Production of Clean Biofuel from Microalgae - KSCST · Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006 Department of Biotechnology, RVCE, Bengaluru i

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

Department of Biotechnology, RVCE, Bengaluru i

R.V. COLLEGE OF ENGINEERING, BENGALURU-560059

(Autonomous Institution Affiliated to VTU, Belagavi)

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 ii

R.V. COLLEGE OF ENGINEERING, BENGALURU – 560059

(Autonomous Institution Affiliated to VTU, Belagavi)

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 iii

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 iv

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 v

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 vi

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 vii

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 viii

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 ix

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

4.4 Yield of Biodiesel from different edible oils based on

theirFFA content by Chemical Trans-esterification 53

4.5 Yield of Biodiesel from different edible oils by Lipase

mediatedTrans-esterification 54

4.6 Yield of Algal Biodiesel 57

4.7 Fuel properties of Biodiesel produced through Chemical

Trans-esterification 58

4.8 Fuel properties of Biodiesel produced through Lipase

mediated Trans-esterification 58


Department of Biotechnology, RVCE, Bengaluru x

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

2.1 Transformation of lipid bodies and vacuoles during the cell

cycle of B.braunii 13

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

4.1 Serial dilution and inoculation of soil and water samples on

LB agar media 34

4.2 Single colony in third dilution of water (A) and colonies in

fourth dilution of soil sample(B) 34

4.3 Colonies in third dilution of soil (A) and light growth of

colonies in fourth dilution of water (B) 35

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

4.8

Colony of P.aeruginosa has produced a zone of hydrolysis

(color change from pale blue to colorless) indicating the

production of lipase.

40


Department of Biotechnology, RVCE, Bengaluru xi

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

4.15 Plot of absorbance v/s concentration of standard BSA

andpurified crude lipase sample 44

4.16 Water sample containing algae traces collected

fromUlsoorlake, Bengaluru 44

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)

45

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

4.23 Morphological features of the pure culture of

B.brauniiobserved under 40X(A) and 10X(B) magnification 48

4.24

Morphological features of the lab grown algae

observedthrough compound microscope under 10X

magnification

48

4.25 Algal mat produced after 4.5 weeks 49

4.26 Dried algal mass 49

4.27 Finely powdered algal mass 49

4.28 Algal oil extraction by Hexane:Petroleum ether

solventextraction 50

4.29 Algal oil extraction by soxhlet apparatus at 70°C 50

4.30 Solvent layer containing algal oil 51

4.31 Chemical method – Formation of 2 phases. Top phase

isBiodiesel 52


Department of Biotechnology, RVCE, Bengaluru xii

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

4.33 Impact of FFA content of oil on biodiesel yield by chemical

method 53

4.34 Impact of FFA content on oil on biodiesel yield by enzymatic

method 55

4.35

Comparison of yield via enzyme method and chemical

method of Trans-esterification of different oils with high FFA

content

56

4.36 Biodiesel from algal oil 57

4.37 Lipase enzyme recovered 58

4.38 Screening of trans-esterification; Pink colour fades in

Standard Petroleum diesel and Algal Biodiesel produced. 58

Production of Clean Biofuel from MicroalgaePPRN: 39S_B_BE_006

Department of Biotechnology, RVCE, Bengaluru 1

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.

In enzyme trans-esterification, biocatalyst required is approximately 1/5,000th

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.



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].

http://en.wikipedia.org/wiki/Bioethanol

http://en.wikipedia.org/wiki/Transesterification



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

http://en.wikipedia.org/wiki/Human_pancreatic_lipase

http://en.wikipedia.org/wiki/Monoglyceride

http://en.wikipedia.org/wiki/Fatty_acid





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

Fig3.1: Workflow of methodology

Culture& isolation of

P.aeruginosa

Screening of P.aeruginosa

Extraction, Isolation and

screening of Lipase

(2 weeks)

Purification of Lipase

Extraction of Algal oil from dried

algal mat

Growth of B.braunii mat

Media optimization & culture of

B.braunii

(3 weeks)

Purification of Algal oil

Microbial Trans-esterification

Screening of Trans-esterification

Qualitative tests of Biodiesel properties



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

Gelatin 10.0 g

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


Gelatin 17.0 g

Proteose peptone (meat and

casein) 3.0 g

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


Casein 10.0 g

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

tube

Sl.no

Volof

standard a

mino acid

Sol. (ml)

Volum

e of

Distille

d

Water

(ml)

Amino

acid

concentra

tion in

each

standard

(µg/ml)

1.0

ml

alkal

ine

copper

rea

gen

t to

eac

h

Lea

ve

for

10 m

inute

s at

room

tem

per

ature

Add 0

.5 m

l of

F.C

Rea

gen

t to

eac

h

Lea

ve

for

20 m

inute

s at

room

tem

per

ature

in d

ark

O.D660

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

7 Given 1.0 ml test

solution

To be

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

COMPONENT STOCK

SOLUTION[g/100ml]

NUTRIENT

SOLUTION[ml]

KNO3 1 20

K2HPO4 0.1 20

MgSO4.7H20 0.1 20

Soil Extract 30

Micronutrient

Solution 5

Distilled Water 905

Table 3.8: Kuhls‘ media Composition- Micronutrient solution

COMPONENT STOCK

SOLUTION[g/100ml]

Applied

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 45oC. 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.



Fig 4.1: Serial dilution (A) and inoculation of soil and water samples onto LB

media(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.

(A) (B)

(A)

(B)



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.

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.

(A) (B)



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

CATALASE TEST Evolution of Oxygen

Bubbles POSITIVE

METHYL RED

TEST No change NEGATIVE

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

After 72h incubation

Fig 4.10: Saturated ammonium

sulphate and stored for 24h at -20°C



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

Fig 4.11: Pellet re-suspended in phosphate buffer to produce enzyme extract



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

tube

Sl.n

o

Volof

standard

amino

acid Sol.

(ml)

Volume of

Distilled

Water

(ml)

Amino

acid

concentrat

ion in each

standard

(µg/ml)

1.0

ml

alkal

ine

copper

rea

gen

t to

eac

h

Lea

ve

for

10 m

inute

s at

room

tem

per

ature

Add 0

.5 m

l of

F.C

Rea

gen

t to

eac

h

Lea

ve

for

20 m

inute

s at

room

tem

per

ature

in d

ark

O.D6

60

1 0 1.0 0.0 0.0

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

7 Given 1.0 ml test

solution

To be

estimated

0.15



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



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]

(A) (B)



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



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.

Fig 4.20:Inoculated with 30ml of Ulsoor lake water sample



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].

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.

(A) (B)



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

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.

Mass

of

crude

Algal

Mat

(g)

Mass of

centrifuged

Algal mat

(g)

Mass of

dried&

powdered

algal mat (g)

Amount of oil

extracted(ml) Yield (%)

14.23 13.07 12.36 7 49.21%



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.

Fig4.32: Microbial Trans-esterification - Formation of 3 clear layers.

Layer 1: Biodiesel

Layer 2: Lipase

Layer 3: Glycerol



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

added(ml)

Methanol

used (ml)

FFA

content

NaOH

used

(g)

Biodiesel

obtained(ml)

Yield(%)

Pongamia

oil

250 125 20% 8.5 90 36%

4% 8.5 225 90%

Cooking

oil

250 125 27.4% 8.5 50 20%

3.96% 8.5 212.5 85%

Rice

Bran oil

250 125 36% 8.5 40 16%

9.78% 8.5 112.5 45%

Fig 4.33:The impact of FFA content of oil on biodiesel yield by Chemical method

Biodiesel yield- 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.

Oil

Quantity

added

(ml)

Methanol

used (ml)

FFA

content

Lipase

used(µl)

Biodiesel

obtained(

ml)

Yield(%)

Pongamia

oil 250 125

20%

4%

25 165 66%

50 183 73.2%

75 182 72.8%

100 170 68%

Cooking

oil 250 125

27.4%

3.96%

25 100 40%

50 125 50%

75 122 48.8%

100 110 44%

Rice Bran

oil 250 125

36%

9.78%

25 105 42%

50 120 48%

75 126 50.4%

100 123 49.2%



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.

0%

20%

40%

60%

80%

0 50 100 150

High FFA- Biodiesel yield by Lipase method

pongamia high ffa

cooking oil

Rice bran

0%

20%

40%

60%

80%

0 50 100 150

Low FFA-Biodiesel yield by Lipase method

pongamia high ffa

cooking oil

Rice bran



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

0

10

20

30

40

50

60

70

80

Pongamia oil Ground nut oil Rice bran oil

Chemical

Lipase mediated

Biodiesel yield- chemical v/s Enzyme



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

obtained

(ml)

Methanol

added (ml)

Lipase

added (µl)

Biodiesel

Obtained

(ml)

Yield (%)

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

Properties

ASTM

standards

Pongamia

Biodiesel

Cooking oil

Biodiesel

Rice bran Oil

Biodiesel

Kinematic

Viscosity (C.st) 1.6 – 6 3.56 4.51 6.2

Flash Point(°C) > 130 153 195 260

Density

(kg/m3)

870 – 900 898 892.4 914

Table 4.8: Fuel properties of Biodiesel produced by Lipase-mediated Trans-esterification

Fuel

Properties

ASTM

standards

Pongamia

Biodiesel

Cooking oil

Biodiesel

Rice bran Oil

Biodiesel

Kinematic

Viscosity (C.st) 1.6 – 6 1.8 2.3 5.3

Flash Point(°C) > 130 147 132 214

Density

(kg/m3)

870 – 900 865 873 917

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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Production of Clean Biofuel from Microalgae - KSCST · Production of Clean Biofuel from Microalgae PPRN: 39S_B_BE_006 Department of Biotechnology, RVCE, Bengaluru i