell wall in these two cases is hard as compared to the Chlorella

vulgaris and Oocystis pusilla due to which the uptake rate is slow.

The uptake of Cu ions by all the algal species is less as compared to K,

Na, Mg, Ca, Mn, Fe and Zn because Copper toxicity is developed in algal

cells after the uptake of a specific concentration of copper.

0.70mg/L of Cu was present in Shahdra waste water samples (Table No.

32) which was reduced to 0.28 mg/L after the growth of Oocystis pusilla

and Maugetia genoflexa while 0.91mg/L after the growth of Chlorella

vulgaris. While the mixed species Chlorella vulgaris and Oocystis pusilla

showed a remarkable result when used together and the amount of Cu

was reduced to0.10.mg/L.The combination of other species did not show

a remarkable effect.

Amount of K and Na was reduced from 0.38mg/L and 0.62 mg/L to 0.14

and 0.20 mg/L after the treatment with Oocystis pusilla, the other species

showed a less reduction as compared to it. Again the mixed species

54

Oocystis pusilla and Chlorella vulgaris when used together reduced the K

and Na to 0.09 mg/L and 0.06 mg/L. Less reduction occurred with the

combination of other species when waste water was analysed after the

growth of algal species. Mg was reduced from 0.44mg/L to 0.18 mg/L

using Oocystis pusilla while the other species did not show equal results.

The amount of Mg remained 0.1 mg/L when Oocystis pusilla and

Chlorella vulgaris were used together for the removal of metal ions.

These were the minimum results compared to the combination of other

species.

The amount of Ca in Shahdra waste water was reduced from 0.5 mg/L to

0.13 mg/L with Oocystis pusilla while the other species showed greater

results as compared to these two species. the combination of Chlorella

vulgaris and Oocystis pusilla mix species reduced the Ca ions to 0.08

mg/L which was the best result as compared to the combination of other

species.

Similarly, Mn ions were reduced from 0.09 mg/L to 0.021 mg/L by both

Chlorella vulgaris and Oocystis pusilla separately and to 0.00mg/L mg/L

when these species were used together while other species and their

combination did not show such results.

Fe and Zn ion concentration in Shahdra waste water was reduced to

0.48mg/L and 0.18 mg/L from 0.72 mg/L and 0.52 mg/L respectively

again in case of the batch which was run with Oocystis pusilla which is

the maximum result as compared to other species. Fe was reduced to 0.21

mg/L and Zn to 0.07 mg/L when Oocystis pusilla and Chlorella vulgaris

55

were used together which is the best result as compared to the

combination of other species (Table No. 32).

When the algal species were grown in the waste waster sample from

Sheikhupura (Table No. 33), a decrease in metal ions was observed

(Table No. 33). The amount of Cu ions was decreased from 0.45mg/L to

0.18 mg/L with Oocystis pusilla and to 0.28 mg/L with Chlorella vulgaris

while the other species did not show a remarkable effect. K was redcued

from 0.20mg/L to 0.18 mg/L with Chlorella vulgaris and to 0.09 mg/L

with Oocystis pusilla while the decrease in Na was also maximum with

Oocystis pusilla which was reduced to 0.38 mg/L from 0.41 mg/L.

Amount of Mg was reduced to 0.18 mg/L from 0.41 mg/L. Mg was

reduced to 0.18mg/L by growing Oocystis pusilla while other species

showed less effect.

Cu and Mn was decreased t 0.14mg/L and 0.00mgL respectively from

0.42 and 0.3 mg/L again in the batch with Oocystis pusilla. Fe and Zn

were also absorbed maximum by Oocystis pusilla which was reduced to

0.02 mg/L in both cases from 0.68 and 0.43 mg/L respectively. Removal

rate of Chlorella vulgaris was less then Oocystis pusilla but more than

other species.

When the Sheikhupura waste water sample was run with mixed species,

the combination of Oocystis pusilla and Chlorella vulgaris reduced all the

metal ions to a level of Cu 0.02 mg/L, K 0.02 mg/L, Na 0.09 mg/L, Mg

0.09 mg/L, Ca 0.06 mg/L, Mn 0.03 mg/L, Fe 0.10 mg/L and Zn 0.13

mg/L respectively. While the analysis result of waste water sample after

the growth of other mixed species were not so encouraging.

56

Waste water samples from Ravi disposal (Table No. 30) on analysis after

the growth of algal species gave encouraging results (Table-30). Amount

of Cu was reduced to 0.12 mg/L, K 0.09 mg/L, Na 0.18 mg/L, Mg 0.24

mg/L, Ca 0.29 mg/L, Mn 0.01 mg/L, Fe 0.02 mg/L and Zn 0.00mgL

mg/L by Oocystis pusilla and Cu to 0.16 mg/L, K 0.15 mg/L, Na 0.22

mg/L, Mg 0.04 mg/L, Fe 0.08 mg/L and Zn 0.04 mg/L by Chlorella

vulgaris.

The mixed species Oocystis pusilla and Chlorella vulgaris reduced the

amount of Cu to 0.09 mg/L, K 0.04 mg/L, Na 0.13 mg/L, Mg 0.19 mg/L,

Ca 0.21 mg/L, and Mn, Fe and Zn to 0.00mg/L. while the combination of

other species did not show such luxurious results.

When waste water smaple from Jallo (Table No. 31) was used as medium

for the growth of algal species (Table-31). the waste water was analysed

for metal ions. The Cu was reduced to 0.04 mg/L, K 0.12 mg/L, Mg 0.21

mg/L, Ca 0.14 mg/L, Mn ....mg/L, Fe 0.02 mg/L and Zn 0.02 mg/L by

Oocystis pusilla while the amount of metal ions left after the growth of

Chlorella vulgaris was Cu 0.08 mg/L, k 0.2 mg/l, Na 0.31mg/L, Mg 0.33

mg/L, Ca 0.22 mg/L, Mn 0.02 mg/l, Fe 0.06 mg/L and Zn 0.04 mg/L.The

amount of metal ions left after the growth of Maugetia genoflexa and

Ulothrix was more than these values. With the combination of Oocystis

pusilla and Chlorella vulgaris amount of metal ions left was, Cu 0.02

mg/L, K 0.06 mg/L, Na 0.15 mg/l, Mg 0.1 mg/l, Ca 0.06 mg/L, Mn, Fe

and Zn 0.00mg/L which are equally considerable results while the other

species did not give such results.

Analytical results of Ferozpur Road bridge waste water samples (Table

No. 34) after the growth of Oocystis pusilla and Chlorella vulgaris are

57

also remarkable. Where the amount of Cu was reduced from 0.19mg/L to

0.09 mg/L, K from 0.41mg/L to 0.12 mg/L, Na from 0.4 mg/L to 0.02

mg/L, Mg from 0.02mg/L to 0.00mg/L and Fe and Zn from 0.11mg/L to

0.00 mg/L ion case of Oocystis pusilla growth while the metal ions left

after the growth of Chlorella vulgaris were Cu 0.2 mg/L, K 0.2 mg/L, Na

0.24 mg/L, Ca 0.32 mg/L, Mn 0.00.mg/L, Fe 0.04 mg/L and Zn 0.02

mg/L which is more than Oocystis pusilla culture. Other species showed

higher values than the two species.

The uptake was greater when Oocystis pusilla and Chlorella vulgaris

were used in combined form as compared to the other algal combinations.

Cu was reduced to 0.02 mg/L, K 0.06 mg/L, Na 0.09 mg/L, Ca 0.1 mg/l

while Mn, Fe and Zn to mg/L by using mixed algal species Oocystis

pusilla and Chlorella vulgaris.

Thokar water sample (Table No. 35) was treated with all the four algal

species and the maximum uptake was observed with Oocystis pusilla and

Chlorella vulgaris as compared to Maugetia genoflexa and Ulothrix.

Considerable reduction in the amount of metal ions occurred. When OP

was grown in Thokar water sample, Cu was reduced from 0.22mg/litre to

0.09 mg/litre, K from 0.36 to 0.14 mg/litre, Na from 0.51 to 0.21 mg/litre,

Mg from 0.42 to 0.09 mg/litre, Ca from 0.38 to 0.12 mg/litre, Fe from

0.10 to 0.02 mg/litre and both Mn and Zn to 0.00 mg/litre.

Uptake of CV was less than O.P in this batch, Cu was reduced to 0.13

mg/litre, K to 0.18 mg/litre, Na to 0.32 mg/litre, Mg to 0.18 mg/litre, Ca

to 0.20 mg/litre, Mn to 0.02 mg/litre, Fe to 0.06 mg/litre and Zn to 0.00

mg/litre.

58

Thokar water sample was also treated with mix combination of the Algal

species and again good results were altoming by a mix culture of C.V. to

P compare to other combination where Cu reduced to 0.02 mg/litre, K

0.09 mg/litre, Na to 0.12 mg/litre, Mg to 0.02 mg/litre, Ca to 0.03 mg/litre

and Mn, Fe and Zn to 0.00 mg/litre.

The other parameters N, P, COD, Chlories, Sulphates TDS and

conductivity are also reduced by C.V. Nitrogen was reduced from 120 to

67, Phosphate from 70 to 52 mg/litre (Tabel No. 5), COD 170 to 110

mg/litre, chlorides from 110 to 62 mg/litre, sulphates from 80 mg/litre to

48 mg/litre, TDS from 1200 mg/litre to 596 mg/litre and conductivity

from 2000 to 1000 by C.V. in Ravi sample.

While with O.P., N was reduced to 86 mg/litre, phosphate to 50 mg/litre,

O.P to 4 mg/litre, chlorides to 38 mg/litre, sulphates to 31 mg/litre, TDS

to 642 mg/litre and conductivity to 1160 while the other two species M.G.

Ulothrix gave high values than O.P and C.V.

Mix species C.V and O.P reduced the N to 22 mg/litre, phosphate to 32

mg/litre, COD to 16 mg/litre, chlorides to 22 mg/litre, sulphates to 20

mg/litre, TDS to 305 mg/litre and conductivity to 590 µs. While the

combination of other species showed higher results than O.P and C.V.

In Jallo Sample, water sample (Table No. 38), N was reduced to 72

mg/litre, phosphate to 59 mg/litre, COD to 119 mg/litre, chlorides to 69

mg/litre, sulphates to 62 mg/litre, TDS to 660 and conductivity to 1170

µs by C.V. (Fig. 38). While O.P reduced N to 78 mg/litre, phosphate to

62 mg/litre, COD to 128 mg/litre, chlorides to 74 mg/litre, sulphates to 66

mg/litre, TDS to 871 mg/litre and conductivity to 1142 mg/litre while the

59

other species did not show remarkable change in the parameters as

compare to C.V. and O.P. The mixed species C.V. and O.P reduced N to

40 mg/litre, phosphates to 48 mg/litre, COD 39 mg/litre, chlorides to 41

mg/litre, sulphate to 58 mg/litre, TDS 475 mg/litre and conductivity to

690 µs while the combination of other species showed less reduction in

these parameters.

Analysis of Thokar waste water (Table No. 39) after the growth of Algal

species and their combination gave following results, N was reduced to

36 mg/litre, phosphate to 32 mg/litre, COD to 94 mg/litre, chlorides to 84

mg/litre, sulphates to 34 mg/litre, TDS to 1120 mg/litre, conductivity to

1800 µs by C.V. The reduction by O.P was N to 38 mg/litre, phosphates

to 34 mg/litre, COD 98 mg/litre, chlorides to 91 mg/litre, sulphates to 34

mg/litre, TDS to 1128 mg/litre, conductivity to 1790 µs while the mix

combination C.V. and O.P. gave remarkable results as compare to other

combination. The N was reduced to 21 mg/litre, phosphate to 19 mg/litre,

COD to 45, chlorides to 51 mg/litre, sulphates to 19 mg/litre, TDS to 675

mg/litre and conductivity to 1650 µs.

The Algae species were cultured in Ferozepur road bridge water sample

(Table No. 40). Broth culture was analysed for the determination of metal

ions and the other factors (Table 40). The results obtained with C.V were

better than O.P where the reduction in N was to 44 mg/litre, phosphate to

48 mg/litre, COD to 86 mg/litre, chlorides to 92 mg/litre, sulphates to 56

mg/litre, TDS to 1160 mg/litre and conductivity to 1910 µs while O.P lift

in the culture, 49 mg/litre of N, 52 mg/litre of phosphate, COD 90

mg/litre, chlorides 96 mg/litre, sulphates 56 mg/litre, TDS 1160 mg/litre

and conductivity 190 µs while the mixed combination of C.V. and O.P

gain encouraging results as compare to other combination where N was

60

reduced to 30 mg/litre, phosphates to 28 mg/litre, COD to 18 mg/litre,

chlorides to 22 mg/litre, sulphates to 17 mg/litre, TDS to 310 mg/litre and

conductivity to 540 mg/litre.

Shahdra water sample (Table No. 41) gave following results after the

growth of Algae species. With C.V, N = 106 mg/litre, phosphate = 18

mg/litre, COD = 18 mg/litre, chloride = 26 mg/litre, sulphates = 78

mg/litre, TDS = 460 mg/litre, conductivity = 780 µs. While in the culture

with O.P, N reduced to 108 mg/litre, phosphate to 138 mg/litre, COD to

21 mg/litre, chlorides to 32 mg/litre, sulphate to 82 mg/litre, TDS to 590

mg/litre and conductivity to 31 µs. The mixed form of C.V and O.P gave

better results than other mixed cultures where N was decreased to 34


mg/litre, sulphates to 26 mg/litre, TDS to 198 mg/litre and conductivity to

406 µs/litre. Medium was separated by centrifugation and subjected to

analysis.

Electrochemical treatment of the waste water studies were carried out to

observe the effect of current vs potential with and without adding H2SO4

as oxidizing agent. Two electrodes of c/pt and pb/sb surface area 2cm and

3cm were used, current was studies in amperes and potential in volts

without H2SO4 maximum potential of 12.30 volts (Graph No. 48) in

Ferozepur road water sample 11.30 in Thokar water samples (Graph No.

49). 13.30 in Sheikhupura water sample (Graph No. 50) 14.10 in Shahdra

water sample (Graph No. 51), 10.60 in Ravi disposal sample (Graph No.

52), 11.20 in Jallo water sample (Graph No. 53) was observed at a current

intensity of 11 ampere with surface area of 3 cm when c/pt electrode was

used.

61

With 2 cm surface area maximum potential was again observed at 11

amperes current and the values were to 6.70 v for Ferozepur road sample

(Graph No. 54), 5.8 v for Thokar sample (Graph No. 55), 8.10 v for

Sheikhupura water sample (Graph No. 56), 7.40 v for Shahdra sample

(Graph No. 57), 5.60 mg/litre for Ravi sample (Graph No. 58) and 5.80

mg/litre for Jallo sample disposal (Graph No. 59).

When the same samples were treated with H2SO4 as oxidizing agent using

c/pt electrode the maximum potential was observed at 9 amperes current.

These values were 16.10 V for Ravi disposal (Graph No. 60) 15.53 V for

Sheikhupura (Graph No. 61), 15.30 V for Shahdra (Graph No. 62) and

11.80 for Ferozepur road (Graph No. 63), 15.10 V for Thokar (Graph No.

64), 15.38 V for Jallo (Graph No. 65) with 3 cm surface area of the

electrode. With 2 cm surface area again the maximum potential was

observed at 9 amperes current and following values were obtained. For

Ravi disposal sample the potential was 9.10 V (Graph No. 66), for Jallo

8.20 V (Graph No. 67) for Thokar water sample 7.40 V (Graph No. 68),

for Sheikhupura sample 8.30 V (Graph No. 69), for Shahdra sample 8.3 V

(Graph No. 70) and for Ferozepur road sample (Graph No. 71) the

maximum potential value observed at 9 ampere current was 7.8 V.

With pb/sb the maximum potential vs current value without using H2SO4

as oxidizing agent was observed at 11 ampere current which were 9.40 V

for Ravi disposal (Graph No. 72), 10.35 V for Jallo sample (Graph No.

73), 10.80 for Ferozepur road sample (Graph No. 74), 10.20 V for Thokar

sample (Graph No. 75), 12.60 V for Shahdra (Graph No. 76) and 11.9 V

for Sheikhpura sample (Graph No. 77) when pb/sb electrode of 3 cm

surface area was used.

62

With 2 cm surface area the maximum potential values were again

observed at 11 ampere current which were 5.10 V for Ravi disposal

(Graph No. 78), 5.90 V for Jallo sample (Graph No. 79), 5.60 V for

Ferozepur road sample (Graph No. 80), 6.80 for Thokar sample (Graph

No. 81), 6.70 V for Shahdra sample (Graph No. 82) and 6.10 V for

Sheikhupura sample (Graph No. 83).

With 3 cm surface area electrode using H2SO4 as oxidizing agent. The

values of current vs potential were low. These values were observed at

current value of 9 amperes which were 7.30 V for Ravi disposal (Graph

No. 84), 8.90 V for Jallo (Graph No. 85), 8.90 V for Ferozpur road

(Graph No. 86), 7.11 V for Thokar (Graph No. 87), 8.10 V for Shahdra

(Graph No. 88) and 8.73 volts for Sheikhupura sample (Graph No. 89).

With the electrode of 2 cm surface area electrode, these values were 3.40

mg/litre for Ravi disposal (Graph No. 90), 5.20 V for Jallo (Graph No.

91), 3.90 V for Ferozepur road (Graph No. 92), 4.60 V for Thokar (Graph

No. 93), 4.11 V for Shahdra (Graph No. 95) and 4.13 V for sheikhupura

sample (Graph No. 96).

Amount of impurities deposited on the electrodes was observed at

different rotation rate by weighing the electrodes before and after the

completion of the process in grams. Maximum deposition in all the cases

occurred at rotation rate of 15 RPM.

When c/pt electrode with surface area 3 cm was used the maximum

deposition which occurred at a rate of 15 RPM was 1.9 gms in Ravi

disposal sample (Graph No. 96), 2.10 gms in Shahdra sample (Graph No.

97), 1.21 gms in Jallo sample (Graph No. 98), 1.86 gms in Ferozepur road

63

sample (Graph No. 99), 2.10 gms in Thokar sample (Graph No. 100) and

2.80 gms in Sheikhupura sample (Graph No. 101).

With 2 cm surface area the amount deposited at rate of 15 RPM was 1.20

gm in Ravi disposal (Graph No. 102) 1.74 gms in Shahdra samples

(Graph No. 103), 1.21 gms in Jallo sample (Graph No. 104), 1.62 gms in

Ferozepur road sample (Graph No. 105), 1.40 gms in Thokar sample

(Graph No. 106) and 1.82 gms in Sheikhupura sample (Graph No. 107).

With pb/sb electrode the deposition at rotation rate of 15 rpm was 1.20

gms with 3 cm area (Graph No. 108), 0.73 gms with 2 cm surface area

(Graph No. 114) in Ravi disposal, 1.21 gms with 3 cm area (Graph No.

109), 0.52 with 2 cm area (Graph No. 115) in Jallo sample (Graph No.

109), 1.34 gms with 3 cm area (Graph No. 110), 1.20 gms with 2 cm area

(Graph No. 116) in Ferozepur road sample. 1.93 gms with 3 cm area

(Graph No. 111), 0.98 gms with cm area (Graph No. 117) in Thokar

sample. 1.72 gms with 3 cm (Graph No. 112) and 0.86 gms with 2 cm

area (Graph No. 118) in Shahdra sample 2.34 gms with 3 cm area (Graph

No. 116) and 1.12 gms with 2 cm area (Graph No. 119) in Sheikhupura

water sample.

The electro-chemically treated waste water with c/pt 3 cm surface and

pb/sb 3 cm area left as dituted water in the central compartment of the

cell was analysed for the determination of metal ions and other

parameters. When c/pt electrode with 3 cm surface area was used (Table

No. 46), Ravi disposal waste water sample gave following results after

electrochemical treatment, Cu = 0.09 mg/litre, K = 0.11 mg/litre, Na =

0.21 mg/litre, Ca = 0.36 mg/litre, Mn = 0.04 mg/litre, Fe = 0.09 mg/litre,

Zn = 0.06 mg/litre, N = 28 mg/litre, phosphate 68 mg/litre, COD to 110

64

mg/litre, chlorides sulphate, TDS = 211 mg/litre, conductivity to 396 µs.

In Jallo water Cu was reduced to 0.06 mg/litre, K to 27 mg/litre, Na to

0.42 mg/litre, Mg to 0.27 mg/litre, Ca to 0.30 mg/litre, Mn to 0.03

mg/litre, Fe to 0.05 mg/litre, Zn to 0.06 mg/litre, N to 36 mg/litre,

phosphate to 67 mg/litre, COD to 98 mg/litre, chlorides to 36 mg/litre,

sulphate to 18 mg/litre, TDS to 252 mg/litre, conductivity to 416 µs.

In Ferozepur road water sample Cu was reduced to 0.12 mg/litre, K to

0.28 mg/litre, Na to 0.31, Ca to 0.35 mg/litre, Mn to 0.00 mg/litre, Fe to

0.04 mg/litre, Zn to 0.00 mg/litre, N to 21 mg/litre, phosphate to 38


mg/litre, TDS to 166 mg/litre and conductivity to 295 µs.

In Thokar water sample the Cu was reduced to 0.11 mg/litre, K to 0.30

mg/litre, Na to 0.34 mg/litre, Mg to 0.22 mg/litre, Ca to 0.22 mg/litre, Mn

to 0.04 mg/litre, Fe to 0.05 mg/litre and Zn to 10, N to 13 mg/litre,

phosphate to 30 mg/litre, COD to 78 mg/litre, chloride 106 mg/litre

sulphates to 17 mg/litre and conductivity to 241 µs. in Sheikhupura water

sample Cu was reduced to 0.23 mg/litre, K to 0.13 mg/litre, Na to 0.26

mg/litre, Cu to 0.22 mg/litre, Mg to 0.31 mg/litre, Mn to 0.17 mg/litre, Fe

to 0.38 mg/litre, Zn to 0.22 mg/litre, N to 26 mg/litre, phosphates to 21


mg/litre, TDS to 141 mg/litre and conductivity to 280 µs and in Shahdra

water sample Cu was reduced to 0.41 mg/litre, K to 0.20 mg/litre, Na to



phosphates to 18 mg/litre, COD to 24 mg/litre, chlorides to 89 mg/litre,

sulphates to 28 mg/litre, TDS to 132 mg/litre and conductivity to 260 µs.

65

Results with pb/sb electrode with 3 cm surface area were not so

encouraging when Ravi water sample was electrochemically treated

(Table No. 47), Cu was reduced to 0.11 mg/litre, K to 0.13 mg/litre, Na to

0.21 mg/litre, Mg to 0.35mg/litre, Ca to 0.38 mg/litre, Mn to 0.08

mg/litre, Fe to 0.10 mg/litre and Zn to 0.08 mg/litre, N to 58 mg/litre,

phosphates to 66 mg/litre, COD 132 mg/litre, chlorides to 88 mg/litre,

sulphates 241 mg/litre, TDS to 413 mg/litre and conductivity to 710 µs.

In Jallo water sample when treatment electrochemically by using pb/sb 3

cm surface area electrode the Cu was reduced to 0.08 mg/litre, K to 0.21


to 0.04 mg/litre, Fe to 0.06 mg/litre, Zn to 0.04 mg/litre, N to 46 mg/litre,


TDS to 465 mg/litre, sulphates to 36 mg/litre and conductivity to 718 µs.

In Ferozepur road water sample Cu was reduced to 0.14 mg/litre, K to

0.30 mg/litre, Na to 0.38 mg/litre, Mg to 0.24 mg/litre, Ca to 0.38

mg/litre, Mn to 0.01 mg/litre, Fe to 0.06 mg/litre, Zn to 0.04 mg/litre, N

to 56 mg/litre, phosphates to 43 mg/litre, COD to 112 mg/litre, chlorides

to 38 mg/litre, sulphates to 32 mg/litre, TDS to 465 mg/litre and

conductivity 718 µs.

In Thokar sample Cu was reduced to 0.13 mg/litre, K to 0.28 mg/litre, Na

to 0.38 mg/litre, Mg to 0.35 mg/litre, Ca to 0.24 mg/litre, Mn to 0.00

mg/litre, Fe to 0.07 mg/litre and Zn to 0.00 mg/litre, N to 0.38 mg/litre,


sulphates to 25 mg/litre, TDS to 372 mg/litre, conductivity to 395 µs.

66

In Sheikhupura water sample Cu was reduced to 0.26 mg/litre, K to

0.15mg/litre, Na to 0.29 mg/litre, Mg to 0.26 mg/litre, Ca to 0.32 mg/litre,

Mn to 0.20 mg/litre, Fe to 0.20 mg/litre, Zn to 0.29 mg/litre, N to 49

mg/litre, phosphates to 24 mg/litre, COD to 27 mg/litre, chlorides to 112


40 µs.

In Shahdra sample Cu reduced to 0.44 mg/litre, K to 0.24 mg/litre, Na to



phosphates to 24 mg/litre, COD to 27 mg/litre, chlorides to 112 mg/litre,


Finally the above electrochemical treated water was again treated

biochemically by using a mixed combination of C.V and O.P as the

maximum reduction in all the parameter and metal ions was observed

with this combination. Remarkable results in the reduction of all the

parameters and metals ions was observed when the electro chemical

treatment was followed by bio chemical treatment (Table 42, 43). In Ravi

disposal waste water sample Cu was reduced to 0.01 mg/litre, K to 0.01


to 0.00 mg/litre, Fe to 0.01 mg/litre, Zn to 0.01 mg/litre, N to 12 mg/litre,



In Jallo waste waer sample the amount of metal ions and other parameter

left was Cu = 0.01 mg/litre, K = 0.08 mg/litre, Na = 0.16 mg/litre,

Mg=0.19 mg/litre, Ca = 0.17 mg/litre, Mn = 0.00 mg/litre, Fe = 0.02

mg/litre, N = 16 mg/litre, phosphates = 26 mg/litre, Cod = 30 mg/litre,

67

chlorides = 16 mg/litre, sulphates = 06 mg/litre, TDS = 132 mg/litre,

conductivity = 200µs.

In Ferozepur road water sample amount left was Cu 0.04 mg/litre, K 0.10

mg/litre, Na 0.18 mg/litre, Mg 0.07 mg/litre, Mn 0.00 mg/litre, Fe 0.00

mg/litre, Zn 0.02 mg/litre, N 11 mg/litre, phosphates 17 mg/litre, COD 30

mg/litre, chlorides 14 mg/litre, sulphates 14 mg/litre, TDS 72 mg/litre and

conductivity 172 µs.

In Thokar water sample, Cu was reduced to 0.00 mg/litre, K to 0.08


to 0.01 mg/litre, Fe to 0.02 mg/litre and Zn to 0.00 mg/litre, N to 04

mg/litre, phosphate 12 mg/litre, COD to 27 mg/litre, chloride ro 21

mg/litre, sulphtes to 10 mg/litre, TDS to 63 mg/litre, conductivity to 69

mg/litre.

In Sheikhupura water sample Cu was reduced to 0.14 mg/litre, K to 0.06


to 0.06 mg/litre, Fe to 0.13 mg/litre and Zn to 0.09 mg/litre, N to 10



128 µs.

In Shahdra water sample Cu was reduced to 0.20 mg/litre, K to 0.02

mg/litre, na to 0.13 mg/litre, Mg to 6.0 mg/litre, Mn to 0.00 mg/litre, Fe

to 0.00 mg/litre, Zn to 0.08 mg/litre, N to 30 mg/litre, phosphate to 05


mg/litre, TDS to 58 mg/litre and conductivity to 133 µs.

69

RESULTS

Table No. 2

Growth Rate of Algae in Bristol media. At 27o-30

oC, pH is

6.5-7 and light intensity is 5000 Lux for culture development

after the identification of the species.

No. of

Days Growth Rate (Millions/ml)

C.V. O.P. M.G. Ulothrix C.V.+O.P. C.V.+M.G.

1 27 18 22 20 68 60

2 48 34 43 41 110 102

3 62 57 57 55 140 132

4 98 72 93 91 171 163

5 116 132 111 109 203 195

6 127 161 122 120 330 322

7 137 163 132 130 330 322

8 131 150 126 124 262 254

9 131 142 126 124 170 162

10 125 118 120 118 102 194

11 118 98 113 111 70 62

12 101 48 196 194 70 62

13 86 31 81 79 40 32

14 57 24 52 50 20 12

15 31 24 26 24 20 12

No. of


C.V.+Ulothrix O.P.+M.G. O.P.+Ulothrix M.G.+Ulothrix

1 62 64 67 68

2 104 106 109 110

3 134 136 139 140

4 165 167 170 171

5 197 199 202 203

6 324 326 329 330

7 324 326 329 330

8 256 258 261 262

9 164 166 169 170

10 196 198 201 202

11 64 66 69 70

12 64 66 69 70

13 34 36 39 40

14 14 16 19 20

15 14 16 19 20

All the results are replicates of three.

70

Table No. 3

Growth of Algae in Chu-10 Median at pH = 6.5-7,

Temp. = 27o-30

o, Illumination = 5000 Lux

No.

of

Days

Growth Rate (Millions/ml)

C.V. O.P. M.G. Ulothrix C.V.+O.P. C.V.+M.G.

1 32 30 35 34 62 52

2 32 31 35 36 62 52

3 36 34 38 38 70 61

4 65 63 62 61 128 118

5 98 96 95 94 155 145

6 102 100 99 98 202 192

7 120 118 117 116 223 213

8 123 121 120 119 211 200

9 122 120 119 118 233 211

10 98 96 95 94 201 185

11 87 85 84 83 180 172

12 74 72 71 70 144 133

13 44 42 41 40 120 112

14 38 36 35 34 99 75

15 36 34 33 32 75 64

No. of

Days



1 70 73 78 73

2 112 110 108 121

3 160 168 166 169

4 202 250 278 255

5 232 265 288 265

6 240 278 289 275

7 240 280 295 277

8 200 220 300 244

9 184 199 222 195

10 172 152 195 155

11 84 111 155 122

12 74 95 142 95

13 62 82 99 71

14 32 62 52 51

15 22 43 32 31

71

Table No. 4

Determination of Metal Ions in Waste Sample

before the Treatment

Amount in mg/liter

Sources of

the Sample Cu K Na Mg Ca Mn Fe Zn

Ravi Disposal 0.20 0.21 0.40 0.61 0.52 0.09 0.12 0.11

Jallo 0.10 0.26 0.61 0.50 0.48 0.07 0.09 0.10

Ferozpur Road 0.19 0.41 0.40 0.31 0.51 0.02 0.11 0.08

Thokar 0.22 0.36 0.51 0.42 0.38 0.07 0.10 0.00

Sheikhupura 0.45 0.20 0.41 0.38 0.42 0.30 0.68 0.43

Shahdara 0.70 0.38 0.62 0.44 0.51 0.09 0.72 0.52

Table No. 5

Analysis of Water Sample before the Treatment

Results in mg/liter

Sources of

the Sample Nitrogen Phosphate COD Chlorides Sulphates TDS pH

µs

Condt.

Ravi Disposal 120 70 170 110 80 1200 7.8 2000

Jallo 138 94 141 80 65 1400 7.4 2400

Ferozpur Road 88 52 127 52 43 1400 7.6 2660

Thokar 40 36 103 96 36 900 7.2 2750

Sheikhupura 180 211 30 105 233 900 5.8 1761

Shahdara 205 189 38 188 50 860 6.3 1610

72

Table No. 6

Effect of pH on Growth Rate of Chlorella Vulgaris (C.V.)

in the Waste Water at Temp. = 27o-30

oC

No. of

Days

Sample No. 1 Sample No. 4 Sample No. 7

Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

1 22 7.0 28 7.0 28 7.0

2 24 7.0 31 7.0 31 7.0

3 27 7.0 91 7.0 31 6.8

4 150 6.9 118 6.9 51 6.8

5 300 6.8 190 6.7 140 6.7

6 221 6.4 188 6.5 140 6.7

7 182 6.2 140 6.1 121 6.5

8 186 6.0 129 5.9 101 6.0

9 126 5.8 124 5.7 90 5.7

10 116 5.4 124 5.2 90 5.5

11 64 5.0 110 5.0 81 5.2

12 39 4.8 89 4.9 81 5.0

13 31 4.5 61 4.7 58 4.7

14 22 4.5 42 4.7 40 4.0

15 27 4.0 36 4.5 29 4.0

Table No. 7

Effect of pH on Growth Rate of Chlorella Vulgaris (C.V.)


oC

No. of

Days


Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

1 25 7.0 31 7.0 31 7.0

2 28 7.0 33 6.9 34 7.0

3 74 6.8 78 6.9 116 6.8

4 189 6.6 121 6.8 204 6.8

5 181 6.6 193 6.6 190 6.6

6 140 6.3 171 6.4 143 6.4

7 185 6.0 160 6.0 193 6.1

8 189 5.7 139 5.8 204 5.9

9 129 5.0 127 5.5 171 5.6

10 119 4.9 126 5.1 130 5.2

11 67 4.9 113 4.8 110 5.0

12 42 4.4 92 4.5 84 4.7

13 34 4.4 64 4.5 61 4.5

14 25 4.0 45 4.0 43 4.1

15 30 4.0 39 4.0 32 4.1


73

Table No. 8

Effect of pH on Growth Rate of Oscystus Pusilla (O.P.)


oC

No. of

Days


Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

1 24 7.0 30 7.0 30 7.0

2 39 6.9 61 7.0 67 6.9

3 73 6.9 120 6.9 142 6.7

4 122 6.7 120 6.8 152 6.7

5 186 6.6 170 6.7 192 6.7

6 139 6.4 190 6.7 203 6.6

7 122 4.0 192 6.6 170 6.4

8 102 5.9 191 6.6 168 6.0

9 96 5.4 126 6.3 140 5.8

10 74 5.4 125 6.0 129 5.4

11 66 5.0 112 5.9 109 5.0

12 41 4.8 91 5.4 83 4.9

13 33 4.6 63 7.0 60 7.5

14 24 4.4 44 6.4 42 6.5

15 29 4.2 38 6.3 31 6.3

Table No. 9

Effect of pH on Growth Rate of Oscystus Pusilla (O.P.)


oC

No. of

Days


Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

1 27 7.0 33 7.0 33 7.0

2 76 6.9 40 7.0 37 6.9

3 103 6.7 103 6.8 98 6.9

4 142 6.7 173 6.7 194 6.7

5 193 6.6 194 6.6 206 6.6

6 187 6.4 182 6.4 206 6.5

7 131 6.0 152 6.0 173 6.2

8 121 5.8 141 6.0 141 5.9

9 102 5.4 129 5.6 124 5.4

10 78 5.3 128 5.3 112 5.0

11 69 5.0 115 5.0 112 8.5

12 44 7.5 94 7.5 86 8.0

13 36 7.0 66 7.0 63 7.5

14 27 6.5 47 6.4 45 6.5

15 32 6.4 41 6.3 34 6.3


74

Table No. 10

Effect of pH on Growth Rate of M.G. in the

Waste Water at Temp. = 27o-30

oC

No. of

Days


Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

1 28 7 34 7 34 7

2 43 7.5 65 8 36 7.5

3 77 8 97 8.5 37 8

4 104 8.5 124 9 57 8.5

5 126 9 174 9.5 76 8.5

6 143 9.5 194 10 146 9

7 188 10 196 10 196 9.5

8 192 10 195 10 207 10

9 132 11 130 9.5 174 9.5

10 122 9 129 9 133 9

11 70 8 116 8 113 8.5

12 45 7.5 95 7.5 84 8

13 37 7 67 7 64 7.5

14 27 6.5 48 6.4 46 6.5

15 33 6.4 42 6.3 35 6.3

Table No. 11

Effect of pH on Growth Rate of M.G. in the


oC

No. of

Days


Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

1 31 7 37 7 37 7

2 46 7.5 68 8 40 7.5

3 80 8 100 8.5 41 8

4 107 8.5 127 9 60 8.5

5 129 9 177 9.5 79 8.5

6 146 9.5 197 10 149 9

7 191 10 199 10 199 9.5

8 195 10 198 10 210 10

9 135 11 133 9.5 174 9.5

10 125 9 132 9 136 9

11 73 8 119 8 116 8.5

12 48 7.5 98 7.5 90 8

13 40 7 70 7 67 7.5

14 31 6.5 51 6.4 49 6.5

15 36 6.4 45 6.3 38 6.3


75

Table No. 12

Effect of pH on Growth Rate of Ulothrix in the


oC

No. of

Days


Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

1 24 7 30 7 30 7

2 39 7.5 61 8 33 7.5

3 73 8 93 8.5 34 8

4 100 8.5 120 9 53 8.5

5 122 9 170 9.5 72 8.5

6 139 9.5 190 10 142 9

7 184 10 192 10 192 9.5

8 186 10 191 10 203 10

9 126 11 125 9.5 166 9.5

10 116 9 125 9 129 9

11 66 8 112 8 109 8.5

12 41 7.5 91 7.5 83 8

13 33 7 63 7 60 7.5

14 24 6.5 44 6.4 42 6.5

15 29 6.4 38 6.3 31 6.3

Table No. 13

Effect of pH on Growth Rate of Ulothrix in the


oC

No. of

Days


Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

Growth Rate

Million/ml

pH

Values

1 32 7 38 7 38 7

2 47 7.5 69 8 41 7.5

3 80 8 101 8.5 42 8

4 108 8.5 128 9 61 8.5

5 130 9 178 9.5 80 8.5

6 147 9.5 198 10 150 9

7 191 10 200 10 200 9.5

8 194 10 209 10 211 10

9 134 11 133 9.5 174 9.5

10 124 9 133 9 137 9

11 74 8 120 8 117 8.5

12 49 7.5 99 7.5 91 8

13 41 7 71 7 68 7.5

14 32 6.5 52 6.4 50 6.5

15 37 6.4 46 6.3 39 6.3


76

Table No. 14

Effect of Temperature on Growth Rate of Chlorella

Vulgaris (C.V.) in the Waste Water with pH = 6.5-7.0

No. of

Days


Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

1 22 25 28 25 31 25

2 136 27 181 27 198 27

3 150 31 190 29 191 30

4 112 29 142 31 160 33

5 136 31 102 33 101 34

6 122 33 90 35 68 35

7 122 36 67 36 62 36

8 110 38 67 38 30 38

9 62 39 18 37 21 37

10 41 40 18 40 12 40

Table No. 15

Effect of Temperature on Growth Rate of Chlorella


No. of

Days


Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

1 24 25 30 25 33 25

2 160 27 192 27 193 27

3 153 29 184 29 225 20

4 130 32 161 31 193 32

5 118 34 104 34 193 34

6 98 35 69 35 106 35

7 66 36 69 36 90 36

8 40 38 23 38 24 38

9 40 39 19 37 20 37

10 22 40 19 40 17 40


77

Table No. 16

Effect of Temperature on Growth Rate of Oocystus

Pusilla (O.P.) in the Waste Water with pH = 6.5-7.0

No. of

Days


Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

1 28 25 34 25 37 25

2 156 27 195 27 229 27

3 144 30 188 31 197 31

4 116 34 122 34 126 34

5 98 34 128 34 124 34

6 80 35 97 35 124 35

7 62 36 52 36 67 36

8 51 38 38 38 36 38

9 29 39 27 37 24 37

10 21 40 22 40 19 40

Table No. 17

Effect of Temperature on Growth Rate of Oocystus


No. of

Days


Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

1 32 25 38 25 41 25

2 159 27 199 27 234 27

3 148 31 187 31 229 31

4 119 34 161 34 130 34

5 90 34 110 34 102 34

6 68 35 82 35 91 35

7 42 36 50 36 48 36

8 27 38 25 38 27 38

9 27 39 21 37 27 37

10 18 40 18 40 20 40


78

Table No. 18

Effect of Temperature on Growth Rate of Maugetia

Genoflex (M.G.) in the Waste Water with pH = 6.5-7.0

No. of

Days


Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

1 34 25 40 25 43 25

2 163 27 159 27 201 27

3 151 31 180 30 235 20

4 124 34 180 32 131 32

5 120 34 114 34 106 34

6 81 35 79 35 80 35

7 72 36 51 36 48 36

8 39 38 29 38 34 38

9 26 39 27 37 25 37

10 20 40 22 40 18 40

Table No. 19

Effect of Temperature on Growth Rate of Maugeria


No. of

Days


Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

1 36 25 42 25 45 25

2 152 27 158 27 133 27

3 165 29 192 30 239 30

4 126 34 129 32 201 32

5 124 34 114 34 180 34

6 70 35 73 35 98 35

7 47 36 60 36 72 36

8 29 38 44 38 52 38

9 21 39 26 37 34 37

10 17 40 24 40 26 40


79

Table No. 20

Effect of Temperature on Growth Rate of Ulothrix in the

Waste Water with pH = 6.5-7.0


Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

Growth Rate

Million/ml

Temp. oC

38 25 40 25 48 25

158 27 169 27 191 27

171 29 190 29 198 29

131 31 186 31 179 31

118 35 148 33 144 33

102 35 121 35 122 35

63 37 62 36 101 36

37 38 51 38 82 38

29 39 42 39 40 39

21 40 31 40 28 40

Table No. 21

Effect of Temperature on Growth Rate of Ulothrix in the



Growth

Rate

Million/ml

Temp. oC

Growth

Rate

Million/ml

Temp. oC

Growth

Rate

Million/ml

Temp. oC

48 25 42 25 48 25

166 27 134 27 134 27

201 29 201 29 202 29

183 30 190 30 178 31

170 32 162 33 118 33

155 34 138 35 99 35

111 35 112 37 81 37

68 36 72 38 42 38

52 38 44 39 31 39

27 40 29 40 22 40


80

Table No. 22

Effect of Illumination on Growth Rate of Chlorella


Light Intensity

in Lux Sample No. 3 Sample No. 6 Sample No. 9

Lux Growth Rate

Million/ml

Growth Rate

Million/ml

Growth Rate

Million/ml

2000 70 82 92

3000 142 128 130

4000 181 174 188

5000 199 194 198

6000 68 73 88

Table No. 23

Effect of Illumination on Growth Rate of Chlorella


Light Intensity


Lux Growth Rate

Million/ml

Growth Rate

Million/ml

Growth Rate

Million/ml

2000 75 87 96

3000 149 133 134

4000 205 179 193

5000 205 199 203

6000 75 77 92


81

Table No. 24

Effect of Illumination on Growth Rate of Oocystus


Light Intensity


Lux Growth Rate

Million/ml

Growth Rate

Million/ml

Growth Rate

Million/ml

2000 79 91 101

3000 149 137 137

4000 190 184 194

5000 208 201 207

6000 77 80 96

Table No. 25

Effect of Illumination on Growth Rate of Oocystus


Light Intensity


Lux Growth Rate

Million/ml

Growth Rate

Million/ml

Growth Rate

Million/ml

2000 82 92 102

3000 152 139 140

4000 191 182 198

5000 209 204 206

6000 78 83 97


82

Table No. 26

Effect of Illumination on Growth Rate of Maugetia


Light Intensity


Lux Growth Rate

Million/ml

Growth Rate

Million/ml

Growth Rate

Million/ml

2000 82 94 101

3000 155 137 146

4000 193 185 198

5000 211 206 208

6000 79 85 99

Table No. 27

Effect of Illumination on Growth Rate of Maugetia


Light Intensity


Lux Growth Rate

Million/ml

Growth Rate

Million/ml

Growth Rate

Million/ml

2000 84 96 103

3000 161 133 144

4000 195 188 199

5000 219 211 208

6000 77 89 99


83

Table No. 28

Effect of Illumination on Growth Rate of Ulothrix in the


Light Intensity


Lux Growth Rate

Million/ml

Growth Rate

Million/ml

Growth Rate

Million/ml

2000 80 84 88

3000 161 164 172

4000 193 201 208

5000 230 227 210

6000 102 98 104

Table No. 29

Effect of Illumination on Growth Rate of Ulothrix in the


Light Intensity


Lux Growth Rate

Million/ml

Growth Rate

Million/ml

Growth Rate

Million/ml

2000 76 80 78

3000 180 172 191

4000 205 196 204

5000 218 223 231

6000 104 96 110


84

Table No. 30

Sample from Ravi Disposal

Amount of Metal Ions in Waste Water after the

Growth of Algae Species

Optimal Conditions: pH = 7, Temp. = 27o-30

o,

Light Intensity = 5000 Lux

Amount in mg/liter

Name of Species Cu K Na Mg Ca Mn Fe Zn

C.V 0.16 0.15 0.22 0.30 0.33 0.04 0.08 0.04

O.P. 0.12 0.09 0.18 0.24 0.29 0.01 0.02 0.00

C.V.+O.P. 0.09 0.04 0.13 0.19 0.21 0.00 0.00 0.00

M.G. 0.16 0.16 0.18 0.15 0.25 0.03 0.06 0.03

Ulothrix 0.14 0.15 0.16 0.14 0.23 0.03 0.05 0.02

C.V.+M.G. 0.10 0.07 0.15 0.17 0.24 0.01 0.01 0.02

C.V.+Ulothrix 0.09 0.06 0.16 0.18 0.22 0.02 0.03 0.03

O.P.+M.G. 0.10 0.01 0.17 0.18 0.26 0.00 0.04 0.01

O.P.+Ulothrix 0.14 0.13 0.14 0.20 0.25 0.02 0.02 0.02

M.G.+Ulothrix 0.13 0.15 0.16 0.23 0.27 0.02 0.02 0.01

Table No. 31

Sample from Jallo


Growth of Algae


o,


Amount in mg/liter


C.V 0.08 0.20 0.31 0.33 0.22 0.02 0.06 0.09

O.P. 0.04 0.12 0.28 0.21 0.14 0.00 0.02 0.02

C.V.+O.P. 0.02 0.06 0.15 0.10 0.06 0.00 0.00 0.00

M.G. 0.04 0.20 0.34 0.36 0.24 0.06 0.08 0.10

Ulothrix 0.10 0.20 0.33 0.35 0.27 0.08 0.08 0.11

C.V.+M.G. 0.05 0.08 0.21 0.14 0.09 0.01 0.01 0.11

C.V.+Ulothrix 0.06 0.07 0.22 0.13 0.10 0.02 0.02 0.13

O.P.+M.G. 0.07 0.08 0.24 0.14 0.12 0.00 0.03 0.11

O.P.+Ulothrix 0.05 0.07 0.17 0.13 0.11 0.02 0.03 0.10

M.G.+Ulothrix 0.11 0.08 0.16 0.14 0.12 0.00 0.04 0.12

85

Table No. 32

Sample from Shahdra


Growth of Algae


o,


Amount in mg/liter


C.V 0.41 0.20 0.41 0.24 0.22 0.02 0.51 0.24

O.P. 0.28 0.14 0.20 0.18 0.13 0.02 0.48 0.18

C.V.+O.P. 0.10 0.09 0.06 0.10 0.08 0.00 0.21 0.07

M.G. 0.47 0.20 0.44 0.28 0.24 0.08 0.27 0.28

Ulothrix 0.45 0.20 0.44 0.26 0.26 0.03 0.21 0.27

C.V.+M.G. 0.13 0.17 0.14 0.17 0.11 0.09 0.20 0.09

C.V.+Ulothrix 0.14 0.18 0.10 0.13 0.11 0.01 0.27 0.10

O.P.+M.G. 0.15 0.14 0.12 0.14 0.15 0.02 0.28 0.14

O.P.+Ulothrix 0.13 0.16 0.10 0.14 0.13 0.01 0.26 0.13

M.G.+Ulothrix 0.13 0.17 0.13 0.17 0.17 0.01 0.24 0.17

Table No. 33

Sample from Sheikhupura


Growth of Algae


o,


Amount in mg/liter


C.V 0.28 0.18 0.38 0.24 0.22 0.08 0.32 0.30

O.P. 0.18 0.09 0.28 0.18 0.14 0.09 0.22 0.20

C.V.+O.P. 0.02 0.02 0.09 0.09 0.06 0.03 0.10 0.13

M.G. 0.26 0.18 0.31 0.29 0.28 0.08 0.35 0.30

Ulothrix 0.28 0.20 0.30 0.28 0.24 0.09 0.33 0.33

C.V.+M.G. 0.30 0.08 0.33 0.18 0.08 0.08 0.31 0.19

C.V.+Ulothrix 0.08 0.06 0.13 0.16 0.14 0.09 0.28 0.16

O.P.+M.G. 0.06 0.07 0.16 0.19 0.16 0.08 0.31 0.18

O.P.+Ulothrix 0.08 0.06 0.12 0.13 0.17 0.08 0.27 0.18

M.G.+Ulothrix 0.07 0.08 0.16 0.13 0.20 0.09 0.30 0.19

86

Table No. 34

Sample from Ferozpur Road

Amount of Metal Ions in Water after the

Growth of Algae


oC,


Amount in mg/liter


C.V. 0.12 0.20 0.24 0.24 0.32 0.00 0.04 0.02

O.P. 0.09 0.12 0.02 0.19 0.20 0.00 0.00 0.00

C.V.+O.P. 0.02 0.06 0.09 0.13 0.10 0.00 0.00 0.00

M.G. 0.17 0.20 0.28 0.30 0.36 0.07 0.05 0.08

Ulothrix 0.16 0.19 0.26 0.28 0.34 0.05 0.11 0.06

C.V.+M.G. 0.14 0.10 0.10 0.16 0.18 0.08 0.10 0.09

C.V.+Ulothrix 0.12 0.09 0.11 0.16 0.16 0.06 0.09 0.07

O.P.+M.G. 0.14 0.08 0.13 0.18 0.15 0.08 0.10 0.07

O.P.+Ulothrix 0.10 0.11 0.12 0.17 0.18 0.06 0.10 0.06

M.G.+Ulothrix 0.10 0.08 0.12 0.16 0.16 0.06 0.09 0.08

Table No. 35

Sample from Thokar


Growth of Algae


o,


Amount in mg/liter


C.V. 0.13 0.18 0.32 0.18 0.20 0.02 0.06 0.00

O.P. 0.09 0.14 0.21 0.09 0.12 0.00 0.02 0.00

C.V.+O.P. 0.02 0.09 0.12 0.02 0.06 0.00 0.00 0.00

M.G. 0.18 0.20 0.30 0.21 0.24 0.08 0.10 0.09

Ulothrix 0.18 0.19 0.29 0.23 0.30 0.08 0.08 0.08

C.V.+M.G. 0.08 0.16 0.14 0.19 0.17 0.04 0.06 0.10

C.V.+Ulothrix 0.08 0.14 0.13 0.18 0.16 0.04 0.08 0.10

O.P.+M.G. 0.06 0.16 0.13 0.18 0.19 0.03 0.08 0.09

O.P.+Ulothrix 0.06 0.17 0.15 0.16 0.18 0.04 0.09 0.10

M.G.+Ulothrix 0.09 0.16 0.16 0.18 0.19 0.04 0.06 0.10

87

Table No. 36

Analysis of the Waste Samples after Growth of

Algae at Optimal Conditions

Samples from Ravi Disposal

Results in mg/liter

Name of

Species Nitrogen Phosphate COD Chlorides Sulphates TDS

Ms

Conduct

C.V. 67 52 110 62 48 596 1000

O.P. 86 50 94 38 31 642 1160

C.V.+O.P. 22 32 16 22 21 305 590

M.G. 75 40 24 30 29 313 598

Ulothrix 77 42 26 32 31 315 600

C.V.+M.G. 26 36 20 26 25 309 594

C.V.+Ulothrix 28 38 22 28 27 311 596

M.G.+Ulothrix 30 40 24 30 29 313 598

O.P.+Ulothrix 32 42 26 32 31 315 600

O.P.+M.G. 31 41 25 31 30 314 599

pH Maintained at 7, Temp. = 27o – 30

oC, Light Intensity = 5000 Lux

Table No. 37



Samples from Sheikhupura

Results in mg/liter

Name of


Ms

Conduct

C.V. 94 132 11 26 70 342 612

O.P. 102 112 10 22 71 513 996

C.V.+O.P. 28 5 12 12 9 172 310

M.G. 103 141 20 35 79 351 621

Ulothrix 105 143 22 37 81 353 623

C.V.+M.G. 100 138 17 32 76 348 618

C.V.+Ulothrix 101 139 18 33 77 349 619

M.G.+Ulothrix 102 140 19 34 78 350 620

O.P.+Ulothrix 99 137 16 31 75 347 617

O.P.+M.G. 103 141 20 35 79 351 621



88

Table No. 38



Samples from Jallo

Results in mg/liter

Name of


Ms

Conduct

C.V. 72 59 119 69 61 660 1170

O.P. 78 62 121 74 66 871 1140

C.V.+O.P. 40 48 39 41 34 475 690

M.G. 76 63 123 73 65 664 1174

Ulothrix 90 67 117 48 58 1170 1840

C.V.+M.G. 48 56 47 49 42 483 698

C.V.+Ulothrix 46 54 45 47 40 481 696

M.G.+Ulothrix 51 59 50 52 45 486 701

O.P.+Ulothrix 47 55 46 48 41 482 697

O.P.+M.G. 49 57 48 50 43 484 699



Table No. 39



Samples from Thokar

Results in mg/liter

Name of


Ms

Conduct

C.V. 36 32 94 84 34 1120 1800

O.P. 38 34 98 91 34 1128 1790

M.G. 36 38 94 95 33 1131 1784

Ulothrix 36 36 96 98 34 1113 1888

C.V.+O.P. 21 19 45 51 19 675 1650

C.V.+M.G. 25 23 49 55 23 679 1654

C.V.+Ulothrix 26 24 50 56 24 680 1655

M.G.+Ulothrix 24 22 48 54 22 678 1653

O.P.+Ulothrix 29 27 53 59 27 683 1658

O.P.+M.G. 27 25 51 57 25 681 1656



89

Table No. 40



Samples from Ferozpur Road

Results in mg/liter

Name of


Ms

Conduct

C.V. 44 48 786 92 56 1160 1910

O.P. 46 52 892 96 48 1210 2010

M.G. 48 54 86 98 59 1290 2210

Ulothrix 48 56 88 106 62 1196 1821

C.V.+O.P. 30 28 18 22 17 310 540

C.V.+M.G. 34 32 22 26 21 314 544

C.V.+Ulothrix 36 34 24 28 23 316 546

M.G.+Ulothrix 38 36 26 30 25 318 548

O.P.+Ulothrix 39 37 27 31 26 319 549

O.P.+M.G. 37 35 25 29 24 317 547



Table No. 41



Samples from Shahdara

Results in mg/liter

Name of


Ms

Conduct

C.V. 106 118 18 26 78 460 780

O.P. 108 138 21 32 82 591 1031

M.G. 120 131 29 38 96 610 1131

Ulothrix 110 101 39 55 88 710 1220

C.V.+O.P. 34 16 24 28 26 198 406

C.V.+M.G. 38 20 28 32 30 202 410

C.V.+Ulothrix 41 23 31 35 33 205 413

M.G.+Ulothrix 43 25 33 37 35 207 415

O.P.+Ulothrix 44 26 34 38 36 208 416

O.P.+M.G. 46 28 36 40 38 210 418



90

Table No. 42

Analysis of Waste Water after Electro-Chemical

Treatment with c/pt Electrode Surface Area 3cm

Results in mg/liter

Sources of

the Sample Nitrogen Phosphate COD Chlorides Sulphates TDS

Ms

Conduct

Ravi Disposal 28 68 110 68 27 211 396

Jallo 36 67 98 36 18 252 416

Ferozpur Road 21 38 92 31 24 166 295

Thokar 13 30 78 61 17 118 241

Sheikhupura 26 21 22 72 38 141 280

Shahdara 27 18 24 89 28 132 260

pH Maintained at 7, Temp. 25oC, Light Intensity 3500 Lux

Table No. 43

Analysis of Waste Water after Electro-Chemical

Treatment with pb/sb Electrode Surface Area 3cm

Results in mg/liter

Sources of


Ms

Conduct

Ravi Disposal 58 66 132 88 41 413 710

Jallo 46 79 126 58 36 465 718

Ferozpur Road 56 43 112 38 32 465 718

Thokar 38 33 91 62 25 372 395

Sheikhupura 49 27 24 89 92 301 421

Shahdara 44 24 24 112 36 298 401

pH Maintained at 7, Temp. 25oC, Light Intensity 3500 Lux

91

Biochemical followed by Electrochemical with c/pt Electrode at

pH = 7, Temp. = 25oC. Samples after the growth of C.V.+O.P.

was treated electrochemically with c/pt, as best results were

obtained with these microbial combination and with c/pt 3cm

area electrode.

Table No. 44

Waste Water Analysis after Biochemical Treatment with

C.V.+O.P. followed by Electrochemical Treatment

using c/pt Electrode

Amount in mg/liter

Sources of


Ravi Disposal 0.01 0.01 0.03 0.16 0.20 0.00 0.01 0.01

Jallo 0.01 0.08 0.16 0.19 0.17 0.00 0.02 0.02

Ferozpur Road 0.04 0.10 0.18 0.07 0.18 0.00 0.00 0.00

Thokar 0.00 0.18 0.16 0.12 0.02 0.01 0.02 0.00

Sheikhupura 0.14 0.06 0.09 0.08 0.14 0.06 0.13 0.09

Shahdara 0.02 0.02 0.13 0.07 0.17 0.00 0.00 0.08

Table No. 45

Waste Water Analysis after Biochemical Treatment with

C.V.+O.P. followed by Electrochemical Treatment

using c/pt Electrode

Results in mg/liter

Sources of


Ms

Conduct

Ravi Disposal 12 24 38 32 14 98 218

Jallo 16 26 30 16 06 132 210

Ferozpur Road 11 17 30 14 13 72 172

Thokar 04 12 27 21 10 63 69

Sheikhupura 10 09 09 28 18 60 128

Shahdara 13 05 08 33 17 58 132



92

Table No. 46

Determination of Metal Ions after Electrochemical

Treatment with c/pt Electrode Surface Area 3cm

Amount in mg/liter

Sources of


Ravi Disposal 0.09 0.11 0.21 0.32 0.36 0.04 0.09 0.06

Jallo 0.06 0.18 0.42 0.27 0.30 0.03 0.05 0.06

Ferozpur Road 0.12 0.28 0.31 0.21 0.35 0.00 0.04 0.02

Thokar 0.11 0.30 0.34 0.32 0.22 0.04 0.05 0.00

Sheikhupura 0.23 0.13 0.26 0.22 0.31 0.17 0.38 0.22

Shahdara 0.41 0.20 0.23 0.20 0.28 0.02 0.30 0.28

Table No. 47

Waste Water Analysis after the Electrochemical

Treatment with pb/sb Surface Area 3cm

Amount in mg/liter

Sources of


Ravi Disposal 0.10 0.13 0.24 0.35 0.38 0.08 0.10 0.08

Jallo 0.08 0.21 0.17 0.31 0.38 0.04 0.06 0.09

Ferozpur Road 0.14 0.30 0.35 0.24 0.38 0.01 0.06 0.04

Thokar 0.13 0.28 0.38 0.35 0.24 0.00 0.07 0.00

Sheikhupura 0.26 0.15 0.24 0.26 0.32 0.20 0.20 0.29

Shahdara 0.44 0.24 0.26 0.23 0.30 0.03 0.38 0.33

93

Table A

Growth of Chlorella Vulgaris (C.V.), Oosystis Pusilla (O.P.),

Maugetia Genoflexa (M.G.) and Ulothrix in the

Sample from Ravi Disposal


o,


No. of


C.V. O.P. C.V.+O.P. C.V.+M.G. M.G. Ulothrix

1 37 40 80 74 32 34

2 48 54 130 124 43 45

3 88 101 188 182 83 85

4 120 130 310 304 115 117

5 140 153 340 334 135 137

6 140 160 348 342 135 137

7 140 160 348 342 135 137

8 160 127 208 202 155 157

9 116 103 162 156 109 111

10 98 88 98 92 93 95

11 66 40 52 46 61 63

12 66 40 52 46 61 63

13 40 22 40 34 35 37

14 26 22 20 14 21 23

15 18 15 20 14 13 15

No. of



1 72 70 68 71

2 122 120 118 121

3 180 178 176 179

4 302 300 298 301

5 332 330 328 331

6 340 338 326 339

7 340 338 326 339

8 200 198 196 199

9 154 152 156 153

10 90 88 86 89

11 44 42 40 43

12 44 42 40 43

13 32 30 28 31

14 12 10 8 11

15 12 10 8 11

94

Table B



Sample from Sheikhupura


o,


Growth Rate Results in mg/liter

No. of



1 36 42 86 90 28 26

2 46 48 102 106 38 35

3 69 1120 168 147 55 64

4 112 130 298 252 90 98

5 136 151 310 294 116 121

6 146 160 340 304 136 130

7 138 152 332 300 136 130

8 128 130 192 168 124 120

9 112 90 101 168 102 94

10 102 80 82 123 91 73

11 72 32 60 102 60 51

12 64 40 62 96 55 42

13 38 98 50 81 38 32

14 30 30 36 78 38 38

15 24 16 20 78 30 28

No. of



1 102 38 31 26

2 128 52 58 48

3 142 78 86 72

4 236 102 116 107

5 283 128 138 126

6 321 231 293 280

7 356 312 333 298

8 300 251 294 212

9 221 162 119 181

10 188 70 101 106

11 119 58 92 91

12 90 38 77 68

13 72 25 60 51

14 60 25 42 32

15 58 20 28 29

95

Table C



Sample from Jallo


o,


No. of



1 29 32 72 66 24 26

2 40 46 122 116 35 37

3 80 93 180 174 75 77

4 112 123 302 296 107 109

5 132 145 332 326 127 129

6 132 152 340 334 127 129

7 132 152 340 334 127 129

8 152 119 200 194 147 149

9 108 95 154 148 101 103

10 90 80 90 84 85 87

11 58 32 44 38 53 55

12 58 32 44 38 53 55

13 32 14 32 26 27 29

14 18 14 12 6 13 15

15 10 7 12 6 5 7

No. of



1 64 62 60 63

2 114 112 110 113

3 172 170 168 171

4 294 292 290 293

5 324 292 320 323

6 332 330 318 331

7 332 330 318 331

8 192 190 188 191

9 146 144 146 145

10 82 80 78 81

11 36 34 32 35

12 36 34 32 35

13 24 22 20 25

14 4 2 3 4

15 4 2 2 3

96

Table D



Sample from Ferozpur Road


o,


No. of



1 19 22 62 56 14 16

2 30 36 112 106 25 27

3 70 83 170 164 65 67

4 102 113 292 286 97 99

5 122 135 322 316 117 119

6 122 142 330 324 117 119

7 122 142 330 324 117 119

8 142 109 192 184 137 139

9 92 85 144 138 93 93

10 80 70 80 74 75 77

11 48 22 34 28 43 45

12 48 22 34 28 43 45

13 12 6 22 16 17 19

14 8 4 5 8 6 9

15 1 2 3 4 3 5

No. of



1 54 52 50 53

2 104 102 100 103

3 162 160 158 161

4 284 282 280 283

5 314 282 310 313

6 322 320 308 321

7 322 320 308 321

8 182 180 178 181

9 136 134 136 135

10 72 70 68 71

11 26 24 22 25

12 26 24 22 25

13 14 12 10 15

14 7 5 7 8

15 4 2 3 4

97

Table E



Sample from Thokar


o,


No. of



1 21 24 64 58 16 18

2 32 38 114 108 27 29

3 72 85 172 166 67 69

4 104 115 294 288 99 101

5 124 137 324 318 119 121

6 124 144 332 326 119 121

7 124 144 332 326 119 121

8 144 111 194 186 139 141

9 94 87 146 140 95 95

10 82 72 82 76 77 79

11 50 24 36 30 45 47

12 50 24 36 30 45 47

13 14 8 24 18 19 21

14 10 6 7 10 8 11

15 3 4 5 6 5 7

No. of



1 56 54 52 55

2 106 104 102 105

3 164 162 160 163

4 286 284 282 285

5 316 284 312 315

6 324 322 310 323

7 324 322 310 323

8 184 182 180 183

9 138 136 138 137

10 74 72 70 73

11 28 26 24 27

12 28 26 24 27

13 16 14 12 17

14 9 7 9 10

15 6 4 5 6

98

Table F



Sample from Shahdara


o,


No. of



1 20 23 63 57 15 17

2 31 37 113 107 26 28

3 71 84 171 165 66 68

4 103 114 293 287 98 100

5 123 136 323 317 118 120

6 123 143 331 325 116 119

7 123 142 330 323 114 117

8 143 110 192 185 137 140

9 93 86 145 139 94 94

10 81 71 81 75 76 78

11 49 23 35 29 44 46

12 48 23 34 27 43 45

13 13 7 23 17 18 20

14 9 5 6 9 7 10

15 2 3 4 5 4 6

No. of

Days



1 55 53 51 54

2 105 103 101 104

3 163 161 159 162

4 285 283 281 284

5 315 281 311 314

6 323 321 309 322

7 322 319 305 321

8 183 180 179 182

9 137 135 137 136

10 73 71 69 72

11 27 25 23 26

12 26 23 21 25

13 15 11 11 16

14 8 6 8 9

15 5 3 4 5

100

GRAPHS

Potential (volts) Current (Ampere) 3Cm 2Cm

3 1.7 0.2

5 2.1 1.1

7 4.4 2.5

9 5.8 3.2

11 12.3 6.7

13 9.8 5.1

15 7.6 3.7

101

Potential(Volts) Current (Ampere)

3Cm 2Cm

3 1.9 1.3

5 5.6 3.3

7 5.9 3.5

9 6.8 3.7

10 8.3 4.3

11 11.2 5.8

13 8.3 4.4

15 5.5 3.1

102

Potential(volts) Current(Ampere)

3Cm 2Cm

3 1.7 0.8

5 2.6 2.4

7 3 1.8

9 5.9 3.4

11 10.6 5.6

13 8 4.3

15 6.6 3.5

103

Potential Current

3Cm 2Cm

3 4.2 2.3

5 6.2 3.6

7 6.9 3.8

9 10.3 5.8

11 14.1 7.4

13 11.8 6.1

15 9.3 5.4

104

Potential Current

3Cm 2Cm

3 3.1 2.6

5 4.7 2.8

7 5.9 3.2

9 8.2 4.6

11 13.3 8.1

13 11.2 5.8

15 9.1 4.7

Sheikhupura Sample without H2SO4

105

Potential Current

3Cm 2Cm

3 2.4 2.4

5 2.7 2.6

7 5.4 2.8

9 6.2 3.3

11 11.3 5.8

13 9 5.1

15 7.3 3.7

Thokar Sample without H2SO4

106

Potential(volts) Current(ampere)

3Cm 2Cm

3 1.5 0.9

5 3.1 1.8

7 5.3 2.7

9 8.1 4.8

11 10.8 5.6

13 9.4 4.7

15 7.6 4.1

107

Potential Current

3Cm 2Cm

3 1.9 1

5 4.7 3.2

7 5.9 3.4

9 7.3 3.8

11 10.3 5.9

13 8.24 4.6

15 7.33 3.8

108

Potential Current

3Cm 2Cm

3 1.3 0.8

5 2.6 1.3

7 4 2

9 5.2 2.6

11 9.4 5.1

13 8 4.3

15 7.4 4

109

Potential Current

3Cm 2Cm

3 1.9 1

5 4.3 2.1

7 5.2 2.5

9 6.8 3.5

11 12.6 6.7

13 10.2 5.1

15 9.3 4.6

110

Potential Current

3Cm 2Cm

3 2.8 2.5

5 5.2 2.8

7 6.8 3.6

9 8.4 4.4

11 11.9 6.1

13 9.8 5.8

15 7.3 3.8

111

Potential Current

3Cm 2Cm

3 1.8 0.9

5 2.9 1.4

7 4.2 2.3

9 8.3 4.3

11 10.2 6.8

13 8.9 4.5

15 7.2 3.7

112

Potential Current

3cm 2cm

3 5.8 3.2

5 9.1 4.6

7 10.9 6.1

9 11.8 7.8

11 11.2 5.6

13 8.3 4.6

15 6.4 3.5

113

Potential Current

3Cm 2Cm

3 5.4 2.8

5 7.3 3.7

7 10.8 5.6

9 15.3 8.2

11 10.9 5.5

13 7.1 3.6

15 5 2.6

114


3Cm 2Cm

3 4.8 2.6

5 6.9 3.5

7 10.9 6.5

9 16.1 9.1

11 11.4 6

13 10.2 5.6

15 5.1 3.1

115

Potential(Volts) Current(Ampere)

3Cm 2Cm

3 5.9 3.4

5 8.8 4.6

7 11.1 5.8

9 15.3 8.3

11 12.4 6.8

13 8.3 4.7

15 5.1 2.8

116


3cm 2Cm

3 6.1 3.4

5 8.2 4.6

7 11.4 6.2

9 15.3 8.3

11 10.2 5.8

13 6.8 4.2

15 4.2 2.3

117

Potential (Volts) Current(Ampere)

3Cm 2Cm

3 4.9 2.5

5 6.7 3.7

7 11.2 5.8

9 15.1 7.4

11 14.8 8.7

13 10.2 5.6

15 6 3

118


3Cm 2Cm

3 0.9 0.41

5 1.1 0.92

7 4.4 3.61

9 8.9 3.4

11 8.1 3.6

13 6.82 3.51

15 5.11 3.11

119


3Cm 2Cm

3 1.2 0.7

5 3 0.71

7 4.1 2.7

9 8.9 5.2

11 7.9 5.8

13 7.1 3.4

15 5.8 2.7

120


3Cm 2Cm

3 1.1 0.07

5 1.8 0.09

7 3.2 1.7

9 7.3 3.4

11 6.9 3.6

13 6.6 3.1

15 6.1 5.7

121


3Cm 2Cm

3 1.1 0.92

5 2.4 1.24

7 2.9 1.26

9 8 4.11

11 7.6 3.7

13 6.3 3.84

15 5.6 3.1

122


3Cm 2Cm

3 1.3 0.94

5 2.71 1.82

7 4.22 2.44

9 8.73 4.13

11 7.16 3.01

13 6.31 3.11

15 4.82 2.73

123


3Cm 2Cm

3 0.81 0.4

5 1.7 0.92

7 3.11 1.21

9 7.11 4.6

11 6.8 3.6

13 6.2 3.4

15 4.9 2.7

124

Rate of Rotation Amount Deposited (gm)

3cm 2Cm

10 1.14 1.02

15 1.86 1.62

20 1.84 1.6

25 1.84 1.6

30 1.72 1.59

35 1.66 1.4

40 1.6 0.9

45 1.58 0.9

50 1.56 0.7

125

Rate of Rotation Amount Deposited(gm)

3cm 2Cm

10 1.4 1.18

15 1.8 1.21

20 1.3 1.2

25 1.3 1.16

30 1.21 1.13

35 1.18 1.13

40 1.14 1.1

45 1.1 1.09

50 1.09 1.04

126


3cm 2Cm

10 0.96 0.42

15 1.2 0.73

20 1.14 0.61

25 1.12 0.58

30 0.93 0.53

35 0.84 0.47

40 0.81 0.41

45 0.77 0.38

50 0.73 0.36

127


3Cm 2Cm

10 2.62 1.74

15 2.8 1.82

20 2.86 1.76

25 2.77 1.7

30 2.4 1.6

35 1.86 1.43

40 1.64 1.31

45 1.62 1.09

50 1.44 1

128


3Cm 2Cm

10 1.93 1.22

15 2.1 1.4

20 1.88 1.38

25 1.84 1.38

30 1.62 1.33

35 1.6 1.26

40 1.54 1.09

45 1.5 1.02

50 1.48 0.92

129

Rate of Rotation Amount deposited

3Cm 2Cm

10 1.3 1.09

15 1.34 1.2

20 1.3 1.13

25 1.24 1.09

30 1.2 1.03

35 1.15 0.09

40 1.09 0.67

45 1.06 0.56

50 0.09 0.05

130

Rate of rotation Amount Deposited

3Cm 2Cm

10 1.85 1.1

15 1.21 0.74

20 1.08 0.53

25 1.04 0.52

30 0.92 0.5

35 0.73 0.45

40 0.62 0.31

45 0.58 0.25

50 0.56 0.23

131


3Cm 2Cm

10 0.96 0.42

15 1.2 0.73

20 1.14 0.61

25 1.12 0.58

30 0.93 0.53

35 0.84 0.47

40 0.81 0.41

45 0.77 0.38

50 0.73 0.36

132

Rate of Rotation Amount Deposited

3Cm 2Cm

10 1.68 1.34

15 1.72 0.86

20 1.53 0.76

25 1.44 0.72

30 0.94 0.42

35 0.87 0.43

40 0.83 0.41

45 0.76 0.38

50 0.63 0.31

133

Rate of Rotation Amont Deposited

3 Cm 2 Cm

10 2.12 1.6

15 2.34 1.12

20 2.83 0.91

25 1.52 0.76

30 0.48 0.24

35 0.36 0.18

40 0.31 0.2

45 0.28 0.15

50 0.22 0.11

134

Rate of Rotation Amount Deposited

3 Cm 2 Cm

10 2.12 1.6

15 2.34 1.12

20 2.83 0.91

25 1.52 0.76

30 0.48 0.24

35 0.36 0.18

40 0.31 0.2

45 0.28 0.15

50 0.22 0.11

135


3Cm 2Cm

10 1.89 0.98

15 1.93 0.94

20 1.84 0.92

25 1.8 0.9

30 1.32 0.66

35 0.92 0.46

40 0.83 0.41

45 0.73 0.36

50 0.62 0.31

136

Deposition v/s Surface Area C/Pt Electrode

Sample Name Amount Deposited (gm)

2cm 3cm

Sheikhupura 1.21 1.91

Jullo 1.3 1.83

Ravi Disposal 1.28 1.47

Ferozpur Road 1.32 1.66

Thokar 1.2 1.6

Shahdra 1.33 1.49

137

Deposition v/s Surface Area Pb/Sb Electrode

Sample Name Amount Deposited(gm)

2cm 3cm

Ravi Disposal 1.08 1.18

Jullo 1.06 1.16

Ferozpur Road 1.04 1.18

Thokar 1.02 1.12

Shahdra 1.06 1.16

sheikhupura 1.03 1.24

142

CONCLUSION

1. Waste water samples were collected from six different points, four

from Lahore canal and two from the industrial area of Sheikhupura

and Shahdara. Water samples were stored in sterilized containers at

10oC for further treatment. Four Algal species were collected from

local habitats namely Oocystis Pusilla, Chlorella Vulgaris,

Maugetia Genoflexa and Ulothrix. First two species are unicellular

while the other two are filamentous. The species were purified by

standard methods and sub cultured in Bristol medium and Chu No.

10 modified medium. Healthy and maximum growth occurred in

Bristol medium as compare to Chu No. 10. This is due to more

availability of nutrients in ionic form in Bristol medium as

compare to Chu No. 10 where some of the nutrients are sparingly

soluble in water.

2. To observe the optimal growth, the growth rate of all the species

was observed at different pH values, temperature and

illuminations. Maximum Algal growth in all the four species was

observed at a pH value between 6-7 above and below which

growth rate decreases due to partial damage of the Algal cell wall.

Moreover the availability of the nutrients to the broth culture in

decreased due to the complex formation of metal ions with buffer

solution. Hence pH = 6.0-7.0 can be consider as optimal value for

the maximum growth of Algae and maximum metal uptake rate by

the Algal cells for all four species.

3. Algal growth rate at different light intensities was observed

between 2500 Lux to 6000 Lux. Maximum Algal growth occurred

143

at a light intensity between 4500 to 5000 Lux. Below and above

this value growth rate decreases. This is due to the variation in

photosynthetic process as all the green Algal species were selected

with chlorophill in them. Moreover the temperature also increases

by the increase in light intensity.

4. Effect of temperature on Algal growth showed that maximum

Algal growth occurred between 27oC to 30

oC, above this

temperature the cell growth decreases due to the hardening of the

cell wall. The uptake rate of nutrients by the Algal cells also

decreases. Due to the evaporation of medium an increase in the

concentration of nutrients in the medium takes place. The death

phase in the growth curve comes earlier due to this reason in C.V.

and O.P. However this phase is bit lengthy in case of M.G. and

Ulothrix due to the soft cell wall in C.V. and O.P. and hard cell

wall in M.G. and Ulothrix.

5. The analysis of waste water samples after the growth Algal species

gave maximum reduction results in all the nutrients. Maximum

uptake of metals ions and other nutrients takes place when

Chlorella Vulgaris and Oosystis Pusilla were grown in the waste

water sample. Same results were obtained when the C.V. and O.P.

were grown in mixed form. Again the reason could be the high

uptake rate of nutrients in these two unicellular Algal species as

compare to the other two species and their mixed combination.

6. In electrochemical treatment, all the six water samples were

electrochemically treated with and without using H2SO4 as

oxidizing agent with c/pt and pb/sb electrode with 2cm and 3cm

144

surface area respectively at variable current/potential between 3-15

ampere and by rotating the electrodes at a rotation speed from 10-

50 rpm. Best results were obtained with surface area 3cm rather

than 2cm with both the electrodes as the amount deposited

increases by the increase in surface. Maximum deposition occurred

by using H2SO4 as oxidizing agent as the H2SO4 is an oxidizer and

the oxidation state of the impurities is changed causing an increase

in deposition rate. The c/pt is an efficient electrode as compared to

pb/sb as pt is placed at the bottom of electrochemical series

causing more attraction of the elements toward it self as compared

to pb/sb electrode. Rate of rotation studies shows that 15 rpm

rotation rate is optimal for the deposition of more amount of

impurities below this rate more time is required for deposition and

above this rate less deposition takes due to the dispersion and

overlapping of the elements. Current vs potential studies show that

maximum oxidation of the impurities takes place at a current of 11

amperes where the sweep stop is obtained. Finally the

electrochemically treated water followed by biochemical treatment.

Results show that the C.V.+O.P. is the best Algal combination for

the maximum removal of impurities.

7. Hence electrochemical treatment with c/pt electrode of 3cm surface

area at a rotation rate of 15 RPM with H2SO4 as oxidizing agent

and a current of 11 amperes followed by biochemical treatment

with mix Algal species Chlorella Vulgaris and Oocystus Pusilla is

the best suggestion for the treatment of waste water.

146

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