Production of Clean Biofuel From Microalgae": Project Proposal Reference No.:39S - B - BE - 006

R.V.
COLLEGE OF ENGINEERING,BENGALURU-560059
(Autonomous Institution Affiliated to VTU, Belagavi)
PROJECT REPORT
“Production of Clean Biofuel from Microalgae”

Submitted in fulfilment for the 39th series SPP Biofuel project
Project Proposal Reference No. :39S_B_BE_006

Supported & funded by
Karnataka State Biofuel Development Board
Submitted By
VIDYASHREE S (USN: 1RV12BT057)

NAVYASHRI(USN: 1RV12BT033)
SHRAVANTHI S KUMAR(USN: 1RV12BT046)
Under the guidance of
Prof. Shivandappa
Asst. Professor
Department of Biotechnology
R.V. College of Engineering, Bengaluru
2016
Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006
R.V. COLLEGE OF ENGINEERING, BENGALURU-560059

DEPARTMENT OF BIOTECHNOLOGY
CERTIFICATE
This is to certify that the project work entitled “Production of Clean Biofuel
from Microalgae” was carried out byNavyashri (1RV12BT033),Shravanthi S
kumar (1RV12BT046) andVidyashree S (1RV12BT057), who are bonafide
students of R.V.College of Engineering, Bengaluru in fulfilment of Biofuel
project sponsored by Karnataka State Council for Science and Technology &
Karnataka State Biofuel Development Board, Bengaluru under 39th series of
Student Project Programme in the year 2015-2016.
Signature of GuideSignature ofHODSignature ofPrincipal

(Prof.Shivandappa) (Dr.Pushpa Agrawal)(Dr. K.N Subramanya)
Department of Biotechnology, RVCE, Bengaluru i

R.V. COLLEGE OF ENGINEERING, BENGALURU – 560059

DEPARTMENT OF BIOTECHNOLOGY
DECLARATION
We, Navyashri,Shravanthi S Kumar and Vidyashree S, students of Eighth

semester B.E.,Department of Biotechnology, R.V. College of Engineering,
Bengaluru-560059, bearing USN: 1RV12BT033, 1RV12BT046 and
1RV12BT057, hereby declare that the project titled “Production of Clean
Biofuel from Microalgae” has been carried outby us and submitted in fulfilment
of Biofuel project sponsored by Karnataka State Council for Science and
Technology & Karnataka State Biofuel Development Board, Bengaluru under
39th series of Student Project Programme in the year 2015-2016.
Further we declare that the content of the dissertation has not been submitted
previously by anybody .
We also declare that any Intellectual property rights generated out of this project
carried out at R.V.C.E. will be the property of R.V. College of Engineering,
Bengaluru and we will only be the authors of the same.
Place: Bengaluru Signature of the students
Date:1. Navyashri
2. Shravanthi S Kumar
3. Vidyashree S
Department of Biotechnology, RVCE, Bengaluru ii

ACKNOWLEDGEMENT
We take this opportunity to express our sincere gratitude to the people who have been
helpful in the successful completion of our project. We would like to show our
greatest appreciation to professors and staff members at the Department of
Biotechnology Engineering.
Our debt of gratitude must go to our guide Prof.Shivandappa for his valuable
suggestions and inspiring guidance throughout the course of this work. We would
also like to thank Dr.Pushpa Agrawal, Professor & Head, Dept. of Biotechnology
Engineering, Dr.K.N.Subramanya, Principal of R.V.C.E, for providing valuable
guidance and facilities to carry out the work. We are grateful to
Ms.Mahalakshmi,Ms.Puneetha&Ms.Smitha, project assistants for being with us,
helping inseveral ways throughout our project.
The study is funded by Karnataka State Biodiesel Development Board(KSBDB),

under 39th series of Student Project Programme. Hence, we would like to extend our
gratitude towards KSBDB, Govt. of Karnataka for the financial support.
Most importantly, we are extremely grateful to our parents for their encouragement,
quiet patience and love which has always helped us sail through obstacles and
complete the project successfully.
Department of Biotechnology, RVCE, Bengaluru iii

TABLE OF CONTENTS
ACKNOWLEDGEMENT………………………………………………………….iii
ABSTRACT………………………………………………………………………….iv
TABLE OF CONTENTS…………………………………………………………….v
ABBREVIATIONS………………………………………………………………...viii
LIST OF TABLES…………………………………………………………………...x
LIST OF FIGURES………………………………………………………………...xii
CHAPTER 1
INTRODUCTION
1.1 Biodiesel Overview………………………………………………………………..1
1.1.1 Advantages of Biodiesel…………………………………………………….2
1.2 Literature survey
1.2.1 Microalgae…………………………………………………………………..3
1.2.2 Algal Biodiesel………………………………………………………………3
1.2.3 Microalgae as source of Biodiesel…………………………………………..4
1.2.4Botryococcusbraunii.………………………………………………………...5
1.2.5 Trans-esterification………………………………………………………….6
1.2.6 Enzyme Trans-esterification………………………………………………...6
1.2.7Pseudomonas aeruginosa…………………………………………………….7
1.3 Problem outcome………………………………………………………………….7
1.4 Objectives………………………………………………………………………….9
1.5 Brief methodology………………………………………………………………...9
1.6 Organization of the report………………………………………………………..10
CHAPTER 2
THEORY AND FUNDAMENTALS
2.1 Motive underlying algae as the biodiesel feedstock……………………………..12
2.2 Principles underlying high oil yield of B.braunii...................................................12
2.3 Bacterial Lipase mediated Trans-esterification…………………………………..13
2.3.1 P.aeruginosa Lipase………………………………………………………..13
2.3.2 Function of P.aeruginosa lipase…………………………………………...14
2.3.3 Structure and catalytic mechanism…………………………………………….14
2.4 Feasibility of production of Biodiesel from algae……………………………….15
Department of Biotechnology, RVCE, Bengaluru iv

CHAPTER 3
MATERIALS AND METHODOLOGY
3.1 Materials used……………………………………………………………………16
3.2 Methodology……………………………………………………………………..17
3.2.1 Preparation of Lipase…………………………………………………………18
3.2.1.1 Serial dilution of soil sample and inoculation on LB agar medium……….18
3.2.1.2 Inoculation on to EMB agar medium……………………………………...19
3.2.1.3Inoculation on to Kings’ B agar medium…………………………………..20
3.2.1.4Inoculation on to Mac-Conkey agar medium……………………………...21
3.2.1.5 Screening of P.aeruginosa by Biochemical tests………………………….21
3.2.1.6 Biochemical screening of lipase by Spirit blue agar medium……………..23
3.2.1.7 Extraction and purification of Lipase……………………………………...24
3.2.1.8 Quantification of purified crude Lipase…………………………………...25
3.2.2 Culture and growth of algae…………………………………………………..26
3.2.2.1 Media Optimization……………………………………………………….27
3.2.2.2 Screening of B.braunii……………………………………………………..28
3.2.2.3 Algal oil extraction………………………………………………………...29
3.2.3 Comparative studies…………………………………………………………..29
3.2.4 Trans-esterification of Algal with lipase isolated from P.aeruginosa………...30
3.2.5 Testing of Fuel properties of Biodiesel……………………………………….30
CHAPTER 4
RESULTS AND DISCUSSION
4.1 Isolation and screening of P.aeruginosa…………………………………………33

4.1.1 Serial dilution of soil sample and inoculation on LB agar medium……………33
4.1.2 Selective media for gram-negative bacterial growth…………………………35
4.1.3 Selective media for Pseudomonas bacteria……………………………………35
4.1.4Differential media for P.aeruginosa…………………………………………...36
4.1.5 Screening of P.aeruginosaby biochemical assay……………………………..37
4.1.6 Screening of P.aeruginosaas lipase producer by Spirit blue agar……………40
4.1.7 Extraction and isolation of lipase……………………………………………..41
4.1.8 Purification of lipase by dialysis method……………………………………..42
4.2 Quantification of Lipase by Lowry’s method……………………………………43
Department of Biotechnology, RVCE, Bengaluru v

4.3 Culture and growth of B.braunii…………………………………………………44

4.3.1 Modified kuhls’ media………………………………………………………..45
4.3.2 Open pond culture…………………………………………………………….46
4.4 Screening of B.braunii……………………………………………………………48
4.5 Algal oil extraction……………………………………………………………….49
4.6 Comparative studies……………………………………………………………...52
4.6.1Effect of FFA content of oil in Chemical Trans-esterification………………..53
4.6.2 Effect of FFA content of oil in Lipase-mediated Trans-esterification………..54
4.7.Trans-esterification of Algal oil with lipase to produce Biodiesel………………56
4.8 Testing of Fuel properties of Biodiesel…………………………………………..57
CHAPTER 5
CONCLUSION AND FUTURE SCOPE OF WORK
5.1 Conclusion……………………………………………………………………….59
5.2 Future prospects………………………………………………………………….60
REFERENCES……………………………………………………………………….61
Department of Biotechnology, RVCE, Bengaluru vi

LIST OF ABBREVIATIONS
LB: Luria Bertani
EMB: Eosin methylene blue
MWCO: Molecular weight cut-off
ASTM: American Society of testing and materials
L ha-1 :Litre per hectare
ACC: Acetyl-CoA Carboxylase
CO2: Carbon dioxide
HPL: Human pancreatic Lipase
MVB: Multi-Vesicular bodies
KDa: Kilo Dalton
H2SO4: Hydrogen sulphate
NaOH: Sodium hydroxide
NaCl: Sodium chloride
µg/ml: Micro gram per mili litre
µl: Micro litre
µm: Micrometre
nm: Nano metre
K2HPO4: Potassium hydrogen phosphate
MgSO4: Magnesium sulphate
Department of Biotechnology, RVCE, Bengaluru vii

H2O2: Hydrogen peroxide
MR: Methyl red
EDTA: Ethylene di-ammine tetra acetic acid
PBS: Phosphate buffer solution
BSA: Bovine serum albumin
FC reagent: Folinciocalteau
OD: Optical density
KNO3: Potassium nitrate
ZnSO4:Zinc sulphate
Co(NO3)2:Cobalt nitrate
Na2.MoO4:Sodium molybdate
CuSO4: Copper sulphate
FeSO4: Ferrous sulphate
Rpm: Revolutions per minute
FFA: Free fatty acid
Department of Biotechnology, RVCE, Bengaluru viii

LIST OF TABLES
Sl. No TABLE NAME Page No.

1.1 Comparison of oil yield between different feedstock 5
1.2 Taxonomy of B.braunii 6
1.3 Comparison of feedstocks with oil yield and land required 7
1.4 Oil content of various microalgal species 8
3.1 Composition of Luria Bertani Agar Media 18
3.2 Composition of EMB agar medium 19
3.3 Composition of Kings’ B agar medium 20
3.4 Composition of Mac-conkey agar medium 21
3.5 Composition of Spirit Blue Agar 23
3.6 Lowry’s method for protein estimation of lipase 26
3.7 Kuhls’ media Composition - Basal media 27
3.8 Kuhls’ media Composition - Micronutrient solution 27
3.9 ASTM range of Biodiesel 30
4.1 Biochemical assay 37
4.2 Lowry’s method of Protein estimation 43
4.3 Algal Oil yield 51
Yield of Biodiesel from different edible oils based on
4.4 53
theirFFA content by Chemical Trans-esterification
Yield of Biodiesel from different edible oils by Lipase
4.5 54
mediatedTrans-esterification
4.6 Yield of Algal Biodiesel 57
Fuel properties of Biodiesel produced through Chemical
4.7 58
Trans-esterification
Fuel properties of Biodiesel produced through Lipase
4.8 58
mediated Trans-esterification
Department of Biotechnology, RVCE, Bengaluru ix

LIST OF FIGURES
Sl. No. FIGURE NAME Page No.

1.1 Trans-esterification of oils to produce Biodiesel 2
1.2 Life cycle of algae 5
1.3 Percentage of Land area for different feedstocks 8
Transformation of lipid bodies and vacuoles during the cell
2.1 13
cycle of B.braunii
2.2 3D structure of Lipase produced by Paseudomonas 14
3.1 Workflow of methodology 17
3.2 Technique of Serial Dilution 19
3.3 Light microscopic observation of B.braunii at 100X 28
3.4 Cannon-Fenske Capillary viscometer tube 31
3.5 Penske-martens flash point apparatus 32
Serial dilution and inoculation of soil and water samples on
4.1 34
LB agar media
Single colony in third dilution of water (A) and colonies in
4.2 34
fourth dilution of soil sample(B)
Colonies in third dilution of soil (A) and light growth of
4.3 35
colonies in fourth dilution of water (B)
4.4 Colonies observed in third dilution of soil sample 36
4.5 Confluent colonies on Mac-conkey agar medium 37
4.6(A) Oxidase test - Purple color development 38
4.6(B) Catalase test - Evolution of O2 bubbles 38
4.6(C) Methyl red test - No colour change of isolate 39
4.6(D) Urease test- No colour change of the isolate 39
4.7 Spirit blue agar plate kept un-inoculated 40
Colony of P.aeruginosa has produced a zone of hydrolysis

4.8 (color change from pale blue to colorless) indicating the 40
production of lipase.
Department of Biotechnology, RVCE, Bengaluru x

4.9 100ml King’s Broth culture after 72h incubation 41

4.10 Saturated ammonium sulphate and stored for 24h at -20°C 41
4.11 Pellet re-suspended in phosphate buffer to produce enzyme 42
4.12 Dialysis in cold condition 42
4.13 Lipase concentrated in dialysis bag. 42
4.14 Purified crude Lipase 42
Plot of absorbance v/s concentration of standard BSA
4.15 44
andpurified crude lipase sample
Water sample containing algae traces collected
4.16 44
fromUlsoorlake, Bengaluru
Kuhls’ media(1X & 2X) inoculated with third and fourth
4.17 dilution of algae samples (A); Growth not observed even after 45
7 days of incubation(B)
4.18 Growth in modified Kuhls’ media after 7 days 46
4.19 Aquarium set up 46
4.20 Inoculated with 30ml of Ulsoor lake water sample 47
4.21 Growth of B.brauniiafter 5 days 47
4.22 Growth ofB.brauniiafter 7 days 47
Morphological features of the pure culture of
4.23 48
B.brauniiobserved under 40X(A) and 10X(B) magnification
Morphological features of the lab grown algae
4.24 observedthrough compound microscope under 10X 48
magnification
4.25 Algal mat produced after 4.5 weeks 49
4.26 Dried algal mass 49
4.27 Finely powdered algal mass 49
Algal oil extraction by Hexane:Petroleum ether
4.28 50
solventextraction
4.29 Algal oil extraction by soxhlet apparatus at 70°C 50
4.30 Solvent layer containing algal oil 51
Chemical method – Formation of 2 phases. Top phase
4.31 52
isBiodiesel
Department of Biotechnology, RVCE, Bengaluru xi

4.32 Microbial Trans-esterification – Formation of 3 clear layers 52
Impact of FFA content of oil on biodiesel yield by chemical

4.33 53
method
Impact of FFA content on oil on biodiesel yield by enzymatic

4.34 55
method
Comparison of yield via enzyme method and chemical

4.35 method of Trans-esterification of different oils with high FFA 56
content
4.36 Biodiesel from algal oil 57
4.37 Lipase enzyme recovered 58
Screening of trans-esterification; Pink colour fades in

4.38 58
Standard Petroleum diesel and Algal Biodiesel produced.
Department of Biotechnology, RVCE, Bengaluru xii

Production of Clean Biofuel from MicroalgaePPRN: 39S_B_BE_006
ABSTRACT
Increasing demand in transport fuel will eventually lead to complete depletion of

fossil fuel and can have severe consequences on human life which has led to
discovery of many alternative energy sources. Jatropa, Pongamia plants along with
few species of Microalgae are being considered as a resource for alternative fuels.
Considering these factors, present work concentrated on production of Biodiesel from
the Microalgae by Lipase mediated trans-esterification process.
Lipase was extracted from the cultures of P.aeruginosa and purified by dialysis
usinga membrane of MWCO-20kDa. The purified crude lipase was found to have
protein concentration of approximately 158 µg/ml. Algal oil obtained from dried
B.braunii algal mat was extracted using Soxhlet apparatus. The final concentrated
Algal oil was separated using separating funnel; yield of algal oil was found to be
approximately 49% of its dried biomass. The extracted algal oil was subjected to
lipase mediated trans-esterification for 6 hours at 35°C-40°C and the yield of
biodiesel obtained from algal oil was approximately 64.32%.
In chemical trans-esterification of pongamia oil with FFA content of 20% & 4%
produced biodiesel yield of 36% & 90% respectively; Groundnut oil with FFA
content of 27.6% & 3.96% produced biodiesel yield of 20% & 85% respectively and
rice bran oil with FFA content of 36% & 9.78% produced biodiesel yield of 16% &
45% respectively while microbial trans-esterification on same oils produced biodiesel
yield of 73.2% for Pongamia oil, 50% for Groundnut oil and 48% for Rice bran oil.
The results indicatethat Fatty Acid content of the feedstock oil does not affect yield of
Biodiesel from microbial trans-esterification which will reduce the two-step FFA
reduction pre-treatment process which is necessary in conventional chemical method.
th
In enzyme trans-esterification, biocatalyst required is approximately 1/5,000 of oil
quantity which is negligible, it is recyclable, waste generation is extremely low and
non-toxic to the environment unlike conventional chemical method. Hence, it can be
suggested that microbial trans-esterification is more beneficial than chemical trans-
esterification process. The positive results paves way for future research work to
focus on Optimization of conditions for Lipase production from P.aeruginosa,
Estimation of optimum quantity of Lipase required for the trans-esterification process
and evaluate experimentally.
Department of Biotechnology, RVCE, Bengaluru 1

CHAPTER 1
INTRODUCTION
Biofuels are a wide range of fuels which are derived from biomass. The term
covers solid biomass, liquid fuels and various biogases.Biofuels are gaining increased
public and scientific attention, driven by factors such as oil price hikes and the need
for increased energy security. Bio-ethanol is an alcohol made by fermenting the sugar
components of plant materials and it is made mostly from sugar and starch crops.
With advanced technology being developed, cellulosic biomass, such as trees and
grasses, are also used as feedstocks for ethanol production. Ethanol can be used as a
fuel for vehicles in its pure form, but it is usually used as a gasoline additive to
increase octane and improve vehicle emissions. Bio-ethanol is widely used in the
USA and in Brazil. Biodiesel is made from vegetable oils, animal fats or recycled
greases. Biodiesel can be used as a fuel for vehicles in its pure form, but it is usually
used as a diesel additive to reduce levels of particulates, carbon monoxide, and
hydrocarbons from diesel-powered vehicles. Biodiesel is produced from oils or fats
usingtrans-esterification and is the most common biofuel in Europe. Biofuels provides
1.8% of the world's transport fuel. Investment into biofuels production capacity
exceeded $4 billion worldwide in 2015 and is growing.
1.1 Overview: Biodiesel

Biodiesel is the fuel which is produced from different feedstock like plant based oils
or animal fat or grease. It is a clean burning and renewable fuel. It is currently used in
blended forms with petroleum diesel in diesel engines. Even a 20% blend of biodiesel
with 80% petroleum diesel reduces emissions significantly and helps the environment.
Diesel engines were designed earlier to run on different kind of fuels like kerosene,
coal dust etc because of its complex injection systems. Due to its high energy content
is bound to be discovered as a sustainable energy source. Biodiesel was used when a
diesel engine was run on peanut oil in 1900 world‘s fair by Otto Company
commissioned by the French government. In 1970‘s during the world war Biodiesel
production saw interest as there was a shortage of conventional fuels. Due to its
numerous advantages it is becoming one the fast growing industry for alternative
fuels[1].

Biodiesel is fuel containing long chain alkyl esters (methyl, ethyl or propyl). Biodiesel
is produced by treating oils or animal fat feedstock with alcohol to produce fatty acid
methyl esters as shown in figure 1.1.
Fig1.1:Trans-esterification of oils to produce biodiesel[1].
1.1.1 Advantages of Biodiesel
Biodiesel can be used in pure form (B100) or can be blended with petro-diesel in the
form of B2 (2% biodiesel, 98% petroleum diesel), B5 (5% biodiesel, 95% petroleum
diesel), B20 (20% biodiesel, 80% petroleum diesel) and B100 (pure biodiesel).
Biodiesel has helped several countries in reducing their dependence on foreign oil
reserves as it is domestically produced and can be used in any diesel engine with little
or no modification to the engine or the fuel system. The advantages of Biodiesel are
as follows
1. Biodiesel is clean burning fuel and has no carcinogenic emissions and gases
which cause global warming.
2. Biodiesel has very low toxicity compared to petroleum biodiesel as it is carbon

neutral.
3. Biodiesel is biodegradable.
4. Feedstock is grown, produced and distributed locally hence reduces cost and
improves economy.
5. Glycerine is produced as by product and can be used in other industries.
6. Biodiesel has a higher flashpoint compared to petroleum diesel.

7. Biodiesel has higher cetane number compared to petroleum diesel hence it has
low idle noise and easy cold start.
1.2 Literature survey
To meet the growing fuel requirements,alternative fuels have been extensively

researched. One of the most recent break through has been biodiesel production from
microalgae. To produce biodiesel from algal oil different microalgae species were
reviewed. The conventional biodiesel production utilizes chemical catalyst in trans-
esterification which poses many drawbacks. Hence, recent research has been focused
on alternative eco-friendly catalyst like enzymes which has been reviewed in the
literature survey.
1.2.1 Microalgae
Microalgae are microscopic photosynthetic organisms that are found in both marine
and fresh water environments. Microalgae are organisms which efficiently convert
solarenergy into biomass.Micro algalfuel has high calorific value, low density and
viscosity and hence makes it a better feedstock than plant based feedstock .The
distinct characteristic of algae is that there are a number of species of algae which can
be used and optimized to produce bio fuels of different characteristics[2-3].
1.2.2 Algal Biodiesel
Algae Biodiesel is an alternative to liquid fossil fuels that uses algae as its source of
energy-rich oils. Also, algae fuels are an alternative to common known biofuel
sources, such as corn and sugarcane. Studies have shown that some species of algae
can produce 60% or more of their dry weight in the form of oil. Because the cells
grow in aqueous suspension, where they have more efficient access to water, CO2 and
dissolved nutrients, microalgae are capable of producing large amounts of biomass
and usable oil in either high rate algal ponds or photo-bioreactors.
Use of algae as feedstock has the following advantages:
1. High photosynthetic efficiency.
2. High production of biomass.

3. Faster growth rate than plant seed feedstock.
4. High CO2 fixation and O2 production.
5. Algae can be harvested throughout the year.
6. Oil yield is very high compared to plant seed feedstock.
7. After oil extraction the biomass can be used as feed for livestock as it is rich in
protein.
8. Algal oil production uses resources which would otherwise be not useful.
9. Relatively harmless to environment, if spilled.
Due to these advantages, in this present work algae was chosen for biodiesel
production [4-5].
1.2.3. Microalgae as a source of Biodiesel
One important way to address dwindling energy reserves is by accessing biomass

fuels. Algal biomass fuels are made by processes similar to fossil fuels where organic
material is converted to oil but the difference is that biomass fuel takes days to
produce compared to millions of years in case of millions of years. The life cycle of
algal biomass is shown in figure 1.2.
Biodiesel can be produced by any organic sources of oil like waste oil, animal fat and
seed oils. Waste oils supply is limited hence it is not a feasible feedstock for biodiesel
production.Using seed oil as feedstock reduces the seeds for food supply hence it is
not viable.There is a need for a feedstock source which is abundant as well as a
productive source of feedstock.The oil yield by different feedstock is shown in Table
1.1.The table shows that microalgae has the highest oil yield, hence it was considered
as feedstock for this present work.
Microalgae are photosynthetic, unicellular and aquatic.Microalgae have high growth

rate as well as population density.Microalgae also have high (>50%) lipid content
Microalgae has very simple media requirement for its growth.Hence it is a very
goodsource of feedstock for biodiesel production.Microalgae have immense potential

to be used as a source of sustainable fuels hence there is vast research and

development in the use to microalgae for biodiesel production [6-8].
Fig1.2: Life cycle of algae [7].
Table 1.1: Comparison of oil yield between different feedstock.
CROP OIL YEILD (L ha-1)
Soybean 446
Canola 1,190
Jatropha 1,892
Palm 5,950
Microalgae 136,900
1.2.4 Botryococcusbraunii
Botryococcusbraunii belongs to kingdom plantae and divisionchlorophyta. The

taxonomy of B.braunii is show in Table 1.2.B.brauniiis a microalgae which is rich in
hydrocarbons(>80%dry weight of cell) and is a potential source for biodiesel hence it
is researched widely. The oil present in B.braunii accumulates in the outer walls of
the cells and can be extracted by dewatering and using non polar solvents like
ammonium sulphate. The growth of B.Braunii requires light which helps in
production of biomass and CO2 fixation.B.braunii also requires macro and
micronutrients for its growth.

Lipid accumulation occurs in B.braunii during stress conditions like environmental

stress.Hence, the lipid content can be enhanced by nitrogen starvation. Acetyl-CoA
Carboxylase (ACCase), involved in fatty acid biosynthesis is involved in process of
lipid accumulation.It is possible to increase lipid accumulation by genetic
manipulation [9].
Table 1.2: Taxonomy of B.braunii[9]
Taxonomy of Botryococcusbraunii
Kingdom Plantae
Division Chlorophyta
Class Chlorophyceae
Order Chlorococcales
Family Dictyosphariaceae
Genus Botryococcus
Species B.barunni
1.2.5 Trans-esterification
Trans-esterification is thechemical reaction in which oils or animal fats are reacted

with alcohols (methanol/ethanol) in the presence of catalyst to produce methyl esters
of long chain fatty acid i.e., Biodiesel. Trans-esterification by chemical methods is
widely used as itis high conversion rate and low time of production.However,
chemical method has drawbacks like energy intensive process, harmful for the
environment and complex recovery of catalyst, product.
1.2.6 Enzyme Trans-esterification
It is the process of conversion of triglycerides into methyl esters in the presence of

enzymes as a catalyst. Enzyme trans-esterification has advantages like (a)Temperature
conditions close to room temperature (b)Process of recovery of catalyst, product
separation and waste water treatment is eliminated. (c)Environment friendly and

biodegradable [10]. Commonly used enzyme for trans-esterification is lipase.

Pseudomonas sp. produces extracellular lipase which can be extracted conveniently as
Pseudomonas is widely researched and validated microbe Lipases are group of
enzymes that hydrolyse oils and fats in biological systems. Most species of animals,
plants,fungi,bacteria produce lipase.Lipase from P.aeruginosa has been most
researched and widely used hence lipase was extracted fromP.aeruginosa in this
present work.
1.2.7Pseudomonas aeruginosa
It is a common Gram-negative bacterium. It is citrate, catalase, and oxidase positive.

It is found in soil, water, skin flora, and most man-made environments throughout the
world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres,
and has, thus, colonized many natural and artificial environments. It uses a wide range
of organic material for food. Due to its versatile growth condition at extreme levels,
wide spread availability and relatively less pathogenicity, it was used as the source of
lipase extraction [12].
1.2 Problem Outcome
There is currently a lot of research to produce biodiesel from different feedstocks to

get better oil content as well as yield of Biodiesel. Vast land area is required usually
to produce feedstock. The comparison between differentfeedstocks with oil yield and
land required is shown in Table 1.3.As observed in the table 1.3 algae has the highest
oil yield and least land requirement hence in the present work algae was considered as
oil feedstock.
Table1.3: Comparison of feed-stocks with oil yield and land required [13].
Crop Oil in Litres per hectare
Jatropa 3400
Castor 1413
Sunflower 952
Safflower 779
Palm 5950

Soy 446
Coconut 2689
Algae 100000
Table1.4: Oil content of various microalgal species [14]

MICROALGA OIL CONTENT
Botryococcusbraunii 25-75
Chlorella sp 28-32
Crypthecodiniumcohnii 20
Cylindrotheca sp. 16-37
Dunaliellaprimolecta 23
Isochrysis sp. 25-33
Monallanthussalina 20
Nannochloris sp. 20-35
Neochlorisoleoabundans 35-54
Fig 1.3: Percentage of Land area for different feedstocks[15].
It is seen that the land required for algae growth is least compared to other feedstock
as shown in Fig 1.3.From the table1.4 it is observed that B. braunii has the highest oil
yield compared to the other microalgae species. Hence in the present work B. braunii
has been used to extract Algal oil.
There are many drawbacks of chemical method of trans-esterification which are

overcome by enzyme trans-esterification hence in this present work we have

compared chemical and microbial trans-esterification of various oils to demonstrate

the feasibility of enzyme trans-esterification over chemical method.
1.4 Objective
As discussed above, to overcome the problems following objectives were chosen for
the present work
1. To isolate P.aeruginosafrom Soil & water
2. To isolate and culture B. braunii
3. To extract and purify the Lipase from P.aeruginosa
4. To extract the algal oil from dried algal mat
5. Microbial trans-esterification of algal oil to produce Biodiesel
1.5 Brief methodology
1) To isolate P.aeruginosafrom Soil &water:P.aeruginosawas isolated by serial

diluting 1gm of soil in distilled water and sample from all the dilutions were plated
onto equal number of petriplates containing LB Agar and incubated overnight. The
colonies obtained in LB Plates were sub-cultured by inoculating colonies onto
EMB agar and kept for overnight incubation. Resultant colonies in EMB plates
were then plated onto petriplates containing Mac-Conkey medium and incubated
overnight. For selective isolation of lipolytic bacteria P.aeruginosa, colonies from
Mac-Conkey plate were then transferred into King‘s B media, which is specific to
lipolytic bacteria and incubated for 24 hours. After the incubation, Biochemical
and spirit blue test were conducted for screening of P.aeruginosa.
2) To isolate and culture B. braunii: After following standard operating procedure,

sample was collected from Ulsoor lake and inoculated into modified kuhl‘s
medium for the growth of B.braunii and kept in tissue culture rack for about 2-
3weeks. Microalgae obtained after 2 weeks of incubation was screened by
microscopic observation. At the end of the 3rd week, fresh medium was added and
continued incubation with aerator and proper light and dark photoperiod. The
culture was then used to extract oil for the production of Biodiesel.

3) To extract and purify the Lipase from P.aeruginosa: Colonies from Mac-
Conkey plate were transferred onto King‘s B broth and incubated overnight, then
broth culture was centrifuged and the supernatant obtained was subjected to
purification by ammonium sulphate precipitation. After precipitation, reaction
mixture was again subjected to centrifugation, pellet was then suspended in
phosphate buffer and dialysis was carried out to obtain crude enzyme extract.
4) To extract the algal oil from dried algal mat: The algal mat obtained in the
aquarium set up was dried and grounded to fine powder form. It was then subjected
for hexane mediated solvent extraction and finally algal oil extraction was done by
using soxhlet apparatus. Later extracted algal oil was used in the process of
biodiesel production.
5) Microbial trans-esterification of algal oil to produce Biodiesel: The algal oil

was subjected to enzyme mediated trans-esterification using the lipase as catalyst
to obtain algal biodiesel. Resulting biodiesel was used for quality studies, the
qualitative parameters studied for the algal biodiesel were reported in result
section.
1.6 Organisation of Report
The report is organized into 5 different chapters. Each chapter begins with a
preamble giving brief introduction of the chapter.
Chapter 1:Introduction- This chapter contains history of origin of biodiesel and brief
introduction to biodiesel trans-esterification. The advantages of biodiesel over
conventional fuels are stated. There is a detailed literature survey on the recent
developments in algal biodiesel and the problem outcomes.
Chapter 2:Theory and Fundamentals - This chapter contains the underlying

principles, motives, science and background of the proposed research work
Chapter 3: Materials and Methodology - The raw materials utilized for the present
work and workflow of the methods followed to meet the objectives are discussed in
this chapter

Chapter 4:Results and discussion- The observations of the experiments conducted in

present work is represented in pictures, tables and graphs. The inferences that can
drawn from the results are discussed.
Chapter 5: Conclusion and future prospects - The conclusions we can draw from
this present work is stated and the advances that can be made further are discussed.

CHAPTER 2
THEORY AND FUNDAMENTALS
Biodiesel from algae is the upcoming third generation renewable fuel. Enzyme trans-
esterification process of producing Biodiesel is proven to be more favourable than
conventional chemical method. This chapter gives detailed explanation of concept
behind B.braunii being used as high oil yield source and Lipase action as catalyst in
trans-esterification.
2.1 Motive underlying algae as biodiesel feedstock
The literature survey shows that microalgae can be used as a good source of vegetable
oil; a recent study suggests that the algae produces and store more oil in their cells.
Fertilizers which are abundant in phosphorous and nitrogen can be obtained by
algae.Algal farms help in recycling nutrient by using nutrient from waste sources like
human sewage,animalwaste,farm wastes.
Biodiesel, Bio-ethanol, Bio-gasoline, Bio-methanol,Bio-butanol etc., are the different

products that can be formed by algal cultivation by using land not suitable for
farming.
2.2 Principle underlying high oil yield of Botryococcusbraunii

B.braunii is noted for the production high amount of hydrocarbons especially in the
form of Triterpenes.Figure 2.1 shows the cell cycle of B.braunii during which there is
transformation of lipid bodies and vacuoles.The first part of figure 2.1 shows the
B.braunii stages of growth. The second and third part of figure 2.1shows the lipid
bodies and vacuole transformation. Because B.braunii produces large mass of lipid
bodies which is present outside the cell, oil extraction from it is more favourable
which adds to the total oil yield. It serves as the promising species to be chosen for the
high oil yield which can be converted to biofuels [15].

Fig 2.1: Transformation of lipid bodies and vacuoles during the cell cycle of
B.braunii [15].
2.3 Bacterial Lipase mediated Trans-esterification
From literature survey related to Lipases, we have been able to realize that bacterial
lipases are more resistant to the methanol used in trans-esterification; more
specifically lipases from P.aeruginosa species are sturdy to the methanol action and
can withstand the reaction temperature up to 45°C.
2.3.1 P. aeruginosa Lipase
Thermo-stable lipases and proteases are produced by Pseudomonas aeruginosa and

other similar pseudomonads. P.aeruginosais an obligate aerobe but certain strains are
capable of using nitrate instead of oxygen as a final electron acceptor during cellular
respiration. It has a very versatile metabolism and can be found in the soil, plants and
in water. It is well adapted to the soil where it was first isolated in agricultural soil. It
is non-pathogenic and the since the lipase is produced extracellularly; extraction of
lipase is also easier. Hence, we chose Pseudomonas aeruginosaspeciesto extract
lipase[16-18].

2.3.2 Function of P. aeruginosa Lipases
Lipases are a part of class of enzymes esterases which initiates hydrolysis of ester
bonds in lipid substrates and it is a water soluble enzyme. Lipases are the most
important factor required for digestion, processing and transport of lipids. Few viruses
have genes which codes for lipases.
Lipases act usually on particular positions like glycerol backbones of lipids. For
example,In case of human pancreatic lipase (HPL), which help in the breakdown of
fats and help digestion, trans-esterifies ingested oils to monoglycerides and free fatty
acids.There are other lipases like phospholipases and sphingomyelinases which are
not ―conventional‖ lipases as their activity type differs.
2.3.3 Structure and catalytic mechanism of P.aeruginosa lipase
Lipase enzymes are found in different forms having several types of protein folds and
mechanism if catalysis.Most of the enzymes are built on hydrolase fold and use a
chymotrypsin like hydrolysis which involves a histidine, serine nucleophile, and an
acidresidue [20].The protein structure of lipase is shown in figure 2.2.
Fig 2.2: Computer generated picture showing 3D Structure of Lipase produced by

Pseudomonas [20].
1. Lipases are water soluble and play an important role in fat digestion. They are
acyl hydrolases enzymes which act by cleaving long-chain triglycerides into
polar lipids. Because of an opposite polarity between the enzyme and their

substrates, the reaction occurs at the interface between the two phases which is
oil and aqueous phase.
2. Lipase which is extracted from Pseudomonas can be used as good catalyst in

various organic reactions. Although many Gram-positive and Gram-negative
bacteria produce lipases, the most widely accepted and researched is the lipase
from Pseudomonas. The lipases from pseudomonas are classified into 3
categories depending on the homology of amino acid sequence
3. Group I: lipases from P.aeruginosa, P.alcaligenes, and P.fragi
4. Group II: lipases from P.cepaciaand P.glumae
5. Group III: lipases from P. Fluorescens
Lipases from group III is secreted by a different mechanism hence is different from
group I and II.Lipase is extracellular produced by P.aeruginosa.It has a molecular
weight of 25.5 kDa. The intracellular lipase produced by Pseudomonas species has a
molecular weights of 35.5, 49 and 70 kDa [21].
Extracellular lipase produced by P. aeruginosa is of molecular weight of 25.5 kDa,

whereas the intracellular lipase produced by other Pseudomonas species showed the
presence of three fractions with molecular weights of 35.5, 49 and 70 kDa[21].
2.4 Feasibility of production of Biodiesel by algae
Biodiesel produced by algalculture is a feasible solution to replace conventional fuels

like petroleum diesel Other feedstock do not have oil yield high enough to produce
large volumes of oil from feedstock hence would require a larger amount of feedstock
or very large percentage of land to produce the feedstock and land has to be used to
produce feedstock instead of food crops which is not feasible.
In practice, biodiesel has not yet been produced on large scale from algalculture,
though large biodiesel production from algae will see a likely rise in the near future
(4-5 years) [22].

CHAPTER 3
MATERIALS AND METHODOLOGY
As discussed in Chapter 2 & 3, after a clear understanding of the theoretical

background and considering all the factors in the production of biodiesel from algae,
we decided on the use of materials and methodology as shown in section 3.1 and 3.2.
3.1 Materials used
The materials used in the present work are summarized and provided in the following
section under different headings.
1. Media: LB Agar (Hi-media laboratories), EMB Agar(Hi-media laboratories),

Spirit Blue agar(Hi-media laboratories), King‘s B Agar(Hi-media laboratories) for
culturing, isolating and screeningof P.aeruginosaand Lipase. Kuhls‘ Basal Media
for culturing B.braunii.
2. Sample collection for algae: water sample containing algae traces was collected
from Ulsoorlake, Bengaluru
3. Culture and growth of B.braunii: Aquarium of 8 liters capacity with aerator was
procured from local stores, Gulmarg Aquarium, vijayanagar, Bengaluru.
4. Sample collection for P.aeruginosa: Soil sample was procured from Football
ground, RVCE and tap water was procured from biotechnology laboratory, RVCE.
Standard culture was procured from Radiant Research Lab Pvt. Ltd, Peenya,
Bengaluru.
5. Experimental setup for Extraction, Purification of Lipase and Trans-

esterification:Remi Refrigeration Centrifuge,Simtronics- Hot Water bath,
RemiBacterial Incubator and 0.5 µm pore size dialysis membrane bag with
MWCO-20kDa
6. Algal Oil Extraction: Closed type Soxhlet extraction apparatus. 4:1 Hexane:
Petroleum Ether (v/v) solvent.
7. Trans-esterification: RankemH2SO4,Methanol, NaOHpellets (fisher), Lipase

enzyme extracted, water bath, phenolpthalein indicator.
8. Oils for comparative studies: Pongamia oil, Groundnut oil and Rice bran oil were
procured from Biodiesel Lab, RVCE.

9. Testing of Biodiesel properties: ASTM D 445 Cannon-Fenske Capillary

Viscometer Tube, ASTM D 93 Pensky-Martens Flashpoint Apparatus, ASTM D –
664 Potientiometric Titration setup
3.2 Methodology
To meet the objectives stated in chapter 1,converting algal oil to biodiesel by

microbial enzyme trans-esterification requires the lipase from the microbe and algal
oil from the algae specie, B.bruanii, so it is necessary to isolate and purify the lipase
from the microbial source as well parallel culture and grow the algae to extract algal
oil for the biodiesel production as shown in figure 3.1.
Preparation of Lipase Preparation of Algal Oil
Culture& isolation of Media optimization & culture of

P.aeruginosa B.braunii
(3 weeks)
Screening of P.aeruginosa Growth of B.braunii mat
Extraction, Isolation and Extraction of Algal oil from dried

screening of Lipase algal mat
(2 weeks)
Purification of Lipase Purification of Algal oil
Microbial Trans-esterification
Screening of Trans-esterification
Qualitative tests of Biodiesel properties
Fig3.1: Workflow of methodology

3.2.1 Preparation of Lipase
P.aeruginosais found in soil, on plant surfaces and in water. The soil sample and
water sample was chosen for the isolation of P.aeruginosa.Soil sample was serially
diluted in distilled water and inoculated onto L B Agar media and incubated at 25 °C
for 24 hours.1ml of tap water sample was directly inoculated onto 5 petriplates of LB
Agar media and incubated at 25 °C for 24 hours.[23]. The colonies observed were
sub-cultured onto differential media for gram-negative bacteria i.e., EMB agar
medium followed by specific media for lypolytic organisms, King‘s B agar medium
and differential media for P.aeruginosa and P.fluorescens and same was confirmed
using biochemical tests. Biochemical screening of the isolated P.aeruginosa for
Lipase production was done by spirit blue agar medium. Eventually, Lipase was
isolated and purified by dialysis. The detailed methodology for the preparation of
lipase is explained in subsequent sections.
3.2.1.1 Serial Dilution of Soil Sample and inoculation onto LB agar medium
Serial dilution is the stepwise dilution of a substance in solutionas shown in Fig 3.2.
Usually the dilution factor at each step is constant, resulting in a geometric
progression of the concentration in a logarithmic fashion. It is an effective way of
obtaining pure cultures.In this case the bacteria present in soil get diluted and hence
decrease in number. The last dilution will have minimum number of microbes and
hence the colonies produced by these inoculums will have number of distinct colonies
with high resolution as compared to the first dilution.1g of soil sample was dissolved
in 10 ml of water, 1ml of this solution is diluted with 9ml water. This process is
carried out up to 10 dilutions. Each of the 10 dilutions are spread plated onto LB agar
plate.The composition of LB agar is shown in table 3.1.
Table 3.1: Composition of Luria Bertani Agar Media

COMPONENT QUANTITY
Tryptone 10.0 g
Yeast extract 5.0 g
NaCl 10.0 g
Agar 15.0 g
Distilled water 1000 ml
pH 7.5 ± 0.5 at 25°C

Fig 3.2:Technique of Serial Dilution
3.2.1.2 Inoculation onto EMB Agar
EMB agar is a differential media for gram negative bacteria and lactose fermenting
bacteria. After thegrowth of colonies on LB agar media, aloopful of culture from LB
agar medium was streaked onto EMB agar medium and incubated for 24h at 25ºC
Table 3.2: Composition of EMB agar medium

COMPONENT QUANTITY
Pancreatic Digest of
10.0 g
Gelatin
Lactose 10.0 g
Dipotassium Phosphate 2.0 g
Methylene Blue 65.0 mg
Eosin Y 0.4 g
Agar 15.0 g
pH 7.2 ± 0.2 at 25°C

The dyes eosin and methyleneblue help in changing color from red to black in case of
bacteria that can break down the lactose sugar (carbon source) present in the media as
shown in table 3.2 Lactose fermenters are blue-black; non-fermenters are colourless
or light purple.
Our bacteria P.aeruginosais gram negative cannot utilize sugar as the carbon source
and requires a lipid as carbon source. Hence their growth can be seen as colourless
colonies in EMB agar.
3.2.1.3 Inoculation onto King’s B media:
King‘s B media, also known as PseudomonasAgar F is the selective media for

Pseudomonas bacteria.The composition of EMB agar is shown in Fig 3.2.The
colorless colonies observed on EMB agar medium has gram-negative bacteria. A
loopful of colonies from EMB agar medium was streaked onto King‘s B agar medium
and incubated for 24h at 25ºC.
Table 3.3:Composition of Kings‘ B agar medium

COMPONENT QUANTITY
Protease peptone 20.0 g
Corn oil 10 ml
K2HPO4 1.5 g
MgSO4 .7H2O 1.5 g
Agar 15.0 g
pH 7.2 ± 0.2 at 25°C
King‘s Agar B enhances the elaboration of the pyocyanin formation; essential for
aeruginosa species. The composition of the media is shown in table 3.3. Peptone
provides the essential nitrogenous nutrients, carbon, sulphur and trace elements. Corn
oil serves as lipid source and Dipotassium hydrogen phosphate buffers the medium.

3.2.1.4 Inoculation onto Mac-conkey agar medium :
Mac-Conkey agar media serves as the differential media for aeruginosa and
fluorescens species of Pseudomonas. A loop full of colonies from King‘s B agar
medium was streaked onto Mac-Conkey agar medium and incubated for 24h at 25ºC.
Table 3.4: Composition of Mac-conkey agar medium

COMPONENT QUANTITY
17.0 g
Gelatin
Proteose peptone (meat and
3.0 g
casein)
Lactose monohydrate 10.0 g
Bile salts 1.5.0 g
Sodium chloride 5g
Neutral red 0.03g
Crystal Violet 0.001g
Agar 13.5.0 g
pH 7.1 +/- 0.2 at 25°C
.aeruginosa gives the confluent growth of greenish-brown colonies which doesn‘t

fluoresce unlike the P.fluorescens specie. The composition of Mac-Conkey agar
medium is shown in Table 3.4
3.2.1.5 Screening of P.aeruginosa by Biochemical Tests
The bacterial colonies isolated fromspecific media was utilized in the biochemical
tests specific for P.aeruginosa. These tests will confirm that isolated colonies are pure
form of P.aeruginosa species. From literature survey, it was observed that

P.aeruginosashows positive results for Catalase and Oxidase test and, negative results
for Urease and Methyl-Red test.
1. Catalase test : Positive
Catalase is an enzyme, which is produced by microorganisms that live in oxygenated

environments to neutralize toxic forms of oxygen metabolites; H2O2. The catalase
enzyme neutralizes the bactericidal effects of hydrogen peroxide and protects them.
Catalase mediates the breakdown of hydrogen peroxide H2O2 into oxygen and water.
The presence of oxygen bubbles indicates the production of Catalase enzyme by the
bacteria.Small inoculums of isolate was mixed into hydrogen peroxide solution (3%)
taken on glass slide and the rapid elaboration of oxygen bubbles were seen.
2. Oxidase test : Positive
The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an
enzyme of the bacterial electron transport chain. When present, the cytochrome c
oxidase oxidizes the reagent to(indophenols) purple color end product. When the
enzyme is not present, the reagent remains reduced and is colourless.Small inoculums
of isolate was smeared on to the filter paper soaked with the reagent (tetramethyl-p-
phenylenediamine). Purple colour was observed within 15-20 seconds.
3. Urease test : Negative
The urease test is used to determine the ability of an organism to split urea,
through the production of the enzyme urease. Urea is the product of decarboxylation
of amino acids. Hydrolysis of urea produces ammonia andCO2. The formation
of ammonia alkalinizes the medium, and the pH shift is detected by the color change
of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1.
Urea agar slant of the composition shown in table 3.5 was prepared. Small inoculums
of the isolate was streaked onto the urease agar slants. Colour change was not
observed in the slants afterincubation at 37ºC for 48h.
4. Methyl-red test : Negative

Methyl Red (MR) test determines whether the microbe performs mixed acids
fermentation when supplied glucose. The bacteria initially metabolise glucose to
pyruvic acid, which is further metabolized through the mixed acid pathway to produce
the stable acid. The type of acid produced differs from species to species and depends
on the specific enzymatic pathways present in the bacteria. The acid so produced
decreases the pH to 4.5 or below, which is indicated by a change in the colour of
methyl red from yellow to red when added to the broth culture.
Inoculums of isolate was grown in a broth medium containing glucose and incubated
for 48h at 37ºC. After 48h, methyl-red indicator was added to the broth culture, to
observe the colour changes.
We performed these biochemical tests on the colonies isolated from Mac-conkey and
favourable results was observed as shown in next chapter [24].
3.2.1.6 Biochemical screening of lipase using Spirit Blue Agar:
Spirit Blue Agar is used for detecting lipolytic bacteria using lipase reagent and lipid
source.A loop full of colonies of isolated pure P.aeruginosawas streaked onto Spirit
blue agar medium and incubated for 24h at 25ºC
Table 3.5: Composition of Spirit Blue Agar
COMPONENT QUANTITY
10.0 g
Casein
Yeast Extract 5.0 g
Agar 20.0 g
Spirit Blue Dye 0.15 g
pH 6.8 ± 0.2 at 25ºC
Spirit Blue Agar contains peptone.Essential nitrogen, Carbonsource,Sulphur and other

elements are provided by peptone.Yeast extract contains B-complex vitamins which
help in bacterial growth. Spirit blue indicates the lipolytic species. Agar is used as a
solidifying agent.

The agar plate containing tributyrin (triglyceride hydrolysed by lipase) is streaked

with bacterial sample. There will be a zone of clearance if the bacteria secrets lipase.
If the bacterial is not lypolytic, the agar will remain opaque.
The molecular weight of extracellular lipase produced by P.aeruginosa is 25.5 KDa.
The organism was inoculated onto the Spirit Blue Agar plate. A large colony was
found after 15 hrs of incubation which turned the color of media from pale blue to
white. Hence the lipase biochemical assay showed positive results for the production
of extracellular lipase by P.aeruginosa [25].
3.2.1.7 Extraction and Purification of Lipase
The extraction involves two steps namely preparation of enzyme extract and
Ammonium sulphate precipitation followed by purification of enzyme by dialysis
method.
1. Preparation of Enzyme Extract
100 ml of 2X King‘s B broth was inoculated with P.aeruginosa and incubated for 72
hours. The incubation time is enough for optimal production of lipase. The broth
culture was centrifuged at 10,000 rpm for 15 minutes at 4°C. The cells in the broth
form a pellet which is discarded. The supernatant contains extracellular lipase
enzyme.
2. Ammonium Sulphate Precipitation
Saturated solution of ammonium sulphate is prepared by adding ammonium sulphate

salt continuously to water on a magnetic stirrer. Equal quantity of supernatant and
saturated ammonium sulphate are mixed and kept in deep freezer at - 20°C for 24
hours. The ice is kept at room temperature for 30 minutes for ice to melt and then this
mixture which contains precipitated enzyme is centrifuged for 30 minutes at 5000 rpm
at 4 °C. The pellet (containing enzyme) was re-suspended in phosphate buffer (pH
7). The solution obtained is the enzyme extract[26].
3. Purification of Lipase by Dialysis
The molecular weight of Lipase is 25kDa hence, the dialysis membrane of 0.5µm
pore size with MWCO of 20KDa was selected for dialysis. This dialysis bag was
immersed in 100 ml of 2% Na2Co3 solution inside boiling water bath for 10 minutes.

The dialysis membrane has to be pre-treated as shown in Fig 3.3. Using forceps the
dialysis bag was transferred to a beaker containing 100 ml distilled water in boiling
water bath and boiled for 10 minutes. After 10 minutes this was transferred to another
beaker containing 100 ml distilled water distilled water in boiling water bath. The
membrane is later transferred to a beaker containing 100ml of 0.05% EDTA solution
inside boiling water bath and boiled for 10 minutes.
Fig 3.3: Steps for pre-treatment of dialysis membrane
The solution is allowed to cool to room temperature. The membrane is taken out and
one end of the membrane is tied with rubber band.
3ml of enzyme extract is poured inside the dialysis bag and the other end is tied with
rubber band. The dialysis bag containing enzyme extract is suspended in a beaker
containing 0.01X PBS (pH 7). The beaker is left on a magnetic stirrer inside the
refrigerator for 24 hrs.
The ammonium sulphate salt attached to enzyme molecules get released into the PBS
solution during dialysis. This process is facilitated by the force provided by the
stirring of solution. The other unwanted ions and impurities also get diffused into the
PBS solution due to difference in ion concentration between the enzyme extract and
the PBS solution (osmosis). The optimal pH is maintained by the PBS solution which
acts as buffer. Hence at the end of dialysis only the enzyme solution is obtained in the
dialysis bag. The purified enzyme is later collected from the dialysis bag and stored at
-4°C until use.
3.2.1.8 Quantification of purified crude Lipase
The basic method of protein estimations such as Lowry‘s method and Bradford
method was studied in the literature survey. Lowry‘s method was adopted to estimate
the concentration of protein in the lipase.BSA stock solution, alkaline copper reagent,
FC reagent required for Lowry‘s method was prepared afresh.DifferentBSA

dilutionswere prepared by mixing stock BSA solution (200µg/ ml). The final volume
in each of the test tubes is made upto 1 ml with distilled water. The BSA range is 40
to 200µg/ ml.
1ml of crude purified lipase is taken as test. All the dilutions with the test was added
with 2ml of alkaline copper sulphate and mixed.It was incubated at room temperature
for 10min. Then, 0.2ml of FC reagent was added and incubated in dark for 20min.The
absorbance was checked at 660nm.The graphof absorbance v/s protein concentration
was plotted as shown in the figure to get a standard calibration curve. The
concentration of test sample was extrapolated from the standard graph.
Table 3.6: Lowry‘s method for protein estimation in lipase

Test Volof Volum Amino O.D660
tube standard a e of acid
Leave for 20 minutes at room temperature in dark

Sl.no mino acid Distille concentra
Leave for 10 minutes at room temperature

Sol. (ml) d tion in 1.0 ml alkaline copper reagent to each
Add 0.5 ml of F.C Reagent to each

Water each
(ml) standard
(µg/ml)
1 0 1.0 0.0
2 0.2 0.8 40
3 0.4 0.6 80
4 0.6 0.4 120
5 0.8 0.2 160
6 1.0 0.0 200
Given 1.0 ml test To be
7
solution estimated
3.2.2 Culture and growth of algae
As discussed in chapter 1& 2, the high oil yielding algae Botryococcusbraunii was
considered for the algal oil extraction. Initially generic algal media; chu13 media was
used to observe the growth in closed system, eventually various ingredients of the
media were modified to get the desirable amount of growth. Then algal mat produces

was subjected to extraction procedures to procure algal oil form the algal mat. The
residual algal biomass serves as the cattle feedstock.
3.2.2.1 Media optimization
The algae sample collected from Ulsoor lake was serially diluted up to 5 dilutions and
inoculated into chu media and then to specific media to observe the growth. The
media specific to the growth of B.braunii specie i.e., Kuhl‘s media was used for the
culture and growth of algae and the composition as shown in table 3.6 & 3.71X & 2X
Basal media was prepared. Third andFourth algae dilution samples (filtered and
diluted) was inoculated separately at 25 ± 10C under 1.2 ± 0.2 Klux light intensity
with 16:8 hrs light photoperiod in sterile condition
Table 3.7: Kuhls‘ media Composition- Basal media

STOCK NUTRIENT
COMPONENT
SOLUTION[g/100ml] SOLUTION[ml]
KNO3 1 20
K2HPO4 0.1 20
MgSO4.7H20 0.1 20
Soil Extract 30
Micronutrient
5
Solution
Distilled Water 905
Table 3.8: Kuhls‘ media Composition- Micronutrient solution

STOCK Applied
COMPONENT
SOLUTION[g/100ml] Solution
ZnSO4.7H20 0.1 1 ml
MnSO4.4H2O 0.1 2 ml
H3BO3 0.2 5 ml

Co(NO3)2.6H20 0.02 5 ml
Na2.MoO4.2H2O 0.02 5 ml
CuSO4.5H20 0.0005 1 ml
FeSO4.7H20 0.7g
EDTA 0.8g
Modified Kuhls‘ media :
Significant growth was not observed even after 7 days, hence Kuhls‘ media was
incorporated with few specific changes as indicated here
 Increased Photoperiod to 17 hours.
 Incorporation of aerator.
 Added excess soil extract to the media.
 Increased the strength of the media from single to double strength.
 Addition ofvitamins.
For all these modifications in kuhls‘ media, a good algal growth was
observed.Although the successful growth was observed only in closed system, an
effort was made to culture the algae in open pond system.
Open pond culture was carried out after the success of growth in modified kuhl‘s
media. Media was poured into a 8 litre capacity aquarium. The media in the aquarium
was inoculated with algae sample, which was obtained in the previous setup & aerator
was provided. Prolific growth was seen after a week from inoculation.
3.2.2.2 Screening of Botryococcusbraunii
Media specific to B.brauniiwas used for the culturing of algae which will permit
onlythe growth of B.braunii specie. To ensure the culture was B.braunii, regular light
microscopic observations were made at 100X. The figure 3.3 shows the significant
morphological characteristics of pure B.braunii culture which was expected in the
cultured algae.
 Cells oval in shape

 Golden-yellow colour lipid droplets
Fig3.3: Light microscopic observation of B.braunii at 100X
3.2.2.3 Algal Oil Extraction
After collecting the algal mass from the aquarium, we centrifuge it at 5000rpm for
5mins. We discard the supernatant and collect the precipitate in a separate beaker.We
keep the beaker containing algal mass in hot air oven (40-500C) for 10mins. This step
helps in removing the cell clumps & facilitates the oil extraction. Then we centrifuge
the mass at 5000rpm in 100ml centrifuge tubes for 5mins at room temperature. We
collect the precipitate and subject it to drying in hot air oven for 24hrs at 45 oC. We
then measure 10gm of the dried algae and put it in a mortar and pestle and add 5gm of
glass fibre to it. We ground it thoroughly and add 20ml of hexane and 5ml of
petroleum ether for the separation of oil exuded from the algal biomass. We then pour
the mixture in a beaker and keep it in a water bath for 10mins at 35-400C. Then we
incubate the mixture at room temperature for 24hrs. This is done to evaporate the
hexane and petroleum ether slowly, thereby leaving behind the oil as a separate layer.
The incubation time depends on the amount of solvents added.
Soxhlet extraction method was used with 5:1 (v/v) Hexane: Petroleum ether as the
solvent at 70°C to extract the algal oil. Soxhlet extraction method will help break up
the polar barriers in trapped lipid bodies of algal mass and allow the solvent to reach
the non-polar compounds and extract them.
3.2.3 Comparative studies of chemical and microbial Trans-esterification on

three different edible oils - Pongamia oil, Groundnut oil, Rice bran oil
The chemical and enzymatic trans-esterification of three edible oils were carried out
to draw more insights about the factors affecting the FFA content of oil, yield of
Biodiesel

Chemical Trans-esterification:
In this stage, six experiments have been conducted by varying FFA contents of oils
(High FFA – 20%, 27.4% and 36% and Low FFA – 4%, 3.96% and 9.78% of
Pongamiaoil, Used Cooking oil and Rice bran oil respectively). Experiments were
conducted by keeping NaOH catalyst concentration (8.5g), Oil: methanol ratio (1:2
v/v) and reaction temperature (75˚C to 80˚C) constant for reaction time of 2h[28].
Microbial Trans-esterification:
In this stage, 12 sets of experiments were conducted for High FFA content Oils (20%,
27.4% and 36%) and Low FFA content oils (4%, 3.96% and 9.78%) by varying the
amount of Lipase (25µl, 50 µl, 75 µl and 100 µl). Experiments were conducted by
keeping Oil: methanol ratio (1:2 v/v) and reaction temperature (35˚C - 40˚C) constant
for reaction time of 6h [29].
3.2.4Trans-esterification of algal oil with Lipase isolated from P.aeruginosa
We take oil extracted from the algae and mix it with methanol in the ratio of (1:2) by
volume. Then the mixture is incubated in a water bath for 5-10mins at 35-400C. We
then remove it from the water bath and keep it in room temperature for 5mins [30].
The lipase is then added to the mixture and then incubated in a water bath for 6hrs at
35-400C. Finally, we test for the presence of methyl ester after incubation time. This
will guarantee the success of the trans-esterification process and indicate towards the
formation of biodiesel [31].
3.2.5 Testing of fuel properties of Biodiesel
The fuel properties of diesel is taken care by United States as ASTM standards. The
standard doesn‘t have any relation with fuel composition or source it only gives the
property values of some parameters to provide acceptable engine operation, safe
storage and transportation. There are ASTM standards for each properties that defines
the method used to measure them. So, the ASTM D975 says permitted value and
measuring methods are given by individual standards [32].

We have tested 3 properties namely Kinematic Viscosity, Flash point and density.
These are the permitted range of the fuel properties.
Table 3.9: ASTM range of Biodiesel [32]

Fuel Properties ASTM standards
Kinematic Viscosity (C.st) 2.4 - 2.6
Flash Point (°C) >150
Density(Kg/m3) 870-900
1. Kinematic Viscosity:
Kinematic viscosity is the resistance offered to the fluid for its flow. Normally it‘s
desirable to have low kinematic viscosity to avoid uneven distribution of fuel in the
engine. Hence, its important parameter of a diesel fuel [33].
ASTDM 445 is the standard test procedure to determine the kinematic viscosity.
Cannon-Fenske Capillary Viscometer Tube was used to note down the time taken by
the fuel to reach the lower mark of the tube as shown in figure 3.4.
2. Flash Point:
It is the temperature at which the fuel starts to vaporize when the ignition source is
given to fuel. ASTDM 93 is the standard test procedure to determine the flash point of
the diesel fuel. Penske-martens flash point cup is filled with the sample fuel and
gradually heated, ignition sources are given at nearest intervals of standard values to
determine the flash point of the sample as shown in figure 3.5 [34].
3. Density:

It‘s usual parameter mass per unit volume. It‘s desirable to have high density value so
that kinematic viscosity will be low and good for fuels. The specific gravity method
was used to measure [35].
Fig 3.4: Cannon-Fenske Capillary Viscometer Tube [33].
Fig3.5: Penske-martens flash point apparatus [34].
Totally 18 samples tests were performed on the biodiesel obtained from Pongamia oil,
Groundnut oil, Rice bran oil via chemical and enzymatic method trans-esterification
and finally algal Biodiesel from enzyme trans-esterification.

CHAPTER 4
RESULTS AND DISCUSSION
The experimentation of biodiesel production was carried out as shown in

methodology section with parallel workflow of isolation of Lipase from P.aeruginosa
and growth of B.braunii to extract algal oil. The algal oil obtained from B.braunii was
subjected to trans-esterification in the presence of Lipase isolated from P.aeruginosa
as the biocatalyst. The Biodiesel from algal oil extracted by B.braunii was produced
using Lipase isolated from P.aeruginosa as biocatalyst in trans-esterification process.
This chapter explains in detail about the culture and growth of P.aeruginosa,
Screening of P.aeruginosa, isolation and purification of Lipase from P.aeruginosa,
quantification of Lipase isolated from P.aeruginosa, Culture and growth of B.braunii,
extraction of algal oil form B.braunii, yield of algal oil from B.braunii culture, yield
of Biodiesel from microbial trans-esterification of algal oil, Comparative studies of
chemical and microbial trans-esterification of 3 different oils namely Pongamia oil,
Groundnut oil and Rice bran oiland qualitative study of the Biodiesel obtained from
microbial trans-esterification.
4.1 Isolation and Screening of Pseudomonas aeruginosa
The isolation of P.aeruginosa from soil and water samples was experimented by
inoculating the samples on the nutrient media, differential media and specific media
used for culture and growth of P.aeruginosa sequentially. The bacterial colonies
obtained from these media were identified and screenedbymicroscopic observation
and biochemical tests specific to P.aeruginosa.
4.1.1Serial dilution and Inoculation on LB agar medium
The soil and water samples were serially diluted up to five dilutions as shown in the
figure 4.1(A).These dilutions were inoculated by spread plating on LB agar medium
as shown in figure 4.1(B). The inoculated plates were incubated for 24h at 25ºC.
Serial dilutions help in isolation of discrete colonies that can be sub-cultured into pure
colonies.

(A) (B)
Fig 4.1: Serial dilution (A) and inoculation of soil and water samples onto LB
media(B)
(A)
(B)
Fig4.2: Single colony seen in fourth dilution culture of water(A) and colonies seen in
third dilution culture of soil sample(B)
The LB medium is the commonly used media for the fast growth of general bacteria.
It has rich source of nutrient which helps in quick growth of all kinds of bacteria
initially. After incubation it was found that the third dilution of soil sample and
fourth dilution of water sample plates contained colonies that matched the colony
characteristics of P. aeruginosa.The other plates contained bacterial colonies that did
not match the colony characteristics ofP.aeruginosaand some were contaminated.
Hence, they were discarded.

4.1.2 Selective media for Gram-negative bacterial growth
EMB agar medium is the selective media for the growth of Gram-negative bacteria.
P.aeruginosa is the gram-negative bacterium. The colonies from LB may contain all
types of bacteria. Culturing on EMB agar media would eliminate the growth of
bacteria other than gram-negative. Hence, a loopful of colonies from LB agar plates
were streaked on to EMB agar medium and incubated for 24h at 25ºC.
(A) (B)
Fig 4.3: Colonies in third dilution of soil (A) and light growth of colonies in fourth
dilution of water (B)
After incubation it was found that the third dilution of soil sample and fourth
dilution of water sampleplates showed thebacterial colonies as shown in figure
4.3(A) and figure 4.3(B). This suggests both colonies contain gram-negative bacteria.
P.aeruginosa could be present in those colonies.
4.1.3 Selective media forPseudomonasBacteria
Kings’ B agar media is thespecific media for Pseudomonasbacterial species.King

Agar B enhances the elaboration of fluoresceinand inhibits the pyocyanin formation. Peptone
provides the essential nitrogenous nutrients, carbon, sulphur and trace elements. Glycerol
serves as a C-source and Dipotassium hydrogen phosphate buffers the medium. The media
contains lipid as the carbohydrate nutrient source, hence the organisms which can
utilize the lipid source as its energy can grow on the media. A loopful of culture from
EMB medium containing only gram-negative bacteria was streaked on to Kings‘ B
agar medium.

The growth was only seen in third dilution of soil which confirms the presence of
Pseudomonas spp as shown in figure 4.4. No growth in water sample suggested that
there are no pseudomonas species, however, lesser colonies in the EMB medium of
water sample can also be indicative of presence of less gram-negative bacteria.
Fig 4.4: Colonies observed only from third dilution of soil sample
P. aeruginosabuild colonies surrounded by a yellow to greenish-yellow zone due to

fluorescein production which fluoresces under UV light. If pyocyanin is also
synthesized, a bright green color is produced. Most pyocyaninproducing
Pseudomonas strain synthesizes also fluorescein and others produce just one pigment.
P. aeruginosacan be differentiated by following cultivation on Mac Conkey Agar

because P.fluorescensdo not show fluorescence under UV light and grow poorly. This
is the main difference between P. Aeruginosaand other Pseudomonas species.
4.1.4 Differential media for P.aeruginosa - Mac-Conkey agar medium
Mac-conkey agar media differentiates between P.aeruginosa and fluorescens

[36].Aloopful of colonies from Kings‘ B agar medium was streaked onto Mac-conkey
agar medium and incubated for 24h at 35ºC. Confluent growth of colonies were
observed after incubation period as shown in figure 4.5.

Fig 4.5: Confluent colonies on Mac-conkey agar medium
P. aeruginosabuild colonies surrounded by a yellow to greenish-yellow zone due to

fluorescein production which fluoresces under UV light. If pyocyanin is also
synthesized, a bright green color is produced. Most pyocyanin-producing
Pseudomonas strain synthesizes also fluorescein and others produce just one pigment.
The temperature can be a determining factor as most fluorescent strains will not grow
at 35°C[37-39].
4.1.5Screening of P.aeruginosa by Biochemical assay
After thorough review of literature, it was found that P.aeruginosashows positive

results for catalase and oxidase test but negative results for urease and methyl red test
as shown in Table 4.1. We performed these biochemical tests on the colonies isolated
from Mac-conkey agar and favourable results were observed as shown in figure 4.6
(A, B, C and D).
Table 4.1:Biochemical assay [40].
Biochemical Test Observation Result

OXIDASE TEST Purple POSITIVE
Evolution of Oxygen
CATALASE TEST POSITIVE
Bubbles
METHYL RED
No change NEGATIVE
TEST
UREASE TEST No change NEGATIVE

Fig 4.6(A):Oxidase test- Purple color development
A smallinoculums of bacterial isolate was smeared on tetramethyl-p-

phenylenediamine reagent, purple color was observed within 20 seconds as shown in
figure 4.6(A) indicating that bacteria produces cytochrome c oxidase enzyme. Since,
the P.aeruginosaspecies show positive results to oxidase test, our resultsindicates that
bacterial isolates belong to P.aeruginosaspecies.
Fig 4.6(B): Catalase test - Evolution of O2 bubbles;
Small inoculums of isolate was mixed into hydrogen peroxide solution (3%) taken on
glass slide and the rapid elaboration of oxygen bubbles were seen as shown in figure
4.6 (B). Since, the P.aeruginosaspecies show positive results for catalase test; our
results indicate that bacterial isolates belong to P.aeruginosaspecies.

Fig 4.6(C): Methyl red test - No colour change of isolate
As the principle explained in methodology section, the isolate did not show any color
changes as shown in figure 4.6(C). Since, the P.aeruginosaspecies show negative
result for methyl red test, our results indicate that bacterial isolates belong to
P.aeruginosaspecies.
Fig 4.6(D): Urease test- No colour change of the isolate
As the principle explained in methodology section, the isolate did not show any color
changes as shown in figure 4.6(D) indicating that isolate did not produce urease
enzyme. Since, the P.aeruginosaspecies show negative result for urease test, our
results indicate that bacterial isolates belong to P.aeruginosaspecies.
These test results confirm that our colonies are pure form of P.aeruginosa. Hence, it
was sub-cultured and used for extraction of Lipase.

4.1.6 Screening of P.aeruginosa as Lipase producer by Spirit blue Agar
It is necessary to confirm the production of Lipase enzyme in the isolated bacterial

cultures before extraction of Lipase at first place. Spirit Blue Agar is for use with
Lipase Reagent or other lipid source for detecting and enumerating lipolytic
microorganisms [41].
Fig 4.7:Spirit blue agar kept uninoculated
Fig 4.8:Colony of P.aeruginosa has produced a zone of hydrolysis (color change from
pale blue to colorless) indicating the production of lipase.
The isolated colonies of P.aeruginosa was cultured on spirit blue agar media which
has lipid as the nutrient source, hence to utilize the lipid source bacteria will produce
Lipase and hydrolyse the lipid source in the media; the hydrolysis is indicated by
spirit blue dye which becomes opaque. If the bacterium secretes lipase, there will be a
zone of clearing surrounding the sample as shown in figure 4.8. If the bacteria does
not produce and secrete lipase, the agar will remain opaque as shown in figure 4.7.

4.1.7 Extraction and Isolation of Lipase – Ammonium Precipitation method
Ideally enzyme purification involves use of chromatography methods such as affinity

chromatography, size exclusion chromatography etc. The resultant proteins are
obtained in extremely pure forms (purity above 95%) [42]. But the studies have
shown that as the number of steps in purification increases, the strain on enzymes
increases which hampers the 3D structure and hence the activity reduces [43]. To
overcome this problem, our bacteria P.aeruginosa were given source of carbon as
glycerol (a lipid) and hence to utilize this carbon source it had to produce lipase in
large quantity. This result in other enzymes being produced in lesser quantity and the
number of purification steps are reduced.
2X King‘s B media which is the selective media for P.aeruginosafulfils all the
requirements for optimal production of lipase in the given media. As discussed in
methodology section, extraction of lipase was carried out by using double
concentration Kings‘ B broth with saturated ammonium sulphate precipitation method
to prepare enzyme extract as shown in figures 4.9, 4.10 and 4.11. The pellet will have
the crude lipase free from other cell debris.
Fig4.9: 100ml King‘s Broth culture Fig 4.10: Saturated ammonium

After 72h incubation sulphate and stored for 24h at -20°C

Fig 4.11: Pellet re-suspended in phosphate buffer to produce enzyme extract
4.1.7.1 Purification of Lipase by dialysis method
As discussed in methodology section, dialysis process in cold condition was used to

isolate the partially purified lipase from the enzyme extract prepared by ammonium-
sulphate precipitation method. The molecular weight of lipase is 25kDa hence,
dialysis bag with MWCO of 20kDa was used as shown in figure 4.12 to ensure that
lipase is retained in the dialysis bag. The other impurities having molecular weight
less than 20kDa pass out of the dialysis bag. After 24hours, 5ml of purified crude
lipase was left in the dialysis bag as shown in the figures 4.13 and 4.14.
Fig4.12: Dialysis in cold condition Fig4.13:Lipase concentrated in dialysisbag
Fig4.14: Purified crude Lipase

4.2Quantification of purified crude Lipase – Lowry’s method
The concentration of protein in the purified crude lipase was estimated by Lowry‘s
method. The table 4.2 shows the experimental values of Lowry‘s method of protein
estimation.
The absorbance of purified crude lipase at 660nm was found to be 0.15. The plot of
absorbance of standard BSA v/s concentration was drawn as shown in figure 4.15.
Then absorbance of purified crude lipase was extrapolated on the standard graph to
estimate the concentration of protein in purified crude lipase. Hence, the
concentration of protein is estimated to be 158 µg/ml.
Table 4.2: Lowry‘s method of Protein estimation
Test Volof Volume of Amino O.D6

tube standard Distilled acid 60
Sl.n amino Water concentrat
o acid Sol. (ml) ion in each
(ml) standard
(µg/ml)
Leave for 20 minutes at room temperature in dark

Leave for 10 minutes at room temperature
0 1.0 0.0 0.0

1.0 ml alkaline copper reagent to each
1
Add 0.5 ml of F.C Reagent to each
2 0.2 0.8 40 0.04
3 0.4 0.6 80 0.07
4 0.6 0.4 120 0.13
5 0.8 0.2 160 0.17
6 1.0 0.0 200 0.18
Given 1.0 ml test To be 0.15

7
solution estimated

Fig 4.15: Plot of absorbance v/s concentration of Standard BSA and purified crude
lipase sample
4.3Culture and growth of B.braunii
Initially we collected algae traces containing water sample from Ulsoor Lake as
shown in figure 4.16 and inoculated into the Chu Media, the growth was not seen, so
we modified the Chu media by reducing the concentration of KI, KNO3 and
MgSO4.H20 to the half of their original concentration. Through review of literature, it
was clear that excess concentration of KI, KNO3 and MgSO4.H20 can inhibit the
growth of Botryococcusbrauniihence we modified the Chu media.
Fig4.16: Water sample containing algae traces collected from Ulsoorlake, Bengaluru

(A) (B)
Fig 4.17:Kuhls‘media(1X & 2X) inoculated with third and fourth dilution of algae
samples(A); Growth not observed even after 7 days of incubation(B).
4.3.1 Modified Kuhls’ media
Following the modification of Chu media, little improvement in the algal growth was
observed, but yet the growth did not persist. Then by literature review, a specific
media for the growth of B.brauniiwas clearly proved, hence we used theKuhls’
Mediawhich is specific for B.braunii growth.
Kuhls‘ media of concentration 1X & 2X Basal media was prepared into which third
and fourth dilution of algae samples (filtered and diluted) were inoculated separately
but there was no growth observed even after 7 days as shown in figure 4.17(A) and
4.17(B). Hence, after literature review we incorporated fewmodifications in the kuhls‘
media which showed very good growth of the algae as shown in figure 4.18. The
modified kuhls‘ media had excess soil extract, vitamins were added. So, the modified
media conditions are as follows
 Increased Photoperiod to 17 hours [44].
 Incorporation of aerator[45].
 Added excess soil extract to the media [46]
 Increased the strength of the media from single to double strength [47]
 Addition ofvitamins[48]

Fig4.18: Growth in modified Kuhls‘ media after 7 days from inoculation
4.3.2 Open pond culture (In Aquarium set up)
For all these modifications in kuhls‘ media, a good algal growth was
observed.Although the successful growth was observed only in closed system, an
effort was made to culture the algae in open pond system.
As discussed in the methodology section, an aquarium setup of 8 litres capacity as

shown in figure 4.19 was used for open pond culturing. As B.brauniimat
formationrequires more surface area and lesser height above the support ground, only
3 litres of media was poured into the aquarium tank with these constituents,
1. Macronutrient (2X)
2. Micronutrient (0.5X)
3. Soil Extract (increased conc. than default)
4. Vitamins (Thiamine & Biotin)
Fig 4.19: Aquarium set up

Fig 4.20:Inoculated with 30ml of Ulsoor lake water sample
Fig4.21: Growth of B.braunii after 5 days
Fig4.22: Growth of B.brauniiafter 7 days
The media was inoculated with 30ml of algae traces containing water sample as
shown in figure 4.20. Confluent growth was observed as shown in figure 4.21 and
4.22 after 5 and 7 days respectively.The growth of algae was allowed till the harvest
time of B.braunii. After 5 weeks, the thick algal mat of B.braunii was grown as shown
in figure 4.25. Then the mat was harvest and prepared for the extraction of oil from it.

4.4Screening of B.braunii
Media specific to B.brauniiwas used for the culturing of algae which will permit only
the growth of B.braunii specie. To ensure the culture was B.braunii, regular light
microscopic observations were made. The morphological characteristics of our culture
as show in figure 4.24 matches well with the morphological characteristics of the pure
culture of B.braunii as shown in figure 4.23(A) and 4.23(B) [49, 50].
(A) (B)
Fig 4.23: Morphological features of the pure culture of B.braunii observed under 40X
(A) and 10X (B) magnification [50-52].
Fig4.24: Morphological features of the lab grown algae observed through compound
microscope under 10X magnification
These morphological observations after using the culture media specific for B.braunii
growth suggests that our algal culture belongs to B.braunii specie. Hence, the growth
was continued by adding fresh culture media every week.

4.5 Algal Oil extraction
After 5 weeks of algal growth, a thick mat as shown in figure 4.25 was dried and
weighed up to 24g as shown in figure 4.26. Then the dried mat was finely powdered
to extract oil from it as shown in figure 4.27. Initially solvent extraction by 3:1(v/v)
hexane: Petroleum ether solvent for 24h was done.The amount of algal oil produced
was not favourable as shown in figure 4.28. This may be due the trapped oil bodies in
the algal mass hence, soxhlet extraction apparatus was used to extract oil from
remaining algal mat as shown in figure 4.29. Then the extraction process was carried
out for 4 continuous days which yielded favourable amount of oil as shown in figure
4.30.
Fig4.25: Algal mat produced after 4.5 weeks
Fig4.26: Dried algal mass
Fig4.27: Finely powdered algal mass

Fig 4.28: Algal oil extraction by Hexane: Petroleum ether solvent extraction
Relatively non-polar molecules, like lipids, can be extracted from a sample using
relatively non-polar molecules. In case of a non-polar solvent, only non-polar
molecules in the sample dissolve while polar ones do not. This claim does not hold
good for lipids on animal and plant cell membranes, which have both polar and non-
polar regions such as triglycerides and phospholipids. These molecules align, with
their polar heads sticking outwards and non-polar tails inwards making it difficult for
extraction. As these molecules are part non-polar and part polar, we need a solvent
that has some of these similar characteristics. This is why we used a mixture of two
solvents, i.e., hexane and petroleum ether. The petroleum ether is polar enough to
interact with the Polar Regions and help relax the cell membrane while also being
non-polar enough to aid the removalof non-polar fats. [53- 55].
Fig4.29: Algal oil extraction by soxhlet apparatus at 70°C

Fig4.30: Solvent layer containing algal oil
Table 4.3: AlgalOil yield

Mass
of Mass of Mass of
crude centrifuged dried& Amount of oil
Yield (%)
Algal Algal mat powdered extracted(ml)
Mat (g) algal mat (g)
(g)
14.23 13.07 12.36 7 49.21%
The algal mat after drying in hot air oven for 24h at 57ºC weighed about 14.23g and
when it was crushed to fine powder it weighed about 12.36g, the reduction in the
mass could be the elimination of air bubbles which weighed more before crushing.
Eventually, 12.36g of finely powdered algal mat produced about 7ml of algal oil as
shown in figure 4.30 and tabulated in table 4.3. After the algal oil extraction, there
was the algal biomass left over which weighed about 6.23g. So, this suggests that
approximately 40% of biomass of B.braunii contains oil in it. The observations made
from our experimentation agrees very well with the literature review made on the
similar research work on oil extraction form B.braunii.

4.6 Comparative studies of chemical and microbial Trans-esterification on three

different oils - Pongamia oil, Groundnut oil and Rice bran oil
Fig 4.31: Chemical method – Formation of 2 phases. Top phase is Biodiesel
To compare various parameters between Chemical trans-esterification and Lipase

mediated trans-esterification, both methods were performed on 3 different oils as
shown in figure 4.31 and figure 4.32. From figure 4.31 it is clear that there is
formation of only two layers namely upper biodiesel and lower glycerol layer whereas
in the case of figure 4.32, three distinct layers are observed; only the middle layer
being lipase enzyme and the rest of the layers are same as in the case of chemical
trans-esterification. This suggests that lipase is regenerated and can be reused for
further conversion of oil into biodiesel.
Layer 1: Biodiesel
Layer 2: Lipase
Layer 3: Glycerol
Fig4.32: Microbial Trans-esterification - Formation of 3 clear layers.

4.6.1 Effect of FFA content of oil in Chemical Trans-esterification
Experiments were made to compare the Lipase-mediated trans-esterification with

chemical method of trans-esterification. One significant parameter i.e., FFA content
of oil that would affect the yield of Biodiesel was considered to represent the
differences between two processes although there are many parameters.
After performing six experiments on chemical Trans-esterification, the readings of
chemical method are tabulated in table 4.4
Table 4.4:Yield of Biodiesel from different edible oils based on their FFA content by
Chemical Trans-esterification
Oil Quantity Methanol FFA NaOH Biodiesel Yield(%)
added(ml) used (ml) content used obtained(ml)
(g)
Pongamia 250 125 20% 8.5 90 36%

oil
4% 8.5 225 90%
Cooking 250 125 27.4% 8.5 50 20%

oil
3.96% 8.5 212.5 85%
Rice 250 125 36% 8.5 40 16%

Bran oil
9.78% 8.5 112.5 45%
Biodiesel yield- chemical method
Fig 4.33:The impact of FFA content of oil on biodiesel yield by Chemical method

The observations in table 4.4 suggests that yield of biodiesel is less when the FFA
content of the oil is high and the yield is high only when the FFA content of the oil is
low as shown in figure 4.33. The less yield of Biodiesel when FFA of oil is high is
because of the catalyst used in the chemical trans-esterification is tending to form
soap readily than forming biodiesel due to high FFA content of oil. This is similar to
acid-base neutralisation process and therefore it can be observed that yield of
biodiesel is increased as the FFA level of oil is reduced. Hence, the FFA content of oil
must be reduced before using chemical Trans-esterification method to produce
biodiesel.
4.6.2 Effect of FFA content of oil in Lipase-mediated Trans-esterification
After performing 12 sets of experiments as mentioned in chapter 3 on Lipase-

mediated Trans-esterificationthe reading observed are as shown in table 4.5.
Table 4.5: Yield of Biodiesel from different edible oils by Lipase-mediated Trans-
esterification.
Quantity Biodiesel
Methanol FFA Lipase
added obtained( Yield(%)
Oil used (ml) content used(µl)
(ml) ml)
25 165 66%
Pongamia 20% 50 183 73.2%

250 125
oil 4% 75 182 72.8%
100 170 68%
25 100 40%
Cooking 27.4% 50 125 50%

250 125
oil 3.96% 75 122 48.8%
100 110 44%
25 105 42%
Rice Bran 36% 50 120 48%

250 125
oil 9.78% 75 126 50.4%
100 123 49.2%

High FFA- Biodiesel yield by Lipase method

80%
60% pongamia high

ffa
40% cooking oil
20%
Rice bran
0%
0 50 100 150
Low FFA-Biodiesel yield by Lipase method
80%
60%
40% pongamia high ffa

cooking oil
20%
Rice bran
0%
0 50 100 150
Fig 4.34: The impact of FFA content of oil on biodiesel yield by enzymatic method.
The observations made in table 4.5 suggest that yield of biodiesel is optimum for 50µl
of the lipase and the yield of biodiesel do not show any significant increase with
increase in the amount of lipase used. This may be due to the competitive inhibition
between enzyme and substrate. Further the readings in table 4.5 and figure 4.34
suggests that yield of Biodiesel is unaffected when the FFA content of the oil is high
also the yield does not show much difference when the FFA content of the oil is low.
This clearly suggests that FFA content of the oil does not have any impact on yield of
Biodiesel during Lipase-mediated Trans-esterification process.
The results also indicates that Yield of Biodiesel from oil having High FFA content is
more through Lipase mediated Trans-esterification than Chemical method of Trans-
esterification as shown in figure 4.35. The lipase is regenerated and can be reused
with zero-residue while the chemical in chemical Trans-esterification will be
completely used and toxic to the environment.

80
Biodiesel yield- chemical v/s Enzyme
70
60
50
40
Chemical
30 Lipase mediated
20
10
Pongamia oil Ground nut oil Rice bran oil
Fig 4.35: Comparison of yield via enzyme method and chemical method of Trans-
esterification of different oils with high FFA content
Observations from above data suggests that in chemical method, the FFA content of
the oil affects the biodiesel yield while in the case of microbial enzyme method, FFA
content of the oil doesn‘t depend on the yield of biodiesel. To get the desirable yield
in chemical method, the FFA content of the oil must be reduced to less than 4% which
is a tedious process. However, in the case of microbial method albeit yield is average,
it is economical in large scale production.
4.7Microbial Trans-esterification of Algal oil with purified crude lipase to

produce Biodiesel
According to observations made from section 4.6, it clearly proved Lipase-mediated

Trans-esterification is more favourable than chemical method. Hence,Algal oil
obtained from algal mat was mixed with methanol and subjected to Trans-
esterification in the presence of optimum lipase of 50µl as experimented in section
4.6.2. The mixture was kept in water bath at 40°C for 6h.
After incubation for 6h, Biodiesel was produced along with glycerol and Lipase
enzyme was recovered as shown in figure 4.36 and 4.37. The Biodiesel obtained was
screened by method mentioned in the methodology section; accordingly the pink

colour in the biodiesel vial and standard petroleum diesel vanishes after 10-
15seconds. The yield of algal biodiesel is 64.32% as shown in table 4.6.
Fig 4.36: Biodiesel from algal oil Fig 4.37: Lipase enzyme recovered
Fig 4.38: screening of trans-esterification; Pink colour fades in Standard Petroleum

diesel and Algal Biodiesel produced.
Table 4.6: Yield of Algal Biodiesel

Algal Oil Biodiesel
Methanol Lipase
obtained Obtained Yield (%)
added (ml) added (µl)
(ml) (ml)
7 3 1.5 4.5 64.32
4.8 Testing of Fuel properties of Biodiesel
Additionally to measure the quality of the algal biodiesel obtained from Lipase
mediated Trans-esterification, we have tested 3 properties namely Kinematic
Viscosity, Flash point and density. Totally 18 samples tests were performed on the
biodiesel obtained from Pongamia oil, Groundnut oil, Rice bran oil via both chemical

and enzymatic method trans-esterification and finally algal Biodiesel from enzyme
trans-esterification. The readings of the experiments performed on Biodiesel obtained
through chemical trans-esterification are tabulated in table 4.7 and the readings of the
experiments performed on Biodiesel obtained through Lipase-mediated trans-
esterification are tabulated in table 4.8
Table 4.7:Fuel properties of Biodiesel produced through Chemical Trans-esterification

Fuel ASTM Pongamia Cooking oil Rice bran Oil
Properties standards Biodiesel Biodiesel Biodiesel
Kinematic
1.6 – 6 3.56 4.51 6.2
Viscosity (C.st)
Flash Point(°C) > 130 153 195 260
Density
870 – 900 898 892.4 914
(kg/m3)
Table 4.8: Fuel properties of Biodiesel produced by Lipase-mediated Trans-esterification

Fuel ASTM Pongamia Cooking oil Rice bran Oil
Properties standards Biodiesel Biodiesel Biodiesel
Kinematic
1.6 – 6 1.8 2.3 5.3
Viscosity (C.st)
Flash Point(°C) > 130 147 132 214
Density
870 – 900 865 873 917
(kg/m3)
The above values indicate that fuel properties of Biodiesel obtained from microbial
trans-esterification are consistent with range of ASTM standard values [56] alike
Biodiesel obtained from chemical trans-esterification.

CHAPTER 5
CONCLUSION AND SCOPE FOR FUTURE WORK
This chapter presents the conclusion drawn about the microbial lipases, B.braunii as
the potential biodiesel feedstock and microbial trans-esterification process when
compared to chemical trans-esterification, and the future scope of this project.
5.1 CONCLUSION
From the observations made in results sections we can conclude that Lipase
mediatedTrans-esterification is advantageous over chemical trans-esterification as
1) Algal oil yield was around 49.21% which is higher than other plant seed oil yield.
The algal biodiesel yield was 64.32%.
2) Crude Lipase extracted from P.aeruginosahad enzyme concentration of

158µg/ml.
3) Fuel obtained from enzyme trans-esterification has fuel properties similar to that
of chemical trans-esterification.
4) The biodiesel fuel properties obtained by enzyme trans-esterification were

consistent with the ASTM standard fuel properties.
5) The enzyme trans-esterification can be performed at 35-40°C while chemical

method needs 80°C which is energy intensive.
6) The two step FFA reduction is waivered in microbial lipase method as it does not
have any effect on FFA content of oil.
7) Lipase from P.aeruginosais environmental friendly catalyst compared to NaOH.
8) The lipase enzyme can be recovered and re-used.
9) These facts show that microbial trans-esterification is more economical for mass
production than chemical method with zero-chemical residue.

5.2 FUTURE PROSPECTS
Further enhancement of lipase production may be achieved by genetic and

computational modelling approach. High levels of expressions of lipases from several
micro-organisms have been successfully achieved usingSaccharomyces cerevisiae as
the host [57]. Among these the lipase cDNA from F.heterosporum increased lipase
productivity 3-fold over that of the original strain. In the light of these findings,
combining a whole cell biocatalyst with the use of a recombinant micro-organism can
be expected to considerably decrease the cost of the lipase production [58]. Such a
novel system along with computational modelling approach offers a promising
processes prospect for industrial Biodiesel Production [59].
Production of algal Biodiesel through microbial Trans-esterification is highly

advantageous over the natural processes, there are few observations made from our
literature survey, they are as below:
1. Since, algae culture uses the atmospheric CO2 for its growth, so it helps to reduce
green gas effect
2. Few algal species (Botryococcus spp.) can be grown in sewage water; therefore it is
useful for sewage treatment, where algal growth can take up some toxic metals for
its growth.
3. Algal biomass obtained after oil extraction is rich with proteins and carbohydrates,
so biomass can be used as cattle and poultry feedstock.
4. Anaerobic fermentation of biomass can yield alcohol, so alcohol obtained can assist
the trans-esterification process.
5. Protein component obtained from algal biomass can be used as an ingredient in

various fruit juices.
6. Petroleum products obtained from microbial trans-esterification of algal oil can be

inter-converted into each other.

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INDEX
Absorbance, 30, 45
Algae, 4, 5, 7, 8, 11, 12, 14, 17, 18, 21, 22, 23, 31, 33, 34, 35, 47, 50, 63, 65
Algal Fuel, 6
Alkaline copper reagent, 30, 46
Ammonium sulphate, 13
ASTM standards, 35, 36, 60, 61, 62
Biochemical tests, 7
Biofuel, 4
Biomass, 11, 13, 34, 63, 64
Botryococcus braunii, 4, 12, 18
Cannon-Fenske Capillary Viscometer Tube, 23, 36
Groundnut oil, 4, 23, 37, 60
Cultures, 4, 24
Density, 36, 37, 60, 61
Dialysis, 4, 14, 16, 22, 29, 30, 44
EDTA, 29, 33
EMB agar medium, 7, 25
Enzyme, 4, 5, 8, 13, 15, 16, 20, 23, 28, 29, 30, 37, 43, 54, 58, 59, 60, 62, 65
Extracellular lipase, 21
FC reagent, 30
Feedstocks, 14, 15
FFA content, 4, 35, 55, 57, 58, 62
Flash Point, 36, 37, 60, 61
Fuel properties, 8, 35, 36, 61
Glycerine, 10
Gram-negative bacteria, 21
Gram-positive, 21
Inoculation, 7, 25, 26, 27, 38, 39, 40
Kinematic Viscosity, 36, 60, 61
King‘s B agar medium, 7, 39
Kuhl‘s basal medium, 4
LB agar medium, 7, 24, 38

Lipase, 4, 5, 6, 7, 8, 13, 15, 16, 17, 19, 20, 21, 22, 23, 24, 28, 29, 30, 35, 37, 42, 43,
44, 45, 55, 59, 62, 65, 66
Lowry‘s method, 30
Mac-conkey agar, 7, 27, 41
Methanol, 23, 55, 59
Microalgae, 6, 10, 11, 18, 64, 65, 66
Microbial, 4, 5, 8, 15, 23, 35, 38, 53, 55, 58, 61, 62, 63
NaOH, 23, 35, 55, 62
P.aeruginosa, 4, 5, 7, 8, 13, 15, 16, 19, 21, 22, 24, 27, 28, 37, 38, 42, 43
Pellet, 16, 28, 29
Penske-martens flash point cup, 37
Petroleum diesel, 9, 10, 21
Pongamia oil, 4, 23, 35, 37, 38, 53, 55, 56, 60
Pseudomonas aeruginosa, 1, 2, 4, 6, 8, 13, 19, 21, 24, 27, 28, 38, 40, 41
Rice bran oil, 4, 23, 37, 60
Screening, 7, 16, 22, 27, 59, 65
Serial dilution, 7, 24, 38
Soxhlet, 4, 22, 34
Spirit Blue Agar, 27, 28, 43
Trans-esterification, 4, 5, 8, 15, 16, 17, 37, 38, 54, 59, 60, 61, 62, 63
Yield, 4, 5, 7, 11, 12, 14, 15, 17, 18, 21, 37, 53, 57, 58, 62, 63

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R.V.

“Production of Clean Biofuel from Microalgae”

Project Proposal Reference No. :39S_B_BE_006

Karnataka State Biofuel Development Board

VIDYASHREE S (USN: 1RV12BT057)

Under the guidance of

R.V. COLLEGE OF ENGINEERING, BENGALURU-560059

Signature of GuideSignature ofHODSignature ofPrincipal

Department of Biotechnology, RVCE, Bengaluru i

R.V. COLLEGE OF ENGINEERING, BENGALURU – 560059

We, Navyashri,Shravanthi S Kumar and Vidyashree S, students of Eighth

Place: Bengaluru Signature of the students

Department of Biotechnology, RVCE, Bengaluru ii

The study is funded by Karnataka State Biodiesel Development Board(KSBDB),

Department of Biotechnology, RVCE, Bengaluru iii

Department of Biotechnology, RVCE, Bengaluru iv

4.1 Isolation and screening of P.aeruginosa…………………………………………33

Department of Biotechnology, RVCE, Bengaluru v

4.3 Culture and growth of B.braunii…………………………………………………44

Department of Biotechnology, RVCE, Bengaluru vi

LB: Luria Bertani

EMB: Eosin methylene blue

MWCO: Molecular weight cut-off

ASTM: American Society of testing and materials

L ha-1 :Litre per hectare

ACC: Acetyl-CoA Carboxylase

CO2: Carbon dioxide

HPL: Human pancreatic Lipase

MVB: Multi-Vesicular bodies

KDa: Kilo Dalton

H2SO4: Hydrogen sulphate

NaOH: Sodium hydroxide

NaCl: Sodium chloride

µg/ml: Micro gram per mili litre

µl: Micro litre

nm: Nano metre

K2HPO4: Potassium hydrogen phosphate

MgSO4: Magnesium sulphate

Department of Biotechnology, RVCE, Bengaluru vii

H2O2: Hydrogen peroxide

MR: Methyl red

EDTA: Ethylene di-ammine tetra acetic acid

PBS: Phosphate buffer solution

BSA: Bovine serum albumin

OD: Optical density

KNO3: Potassium nitrate

CuSO4: Copper sulphate

FeSO4: Ferrous sulphate

Rpm: Revolutions per minute

FFA: Free fatty acid

Department of Biotechnology, RVCE, Bengaluru viii

Sl. No TABLE NAME Page No.

Department of Biotechnology, RVCE, Bengaluru ix

Sl. No. FIGURE NAME Page No.

Colony of P.aeruginosa has produced a zone of hydrolysis

Department of Biotechnology, RVCE, Bengaluru x

4.9 100ml King’s Broth culture after 72h incubation 41

Department of Biotechnology, RVCE, Bengaluru xi

4.32 Microbial Trans-esterification – Formation of 3 clear layers 52