ManendraSinghNegi PDF

EFFICIENT PRODUCTION OF BIODIESEL BY
ESTERIFICATION/TRANSESTERIFICATION REACTION
USING IMMOBILIZED ENZYME AND CHEMICAL CATALYST
Thesis
Submitted to the
G.B. Pant University of Agriculture & Technology

Pantnagar – 263145, U.S. Nagar, Uttarakhand, INDIA
By
Manendra Singh Negi

B.Sc. (ZBC)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE DEGREE OF
Master of Science
(Biochemistry)
July, 2017
ACKNOWLEDGEMENT
On the occasion of accomplishment of the present study I would like to express my

sincere gratitude to all who helped me directly or indirectly for the pursuit of this study. The
precious gift of learning is a debt that is difficult to pay. Only gratitude and veneration can be
felt. Indeed the work at my command are insufficient in form or in the spirit to express my
heartfelt, indebtedness to Dr. A. K. Verma, JRO Department of Biochemistry and Chairman
of my Advisory Committee for his sincere guidance, constant encouragement, constructive
criticism and supporting attitude throughout the investigation of the present research problem
and preparation of this manuscript.
With deep sense of gratitude I extend my warmest thanks to the members of my

advisory committee Dr. T.K. Bhattacharya, Professor and Head Department of Farm
Machinery, Dr. B.R. Singh, Assistant Professor in department of Molecular Biology and
Genetic Engineering and Dr. Ashutosh Dubey JRO, Department of Biochemistry for their
kind, cooperative, authentic Guidence, keen interest in valuable criticism during course of
investigation and thesis writing.
This thesis would not be completed without thanking people who genuinely helped me
and supported me through my dissertation. This journey wouldn’t have been so easy and
exciting without showing my gratitude to all of them.
I express my sincere thanks to Head Department of Biochemistry, Dr. Sanjeev

Agarawal for his guidance providing adequate facilities in the Department. I would like to
take this opportunity to express my gratitude for support and guidance by all faculty members
of Department of Biochemistry.
Words are too hard to express my feelings toward my seniors Pratima Ma’am Manali
Ma’am, Archana Ma’am, Himansu Sir, Vivek Sir, Sonia Ma’am for their affection, support
and inspiration during course of study. My daily work I have been blessed with a friendly and
cheerful batch mates cum family. I would like to thank Jyoti Bhatt, Jitendra Bisht and Swati
Panwar for making such a home environment even in the lab during the course of study. And
I also thank my best friends who supported and encouraged me throughout my degree
programme, who have been a source of moral Aamir khan, Asshish raturi, Divyansh
panthari, Abhay Gautam, Sumant Tarfdar, Arnav saha, Roshan jadav, Sagnik Dhar, Akash
Sinha and all other my friends.
This acknowledgment will be incomplete my love and gratitude toward my Idol my

Grand Father Mr. Chandra Singh, Father Mr. Rajendra Singh Mother Mrs. Sateshwari
Devi, Grandmother Mrs. Sushela Devi who have always been a source of inspiration and
always stood by my side. Without their support, trust and faith this wouldn’t have been
possible. Also thanks to my sister Manorama Negi, Pooja Negi, Ankita Negi my brother Amit
Negi who always supported me and loved me. I would like to acknowledge for their help,
support, and guidance. I could not have done what I was able to do without them.
Special thanks goes to my buddies of all time Pradeep, Raunak, Kirty, Anmol and
Shubham for being such a nice friends with constant support encouragement crazy suggestions
and entertainment during the ups and down of my dissertation.
Last but not the least, I express my heartfelt thanks to all beloved and respected who
helped me but could not find separate mention.
Pantnagar (Manendra Singh Negi)

July, 2017 Author
CERTIFICATE
This is to certify that the thesis entitled “EFFICIENT
PRODUCTION OF BIODIESEL BY ESTERIFICATION/
TRANSESTERIFICATION REACTION USING IMMOBILIZED
ENZYME AND CHEMICAL CATALYST”, submitted in partial
fulfillment of the requirements for the degree of Master of Science with
major in Biochemistry and minor in Molecular Biology and
Biotechnology, of the College of Post Graduate Studies, G. B. Pant University
of Agriculture & Technology, Pantnagar, is a record of bona fide work carried
out by Mr. Manendra Singh Negi, Id. No. 49364, under my supervision and
no part of the thesis has been submitted for any other degree or diploma.
The assistance and help received during the course of this
investigation has been acknowledged.
Pantnagar (A. K. Verma)

July, 2017 Chairman
Advisory Committee
CERTIFICATE
We, the undersigned, members of the Advisory Committee of
Mr. Manendra Singh Negi, Id. No. 49364, a candidate for the degree of
Master of Science with major in Biochemistry and minor in Molecular
Biology and Biotechnology agree that the thesis entitled “EFFICIENT
PRODUCTION OF BIODIESEL BY ESTERIFICATION/
ENZYME AND CHEMICAL CATALYST” may be submitted in partial
fulfillment of the requirements for the degree.
(A.K. Verma)
Chairman
Advisory Committee
(T. K. Bhattacharya) (B. R. Singh) (Ashutosh Dubey)

Member Member Member
CONTENTS
S. No. Chapters Pages
1. Introduction
2. Review of Literature
3. Materials and Methods
4. Results and Discussion
5. Summary and Conclusion
Literature Cited
Annexure
Vita
Abstract
LIST OF TABLES
Table No Table Title Page No
2.1 Fuel property of petroleum diesel and biodiesel.
2.2 Oil seed plants as potential alternative feedstock of biodiesel.
2.3 Summary of various acid catalyzed transesterification
3.1 Fat components of different Tree Oilseed
3.2 Reaction mixtures of various Acid to Acohol molar ratio
3.3 Catalyst amount for various mol% of catalyst
3.4 Reaction mixtures for transesterification of various tree seed

oils
3.5 Reaction mixtures of different volume ratio of Oil to Aqueous

Phase for Lipase catalyzed biphasic reaction
3.6 Reaction mixtures of different Substrate concentration for

Lipase activity
3.7 Reaction mixtures of different molar ratio Substrate

concentration for Lipase catalyzed esterification of Lauric Acid
and Methanol.
4.1 Summery of GC-MS analysis obtained from esterified

sugarcane bio oil.
4.2 Summery of GC-MS analysis of biodiesel obtained by alkali

catalyzed transesterification of tree seed oil.
4.3 Change in kinetic parameters of lipase after immobilization
4.4 Summery of GC-MS analysis of biodiesel obtained by lipase

catalyzed transesterification of tree seed oil.
LIST OF FIGURES
Table Figure Title Page

No No
2.1 Schemes of esterification andTranesterification
3.1 Bovine Serum Albumin (BSA) standards for estimation of protein
4.1 HClO4-SiO2 catalyzed esterification of Lauric Acid with Propanol
4.2 Esterification of lauric acid with propanol at different molar ratio
4.3 Effect of HClO4-SiO2 amount on esterification reaction
4.4 Effect of Temperature on Esterification of Lauric Acid with Propanol
4.5 Reaction progress with time at different Temperatures.
4.6 Reusability of HClO4-SiO2 up to five cycles.
3.7 Iron oxide Nanoparticle (A) In phosphate buffer (B) Under influence
of magnetic field
4.8 Hydrodynamic property of Iron Nanoparticles: (A), (B) Zeta size of

bare and enzyme bound nanoparticles and (C), (D) Zeta potential of
bare and enzyme bound nanoparticles.
4.9 4.9 Dispersity of iron nanoparticles in different solvent (A) just after
dispersion, (B) after 10 min.
4.10 Effect of Oil to Aqueous Phase Volume ratio on Lipase Activity
4.11 Michaelis-Menten curves for free and immobilized lipase
4.12 Lineweaver-Burk plot for free and immobilized lipase
4.13 Effect of Temperature on Lipase Activity
4.14 Effect of pH on Lipase Activity
4.15 Storage stability of immobilized lipase
4.16 Organic solvent stability of immobilized Lipase
4.17 Reusability of immobilized Lipase
4.18 Immobilized lipase catalyzed esterification of lauric acid with

methanol
4.19 Comparative analysis of transesterification and biodiesel yield in

alkali and lipase catalyzed method.
ABBREVATIONS
AIRF Advanced Research Instrumentation Facility
APTES 3-Aminopropyltriethoxysilane
BSA Bovine Serum Albumin
CBB Coomassie Brilliant Blue
DAG Di Acyl glycerol
FFA Free Fatty Acid
GC-MS Gas Chromatography and Mass Spectroscopy
mM mili molar
µM Micro molar
MAG Mono Acyl gycerol
TAG Tri Acyl Glycerol
TAN Total Acid Number

Introduction
Chapter 1 INTRODUCTION
Current global energy consumption is heavily dominated by fossil fuels derived

from the earth’s crust, including gaseous and liquid hydrocarbons (methane and crude
oil) and solid materials (coal), and is likely to remain so into the foreseeable future.
Indeed, world energy consumption is projected to increase by 48% from 2012 to 2040,
with over 80% of this demand derived from fossil fuels (EIA, 2016). Similar prediction
registered by International Energy Agency (IEA-2009) stating 1.6% per year increase
in global energy demand. Further, accelerating oil prices and exhaustion of oil assets
demand improved substitutes of energy from fossil fuels. Considerable efforts have
already been made in order to reduce the use of fossil fuels through first-generation
biofuels (i.e. biodiesel and bioethanol), which use crude biomass for their production.
However, these processes may be considered as non-ideal in some parts of the globe,
once they demand great areas of the plantation, and it is claimed that the reduction of
CO2 emissions does not compensate the competition with industry. Therefore, the first-
generation biofuels should be associated with more efficient generation techniques or
substituted by the so-called second-generation fuels, i.e. those from lignocellulosic
biomass waste.
An increase alarm for pollution produced by fossil fuels such as petroleum, coal
and natural gas, alternative fuels and renewable sources of energy such as biodiesel are
coming in vogue (Singh and Singh, 2010). Renewable fuels such as biodiesel are one
of the potential contributors for solving the energy problem. Biodiesel a non-toxic,
biodegradable and a low air pollutant emitting fuel over petroleum diesel
(Abbaszaadeh et al., 2012), can be produced from vegetable oil, animal oil/fats, tallow
and waste cooking oil. The leading advantage of biodiesel is that it can be described as
‘carbon neutral’. Biodiesel is a mixture of Fatty Acid Methyl Esters (FAMEs) which is
produced from renewable resources. Different procedures are currently accessible to
achieve transesterification of oils for the production of biodiesel, including chemical,
enzymatic catalysis, and supercritical alcohol. However, fats and oils are often used
interchangeably referring to the feedstock employed in biodiesel production. The raw
Introduction............
materials used for the production of biodiesel can be crude, refined or waste such as
frying oils/fats (Marchetti et al., 2008). The feedstock can also be classified as plant
derived, animal derived, microbial or waste materials (Akoh et al., 2007). More than
300 oil-bearing plants/trees have identified that can be utilized to make biodiesel
(Subramanian et al. (2005). The most popular plant derived oils applied for
production of biodiesel are: canola, Karanj, olive, palm, jatropha, coconut, cottonseed,
groundnut, peanut, rapeseed, safflower, soybean and sunflower oils (Demirbas, 2007;
Akoh et al., 2007 and Robles et al., 2009). Waste oils and fats (beef tallow, lard and
yellow grease), hemp oil, waste cooking oil, the greasy by-product from omega-3 fatty
acids production from fish oils and microalgae oil are also considered as potential
alternative feedstocks for biodiesel production (Demirbas, 2003; Marchetti et al.,
2008; Ranganathan et al., 2008 and Antczak et al., 2009). The technology for
biodiesel production from Jatropha has been developed but due to some lack of
government policies and a high cost of biodiesel production the technology could not
sustain in India. Hence it is essential to optimize biodiesel production from the other
Indian tree’s seed oil. Although biodiesel can be effectively produced by the chemical
approach, there are several associated complications, such as glycerol recovery and
removal of inorganic salts (Basri et al., 1997 and Mittelbach, 1990). Use of
biocatalysts (lipases) in the transesterification of oils for biodiesel production addresses
these (Shimada et al., 2002) and proposes an environmentally more attractive
opportunity to the conventional processes. However, a high cost of the enzymes often
presents the biggest obstacle. The key development in enzymatic processes resides in
the successful immobilization of the enzyme, which enables its easy recovery and reuse
(Balcao et al., 1996).
Heterogeneous solid catalyst also seeks attention for biodiesel in economic as well
as environmental point of view because of their ease in separation, reusability and non-
corrosive nature (Helwani et al., 2009). However, it is also associated with limitations like
slow reaction rate, high temperature, and high alcohol to oil ratio. Immobilization of
homogeneous liquid catalyst with solid support, such as alumina, silica, zinc oxide and
zirconium oxide overcomes these limitations. (Zabeti et al., 2009).
Immobilized lipase catalysis has been found to be susceptible against
deactivation by lower alcohols such as methanol and ethanol conventionally applied in
transesterification for biodiesel production (Chen and Wu, 2003 and Samukawa et
al., 2000). The degree of deactivation has been found to be inversely proportional to the
number of carbon atoms in the linear lower alcohols (Chen and Wu, 2003). Several
strategies have been adopted to overcome this problem. Samukawa et al. (2000)
suggested upholding a very low concentration of methanol (oil to methanol molar ratio
of 1:0.33) during the reaction. Nelson et al. (1996) used hexane as a diluent to prevent
the deactivation by a lower alcohol. However, diluent decreases the reaction rate and
upholding a very low concentration of methanol in the reaction mixture by a precise
control is not a practical approach for industrial production of biodiesel.
As a renewable energy source biomass can be converted into bio-oil that could
be another source of fuel. Bio-oil is a highly oxygenated, a dark brown organic liquid,
easily transportable and storable material obtained by thermal degradation (> 400 °C)
of biomass by technique pyrolysis (Bridgwater, 2012 ). Bio-oil can be burned to
produce heat and electricity or upgraded to higher value products, such as
transportation fuels or chemicals (Sadaka and Boateng, 2009). Thus it is another
probable candidate for renewable fuel alternative fossil fuel. However, fast pyrolysis
bio-oil has challenging chemical and physical properties such as low heating value,
corrosiveness, instability, and high water and solids content, that currently prevent its
economical use in most stationary and transportation fuel applications (Xiu and
Shahbazi, 2012). Various upgrading methods have been introduced to improve those
properties like hydrotreatment, supercritical water treatment, and catalytic cracking and
esterification bio-oils (Isahak et al., 2012). Esterification of bio-oil decrease viscosity,
aging rate, acidity, and corrosiveness as well as improve volatility and heating value
and miscibility with diesel fuels (Sundqvist et al., 2015). But the chemical composition
of bio-oil varies from one type of biomass to another. Bio-oil obtained from sugarcane
residue contain a large amount of free fatty acid. Hence that bio-oil could be the good
source for esterification with alcohol to obtain biodiesel for fuel compatible.
Hence in view of the above oil of three tree plant seeds Cheura (Diploknema
butyracea), Wild Apricot (Prunus armeniaca), Karanj (Pongammia pinnata) and bio-
oil of the sugarcane residues was used for the chemical and enzymatic
esterification/transesterification under following objectives:
1. Immobilization of perchloric acid on Silica gel and lipase and on Iron

nanoparticle.
2. Optimization of esterification/transesterification reaction with both the

immobilized perchloric acid and lipase enzyme.
3. Comparative analysis of esterification/transesterification of oil of seeds of

three tree species with both the immobilized perchloric acid and lipase
enzyme.
Review
of
Literature
Chapter 2 REVIEW OF LITERATURE
Energy is a fundamental necessity for fulfilling social economic and

developmental aims of current world (Vimala, 2016). Energy is considered to be one of
the vital driving forces of economic growth in all economies (Pokharel, 2006).
Subjecting this interrelation to greater study, energy is universally identified as the
most important catalyst for development, a strong two-way relationship between energy
consumption and economic development has been observed. On one hand, the level of
economic development has been depended on the energy demand, and on the other
hand economic growth, with its global competitiveness, hinges on the availability of
cost-effective and environmentally benevolent energy resources (EIA, 2010). Energy
intensity shows how efficiently energy is implicated in the economy. The energy
intensity of developing economy like India is over twice than that of the matured
economies, which has been decreasing since 1999 and is expected to continue to
decrease (OECD-2017). The largest contribution to global energy consumption given
by oil based fuel (33%), followed by coal (30%), natural gas (24%), hydroelectricity
(6%), and nuclear power (5%). Entire fossil fuels provided 87% of the world’s energy
in 2011 (Swain, 2014).
India is world’s fifth largest consumer of energy and has only 0.4% of the
world’s oil reserves. In 2012 the estimated demand of biofuel in India was 0.5 billion
gallons and it is expected to reach 6.8 billion gallons by 2022. India produces only 1%
of biofuel of global production which includes 380 million liters of ethanol and 45
million liters of biodiesel (Patni et al., 2011).
Over 50% of global energy demand is fulfilled by fossil derived fuels either
coal petroleum and natural gas (Vimala, 2016) is neither sustainable nor
environmentally benevolent. One hand elevated consumption of fossil-derived fuel
resulting exhaustion of fuel resources other hand causing savior environmental issue
through emission of greenhouse gases such as CO2, SOX, and NOX (Patil et al., 2008).
Renewable energy sources such as biofuels shift the dependence of modern world away
from fossil fuel and petroleum and contribute to the development of eco-friendly and
sustainable world (Demirbas and Demirbas, 2007). First generation biofuels
generated from agriculture and feedstock such as corn and sugarcane for ethanol,
Review of Literature............
vegetable oil such as soybean and palm oil for biodiesel, effectively contributed to
renewable energy production but also gained criticism for competing with agriculture
and food production (Havlík et al., 2011). Second generation biofuel produced from
non-agriculture biomass and agriculture waste, utilizing lignocellulosic biomass and,
agriculture and organic waste and some specialized energy crops appears to be a more
sustainable option for green energy. Although a different type of biomass requires
multiple biorefinery procedures, resulting from higher production cost (Sims et al.,
2010). Three types of biofuel produced from biomass are bioethanol, biodiesel, and
biodiesel through the process esterification, transesterification, and digestion and
upgrading of organic waste (Balat, 2007). Generally, a blend of 5% to 20% of biodiesel
(B5 to B2) is used in India (Patni et al., 2011).
2.1 Biodiesel
Biodiesel is an alternative to diesel fuel derived from animal or vegetable oil

and fat through transesterification in the form of mono-alkyl ester (Knothe, 2010). The
interesting features of biodiesel are high biodegradability, minimal toxicity, zero
emissions of pollutants (S0X, NOX, aromatic compounds) and carbon dioxide (CO2)
neutrality replace diesel fuel in many engines without major modifications (Atabani et
al., 2012). Rudolf Diesel was the first who tested vegetable oil as fuel for the engine,
about one hundred years ago (Shay, 1993) but, it’s full examination for biodiesel only
came to light in the 1980s as a renewable energy source for reducing greenhouse gas
emissions (Shahid and Jamal, 2011). In the decade of 30s and 40s vegetable oils were
also used as diesel at the time of emergency situations (Ma and Hanna, 1999). Direct
use of triglyceride as alternative fuel was challenged by high viscosity, low volatility
and reactivity and condensation due to polyunsaturation (Srivastava, 2000).
Transesterification with methanol decrease the viscosity and increases the volatility by
forming FAME (Fatty Acid Methyl Ester) and byproduct glycerol also can be used in
industry. Fuel property of biodiesel as compared to petrodiesel is compared in Table
2.1 (Demirbas, 2007). Due to a rapid elevation in a price of the petroleum and rapid
depletion of fossil fuel, biodiesel production got vast interest and boost across the world
after 2001 (Swain, 2014). Biodiesel blended with petroleum diesel creating a stable
fuel blend with premium features that could be used without needing for any engine
modifications (Hajjari et al., 2017).
Table 2.1 Fuel property of petroleum diesel and biodiesel.
PROPERTY PETROLIUM DIESEL BIODIESEL

Flash Point 325K 403K
Catane Number 40 47
Water and Sediment 0.05 max vol% 0.05 max vol%
Kinematic Viscosity (313K) 1.3–4.1 mm2 /s 1.9-6.0 mm2 /s
Copper strip Corrosion NO3 max NO3 max
Aromaticity 35 max vol % -
Distillation Temprature 511-611K -
2.2 Feedstock for Biodiesel Production
Major feedstock for biodiesel are algae, oilseeds, animal fats, and various low-
value materials such as used cooking oils, greases, and soapstocks (Moser, (2009).
Oilseeds are the traditionally used for biodiesel production includes soybean,
rapeseed/canola, palm, corn, sunflower, cottonseed, peanut, and coconut oils. Choice of
feedstock is the varied depending place, production and quality of oil like
saponification number, iodine value and cetane number of fatty acid methyl esters of
the oil (Sharma and Singh, 2010). Soybean oil is the major feedstock in the United
States whereas Europe uses rapeseed and sunflower oils. Palm oil used as major
feedstock in Southeast Asia and the Philippines uses coconut oil (Bhatti et al., 2008;
Murugesan et al., 2009). India depends on import of large quantity of edible vegetable
oils to fulfill the domestic demand. So India does not use vegetable oils derived from
rapeseed, mustard or palm oil for production of biodiesel (Patni et al., 2011).
Increasing biodiesel sector meet the demand of world energy in increasing

biodiesel industry require alternative crops as a feedstock (Ribeiro et al., 2013). Non edible
oil plants like Jatropha (Jatropha curcas), Karanj (Pongamia pinnata), Mahua (Madhuca
indica), Moringa (Moringa oleifera), Wild Apricot (Prunus armeniaca), Simarouba
(Simarouba glauca), Kokum (Garcinia indica), Cheura (Diploknema butyracea), Tung
(Aleurites fordii), Olive (Olea europaea), Jojoba (Simmondsia chinensis), Neem
(Azadirachta indica) etc, cultivated in different agro-climatic zones of India (Dhyani et al.,
2015) are potential candidate as alternative feedstock of biodiesel (Table 2.2).
Table 2.2 Oil seed plants as potential alternative feedstock of biodiesel.
TBOs Scientific Habit Flowering Harvest Fruiting Seed Oil (%)

Name Season Season Starts Yield
(Years) (Kg)
JATOPHA Jatropha Large May-Aug Aug.-Oct 3 01-02 30-40

Curcas shrub
Karanj Pongamia Medium Mar-May Jan-Mar 5 15-40 27-39

Pinnata sized tree
Mahua Madhuca Tree Mar-Apr June-Aug 8-10 10-40 40-43

indica
Wild Apricot Prunus Medium March May-June 7-8 30-173 22-25

armeniaca sized tree
Simarouba Simarouba Medium Dec-Feb Feb-April 4 10-25 60-70

glauca sized tree
Kokum Garcinia Tree Oct-Feb Mar-June 7-8 30-173 22-25

indica
Cheura Diploknema Tree Oct-Nov June-July 8-10 2-12 42-47

butyracea
Tung Aleurites Tree March Sept-Nov 3-4 1-2 30-40

fordii
Olive Olea Small tree Mar-Apr Oct 5-10 2.5-4.0 60-70

europaea
Jojoba Simmondsia Shrub Dec-Jan June 4 3-5 50

chinensis
Neem Azadirachta Tree Mar-May June-Nov 8 20-40 30

indica
Sunflower (Helianthus annus) belongs to Family Asteraceae is a high oil

content seed and average yields can produce 600 pounds of oil per acre, more than
soybean considered as an important crop for biodiesel production
(Thirumarimurugan et al., 2012). Sunflower oil was once the second feedstock for
biodiesel production after rapeseed oil. But the high cost of the sunflower oil is a
problem in order to obtain an economical biodiesel (USDA and Census Bureau,
2010). Due to good properties of Sunflower oil biodiesel in 2002, 13% of the world’s
biodiesel production contributed by sunflower oil (Sales, 2011). Biodiesel production
from sunflower oil via alkali (NaOH) catalyzed transesterification optimized with
98.181% yield under 48°C, molar ratio of methanol to oil of 6.825:1, catalyst (NaOH)
concentration 0.679 % w/w, at 290 rpm stirring for 2 hours (Mansourpoor et al.,
2012). Fuel properties of sunflower biodiesel found to be in the range of European pre-
legislation standards fulfills good quality fuel requirements (Antolın, et al., 2002).
Jatropha (Jatropha curcas) belongs to Family Euphorbiaceae, is cultivated in

Central and South America, South-east Asia, India and Africa (Koh and Ghazi, 2011).
Ease in world wild cultivation long fruiting time (50 years) with a high oil content
(>37%) valuable properties of oils (low acidity, good stability, low viscosity) and
higher a cetane number makes it a good alternative fuel with no modifications required
in the engine (Tapanes et al., 2008; Lu et al., 2009 and Divakara et al., 2010).
Biodiesel production from Jatropha in India started 2003 with estimated target of 20%
blending in petroleum diesel for biodiesel by 2010 and 30% by the year 2030 (Fairless
et al., 2007 and Kant and Wu, 2011). The expected ability of Jatropha like ability to
grow anywhere without irrigation and agricultural inputs, having high seed oil content,
drought tolerance, pest resistance, rapid growth, easy propagation, small gestation
period, adaptation to a wide range of environmental conditions were lacking scientific
agronomical study in Indian context (Singh et al., 2014). Positive claims, popularity
convinced an investor to make policy on Jatropha without scientific backup resulted in
failure of the project in India.
Wild Apricot (Prunus armeniaca), belongs to the family Rosaceae locally

called Chullu is an important tree borne oilseed distributed in the dry temperate regions
of North-Western Himalayas like valleys of Jammu and Kashmir, Chenab; Kullu and
Shimla regions of Himachal Pradesh and Garhwal hills of Uttrakhand at altitudes and in
all the three districts of Kumaon region Nainital, Almora and Pithoragarh above the
altitude of 3000m (Kureel et al., 2007). Wild Apricot kernels oil containing (>53%) oil
makes it a promising feedstock for biodiesel production (Zhang, 2003; Luo and Liu,
2007 and Ozcan, 2000). Very low acid value and water content of apricot seed kernels
oil enable direct base-catalysed transesterification for biodiesel production without acid
pretreatment and the yield of biodiesel was obtained at 88.7% (Wang and Yu, 2012). It
is expected that apricot biodiesel will increase the energy sustainability and will replace
the fossil diesel in the near future in high altitude areas of India (Gurau et al., 2016).
Biodiesel production using Wild Apricot oil via alkali-catalyzed transesterification
optimized with maximum yield under Methanol/oil molar ratio 1:6, reaction time 3h,
Catalyst (NaOH) 2%w/w and at 85°C (Singh et al., 2015).
Karanj (Pongamia pinnata) is a species of tree in the pea family, Fabaceae,

native to tropical and temperate Asia including parts of India, China, Japan, Malaysia,
Australia & Pacific Islands (Alam, 2011). Karanj fruits have viability period of one
year; seeds number varies between 9–90 kg per tree containing 27–39% of the non-
edible toxic oil used in crude drugs, veterinary medicines, soap making, fuel for the
lamp and finishing and tanning in leather industries (Patel and Sankhavara, 2016).
Karanj oil is suitable feedstock for the production of biodiesel in India. Biodiesel
produced from Karanj cost effective to find the suitability of Karanj (non-edible oil)
biodiesel as an alternative fuel to diesel (Mane, 2013). The biodiesel was developed
from oil through acid as well as alkaline esterification and NaOH was found to be a
better catalyst than KOH with maximum yield 89.5% at 9:1 molar ratio, 0.5% (w/w)
catalyst (Sharma et al., 2008). Karanj oil biodiesel blends found to be having higher
brake thermal efficiency and lower NOx emission than normal diesel (Agrawal and
Rajamanmohan, 2009).
Cheura (Diploknema butyracea), belong to Family- Sapotaceae is a tree

distributed from India to the Philippines. The species is distributed from Kumaun
eastwards to Sikkim and Bhutan (sub-Himalayan tracts and outer Himalayan ranges),
and it also occurs sporadically in the tropical moist deciduous forest (Tewari et al.,
2015). It contains high quantity (> 60%) of oils/fats in kernel including high palmitic
acid, oleic acid, linoleic acid and stearic acid. Oil/fat is used for edible purposes,
medicine, and soaps by locals (Bandyopadhyay et al., 2017). High oil content of
Cheura makes it a strong candidate for biodiesel feedstock. Biodiesel production from
Cheura oil can be a good research topic.
2.3 Biodiesel from Bio-Oil
Bio-oil is another alternative energy source besides biodiesel, derived mainly

from plant-based biomass. Bio-oil is a dark brown liquid containing hundreds of
organic compounds (alkanes, aromatic hydrocarbons, phenol derivatives and little
amounts of ketones, esters, ethers, sugars, amines and alcohols), produced by pyrolysis
(thermal cracking) of biomass at high temperature (>400˚C) and absence of oxygen
(Isahak et al., 2012) followed by rapid condensation. Bio-oil satisfies the demand of
second generation biofuel using biomass of agricultural waste and non-food crops,
wood and other organic waste (Jacobson et al., 2013). The chemical composition of
bio-oil varies with the composition of biomass. Whereas bio-oil exhibit high energy
content but still having a reservation with its properties for engine fuel. Because of its
low pH, high water content, corrosiveness, low calorific value (high oxygen content),
high viscosity bio-oil cannot be directly used as engine fuel (Bulushev and Ross,
2011). Hydrotreatment of biodiesel in presence of catalyst reduce its oxygen content
and increase calorific value (Zheng and Wei, 2011). Esterification of free fatty acid of
bio-oil with low molecular weight alcohols has been an effective approach to overcome
the deleterious properties of bio-oil. In the past few years, a number of dedicated
endeavors have been devoted to modifying and upgrade bio-oils by esterification
(Isahak et al., 2012).
2.4 Transesterification
The condensation reaction of an alcohol with fatty acid along with the removal
of water molecules forming esters called esterification (Lunagariya and Vekariya,
2017). Whereas switching of alkyl group between one alcohol and ester, called
transesterification (Romanski et al., 2012). Triglycerides are esters of long chain fatty
acid with glycerol, which can react with alcohol in the presence of a catalyst or
enzyme-producing fatty acid alkyl ester (Biodiesel) and glycerol by the process
transesterification Methanol is the preferred alcohol for producing biodiesel because of
its low cost (Leung et al., 2010).
Figure 2.1 Schemes of esterification and Tranesterification
There are basic routes of catalyzed transesterification to biodiesel production
from oils and fats. 1. Alkali catalyzed transesterification, 2. Direct acid catalyzed
transesterification, 3 Transesterification using heterogeneous catalysis and 4 Enzyme
(lipase) catalyzed transesterification.
2.4.1 Alkali-Catalyzed Transesterification
Alkali-catalyzed transesterification process that catalyzed by alkaline metal

alkoxides (Schwab et al., 1987) hydroxides (Mehar, 2006), or sodium or potassium
carbonates demonstrated high catalytic activity resulted in the production of high-
quality biodiesel at 50-70˚C. Methanol used in the process can be recovered and the
glycerin byproduct is suitable and safe for use in pharmaceutical, cosmetics, and other
applications. However, the glycerin needed to be separated and removed completely
from the biodiesel as it would form formaldehyde or acetaldehyde on combustion
(Helwani et al., 2009). Usually used the catalyst of alkali-catalyzed transesterification
are Sodium hydroxide, potassium hydroxide, and sodium methoxide among which
Sodium hydroxide is preferable owing to its intermediate catalytic activity and a much
lower cost followed by sodium methoxide and potassium hydroxide (Leung and Guo,
2006). However, a drawback associated with base-catalyzed transesterification is the
presence of significant amounts of free fatty acids in the oil that contained could not be
converted into biodiesel completely and remained as soap in huge quantities (Furuta et
al., 2004). Soaps inhibit the separation of biodiesel, glycerin and wash water (Canakci
et al., 2003). Removal of catalyst from final product is technically challenging and
surely brings additional cost. Though base catalyzed transesterification reaction is fast,
the disadvantages are mainly due to the energy intensiveness and the difficulty in
separating the glycerol from methyl esters. In addition to alkaline wastewater that
requires further treatment, the free fatty acid and water inhibit the transesterification
reaction (Ejikeme et al., 2010). Overall alkaline catalyst exhibit higher catalytic
activity and found to be suitable for biodiesel production from refined oils having
negligible free fatty acids content (< 0.5%) (Gebremariam and Marchetti, 2017 ).
2.4.2 Acid Catalyzed Transesterification
Catalytic transesterification using acid catalyst, the reaction is catalyzed by

sulphuric (Lopez et al., 2005), hydrochloric (Goff et al., 2005) or sulphonic acids
(Stern and Hillion et al., 1990). Generally, acid-catalyzed reactions are performed at
high alcohol-oil molar ratios, low to moderate temperatures, pressures and high catalyst
concentrations (Narasimharao and Wilson, 2007). These reactions require the high
alcohol-oil molar ratios to obtain good product yields within reasonable reaction time
(Freedman et al., 1984). However, methyl ester yields do not increase proportionally
with the molar ratio. For instance, sulfuric acid catalyzed transesterification of soybean
oil show sharply improvement ester formation was from 77% under methanol to oil
ratio of 3.3:1 to 87.8% with a ratio of 6:1. The only moderate improvement observed
with the higher molar ratio to a maximum of 98.4% at ratio 30:1 (Lotero et al., 2006).
Acid catalysts are attractive due to insensitiveness for free fatty acids present in the
feedstock but still been largely ignored due to slow reaction rate (Zhang et al., 2003).
However, the homogeneous acid catalyst is still appreciable and provides opportunities
for specific applications. Caprolactam-based bronsted acidic ionic liquid found to be
simpler cost-effective and less toxic and reusable acid catalyst with a higher activity for
transesterification of Jatropha oil having above 95% yield at a 140˚C temperature (Luo
et al., 2017). Transesterification catalyzed by various acid catalysts summarized in
Table 2.3.
Table 2.3 Summary of various acid catalyzed transesterification (Gebremariam

and Marchetti, 2017).
Reaction Condition
Yield
S.N. Feedstock Alcohol
Molar Temp Time (%)
Catalyst
Ratio (˚C) (hour)
1 Mixed oil Methanol 6:1 60 - H2SO4 2.5% 96.6

-
2 Soybean oil Methanol 20:1 120 5 trifluoroacetic acid 98.4

2.0 M
3 Canola oil Methanol 24:1 110 18 h AlCl3 5% 98
4 Corn oil Methanol 6:1 80 2 p-toluenesulfonic acid 100%

4 wt.%
5 Canola oil up to Methanol 9:1 200 12 Tungstophosphoric acid 90

20% FFA 3 wt.% wt.%
2.4.3 Transesterification Using Heterogeneous Catalysts
Heterogeneous catalysts are environmentally benevolent, non-corrosive, easily

recovered from the reaction mixture, have higher activity and longer catalyst lifetime
(Venkateswarulu et al., 2014). Recovery of the conventional homogeneous catalyst
after the reaction is technically difficult and large volumes of waste water are produced
to separate and clean the catalyst and the products. Conventional homogeneous
catalysts are expected to be replaced with environmentally friendly heterogeneous
catalysts (Xuejun et al., 2008). Heterogeneous catalysts can easily be separated from
reaction mixtures even by coarse filtration technique and reduce waste water pollutants.
The easier working procedure, activity, stability, ease of recovery and resistance to
poisoning provide economic benefits (Demirbas, 2007).
Heterogeneous base catalysts like calcium oxide (CaO) treated with

ammonium carbonate solution and calcinated at high temperature shows high
catalytic activity in transesterification with 93% conversion of Jatropha oil
(Huaping et al., 2006), 96.3% with Jojoba oil at high-pressure reactor (Sánchez et
al., 2016). The occlusion of alkali metal oxide clusters in zeolite cages results in an
increase in the basicity of these materials (Philippou et al., 2000). Methyl ester
production using NaX faujasite zeolite catalyst was higher than that of the pure
zeolite. Hydrotalcites (HT) are anionic and basic clays known as layered double
hydroxides (LDH) with the formula Mg6Al2(OH)16CO3.4H2O. Transesterification of
rapeseed oil with methanol using Mg-Al HT achieved about 90.5% yield. Alkaline
earth oxides are potential base catalysts for use in triglyceride transesterification
(Shumaker et al., 2008). Table 2.4 summarizes different types of heterogeneous
catalysts supported on porous substrates and their conversion rate at different
operating parameters (Helwani et al., 2009).
Solid base catalyst are also found sensitive to free fatty acid and water content
limiting its application to refined oils but not crude oils (Melero et al., 2009).
Replacing strong liquid acids with solid acid catalysts can eliminate the
corrosion problems and consequent environmental hazards posed by the liquid acids
(Zhang et al., 2014). Solid acids are able to esterify FFAs to FAME in parallel with
transesterification of major TAG components, without saponification, and reduce the
numbers of pretreatment and processing steps present in the case of solid base catalyzed
transesterification (Kouzu et al., 2011). However, solid acid catalysts for
transesterification are expected to be possibly having low activity and adverse side
reaction (Lotero et al., 2005). Zeolites, heteropoly acids, functionalized zirconia and
silica, tungsten oxides, sulphonated zirconia, Amberlyst, sulphonated saccharides,
Nafion1 resins, organosulphonic functionalized mesoporous silica etc are solid acid
catalysts that were exploited for use in esterification and transesterification reactions
(Helwani et al., 2009).
Table 2.4 Different heterogeneous catalysts used for transesterification of

vegetable oils.
Reaction Parameters
Conversion,
Vegetable oil Catalysts
Methanol : Time Temp %
Oil (molar) (hour) ˚C
Blended Mesoporous silica loaded 8 5 220 96

vegetable oil with MgO
Soybean oil WO3/ZrO2, Zirconia- 40 20 200-300 90

alumina and
sulphated tin oxide
Soybean oil La/Zeolite beta 14.5 4 160 48.9
Palm oil Mg–Al–Co3 (Hydrotalcite) 30 6 100 86.6
Rapeseed oil CaTiO3, CaMnO3, 6 10 60 90

Ca2Fe2O5,CaZrO3,
CaO-CeO2
Soybean oil MgO.MgAl2O4 3 10 65 57
Sunflower oil CaO/SBA-14 12 5 160 95
Soybean oil MgO, ZnO, Al2O3 55 7 70-130 82
Soybean oil CaO, Ca(OH)2, CaCO3 2 65 99
Jatropha curcas CaO 9 2.5 70 93

oil
Rapeseed oil Mg–Al HT 6 4 65 90.5
Soybean oil CaO, SrO 12 3 65 95
Cotton seed oil Mg–Al–CO3 HT 6 12 180-210 87
Immobilization of acids catalysts on solid silica support has been proved an
efficient mechanism enabling their reusability, easy handling, large-scale operation,
preventing associated environmental hazards (Kaur et al., 2015). Perchloric acid
immobilized on silica support (HClO4-SiO2) observed is an extremely efficient
heterogeneous acid catalyst system for esterification reaction (Chakraborti et al.,
2009), synthesis of thioesters (Bandgar et al., 2010) and many other condensation
reactions of organic synthesis (Wu et al., 2010). HClO4-SiO2 exhibit superior catalytic
activity, high conversions, operational simplicity, enhanced reaction rates, cleaner
reaction profiles, ease of isolation of products and catalyst reuse. The potential of
HClO4-SiO2 can be applied on esterification/transesterification of bio-oil.
2.4.4 Enzyme Catalyzed Transesterification
Transesterification catalyzed by biological catalyst called enzymes is carried out

at a lower temperature, having no issues of saponification, purification, washing, and
neutralization and has a more preferred method for biodiesel production (Leung et al.,
2010). The enzymatic transesterification is typically catalyzed by lipases Candida
antartica (Royon et al., 2007), Candida rugasa (Linko et al., 1998), Pseudomonas
cepacia (Shah and Gupta, 2007), Pseudomonas spp. or Rhizomucar miehei (Ming et
al., 1999) and immobilized lipase (Lipozyme RMIM) (Bernardes et al., 2007). The
yield of biodiesel in enzymatic transesterification can vary according to on the type of
enzyme used. Enzymatic transesterification of soybean oil with methanol and ethanol
using a commercially available immobilized lipase (Lipozyme RMIM) (Bernardes et
al., 2007) with 60% yield at optimum conditions in a solvent-free system using
ethanol/oil molar ratio of 3.0, temperature of 50˚C and enzyme concentration of 7.0%
(w/w). Shah and Gupta (2007) showed a yield of as high as 98% was obtained using
P. cepacia lipase immobilized on celite at 50.8˚C in the presence of 4-5% (w/w) water
for 8 h. The enzyme-catalyzed system requires a much longer reaction time than the
base catalyzed systems. Transesterification of algae oil with commercial lipase
(Lipolase 100) was optimized with 96.9% yield after 26 hours at 30˚C, molar ratio 3:1
and lipase amount 10 % (w/w) (Makareviciene et al., 2017).
Both extracellular and intracellular lipases are able to effectively catalyze the
transesterification of triglycerides in either aqueous or non-aqueous system
(Stamenkovic et al., 2007). Intracellular lipase implies an attractive process for the bulk
production of biodiesel and polyesters using whole cells as biocatalysts (Iftikhar et al.,
2008 and Ghaly et al., 2010). Microorganisms like Candida antarctica, Rhizopus
chinensis, Rhizopus oryzae and Saccharomyces cerevisiae, (Fukuda et al., 2008, 2009
and Robles et al., 2009) able to be immobilized spontaneously on certain supports give
advantage over the costly process of purification and immobilization required with
extracellular lipase (Fukuda et al., 2001). Intracellular lipases slow down the
transesterification process (Robles et al., 2009), but its higher stability increases the
conversion efficiency by longer use (Klibanov, 1983 and Ranganathan et al., 2008).
Rhizopus oryzae whole cells efficiently catalyze the tranesterification of vegetable oils
and Rhizopus chinensis whole cells are efficient in the transesterification of short chain
fatty acids (Qin et al., 2008). Rhizopus chinensis also show the higher transesterification
activity than Candida antarctica, Candida rugosa, Pseudomonas cepacia and porcine
pancreas lipase for soybean bean oil in a solvent free system (Qin et al., 2008).
The presence of free fatty acids did not affect the lipase activity (Narasimharao
et al., 2007). Babassu cake with Rhizomucor miehei lipase activity is capable of
transesterification and esterification reactions simultaneously in acid oil getting 85%
yield after 96 h at 40 °C and molar ratio 6:1 (Aguieiras et al., 2017). Enzymatic
reactions are highly specific and chemically clean. The problem associated with lipase-
catalyzed transesterification process is the high cost of the lipases (Royon et al., 2007).
To deal with the inhibitory effect of most lipases to lower alcohol, a typical
strategy is to feed the alcohol into the reactor in three steps of 1:1 mole ratio each
(Shimada et al., 1999). The reactions rate is very slow in nature, three step sequence
takes 4 to 40 h or more to complete. The reaction conditions become modest as the
temperature was raised, for example, from 35 to 45˚C (Saka and Kusdiana, 2001).
2.4.4.1 Lipase Immobilization
Biodiesel production using free lipase is costly with slow reaction rate
(Fjerbaek et al., 2009). Immobilization of lipase by physically localizing in a certain
support improves lipase stability and reusability enable its repeated and continuous use
(Tan et al., 2010). Lipase from various sources either microbes or animals such as
porcine pancreatic, Burkholderia cepacia, Pseudomonas spp., and Candida spp. can be
subjected to immobilization (Dumri and Hung, 2014). Enzymes can be immobilized
through various methods like adsorption (on acrylic resin, celite, and anion resin),
covalent bonding (using silica-PVA and styrene-divinylbenzene), entrapment (using
hydrophobic sol-gel support), and cross-linking (using glutaraldehyde cross-linker)
(Ahmad and Sardar, 2015). Immobilised Pseudomonas fluorescence lipase on
macroporous polypropylene via physical adsorption was found to be active biocatalyst,
followed by immobilised Pseudomonas cepacia lipase with an ester yield of 98 mol%
after 70 hour at 30 °C, water content = 0.5 mg water/mg of biocatalyst and enzyme
loading 600 mg lipase /g support (Salis et al., 2008). Pseudomonas cepacia lipase
immobilized on accurel support via cross-linking used for biodiesel production from
Madhuca indica oil gave 98% yield at 6 hours using 50 mg of lipase (Kumari et al.,
2007). Thermotolerant lipase isolated from Bacillus aerius was immobilized on
inexpensive support silica gel matrix used for biodiesel production from castor oil in a
solvent free system at 55°C gave the highest yield was up to 78.13% (Narwal et al.,
2015). Immobilized lipase enzyme (CALB - DILBEADSTM (10000)) used for
biodiesel production from waste cooking oil giving 99.71% conversion with 5% (w/w)
enzyme concentration, 3:1 methanol:oil molar ratio and 8 hours reaction time at 37°C
and 150 rpm (Chourasia et al., 2015). Christopher et al (2014) reviewed enzymatic
esterification of many oils with lipase listed in Table 2.5.
Table 2.5 Lipase catalyzed esterification of different oils for biodiesel production
Catalytic Condition Yield

S.N. Oil Source Organism Alcohol
Temp. (˚C) Time (hour) (%)
1 Jatropha oil E. aerogenes Methanol 55 48 94

2 Sunflower oil Pseudomonas spp Methanol 45 5 79
3 Rice bran oil Cryptococcus spp Methanol 30 96 80
4 Rapeseed oil C. rugosa 2-Ethyl-1-hexanol 37 94 97
5 Soybean oil C. rugosa Methanol 35 90 80
6 Sunflower oil R. miehei Methanol 40 48 95.5
7 Corn oil P. expansum l Methanol 40 24 86

8 Soybean oil P. fluorescens Methanol 35 90 80
9 Microalgae Candida sp Methanol 38 12 98
10 Plant oils R. oryzae Methanol 35 5 80-90
Methanol 67
11 Soybean oil P. cepacia 37 1
Ethanol 65
12 Palm oil R. oryzae Methanol 35 96 55
Nanoparticles found to be very proficient support for enzyme immobilization,
because of their ideal characteristics including specific surface area, mass transfer
resistance and effective enzyme loading (Ahmad and Sardar, 2015). Brownian movement
of enzyme bound nanoparticles solves diffusion problem with the macromolecular
substrates by dispersing in aqueous solutions, showing better enzymatic activities.
Magnetic nanoparticles such as Iron Oxide Nanoparticle hold the additional advantage of
easy separation using an external magnetic field (Gupta et al., 2011). Studies suggest
binding with nanoparticle prevents enzyme unfolding and increases enzyme stability
(Gupta et al., 2011 and Li et al., 2017). Lipase from Pseudomonas cepacia covalently
attached to polydopamine coated magnetite nanoparticles employed in the conversion of
soybean oil into biodiesel, by stepwise addition of methanol show yield of 90% was
achieved at 37˚C and allow efficient recycling with the use of an external magnetic field
(Andrade et al., 2016). Lipase from genetically modified Aspergillus niger immobilized
on Fe3O4 magnetic nanoparticles, using glutaraldehyde cross-linking found the yield of
89% and 81% biodiesel with two different amino functionalizing reagents APTES and
MMPTES respectively, at 12 hours with molar ratio 1:6, 45˚C temperature (Thangaraj et
al., 2016). Candida rugosa lipase co-immobilized magnetic nanoparticles using metal
coordinated hydrogel nanofibers shows 8-fold higher activity at pH 10.0 and nearly 1-fold
in a 50 ◦C water bath after 30 min, compared to free enzyme, with excellent storage
stability (80% residual activity after13 days) (Li et al., 2017).
Materials
and
Methods
Chapter 3 MATERIALS AND METHODS
The present study was conducted in the Department of Biochemistry, College of

Basic Sciences and Humanities, G. B. Pant University of Agriculture and Technology,
Pantnagar, Uttarakhand.
3.1 Materials
Following materials were used in the present study.
3.1.1 Oil Samples
In the present investigation oils of three tree species procured from local market.
Cheura (Diploknema butyracea, Family: Sapotaceae) seed oil bought from local market of
Pithoragarh district in its popularly known form as “Phulwara Ghee” an alternative for
ghee or butter used by the local people. Wild Apricot (Prunus armeniaca, Family:
Rosaceae) kernel oil bought from Sos Organics Pvt. Ltd., in cold, pressed from
commercially available in the market. Karanj (Pongammia pinnata, Family: Fabaceae)
seed oil bought from Devinez Lvt. Ltd., in undiluted pure natural form. Commercially
available edible refined Sunflower (Helianthus annuus, Family: Asteraceae) seed oil
bought from the local market. Average molecular weight and composition of oils
calculated from their fat component described table 3.1. Bio-oil obtained by pyrolysis of
sugarcane residue procured from Department of Farm Machinery, College of Technology,
G.B. Pant University of Agriculture and Technology, Pantnagar. This bio-oil contains long
and short chain free fatty acid used for esterification.
3.1.2 Chemicals
All the chemicals and solvents used in the experiments were of molecular biology
grade and analytical grade. These were obtained from standard manufacturers as Merck
(India) Ltd., Himedia laboratories Pvt. Ltd., SRL (Sisco research laboratories) Pvt. Ltd.
3.1.3 Glassware and Equipments
All the glassware and plastic wares viz. glass beakers, conical flasks, round
bottom flasks, flat bottom flasks, reflux condenser, culture tubes, glass pipette,
eppendorf tubes, micropipette tips, test tubes, etc. utilized were supplied by Scott and
Materials and Methods ...........
Duran, Brand (Germany) , Borosil glass works Ltd., Tarson product Pvt. Ltd., Axygen
scientific Pvt. Ltd. etc. The list of equipment used during the experimentation is given
in Annexure I.
Table 3.1 Fat components of different Tree Oilseed
Oils Samples Cheura oil Wild Apricot oil Karanj oil Refined
(%) (%) (%) Sunflower oil
Components (%)
Myristic acid - 1.18 --
Palmitic Acid 56.6 3.31 11.65 5
Stearic Acid 3.6 2.68 7.5 6
Oleic Acid 36.0 73.58 51.59 30
Linoleic Acid 3.8 19.26 16.64 59
Linolenic Acid - - 5.73 -
Dosocasnoic Acid - - 4.45 -
Eicosenoic Acid - - 1.35 -
Tetracosanoic Acid - - 1.09 -
Average Mr (g mol-1) 267.37 280.65 282.91 280.09
References (Ghosh et (Bachheti et al., (Bobade, et (Alfred T.,

al., 2007) 2007) al., 2012) 2002)
3.2 Experimental Design
The experiments were designed in following orders to fulfill the objectives.
1. Immobilization of perchloric acid (HClO4) on silica gel (SiO2)
2. Kinetic analysis of esterification/ transesterification of Dodecanoic acid (Lauric

acid), tree-seed- oils and bio-oil by HClO4-SiO2.
3. Alkali-catalyzed transesterification of Tree-seed- oils.
4. Immobilization of lipase on Iron Oxide nanoparticles and characterization.
5. Kinetic analysis of immobilized lipase and it's used for transesterification of Tree
seed oil.

3.3 Immobilization of Perchloric Acid (HClO4) on Silica Gel (SiO2)
Immobilization of perchloric acid on silica gel was done by the method

descriced by Chakraborti et al. (2009).
Reagents
1. Silica Gel (SiO2) : 23.75 g (mesh no. 60-200)
2. Perchloric Acid (HClO4) : 12.5 mmol (1.25 g or 1.78 mL from a 70%

solution of HClO4)
3. Diethyl Ether (Et2O) : 50mL
Procedure
Silica gel (23.75g) suspended in 50 mL of diethyl ether (Et2O) and added 1.5
mmol perchloric acid (HClO4) to the mixture and stirred magnetically for 30 min at
room temperature. The Et2O was removed under reduced pressure in a rotary
evaporator and the residue heated and dried at 100 ˚C for 72 h under vacuum. The
resulting free flowing powder stored in a desiccator and kept in dark.
3.4 HClO4-SiO2 Catalyzed Esterification of Dodecanoic acid and Propanol

(Preliminary Experiment)
Reagents
1. Dodecanoic acid (Mr=200.32 g/ mol) : 8.91g (44.46 mmol)
2. 1-propanol (Mr=60.09g/mol, 99.9%, 19g) : 20mL (266.67 mmol)
3. Catalyst (HClO4-SiO2) : 5.21g
Procedure
A reaction mixture was prepared by dissolving 8.91g of Dodecanoic acid in 20

mL 1-propanol in a 100mL conical flask and 1 mol% (5.21g), of catalyst, was added.
The esterification reaction was carried out at 80˚C on a magnetic stirrer at 600 rpm for
4 hours. Two control reactions also carried out in presence of only silica gel (SiO2) and
one without a catalyst. Reaction progress was analyzed by measuring the decrease in
free fatty acid per mL of the reaction mixture by titration with KOH.

3.4.1 Measurement of Free Fatty Acid
Reagents
1. 0.1N Oxalic acid : 0.63g of Oxalic acid was dissolved in 50 mL of distilled

water and the volume was made up to 100mL.
2. 0.1N KOH solution : 5.611g of KOH was dissolved in 900mL distilled water
and solution was standardized by 0.1N oxalic acid and
required volume make up is done to the 0.1N
concentration.
3. 1% Phenopthaliein : 1g of phenopthalein was dissolved in 100mL of 95%

ethanol.
Procedure
The acid number of the reaction mixture was measured by KOH titration using
phenolphthalein indicator. One mL of the sample was taken in 50 mL conical flask and
diluted with 4 mL of isopropanol. 3-4 drops of phenolphthalein indicator were added
and titrated with 0.1N KOH. The volume at which light pink color appears was
considered end point titer volume. Reaction progress was calculated through following
formula:-

Reaction Progress (%) = 100 -

X 100
3.5 Kinetic Parameters for HClO4-SiO2 Catalyzed Esterification Reaction
Following parameters were investigated for silica supported parchloric acid

catalyzed esterification reaction.
1. Acid to alcohol molar ratio
2. Reaction temperature
3. Catalyst amount
4. Reaction progress with time
3.5.1 Effect of Acid to Alcohol Molar ratio on Esterification Reaction
The various molar ratio of Dodecanoic acid and propanol was taken for
standardization of esterification reaction. Dodecanoic acid dissolved in 20mL 1-propanol in
the 100ml conical flask as described in Table 3.2. The esterification reaction was carried

out at 80˚C on a magnetic stirrer (600 rpm) for 3 hours. Reaction progress was analyzed by
measuring the decrease in acid number per mL of the reaction mixture as described in
section 3.4.1.
Table 3.2 Reaction mixtures of various Acid to Acohol molar ratio
MOLAR DODECANOIC ACID PROPANOL

S/N
RATIO grams mol mL Mol
1 1:3 17.82 0.089 20 0.267
2 1:4 13.37 0.067 20 0.267
3 1:5 10.69 0.053 20 0.267
4 1:6 8.91 0.044 20 0.267
5 1:7 7.64 0.038 20 0.267
6 1:8 6.68 0.033 20 0.267
7 1:9 5.94 0.028 20 0.267
8 1:10 5.34 0.0267 20 0.267
3.5.2 Effect of HClO4-SiO2 Amount on Esterification
Esterification reaction carried out with optimum substrate molar ratio with
80˚C temperature and different mol% (0.5 mmol/gram) of catalyst was taken for
esterification reaction as described in Table 3.3. The reaction was carried out for two
hours and reaction progress was calculated as described in section 3.4.1.
Table 3.3 Catalyst amount for various mol% of catalyst
CATYLYST
S/N
mol% mmol Weight (g)
1 1.00 2.66 5.32
2 0.50 1.33 2.66
3 0.25 0.66 1.33
4 0.125 0.33 0.66
5 0.10 0.266 0.33

3.5.3 Effect of Reaction Temperature on Esterification Reaction
To check the effect of reaction temperature on the esterification of Dodecanoic

acid with 1-propanol was carried out at different temperature (70, 80, 90 and 100 ˚C)
with optimum substrate molar ratio and catalyst amount. The reaction mixture was
placed on a magnetic stirrer (600rpm). The reaction progress was checked in one mL of
sample withdrawn from the reaction mixture at half hour interval and acid number per
mL of reaction mixture assayed through KOH titration as described in section 3.4.1.
3.5.4 Reusability of HClO4-SiO2 Catalyst System
The reusability of HClO4-SiO2 was determined by each cycle where the HClO4-
SiO2 catalyst was separated by filtration, washed 3-4 time with di-ethyl ether (Et2O),
dried and reintroduced into a fresh reaction mixture and this procedure was repeated up
to five cycles in the same optimum conditions.
3.6 HClO4-SiO2 Catalyzed Esterification of Bio-Oil
Esterification of bio-oil obtained from sugarcane residues was done using

optimum parameters (molar ratio, temperature, catalyst amount, time) for HClO4-SiO2
catalyst system. 10g of bio-oil mixed with 6 mL of 1-propanol. Esterification reaction
carried out in a 250mL flat bottom flask connected with reflux condenser by heating on
temperature controlled magnetic stirrer (600 rpm) at 90˚C, for 2 hours. Conversion of
oil to biodiesel (fatty acid propyl esters) was analyzed by GC-MS.
3.7 HClO4-SiO2 Catalyzed Transterification of Tree Seed-Oils (Preliminary

Experiment)
Oil Samples:-
1. Cheura (Diploknema butyracea) : average Mr.-267.37
2. Wild Apricot (Prunus armeniaca) : average Mr.-280.65
3. Karanj (Pongammia pinnata) : average Mr.-282.91
4. Sunflower (Helianthus annuus) : average Mr.-280.086
Procedure:-
For transesterification 10g of each oil sample mixed with the appropriate
amount (described in table) of methanol in the molar ratio 1:6 and 1:9 as described in

Table 3.4. Transesterification reaction carried out in a flat bottom flask connected with
reflux condenser on a magnetic stirrer (600-800 rpm) keeping same reaction parameters
optimized for esterification in section 3.6.
Table 3.4 Reaction mixtures for transesterification of various tree-seed- oils
OIL METHANOL(1:6) METHANOL(1:9)

S/N TREE SPECIES
grams mol mL grams mol mL Grams Mol
1 Cheura 10 0.037 8.88 7.11 0.222 13.47 10.78 0.333

(Diploknema butyracea)
2 Wild Apricot 10 0.0363 8.72 6.98 0.218 13.10 10.48 0.324

(Prunus armeniaca)
3 Karanj 10 0.035 8.41 6.73 0.21 12.61 10.09 0.315

(Pongammia pinnata)
4 Sunflower 10 0.0357 8.56 6.85 0.214 12.98 10.38 0.324

(Helianthus annuus)
3.8 Alkali Catalyzed Transesterification of Tree Oils (Preliminary Experiment)
Base catalyzed transesterification was done as describes by the method

Freedman et al. (1986) with slight modification in temperature molar ratio and time.
Procedure
For base catalyzed transesterification 1 % (w/w), NaOH catalyst was dissolved

in required amount of methanol and reaction mixture prepared as described in table 3.4.
Transesterification reaction carried out in 250mL flat bottom flask connected with
reflux condenser on a magnetic stirrer (600-800rpm) at 70˚C for 3 hours. After reaction
completion, the flat bottom flask subjected to running water to cool down and stop the
reaction. The reaction product kept overnight in a separatory funnel to separate glycerol
as a lower layer from fatty acid methyl ester (biodiesel). Conversion of oil to biodiesel
(FAME) was analyzed by GC-MS.
3.9 GC-MS Analysis of Biodiesel Samples
All esterified or transesterified samples were analyzed by GC-MS in Advanced

Instrumental Research Facility (AIRF) JNU, New Delhi. Samples were diluted 10 times
prior to analysis. Phulwara biodiesel diluted 100 times due to its high viscosity and
concentration. 1 µL sample of biodiesel was injected GC-MS analysis. FAME analysis
was done using RTX-5 MS equipped with an auto sampler, inlet and detector was used
under following condition column oven temperature 140˚C, injection temperature 260
˚C, pressure 107.4KPa, total flow 101.0mL/min, column flow 1.21mL/min, purge flow
3.0mL/min linear velocity 41.6cm/sec and split ratio 80.0. The compounds were
identified against NIST database.
3.10 Preparation of Immobilized Enzyme (Lipase- Fe3O4)
Lipase was immobilized on iron oxide nanoparticle through glutaraldehyde

linkage by amino functionalizing the nanoparticle.
3.10.1 Synthesis of Iron Oxide (Fe3O4) Nanoparticle
Iron oxide nanoparticle synthesized by previously reported co-precipitation

method (Shaw et al. 2006) and characterized (Juyal et al., 2016).
Reagents
1. Ferric chloride anhydrous (FeCl3 Mr. 162.2) : 14.074g (86.6 mmol)
2. Ferrous chloride dihydrate (FeCl2.2H2O Mr. 162.78) : 7.069g (43.42 mmol)
Procedure
Iron oxide (Fe3O4) precursor’s materials of FeCl2·2H2O and FeCl3 (molar ratio
1:2) were dissolved in 600 mL deionized water (heated at 30°C) under the protection of
continuously flowing nitrogen gas. The mixture was continuously stirred (600-800
rpm) and Ammonia solution (25%) added drop wise until the pH reached to 10. The
obtained black solution was precipitated and resulting Fe3O4 magnetic nanoparticles
were collected by applying external magnetic field. The particles were washed with
deionized water and ethanol. Finally, the resultant magnetic nanoparticles were
lyophilized under vacuum for 48 hours to form a powder.
3.10.2 Amino functionalization of Iron Oxide Nanoparticles
Reagents
1. Fe3O4 Nanoparticles : 0.5g
2. 3- aminopropyltriethoxysilane (APTES) : 0.3mL
3. Ethanol : 10mL

Procedure
Fe3O4 magnetic nanoparticles (0.5 g) were dispersed in 10 mL of ethanol

through ultrasonication for 10 minutes. APTES solution (0.3 mL) was added and the
mixture was again dispersed by ultrasonication sufficiently. Then the mixture kept in
rolling at 25-30°C for 3 hours. The obtained amino functionalized magnetic
nanoparticles were recovered by external magnetic field and rinsed with deionized
water and ethanol to remove the excess organosilane reagents.
3.10.3 Immobilization of Lipase on Iron Oxide Nanoparticle
Preparation of solutions
1. Glutaraldehyde Solution (10%) 8 mL of 25% glutaraldehyde solution obtained

from Thomas Baker Pvt. Ltd. was diluted to 20 mL by deionized water.
2. Phosphate Buffer (0.1M)
Solution 1: 0.1M Na2HPO4 : 7.1g of disodium hydrogen phosphate was dissolved in

200 mL of distilled water and volume was made up to 500mL.
Solution 2: 0.1M NaH2PO4 : 204g of sodium dihydrogen phosphate was dissolved in

50 mL of distilled water and volume was made up to 200mL.
Solution 2 was added solution 1 until pH of mixture adjusted to 7.4.
3. Lipase Solution (1mg/mL) 50 mg of Lipase Steapsin ex. Microorganism obtained

from Sisco Research Laboratories Pvt. Ltd. was dissolved in little amount of
phosphate buffer and volume was made up to 50mL
Procedure
Amino functionalized iron oxide nanoparticles were activated by addition of 20

mL coupling reagent glutaraldehyde (10%) solution. The mixture was kept on rolling at
25°C for 2 hours. Finally, the activated iron oxide nanoparticles were recovered by
external magnetic field and washed well with deionized water to remove the unreacted
glutaraldehyde solution. The obtained nanoparticles were applied for lipase
immobilization by the addition of 10 mL lipase solution in phosphate buffer. The
mixture was shaken sufficiently at 25°C and incubated at 4°C overnight. Then the
lipase immobilized magnetic nanoparticles recovered, washed with phosphate buffer
solution for several times.

3.10.4 Characterization of Bare Nanoparticle and Enzyme linked Nanoparticle
Bare and enzyme linked nanoparticle characterization was done by following

techniques.
a. Zeta Potential
Zeta potential of Iron Oxide (Fe3O4) nanoparticle was analyzed in Department

of M.B.G.E., C.B.S.H., Pantnagar. The iron oxide nanoparticle (0.5g) was first
dissolved in 10mL of distilled water and then centrifuged. After centrifugation, the
supernatant was taken in the cuvette and put in the zeta-sizer for determining zeta-
potential.
b. Dispersity of Nanoparticles in Different Solvents
To examine the dispersity of bare nanoparticle and enzyme coated nanoparticle

in various solvents, 25 mg of nanoparticles were dissolved in 5 mL of organic solvents
methanol, ethanol, n hexane and phosphate buffer and their sedimentation was
examined after 2 minutes and 10 minutes of dissolving.
3.10.5 Immobilization Efficiency and Activity Recovery
Amount of lipase bounded on the particles was measured by quantifying protein in the
solution before and after immobilization.
a. Protein Estimation
Protein estimation was done by spectrophotometric method (Bradford 1976).
Solutions
1. Bradford Dye
Coomassie Brilliant Blue (CBB) G-250 : 10mg
Ethanol : 5 mL
Ortho-Phosphoric Acid (85%w/w) : 10 mL
Bradford reagent was prepared by dissolving 10mg of CBB G-250 in 5 mL of

ethanol. Then 10 mL of 85% orthophosphoric acid was added and made volume up to
100 mL. The resulting solution was filtered through Whatman Filter Paper no. 1 in and
stored in dark.

2. Phosphate Buffer (0.1M, pH 7.4) was prepared as described in section 3.11.2
3. Bovine Serum Albumin (BSA) stock (100µg/mL) was made by dissolving 5mg BSA
in small amount of phosphate buffer and volume was made up to 50 mL.
Procedure
To make BSA standard curve appropriate aliquots (100, 200, 400, 600,800 and
1000 µL) of BSA solution (100µg/mL), taken in different test tubes in duplicate. The
volume was made up to 1mL with phosphate buffer. 200 µL of lipase solution (before
and after immobilization) also taken as unknown sample and made up volume up to 1
mL. then 5 mL of Bradford dye was added to each tube and mixed thoroughly. The
absorbance of each sample recorded at 595nm against a blank containing phosphate
buffer (1ml) and Bradford dye only (5 mL). A standard curve of A595 versus µg protein
plotted for BSA (figure 3.1). The amount of protein in lipase solution estimated from
the BSA standard curve.
0.3
0.25
Absorbance (A595)
0.2
y = 0.0026x
R² = 0.9978
0.15
0.1
0.05
0
0 20 40 60 80 100 120
BSA amount (µg)
Figure 3.1 Bovine Serum Albumin (BSA) standards for estimation of protein

b. Immobilization Efficiency
Immobilization efficiency was calculated by following formula (Hu et al., 2009).

IE (%) =
X 100
Where
Ci = initial is the amount of the lipase protein existing in solution before

immobilization
Cf = final is the amount of the lipase protein remaining in solution after

immobilization.
c. Lipase Assay
The activity of free and immobilized lipase was measured by hydrolysis of

refined sunflower oil.
Reagents
1. Phosphate buffer (0.1M) : As described in section 3.11.2
2. Refined sunflower oil : 10mL
3. Potassium Hydroxide (0.1M) : As described in section 3.4.1.
Procedure
2.5 mL of refined sunflower oil mixed with 2 mL of phosphate buffer and 0.5
mL of enzyme (immobilized or free) then incubated for two hours. Hydrolysis of
triglycerides analyzed, by measuring amount of free fatty acid in the reaction mixture
by KOH (0.1M) titration as described in section 3.4.1.
Lipase activity was calculated by following formula

.
Lipase activity A=
()
Where
0.1 is normality of KOH
1000 is conversion factor to micromole

d. Activity Recovery
Activity recovery was calculated from the following equation (Hu et al., 2009).

R (%) = X 100

Where
R= Activity recovery
Ao = Activity of free lipase solution before immobilization
A= Activity of free lipase solution after immobilization
Ai = Activity of immobilized lipase
3.11 Kinetic Analysis of Immobilized Lipase
Hydrolysis and methanolysis (transestrification) of triglycerides of tree-seed-

oils are similar kind of biphasic reaction containing one hydrophilic (water or
methanol) and one hydrophobic (oil) substrate and reaction occur at the interphase in
emulsion form of two substrate.
Hence following kinetic parameters of lipase activity were studied for hydrolysis of
triglyceride and their optimum value applied for transesterification (methanolysis)
reaction.
1. Oil to Aqueous Phase volume ratio
2. Effect of Substrate Concentration on Enzyme Activity
3. Optimum Temperature
4. Optimum pH
3.11.1 Effect of Oil to Aqueous Phase Volume ratio on Lipase Activity
For 5 mL reaction mixture different oil to aqueous phase volume ratio prepared
in 100 mL conical flasks as described in table 3.5 and lipase bound nanoparticle
(5.25U/0.5mL) was added to each reaction mixture. Hydrolysis reaction was carried out
at 37˚C for two hours in an incubator shaker (170 rpm). Hydrolysis was analyzed by
estimating free fatty acid in reaction mixture through KOH titration as described in
section 3.4.1 and activity was calculated using the same formula in section 3.10.5c.

3.11.2 Effect of Substrate Concentration on Lipase Activity
Reagents
1. n-Hexane : 100mL
2. Refined Sunflower Oil (2M) : Prepared by diluting 28.009g refined

sunflower oil to 50 mL by n-Hexane
3. Phosphate Buffer (0.1M) : Prepared as described in 3.10.2
Procedure
Different concentration of oil phase (Substrate) prepared by diluting the refined

sunflower oil with n-hexane. The reaction mixtures were prepared as described in table
3.6. Hydrolysis reaction carried out at 37˚C for two hours. Reaction progress and lipase
activity analyzed as described in in section 3.10.4. Km and Vmax value for free and
immobilized lipase were determined by Michaelis Menten and Lineweaver Burk plot
Table 3.5 Reaction mixtures of different volume ratio of Oil to Aqueous Phase for
Lipase catalyzed biphasic reaction
Oil Phase Aqueous Phase

S/N Volume Ratio
Oil Volume(mL) Phosphate Buffer (mL) Enzyme(mL)
1 1:6 0.714 4.12 0.5
2 1:4 1.00 3.50 0.5
3 1:2 1.667 2.833 0.5
4 1:1 2.5 2.0 0.5
5 1:0.8 2.778 1.722 0.5
6 1:0.6 3.125 1.375 0.5
7 1:.04 3.571 0.929 0.5
8 1:0.2 4.167 0.333 0.5
3.11.3 Effect of Reaction Temperature on Lipase Activity
To determine temperature optima of immobilized lipase, refined Sunflower was

used as substrate and the reaction were oil carried out at different temperature 25, 30,
35, 40, 45, 50˚C.

Procedure
2.5 mL of refined sunflower oil mixed with 2.5mL of phosphate buffer and
25mg lipase bound nanoparticle (10.5 U) added. Hydrolysis reaction was carried out at
different temperatures for two hour. Reaction progress was calculated as described in
section 3.12.4.
Table 3.6 Reaction mixtures of different Substrate concentration for Lipase

activity
Oil Pahse 0.1M

2M Oil Hexane Enzyme
S/N Concentration Phosphate
(mL) (mL) (mL)*
(mM) Buffer (mL)
1 200 0.250 2.250 2.0 0.5

2 400 0.500 2.000 2.0 0.5
3 600 0.750 1.750 2.0 0.5
4 800 1.000 1.500 2.0 0.5
5 1000 1.250 1.250 2.0 0.5
6 1200 1.500 1.000 2.0 0.5
7 1400 1.750 0.750 2.0 0.5
8 1600 2.000 0.500 2.0 0.5
9 1800 2.250 0.250 2.0 0.5
10 2000 2.500 0.000 2.0 0.5
*0.5 mL enzyme solution in 0.1M phosphate buffer contains 5.25U lipase
3.11.4 Effect of pH on Lipase Activity
To analyze pH optima of immobilized lipase, hydrolysis reaction of triglycerides

was carried out at different pH with suitable buffers.
Buffers
1. Acetate buffer (0.1M) : pH 5.0 and 5.5, Prepared by mixing Acetic acid (0.1M)
to sodium acetate (0.1M) until require pH was achieved.
2. Phosphate Buffer (0.1M) : pH 6.0, 6.5, 7.0 and 7.4, Prepared by mixing sodium
dihydrogen phosphate (0.1M), to disodium hydrogen phosphate (0.1M) until
require pH was achieved.
3. Tris Buffer (0.1M) : pH 8.0, Prepared by dissolving 1.21g in of tris in 800mL
distilled water and pH was adjusted according to requirement by HCl (6N) and
volume made up to 1L.
4. Borate buffer : pH 8.5 and 9.0, Prepared by mixing sodium tetraborate

(Na2B4O7, 0.1M) to boric acid (H3BO3, 0.1M) until required pH was achieved.
Procedure
Refined sunflower oil 2.5mL mixed with 2.5mL of each buffer and 25 mg lipase
bound nanoparticle (10.5 U) was added. Hydrolysis reaction carried out at 40˚C for two
hours. A reaction without lipase was used as a control. Reaction progress and lipase
activity were determined by KOH titration as described in section 3.10.5.
3.12 Operational Stability of Immobilized Lipase
Operational stability of immobilized lipase was analyzed through following

parameters.
1. Storage Stability
2. Organic Solvent Stability
3. Reusability
3.12.1 Storage Stability
The storage stability of immobilized lipase was determined by the incubation at

4°C for the period of (0-72 hour) in phosphate buffer (pH 7.4). Residual activity was
measured at different intervals (12, 36, 60 and 72 hours) as described in section 3.10.5.
3.12.2 Organic Solvent Stability
Organic Solvents
1. Methanol
2. Ethanol
3. Isopropanol
4. n- Hexane
Immobilized lipase (25 mg) was incubated with 1.0 mL of organic solvents at 30
°C for 2 h. Subsequently, the organic solvent was removed and lipase bound nanoparticles
were collected by external magnetic field. Collected lipase bound nanoparticles were
washed 3 times with 1 mL of PBS (0.1 M, pH 7.4). Residual enzyme activity was
measured as described in the section 3.10.5. Immobilized lipase (steapsin) which was not
exposed to the organic solvent was used as the control.
3.12.3 Reusability of Immobilized lipase
The reusability was studied by determining the activity of immobilized

lipase (steapsin) in subsequent reactions relative to that of the first reaction. After
each cycle, the immobilized lipase was washed with hexane then with 0.1M
phosphate buffer (pH 7.4) and reintroduced into a fresh reaction medium for another
assay run and this procedure was repeated up to five cycles in the same condition as
described in section 3.10.5.
3.13 Esterification of Dodecanoic acid with Methanol by Immobilized Lipase.
Reagents:-
1. Dodecanoic acid : 4g
2. Methanol : 20mL
3. n-Hexane : As per requirement
Procedure
Dodecanoic acid and methanol in different molar ratio prepared by mixing 0.4g
of Dodecanoic acid in a different amount of methanol as described in table 3.7 and
volume made up to 5 mL by n-Hexane. 25mg (10U) of the enzyme was added to each
reaction mixture and esterification reaction was carried out at 40˚C for 12 hours.
Reaction progress was analyzed by measuring the amount of free fatty acid remaining
in the reaction mixture through titration by KOH as described in section 3.5.1.
3.15 Immobilized Lipase Catalyzed Transterification of Tree Seed-Oil
Procedure
Different oil samples (sunflower oil, Phulwara oil, Wild Apricot oil and Karanj
oil) mixed with 4 mL of methanol and 1 mL (10.5 U, 50mg) of immobilized enzyme
suspended in phosphate buffer (pH 7.4) in a 100 mL conical flask and
transesterification reaction carried out at 40˚C for 24 hour in incubator shaker at 170

rpm. After completion of the reaction, the enzyme separated from the product by a
magnet. Conversion of oil to biodiesel (FAME) was analyzed by GC-MS as described
in section 3.9.
Table 3.7 Reaction mixtures of different molar ratio Substrate concentration for
Lipase catalyzed esterification of Dodecanoic acid and Methanol.
S/N Dodecanoic acid Dodecanoic acid Methanol Methanol

(g) (mmol) (µL) (mmol)
1 0.4 2.0 80.9 2.0
2 0.4 2.0 161.8 4.0
3 0.4 2.0 242.7 6.0
4 0.4 2.0 323.7 8.0
5 0.4 2.0 404.5 10.0
6 0.4 2.0 485.4 12.0
7 0.4 2.0 566.3 14.0
8 0.4 2.0 647.7 16.0
9 0.4 2.0 728.1 18.0
10 0.4 2.0 809.0 20.0
3.15 Statistical Analysis
Data obtained after study of all parameter was analyzed on MS excel and their
mean value and respective standard error (n=3) were tabulated. The graphs were plotted
with the help of Prism 13 (Graphpad Software, Inc.).

Results
and
Discussion
Chapter 4 RESULTS AND DISCUSSION
Biodiesel obtained from transesterification of plant and animal fat/oil is a strong

alternative to petroleum based diesel fuel. Biodiesel production from oil crops like
soybean, sunflower, palm, corn etc. is already in practice but criticized due to its
competition with food resources. A variety of non-edible oil plants cultivated on the
agro-climatic zone of India is promising feedstock for biodiesel production.
Transesterification through the alkaline catalyst in biodiesel production is already
studied with a high rate of reaction but associated with a limitation like a high energy
(temperature) requirement, the sensitivity with free fatty acid (soap formation). Acid
catalysts overcome the problem of soap formation but require a higher temperature,
high methanol to oil molar ratio and have low reaction rate. Enzymes such as lipase
work at low temperature with substrate specificity, able to catalyze transesterification
of triglyceride as well as esterification of free fatty acid. So there is no problem
associated with the enzyme related to product separation, or energy requirement.
In this study Oil of three oil tree species of Uttarakhand, Cheura (Diploknema
butyracea), Wild Apricot (Prunus armeniaca), Karanj (Pongammia pinnata), along
with Sunflower (Helianthus annuus) was transesterified. Additionally, bio oil obtained
from pyrolysis of sugarcane residues containing free fatty acid was esterified with
propanol. Transesterification by lipase enzyme was compared the chemical catalyst. To
overcome difficulty associated with the use of free lipase such high cost and separation
from the product, immobilization technique was employed by taking the iron
nanoparticles because of their small size providing large surface area and magnetic
property enabling ease in separation. Acid catalyst perchloric acid was also
immobilized on silica support was found an efficient reusable catalyst for esterification.
Esterified/transesterified product was analyzed by GC-MS.
Detailed tables of results of all experiments are given in Annexure 2.
4.1 Yield of Immobilized Perchloric Acid (HClO4-SiO2)
A white free-flowing powder obtained containing HClO4-SiO2 (0.5 mmol/g).

The yield of catalyst was 24.8g (99.2%) due to minute loss of catalyst during transfer
and weighing.
Results and Discussion............
4.2 Esterification of Dodecanoic acid with Propanol by HClO4-SiO2
A preliminary experiment conducted to know the ability of Silica Supported

perchloric acid (HClO4-SiO2) to esterify fatty acids are reported in Figure 4.1.
Approximately 75.35% of Dodecanoic acid esterified into propyl esters in 3.5 hours in a
reaction mixture containing HClO4-SiO2. Silica gel as control and reaction mixture
without any catalyst did not show esterification as no significant reaction observed. Silica
Supported Perchloric Acid (HClO4-SiO2) as an efficient catalyst for esterification of short
chain and long chain fatty acids (Chakraborti et al., 2009 and Kaur et al., 2015).
control HClO4-SiO2 Silica Gel

90.00
80.00
70.00
Reaction Progress (%)
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0 1 2 3 4 5
-10.00
-20.00
Reaction Time (hours)
Figure 4.1 HClO4-SiO2 catalyzed esterification of Dodecanoic acid with Propanol
4.3 Kinetic Parameters for HClO4-SiO2 Catalyzed Esterification Reaction.
4.3.1 Effect of Acid to Alcohol Molar on Esterification
Acid to alcohol molar ratio one of the important factors determines reaction
progress and conversion efficiency (Reşitoğlu et al., 2012). Esterification is a
reversible reaction which can proceed backward as the amount of product increases.
Since replenishing of the product is not possible in the reaction condition, a higher
amount of alcohol than acid needed to shift reaction forward direction to achieve
maximum esterification of acid (Meher et al., 2006). The rate of esterification reaction

increases as the mole ratio was increased from 1:1 to 1:5 (Manjare et al., 2015). To
study the effect of molar ratio on Dodecanoic acid esterification acid to alcohol molar
ratio varied from 1:3 to 1:10. The reaction mixture of the molar ratio of 1:2 and 1:1
were not possible due to limited solubility of Dodecanoic acid in propanol. Increased
ester conversion was observed with increase in alcohol amount from molar ratio 1:3 to
1:6, and then it slightly decreases with the optimum molar ratio of 1:6 (Figure 4.2).
The decrease in activity beyond molar ratio 1:6 may be due to change in the kinetics of
reaction from second order to the first order in presence of excess alcohol and dilution
of Dodecanoic acid and less available at the reactive site of a heterogeneous catalyst
(Suryawanshi et al., 2014).
94
92
Percentage Conversion
90
88
86
84
82
80
78
1:3 1:4 1:5 1:6 1:7 1:8 1:9 1:10
Molar ratio( Dodecanoic acid to Propanol)
Figure 4.2 Esterification of Dodecanoic acid with propanol at different molar ratio
4.3.2 Effect of HClO4-SiO2 Amount
Esterification is a very slow reaction at room temperature, so carried out at a

higher temperature near the boiling point of alcohol in presence of chemical catalysts.
Catalysts decrease the activation energy and enable the reaction at a lower temperature
(Copper and Koubek., 1999). Generally, there is a linear ratio between the rate of
reaction and catalyst amount. Higher the amount of catalyst more the reactive site
available to catalyze the reaction (Khudsange and Wasewar, 2017). It was also
observed after a particular range, increasing catalyst amount may cause unwanted
reactions (Berchmans et al., 2008). Hence increasing the amount of catalyst from 0.1

mol% to 0.25mol % leads to significant increase in reaction rate, while the reaction
progress decreased by increasing the amount of catalyst beyond 0.25 mol % (Figure
4.3). Slower reaction at higher catalyst amount could be due to interruption of solid
heterogeneous catalyst with miscibility of reactants or stimulation of unwanted
reactions which inhibiting the esterification reaction.
100
90
80
70
60
50
40
30
20
10
0
0.10% 0.13% 0.25% 0.50% 1%
Catalyst (mol%)
Figure 4.3 Effect of HClO4-SiO2 amount on esterification
4.3.3 Effect of Reaction Temperature
The rate of reaction increases, with an increase in temperature (Patil and

Kulkarni., 2014). Increased temperature provides more kinetic energy to reactant
molecule for faster movement and more effective collision enabling faster chemical
reaction (Khudsange and Wasewar, 2017). The reaction rate was increased with
increasing temperature and 91.7% conversion was achieved in 2 hours at 90˚C while at
100 ˚C 94.8% conversion was achieved in two hours (Figure 4.4). The reaction
temperature was not increased beyond 100 ˚C due to unfavorable operating conditions
above the boiling point of propanol. Reaction progress with time suggests that at 90˚C
temperature the maximum conversion 92.19% achieved at 2 hours. However, at 100 ˚C
comparative maximum conversions, 95.07% was achieved at 1.5 hours but this product
composition was stable only at high temperature and percent progress was reduces to
92% after cooling down product (Figure 4.5).

100
95

90
85
80
75
70
70 80 90 100
Temperature (˚C)
Figure 4.4 Effect of Temperature on Esterification of Dodecanoic acid with

Propanol
120.00
100.00
80.00
70
60.00
80
40.00 90
100
20.00
0.00
0.00 0.50 1.00 1.50 2.00 2.50
Reaction Time (hours)
Figure 4.5 Reaction progress with time at different Temperatures.
4.3.5 Reusability of HClO4-SiO2 Catalyst
The heterogeneous catalyst could be separated from the reaction after reaction
completion and reused for next cycle of reaction (Venkateswarulu et al., 2014). The
advantage of immobilizing liquid catalyst perchloric acid on silica support is its easy
recovery and reusability (Bandgar et al., 2010). After completion of esterification of

Dodecanoic acid HClO4-SiO2 catalyst separated from the product by filtration and
washed several time with diethyl ether (Et2O), dried and subjected to next cycle of
reaction. Decrease from 92.19% (cycle one) to 62.72% (cycle 5) in catalytic activity
was observed with successive cycles of reactions (Figure 4.6). This decrease was
attributed to the desorption of perchloric acid from silica due to stirring a reaction and
in washing step during recovery. Moreover, catalyst loosed after each cycle during
separation and transfer also resulted in a decrease in activity (Chakraborti et al.,
2009). A better reactor with smooth stirring and methodology of separation could
increase the reusability of the catalyst.
100
90
80
Reaction Progress(%)
70
60
50
40
30
20
10
0
1 2 3 4 5
Number of Cycles
Figure 4.6 Reusability of HClO4-SiO2 up to five cycles.
4.4 HClO4-SiO2 Catalyzed Esterification of Bio-Oil and GC-MS analysis
Sugarcane bio oil sample esterified with propanol through HClO4-SiO2 catalyst
using optimized parameters 1:6 molar ratio, 1.25mol% catalyst, the 90˚C temperature
for 2 hours. This esterified bio oil is analyzed by GC-MS analysis (Annexure 3).
Esterified sugarcane bio oil contain 9.75% fatty acid propyl ester, 2.27% other
alkyl esters, 62.47% Free Fatty acid and 25.53% other compounds. Very less amount of
propyl ester and high amount of free fatty acid indicate insignificant esterification of bio
oil through HClO4-SiO2 (Table 4.3). Probable reason behind this insignificant

esterification may be due to inhibition of esterification in presence of water and other
complex compounds in the sugarcane bio oil or may be due to less molar combination of
propanol used. Since HClO4-SiO2 found to be efficient in esterification of Dodecanoic
acid further study is regarded for its catalysis for bio oil esterification. Using dicationic
ionic liquid C6(mim)2-HSO4 for esterifying bio-oil organic acids resulted in an increase
in pH from 2.9 to 5.1 and free fatty acid left only 0.05% (Xiong et al., 2009). Employing
Solid acid 40SiO2/TiO2-SO42- on bio oil upgrading was successful in esterifying free
fatty acid with 20 fold increase in fatty esters in the bio oil (Zhang et al., 2006). La0.06-
SO42-/TiO2-SiO2 catalyst developed for esterification and implemented for bio oil
esterification with 91.7% esterification achieved (Gu et al., 2010).
Table 4.1 Summery of GC-MS analysis obtained from esterified sugarcane bio oil.
S.N. Components Percentage
1 Fatty acid propyl esters 9.75
2 Other alkyl esters 2.27
3 Free Fatty Acid 62.47
4 Other compounds 25.53
4.5 HClO4-SiO2 Catalyzed Transesterification of Tree-seed- oils
A preliminary experiment conducted for transesterification of tree-seed- oils

with methanol through HClO4-SiO2 using optimum parameters of esterification
reaction 1:6 molar ratio, 1.25mol% catalyst, the 90˚C temperature for 2 hours. No
significant reaction appeared after 2 hours as two clear phases of oil and methanol
appeared. The inefficiency of HClO4-SiO2 for transesterification is due to its inability
to break ester bond of tri-acyl glycerol and previously HClO4-SiO2 was found efficient
catalyst only for organic condensation reactions like a condensation of free fatty acid
with alcohol i.e. esterification (Kaur et al., 2015).
4.6 Alkali Catalyzed transesterification of Tree-seed- oils and GC-MS Analysis
Alkali catalyzed transesterification of tree-seed- oils carried out on previously

practiced parameter with slight modification molar ratio 1:9 (instead of 1:6),
temperature 70˚C (instead of 60˚C) and time 3 (instead of 2) hours. Condition
modified due to properties of Cheura oil containing a high amount of saturated palmitic
acid was increasing its melting point and reducing miscibility in methanol. Methyl ester
conversion was investigated by GC-MS analysis of reaction product.
Biodiesel produced from alkali catalyzed transesterification of tree seed oil

subjected to GC-MS analysis contain a high amount of fatty acid methyl ester (FAME)
(above 90%) indicates significant transesterification (Table 4.2).
GC-MS chromatogram of cheura oil biodiesel (Annexure 3) contains 16 peaks

showing 99.71% of FAME of which 54.35% is hexadecanoic acid methyl ester, a
saturated FAME and 39.53% octadecenoic acid, methyl ester, (e)-. Fewer amounts of
unwanted compounds and no free fatty acid increases storage stability of biodiesel and
make it good quality biodiesel for blending with petroleum (Knothe, 2005).
GC-MS chromatogram of biodiesel from wild apricot kernels oil (Annexure 3)

contains 30 peaks showed 93%.7% of FAME among which 71.11% 9-octadecenoic
acid (z)-, methyl ester, a monounsaturated FAME. A negligible amount of free fatty
acid (1.43%) and other compounds (1.57%) cannot affect the quality of biodiesel
(Singh et al., 2016).
GC-MS chromatogram of biodiesel from Karanj seed oil (Figure 4.19) showed
38 peaks contain 90.47 % of FAME consisted a variety of long chain FAME majorly
38.6% 9-octadecenoic acid, methyl ester, (E)- and 13.49% Hexadecanoic acid, methyl
ester. It also contains 8.63% of other compounds. Although FAME content is lesser
than cheura oil and wild apricot oil still it persists its place as a good source of biodiesel
(Mamilla et al., 2016).
GC-MS chromatogram of biodiesel from sunflower seed oil (Figure 4.20)

shows 41 peaks containing 98.5% of FAME among which 74.64% 9-octadecenoic acid
(Z)-, methyl ester, (E)- and 11.04% Hexadecanoic acid, methyl ester. An insignificant
amount of free fatty acid (0.48%) and other compounds (2.07%) makes it good quality
biodiesel and explain the why it is conventional feedstock of biodiesel in Europe
(Antolın et al., 2002).

Table 4.2 Summery of GC-MS analysis of biodiesel obtained by alkali catalyzed
transesterification of tree seed oil.
Biodiesel Components (%)

S.N. Tree-seed- oils
FAME FFA MAG DAG TAG OTHER
1 Cheura oil 99.71 0.00 0.13 0.11 0.00 0.05
2 Wild Apricot oil 93.7 1.43 3.3 0.00 0.00 1.57
3 Karanj oil 90.47 1.43 0.57 0.00 0.00 7.53
4 Sunflower oil 98.45 0.48 0.00 0.00 0.00 1.07
4.7 Immobilization of Enzyme on Iron Nanoparticle and Characterization
4.7.1 Yield of Iron Oxide (Fe3O4) Nanoparticles
Iron oxide (Fe3O4) nanoparticles synthesized by coprecipitation method had

yield of 78.04% (16.5g) (Figure 4.7).
(A) (B)
Figure 4.7 Iron oxide Nanoparticle (A) In phosphate buffer (B) Under influence of
magnetic field
4.7.1 Particle Size and Zeta Potential
Zeta potential of nanoparticle indicates its stability in colloidal solution. The

high magnitude of zeta potential specifies higher electrostatic repulsion between
adjacent nanoparticles due to similar charge which prevents aggregation of
nanoparticles. It also evaluates the magnitude and nature of charge on the surface of
the nanoparticle (Sun et al., 2006). Generally Zeta potential higher than 30 mV
indicates a suspension is electrostatically stabilized (Goldstein and Greenlee,
2012). As described in Figure 4.8 Zeta size of bare iron nanoparticle was 240.7
±94.38 and enzyme bound nanoparticles is 267.4±111nm. Zeta potential of bare iron
nanoparticle was -24.5±15.9 mV and enzyme bound nanoparticles is 26.8±17.7 mV.
The Higher size of enzyme bound nanoparticle is due to enzyme binding. Higher
zeta potential indicates an increase in dispersity of nanoparticle in the aqueous
buffer after enzyme binding. The size of enzyme bound and bare nanoparticle was
higher than normal and in same way zeta potential was also lower than normal
indicating incipient stability. The reason behind this behavior is an aggregation of
nanoparticles after storage.
4.8.2 Dispersity of Nanoparticles in Different Solvents
Since Iron nanoparticle has negative charge on the surface they tend to disperse
in the polar solvent and remain aggregated in a nonpolar solvent (Kharisov et al.,
2014). Among different solvents like phosphate buffer, methanol, ethanol and n-
Hexane, a dispersity of nanoparticles was found the maximum in phosphate buffer
followed by methanol, ethanol. No dispersity of nanoparticle observed in a nonpolar
solvent like hexane.
4.8.3 Immobilization Efficiency and Activity Recovery
Protein Content Activity

Enzyme
(µg/ml) (µmol/min)
Free Enzyme before immobilization 371.35 6.69±0.17
Free residual Enzyme after immobilization 55.77 0.67±0.08
Immobilized Enzyme - 5.33±0.08

(A)
(B)
(C)
(D)
Figure 4.8 Hydrodynamic property of Iron Nanoparticles: (A), (B) Zeta size of
bare and enzyme bound nanoparticles and (C), (D) Zeta potential of
bare and enzyme bound nanoparticles.

(A)
(B)
Phosphate Methanol Ethanol n-Hexane

Buffer
Figure 4.9 Dispersity of iron nanoparticles in different solvent (A) just after
dispersion, (B) after 10 min.
..
Immobilization efficiency IE (%) = .
X 100
=85.75%
.
Activity recovery R (%) = X 100
.
.
=88.47%
Lipase was immobilized with 85.75% efficiency out of which 88.47%

activity was recovered. Hence immobilized lipase solution contain 0.857mg/mL
enzyme with about 5.25 units /mL or 0.525 unit /mg. In previous reports, maximum
immobilization efficiency (97%) and activity recovery (86%) were achieved by
similar procedure and parameters with microbial lipase Aspergillus niger
(NS81006) (Thangraj et al., 2016).

4.9 Kinetic Analysis of Immobilized Lipase
4.9.1 Effect of Oil to Aqueous Phase Volume ratio on Lipase Activity
Hydrolysis of triglyceride via lipase is a biphasic reaction containing aqueous

phase (water or buffer) and triglyceride (non-aqueous phase) in which lipase remain
dissolved in the aqueous phase (Tweddell et al., 1997). The chemical reactions take
place at the interphase of aqueous and organic phase (Van et al., 1995). The fascinating
property of lipase is its activation at the interphase to perform hydrolysis reaction (Tsai
and Chang, 1993). The rate of hydrolysis is limited by the interfacial area of aqueous-
organic phase in the emulsion (Kim and Chung, 1989). So a proper volume ratio
between aqueous and organic phase required in order to achieve maximum interfacial
area. The activity of lipase increased with increasing the volume ratio from 1:6 to 1:1
and then decrease from 1:1 to 1: 0.2 (Figure 4.10). These results clearly indicated that
lipase activity increased with increasing interfacial area of oil and aqueous phase with
optimum at 1:1 volume ratio (Tsai et al., 1991).
5.00
4.50
Enzyme Activity (µmol/min
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
1:6 1:4 1:2 1:1 1:0.8 1:0.6 1:0.4 1:0.2
Volume Ratio
Figure 4.10 Effect of Oil to Aqueous Phase Volume ratio on Lipase Activity
4.9.2 Effect of Substrate Concentration on Lipase Activity
Effect of substrate concentration of lipase activity analyzed, by varying the

concentration of one substrate (oil) in the organic phase. The concentration of oil varied
from 0.2M to 2M by its dilution with n-hexane. On the basis of the result shown in
figure4.8 and 4.9 through Michaeles Menten curve and Lineweaver- Burk plot, Km and

Vmax of free and immobilized enzyme calculated. A Higher value of Km (869.4 mM)
and lower value of Vmax (5.13 µmol/min) observed after immobilization than free
enzyme having Km (668.8 mM) and Vmax (8.45). Alteration in Km and Vmax after
immobilization is observed due to structural rigidity and limited access to active site,
which lowers the affinity of the immobilized enzyme for the substrate. This typical
phenomenon always observed after immobilization. Sometimes there is 2.8 time (Chen
et al., 2011) or 2.5 time (Tavano et al., 2013) increase in Km after immobilization due
to diffusional restriction and change in orientation.
Table 4.3 Change in kinetic parameters of lipase after immobilization
Parameter Free Enzyme Immobilized Enzyme
Km 668.8 mM 869.4 mM
Vmax 8.45 µmol/min 5.13 µmol/min
Kcat 7.5 X 10-3 s-1 4.95 X10-3 s-1
Kcat/Km 0.61mM-1s-1 0.341 mM-1s-1
The Kcat value for immobilized enzyme was 4.95 X10-3 s-1which was also
found less than free enzyme 7.5 X 10-3 s-1 ultimately resulting lesser enzyme efficiency
of immobilized lipase (Table 4.3).
Figure 4.11 Michaelis-Menten curves for free and immobilized lipase

1.4
1.2
1
1/Vo (min/µmol)
0.8
0.6
0.4
0.2
0
-0.002 -0.2 0 0.002 0.004 0.006
-0.4 1/[S](mM-1)
Free Enzyme Immoblized Enzyme
Figure 4.12 Lineweaver-Burk plot for free and immobilized lipase
4.9.3 Optimum Temperature and pH
Generally, enzyme activity increases with increase in temperature. More

activation energy for the reaction at a higher temperature increase reaction rate till an
optimum temperature achieved after which rate of rate of reaction starts decreasing.
Enzymes are temperature sensitive. High temperature degrades enzyme structure by
disrupting hydrogen bonds (Daniel et al., 2010). Immobilized lipase activity was
investigated at a different range of temperatures from 25 to 50˚C (Figure 4.13). The
optimum temperature for immobilized lipase was observed at 40˚C which was higher
than normal lipase taken from SRL Pvt. Ltd. (37 ˚C) which showed thermal stability of
immobilized lipase was increased. It had also observed that steapsin lipase from the
porcine pancreas is generally unstable at 40˚C above and loose activity faster (Linsha
et al., 2016). However at 45 ˚C rapid decreases in lipase activity observed.
Immobilization technique with other microbial lipase achieved more thermostability
above than 50˚C (Nawani et al., 2006 and Zhang et al., 2016).
Catalysis of enzyme relies on charged amino acid at the active site and their
particular ionized state. This ionized state is pH dependent as every amino acid has
characteristic pKa. So every enzyme has pH optima at which they showed the highest
activity. Changes in pH not only affect enzyme activity but also the shape of the
enzyme by altering hydrogen bonding in the enzyme. As clearly indicated in figure
4.14 maximum activity of lipase was observed at pH 7.4, which is optimum pH also for

free, lipase. But Immobilized lipase also retained sufficient activity at pH 8.0 as well as
pH 7.0. Many other studies found to increase in pH stability over alkaline pH range
achieved by immobilized lipase (Biró et al., 2005 and Sun et al., 2015).
Enzyme Activity (µ mol/min) 4.00

3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
25 30 35 40 45 50
Temperature (˚C)
Figure 4.13 Effect of Temperature on Lipase Activity
4.00
3.50
3.00
Enzyme Activity
2.50
2.00
1.50
1.00
0.50
0.00
5.00 5.50 6.00 6.50 7.00 7.40 8.00 8.50 9.00
pH
Figure 4.14 Effect of pH on Lipase Activity
4.10 Operational Stability of Immobilized Lipase
4.10.1 Storage Stability
Storage stability of immobilized lipase was examined over the time period of
12-72 hours (Figure 4.15). However, steapsin lipase of the digestive system is less
stable and loose activity at room temperature. Immobilized steapsin lipase also showed

less storage stability and loose activity in 72. After 60 hours of incubation residual
activity, 17.85% remained which decreased to 15.78% at 72 hours incubation. High
storage stability of 30 days observed in Candida rugosa lipase immobilized on chitosan
beads (Chiou and Wu, 2004). Immobilized Candida parapsilosis lipase also found
operationally stable in a batch reactor at 60˚C with a half-life of 18 hours (Osório et
al., 2009). Replacing steapsin with microbial lipase with high stability can be utilized in
immobilization technique for industrial purposes.
8.00
7.00
Residual Activity (µmol/min)
6.00
5.00
4.00
3.00
2.00
1.00
0.00
12 36 60 72
Time (Hours)
Figure 4.15 Storage stability of immobilized lipase
4.10.2 Stability in Organic Solvent
Stability of lipase in organic solvent was examined using four organic

solvents methanol, ethanol, isopropanol and hexane. Lipase showed the highest
activity in n-Hexane and very low activity observed in ethanol, methanol, and
isopropanol. Lipase activity in hydrophobic organic solvent observed higher than
hydrophilic solvents, which was also observed in other studies. Hernández et al.
(2009) observed higher lipase activity in an aprotic polar solvent such as
acetonitrile, acetone and ethyl acetate and lower in protic polar solvents such as
ethanol and isopropanol etc. Inactivation of lipase due to methanol was also
observed during enzymatic biodiesel production (Lotti et al., 2015). Nonpolar
solvents like hexane found as a suitable solvent to process lipase can be used during
its separation from the reaction mixture.

3.50
3.00
2.50
Residual Activity
2.00
1.50
1.00
0.50
0.00
Methanol Ethanol Isopropanol Hexane
Organic Solvents
Figure 4.16 Organic solvent stability of immobilized Lipase
4.10.3 Reusability of Immobilized lipase
The main advantage of lipase immobilization is it’s separation from the reaction
mixture and reusability to make the enzymatic process more economical (Tan et al.,
2010). Reusability of lipase examined over five cycles of 2-hour hydrolysis reaction.
Gradual loss of lipase activity was observed with each successive cycle with 31.91% of
residual activity at the fifth cycle. The loss of activity with reusability may attribute to
dissociation loss of lipase from nanoparticle due to mechanical shaking. Other forms of
bioreactor can be explored in the future study to avoid such loss during the repeated
uses (Thangaraj et al., 2016).
5.0
4.5
4.0
3.5
Residual Activity
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1 2 3 4 5
Reaction Cycles
Figure 4.17 Reusability of immobilized Lipase

4.11 Esterification of Dodecanoic acid with Methanol by Immobilized Lipase
Esterification of Dodecanoic acid with methanol carried out with the different
molar ratio from 1:1 to 1:10 and found that 12 hour reaction time lipase was found
efficiently esterifying Dodecanoic acid with the maximum conversion of 87.83% at
molar ratio 1:6 (Figure 4.18). Effect of the molar ratio in esterification reaction was
similar as in HClO4-SiO2 catalyzed esterification. The increase in ester conversion
observed with increase in methanol from molar ratio 1:3 to 1:6 due to shifting of
reaction equilibrium toward forwarding direction. Beyond molar ratio 1:6 the decrease
in ester conversion may be inactivation of lipase due to excess methanol inhibition.
Molar ratio 1:6 was found to be best for both enzyme as well as chemical catalyzed
esterification reaction. Linsha et al. (2016) also found maximum esterification of oleic
acid with methanol using immobilized lipase at molar ratio 1:6.
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
1:1 1:2 1:3 1:4 1:5 1:6 1:7 1:8 1:9 1:10
Molar ratio (Acid:Alcohol)
Figure 4.18 Immobilized lipase catalyzed esterification of Dodecanoic acid with

methanol
4.12 Lipase Catalyzed Transesterification of Tree-seed- oils and GC-MS Analysis
Lipase-catalyzed transesterification of tree seed oil carried out using optimized

parameter for immobilized enzyme oil to methanol volume ratio 1:1, temperature 40˚C
with 10.5 U enzymes for 24 hours at 170 rpm. Biodiesel obtained by lipase-catalyzed
transesterification examined by GC-MS analysis (Annexure 3).

Biodiesel obtained from an enzyme catalyzed transesterification also contain a
high amount of FAME but comparatively lesser than observed in alkali-catalyzed
transesterification (Table 4.4). Sufficient FAME yield 74.46% for cheura oil, 74.48%
for wild apricot oil, 76.15% for Karanj oil and 85.66% for sunflower oil observed in 24
hour reaction which might increase with in reaction time.
The Higher amount of free fatty acid was observed in biodiesel produced from
enzyme catalyzed transesterification as compared to alkali-catalyzed transesterification.
This could be due to water associated with immobilized enzyme solution induces
lipase-catalyzed hydrolysis of triglyceride to free fatty acid (Figure 4.19). Maximum
yield of biodiesel obtained from sunflower oil through enzyme catalyzed transesterification
with 85.66% FAME yield with 1% free fatty acid.
Table 4.4 Summery of GC-MS analysis of biodiesel obtained by lipase catalyzed

transesterification of tree seed oil.
Biodiesel Components (%)
S.N. Tree-seed- oils
FAME FFA MAG DAG TAG OTHER
1 Cheura oil 76.46 19.9 0.86 1.96 0.00 0.82
2 Wild Apricot oil 74.48 10.57 0.00 0 0.00 14.95
3 Karanj oil 76.15 5.69 0.00 1.85 0.00 16.31
4 Sunflower oil 85.66 1.00 0.00 1.68 0.00 11.66
100%
90%
80%
70%
OTHERS
60%
TAG
50%
DAG
40%
MAG
30%
FFA
20%
FAME
10%
0%
A L A L A L A L
Cheura oil Wild Apticot Oil Karanj Oil Sunflower oil
Figure 4.19 Comparative analysis of transesterification and biodiesel yield.

A= Alkali catalyzed transesterification, L= lipase catalyzed
transesterification, of different tree-seed- oils

Biodiesel production using alkali catalyst was faster and efficient process which
gave high yield in less time but also associated with limitations like high energy
requirement and, negative effect soap formation with free fatty acid present in oil. Use
of lipase in bio diesel production was found efficient in terms of energy requirement
and ability to esterify free fatty acid overcomes the issues associated with alkali
catalysis. However due to low activity and longer reaction time it could take a more
investment and patience to employ enzyme catalysis in commercial biodiesel
production. But after establishment of plant and continuous production for some time
this technique might be energy efficient sustainable in fuel production. Employing
immobilization technique in both enzyme and chemical catalysis enabled reusability of
enzyme and catalyst and makes the process more economic. Increased stability of
enzyme after immobilization enabled it’s longer and repeated use can compensate the
high cost of enzyme.

Summary
and
Conclusions
Chapter 5 SUMMARY AND CONCLUSION
Current global energy consumption projected to increase by 48% from 2012 to

2040, with over 80% of this demand derived from fossil fuels. Accelerating oil prices
and exhaustion of oil assets and savior environmental issues associated with them
demand a better alternative of energy. The considerable contribution has already been
made in order to reduce the use of fossil fuels through first-generation biofuels (i.e.
Biodiesel and bioethanol), using crude biomass for their production but unable to
compensate the competition with the food industry. Hence there is a requirement of
more efficient biofuel generation techniques or so-called second-generation fuels, i.e.
those from the non-food crop, lignocellulosic biomass, and agriculture waste. Biodiesel
is a carbon-neutral, non-toxic, biodegradable and a low-pollutant emitting alternative to
diesel fuel derived from animal or vegetable oil and fat through transesterification in
the form of a mono-alkyl ester. A variety of non-edible oil plant species cultivated in
agro-climatic zones of India is promising feedstock for biodiesel. Hence seed oil of
three tree species Cheura (Diploknema butyracea), Wild Apricot (Prunus armeniaca),
Karanj (Pongammia pinnata) and one edible crop Sunflower (Helianthus annuus) was
used for chemical and enzymatic transesterification. Bio-oil of sugarcane residue
contains free fatty acid was also used for the esterification. For the catalysis of
transesterification/esterification, the chemical catalyst perchloric acid (HClO4)
immobilized on silica gel and lipase immobilized on iron nanoparticles. The kinetic
parameters of esterification/transesterification were optimized for immobilized
perchloric acid and immobilized enzyme and applied them for esterification/
transesterification of tree seed oil and bio-oil and analyzed by GC-MS. Chemical and
enzymatic transesterification were compared with alkali-catalyzed transesterification.
Perchloric acid (HClO4) immobilized on silica gel (SiO2) containing 0.5mmol

HClO4-SiO2/g of the catalyst. This catalyst (1mol %) was efficient to esterify
dodecanoic acid (lauric acid) with propanol with 75.35 % conversion at 80˚C within 3
hours. Whereas no significant transesterification was observed with the tree seed oil in
the same condition.
Summary and Conclusion............

Analysis of kinetic parameter for HClO4-SiO2 revealed the optimum molar ratio
for esterification of dodecanoic acid with propanol was 1:6 resulting 92.62%
conversion in 3 hours at 80˚C. The varying molar ratio in increasing or decreasing
order resulted in a gradual decrease in conversion. The activity of HClO4-SiO2
increased with decreasing catalyst amount from 1mol% to 0.25% with the higher
conversion of 88.67% in 2 hours which was 66.7% in case of 1 mol% HClO4-SiO2
amount. Decreasing catalyst amount further resulted in a decrease in reaction rate. The
rate of HClO4-SiO2 catalyzed reaction increased with increase in temperature and
91.7% conversion was achieved in 2 hours at 90 ˚C and 94.8% at 100 ˚C. However,
reaction equilibrium of 94% conversion was stable only at a higher temperature and
reduced after cooling down of the product. So 90˚C temperature was optimum for
HClO4-SiO2. The heterogeneous HClO4-SiO2 catalyst was easily separable from
reaction product and reused up to five times in reaction cycles. A gradual decrease in
reaction conversion was observed after each reaction cycle with 68.03% residual
activity.
Esterification of bio-oil with propanol carried out by 0.25 mol% HClO4-SiO2

catalyst at optimized parameter i.e. molar ratio 1:6, temperature 90 ˚C for 3 hours and
the product analyzed by GC-MS. Efficient results were not obtained as bio-oil
esterified contain a high amount of free fatty acid (54%) the esterified product was
approximate 10%. The reason behind the insufficient conversion could be water in bio-
oil inhibiting esterification. Hence higher amount of alcohol (propanol) is required and
further optimization required for HClO4-SiO2 catalyzed the esterification of bio-oil.
HClO4-SiO2 was unable to transesterify tree seed oil in the preliminary experiment and
different phases of methanol and oil were observed in reaction product after 2-hour
reaction on optimized parameters. The reason might be due to non-reactivity of HClO4-
SiO2 on ester bond of tri-acyl glycerol as it had also reported that HClO4-SiO2 only
efficient in condensation of an alcohol with acid i.e. esterification.
The optimized parameters were also found to be efficient with alkali NaOH
bound catalysis and slider modification was found was efficient. It transesterified with
oils with a high yield of FAME 99.71% in Cheura oil, 93.7% Wild Apricot, 90.47% in
Karanja oil and 98.45% in Sunflower oil.

Iron nanoparticles were synthesized with 78.04% yield, and lipase was
successfully immobilized on nanoparticle with 85.75% immobilization efficiency and
88.47% activity recovery. The size of bare and lipase bound nanoparticles on zeta sizer
was 240.7nm and 267.4 nm respectively. The large size of nanoparticle might be due to
aggregation of nanoparticles by providing 3 month of storage. The zeta potential of
bare and lipase bound nanoparticle was -24.5mV and -26.8mV respectively which
show incipient stability in the solution due to large size.
Immobilize lipase was able to hydrolyze sunflower oil in biphasic reaction with
oil to aqueous phase volume ratio 1:1 on which higher interfacial ratio for lipase
activity available. Kinetic parameter show increase in Km of immobilized lipase (Km=
668 mM) as compared to free lipase (Km=869.4mM). Whereas Vmax of immobilized
lipase decreased (Vmax= 5.31µmol/min) than free lipase (Vmax= 8.45µmol/min)
resulting lower turnover number of immobilized lipase (Kcat=4.95 X10-3 s-1) than free
lipase (Kcat= 7.5 X 10-3 s-1). Total enzyme efficiency of immobilized lipase was
decreased as compared to free lipase. The reason behind the decrease in enzyme
efficiency was the diffusional resistance of lipase after immobilization on large size
nanoparticles. The optimum temperature for immobilized lipase was 40˚C which was
higher than free lipase (37 ˚C) and optimum pH of immobilized lipase was same as free
lipase 7.4.
Immobilized lipase was found less stable and lost its activity with increasing
storage period. A gradual decrease in lipase activity observed with 58% residual
activity after 36-hour storage which decreased to 15% after the time period of 72 hours.
Immobilized lipase was found stable with high activity in nonpolar organic solvent n-
hexane and showed lower activity in a polar organic solvent like methanol, ethanol, and
isopropanol. Immobilized lipase showed reusability up to five cycles. However
decreased in the activity of lipase was observed after each reaction cycle with 31.91%
residual activity.
Immobilized lipase was efficient to transesterify tree-seed-oils on an optimized

parameter in 24 hours with the yield of 76.46% in Cheura oil, 74.48% in wild apricot
76.15 in Karanj and 85.66% in sunflower oil which could be increased with increase in
reaction time. However higher amount of free fatty acid observed in each sample due
to hydrolysis of triglyceride by lipase.
Hence it can be concluded from above study that oil tree species Cheura
(Diploknema butyracea), Wild Apricot (Prunus armeniaca), Karanj (Pongammia
pinnata) are a promising source of biodiesel having high-fat content which can be
converted to FAME and for biodiesel production. Immobilization of chemical catalysis
and lipase is a useful strategy in biodiesel production with increased reusability of
chemical catalyst and lipase which makes the process more economic. Perchloric acid
bound to silica support (HClO4-SiO2) was found more effective in esterification of
dodecanoic acid and could be implemented for esterification of bio-oil containing free
fatty acid. Alkaline catalysis in biodiesel production was found very efficient for high
yield of biodiesel in less time but require high energy. Lipase-mediated catalysis for
biodiesel production gives a high yield. However, reaction time was longer in lipase-
catalyzed biodiesel production but with continuous use, it can compensate for high
energy cost required in alkaline catalysis.

Literature
Cited
LITERATURE CITED
Abbaszaadeh, A., Ghobadian, B., Omidkhah, M. R. and Najafi, G. 2012. Current

biodiesel production technologies: a comparative review. Energy Conversion
and Management. 63: 138-148.
Agarwal, A. K. 2007. Biofuels (alcohols and biodiesel) applications as fuels for

internal combustion engines. Progress in Energy and Combustion Science.
33(3): 233-271.
Agrawal, A. K. and Rajamanoharan, K. 2009. Experimental investigations of

performance and emission of Kranja oil and its blend in a single cylinder diesel
engine. Applied Energy. 86: 106-112.
Aguieiras, E. C., Cavalcanti-Oliveira, E. D., de Castro, A. M., Langone, M. A. and

Freire, D. M. 2017. Simultaneous enzymatic transesterification and
esterification of an acid oil using fermented solid as biocatalyst. Journal of the
American Oil Chemists Society. 94(4): 551-558.
Ahmad, R. and Sardar, M. 2015. Enzyme immobilization: an overview on

nanoparticles as immobilization matrix. Biochemistry and Analytical
Biochemistry. 4(2): 1-8.
Akoh, C. C., Chang, S. W., Lee, G. C. and Shaw, J. F. 2007. Enzymatic approach
to biodiesel production. Journal of agricultural and food chemistry. 55(22):
8995-9005.
Alam, R. 2011. Studies on the properties of Karanja oil for probable industrial application
Doctoral dissertation, National Institute of Technology, Rourkela. 28p.
Alfred, T. 2002. Fats and Fatty Oils. Ullmann’s Encyclopedia of Industrial Chemistry.
7th ed. Print and Online. 148p.
Andrade, M. F., Parussulo, A. L., Netto, C. G., Andrade, L. H. and Toma, H. E. 2016.
Lipase immobilized on polydopamine-coated magnetite nanoparticles for biodiesel
production from soybean oil. Biofuel Research Journal. 3(2): 403-409.
Antczak, M. S., Kubiak, A., Antczak, T. and Bielecki, S. 2009. Enzymatic biodiesel
synthesis–key factors affecting efficiency of the process. Renewable Energy.
34(5): 1185-1194.
Literature Cited............
Antolın, G., Tinaut, F. V., Briceno, Y., Castano, V., Perez, C. and Ramırez, A. I.
2002. Optimisation of biodiesel production by sunflower oil transesterification.
Bioresource Technology. 83(2): 111-114.
Atabani, A. E., Silitonga, A. S., Badruddin, I. A., Mahlia, T. M. I., Masjuki, H. H.

and Mekhilef, S. 2012. A comprehensive review on biodiesel as an alternative
energy resource and its characteristics. Renewable and Sustainable Energy
Reviews. 16(4): 2070-2093.
Bachheti, R. K., Rai, I., Joshi, A. and Rana, V. 2012. Physico-chemical study of seed
oil of Prunus armeniaca L. grown in Garhwal region (India) and its comparison
with some conventional food oils. International Food Research Journal. 19(2):
577-581.
Balat, M. 2007. Global bio-fuel processing and production trends. Energy Exploration
and Exploitation. 25(3): 195-218.
Balcão, V. M., Paiva, A. L. and Malcata, F. X. 1996. Bioreactors with immobilized

lipases: state of the art. Enzyme and Microbial Technology. 18(6):392-416.
Bandyopadhyay, D., Manchanda, K., Nayak, B. and Basu, N. Related items.

Academy of Agriculture Journal. 2(01): 1-4
Basri, M., Heng, A. C., Razak, C. N., Wan Yunus, W. M., Ahmad, M., Rahman, R.
N. and Salleh, A. B. 1997. Alcoholysis of palm oil mid-fraction by lipase from
Rhizopus rhizopodiformis. Journal of the American Oil Chemists' Society.
74(2): 113-116.
Berchmans, H. J. and Hirata, S. 2008. Biodiesel production from crude Jatropha

curcas L. seed oil with a high content of free fatty acids. Bioresource
Technology. 99(6): 1716-1721.
Biró, E., Németh, Á. S., Sisak, C., Feczkó, T. and Gyenis, J. 2008. Preparation of
chitosan particles suitable for enzyme immobilization. Journal of Biochemical
and Biophysical Methods, 70(6): 1240-1246.
Bobade, S. N. and Khyade, V. B. 2012. Detail study on the Properties of Pongamia

Pinnata (Karanja) for the Production of Biofuel. Research Journal of Chemical
Sciences. 2(7): 16-20.
Bridgwater, A. V. 2012. Upgrading biomass fast pyrolysis liquids. Environmental

Progress & Sustainable Energy. 31(2): 261-268.
Budžaki, S., Šalić, A., andZelić, B. 2015. Enzyme-catalysed biodiesel production
from edible and waste cooking oils. Chemical and Biochemical Engineering
Quarterly. 29(3): 329-333.
Bulushev, D. A. and Ross, J. R. 2011. Catalysis for conversion of biomass to fuels via
pyrolysis and gasification: a review. Catalysis Today. 171(1): 1-13.
Chakraborti, A. K., Singh, B., Chankeshwara, S. V. and Patel, A. R. 2009. Protic

acid immobilized on solid support as an extremely efficient recyclable catalyst
system for a direct and atom economical esterification of carboxylic acids with
alcohols. The Journal of Organic Chemistry. 74(16): 5967-5974.
Chen, J. W. and Wu, W. T. 2003. Regeneration of immobilized Candida antarctica

lipase for transesterification. Journal of Bioscience and Bioengineering. 95(5):
466-469.
Chiou, S. H. and Wu, W. T. 2004. Immobilization of Candida rugosa lipase on

chitosan with activation of the hydroxyl groups. Biomaterials. 25(2): 197-204.
Chourasia, V. R., Gawas, A. S., Menon, A. S. and Shinde, P. M. 2015. Production of

biodiesel by enzymatic transesterification using immobilized lipase.
International Journal of Engineering Research and General Science. 3: 1-9.
Copper, C. L. and Koubek, E. 1999. An experiment to demonstrate how a catalyst

affects the rate of a reaction. Journal of Chemical Education. 76(12): 1714.
Daniel, R. M. and Danson, M. J. 2010. A new understanding of how temperature

affects the catalytic activity of enzymes. Trends in Biochemical Sciences.
35(10): 584-591.
Demirbas, A. 2007. Importance of biodiesel as transportation fuel. Energy Policy.

35(9): 4661-4670.
Demirbas, A. 2007. Progress and recent trends in biofuels. Progress in energy and
combustion science. 33(1): 1-18.
Demirbas, A. H. and Demirbas, I. 2007. Importance of rural bioenergy for developing

countries. Energy Conversion and Management. 48(8): 2386-2398.
Dhyani, S. K., Vimala Devi, S. and Handa, A. K. 2015: Tree Borne Oilseeds for Oil
and Biofuel. Technical Bulletin 2/2015. ICAR-CAFRI, Jhansi. pp50.
Divakara B., Upadhyaya H.D., Wani S.P. and LaxmipathiGowda C. 2010. Biology
and genetics improvement of Jatropha curcas L.: a review. Applied Energy.
87(3): 732–742.
Dumri, K. and Hung Anh, D. 2014. Immobilization of lipase on silver nanoparticles

via adhesive polydopamine for biodiesel production. Enzyme Research. 38(73):
1-9.
Ejikeme, P. M., Anyaogu, I. D., Ejikeme, C. L., Nwafor, N. P., Egbuonu, C. A. C.,
Ukogu, K. and Ibemesi, J. A. 2010. Catalysis in biodiesel production by
transesterification processes-an insight. Journal of Chemistry. 7(4): 1120-1132.
Fairless D. 2007. The little shrub that could-maybe. Nature. 449: 652–655.
Fjerbaek, L., Christensen, K. V., and Norddahl, B. 2009. A review of the current
state of biodiesel production using enzymatic transesterification. Biotechnology
and Bioengineering. 102(5): 1298-1315.
Freedman, B. E. H. P., Pryde, E. H. and Mounts, T. L. 1984. Variables affecting the

yields of fatty esters from transesterified vegetable oils. Journal of the American
Oil Chemists' Society, 61(10): 1638-1643.
Freedman, B., Butterfield, R. O. and Pryde, E. H. 1986. Transesterification kinetics of

soybean oil. Journal of the American Oil Chemists' Society. 63(10): 1375-1380.
Fukuda, H., Kondo, A. and Tamalampudi, S. 2009. Bioenergy: Sustainable fuels

from biomass by yeast and fungal whole-cell biocatalysts. Biochemical
Engineering Journal. 44(1): 2-12.
Gebremariam, S. N., and Marchetti, J. M. 2017. Biodiesel production technologies.

AIMS Energy. 5(3): 425-457.
Ghaly, A. E., Dave, D., Brooks, M. S. and Budge, S. 2010. Production of biodiesel by
enzymatic transesterification. American Journal of Biochemistry and
Biotechnology. 6(2): 54-76.
Ghosh, S. K., Bhattacharjee, A., Jha, J. K., Mondal, A. K., Maiti, M. K., Basu, A.
and Sen, S. K. 2007. Characterization and cloning of a stearoyl/oleoyl specific
fatty acyl-acyl carrier protein thioesterase from the seeds of Madhucalongifolia
(latifolia). Plant Physiology and Biochemistry. 45(12): 887-897.
Goldstein, N. and Greenlee, L. F. 2012. Influence of synthesis parameters on iron

nanoparticle size and zeta potential. Journal of Nanoparticle Research. 14(4): 1-15.
Gupta, M. N., Kaloti, M., Kapoor, M., and Solanki, K. 2011. Nanomaterials as
matrices for enzyme immobilization. Artificial Cells, Blood Substitutes and
Gurau, V. S., Sandhu, S. S. and Sarma, A. K. 2016. Review and prospects of bitter apricot
oil as an alternative feedstock for biodiesel production-an Indian perspective.
International Journal of Oil, Gas and Coal Technology. 12(4): 425-439.
Hajjari, M., Tabatabaei, M., Aghbashlo, M. and Ghanavati, H. 2017. A review on

the prospects of sustainable biodiesel production: A global scenario with an
emphasis on waste-oil biodiesel utilization. Renewable and Sustainable Energy
Reviews. 72: 445-464.
Havlík, P., Schneider, U. A., Schmid, E., Böttcher, H., Fritz, S., Skalský, R. and
Leduc, S. 2011. Global land-use implications of first and second generation
biofuel targets. Energy Policy. 39(10): 5690-5702.
Helwani, Z., Othman, M. R., Aziz, N., Kim, J. and Fernando, W. J. N. 2009. Solid
heterogeneous catalysts for transesterification of triglycerides with methanol: a
review. Applied Catalysis A: General, 363(1): 1-10.
Hernández-Rodríguez, B., Córdova, J., Bárzana, E. and Favela-Torres, E. 2009.

Effects of organic solvents on activity and stability of lipases produced by
thermotolerant fungi in solid-state fermentation. Journal of Molecular Catalysis
B: Enzymatic. 61(3): 136-142.
http://planningcommission.nic.in Planning commission 15/05/2017
Hu, B., Pan, J., Yu, H. L., Liu, J. W. and Xu, J. H. 2009. Immobilization of
Serratiamarcescens lipase onto amino-functionalized magnetic nanoparticles for
repeated use in enzymatic synthesis of Diltiazem intermediate. Process
Biochemistry. 44(9): 1019-1024.
Huaping, Z., Zongbin, W. U., Yuanxiong, C., Zhang, P., Shijie, D., Xiaohua, L. I.
U. and Zongqiang, M. A. O. 2006. Preparation of biodiesel catalyzed by solid
super base of calcium oxide and its refining process. Chinese Journal of
Catalysis. 27(5): 391-396.
Iftikhar, T., Niaz, M., Ikram-ul-Haq, M.A. and M.I. Rajoka. 2008. Maximization of
intracellular lipase production in a lipase-overproducing mutant derivative of
Rhizopus oligosporus DGM 31: A Kinetic Study. Food Technol. Biotechnol.
46: 402-412.
International Energy Agency. 2009. World energy outlook 2009. 698p.
Isahak., Wan, N. R. W., Mohamed, W. M. H., Ambar, Y. and Taufiq, Y. H. 2012.

A review on bio-oil production from biomass by using pyrolysis method."
Renewable and Sustainable Energy Reviews. 16(8): 5910-5923.
Jacobson, K., Maheria, K. C. and Dalai, A. K. 2013. Bio-oil valorization: a review.

Renewable and Sustainable Energy Reviews. 23: 91-106.
Juyal, M. 2016. Immobilization of lipase on Iron Nanoparticles and Kinetic

Characterization. Thesis, M.Sc. G.B. Pant University of Agriculture and
Technology, Pantnagar. 77p.
Kant, P. and Wu, S. 2011. The extraordinary collapse of Jatropha as a global biofuel.
Environmental Science and Technology. 45: 7114–7115.
Kaur, M., Sharma, S. and Bedi, P. M. 2015. Silica supported Brönsted acids as
catalyst in organic transformations: A comprehensive review. Chinese Journal
of Catalysis. 36(4): 520-549.
Kaya, C., Kola, O., Ozer, M. S. and Altan, A. 2008. Some characteristics and fatty
acids composition of wild apricot (Prunuspseudoarmeniaca L.) kernel oil.
Asian Journal of Chemistry. 20(4): 2597-2602.
Kharisov, B. I., Dias, H. R., Kharissova, O. V., Vázquez, A., Pena, Y. and Gomez,
I. 2014. Solubilization, dispersion and stabilization of magnetic nanoparticles
in water and non-aqueous solvents: recent trends. RSC Advances. 4(85):
45354-45381.
Khudsange, C. R. and Wasewar, K. L. 2017. Process intensification of esterification

reaction for the production of propyl butyrate by pervaporation. Resource-
Efficient Technologies. 3(1): 88-93.
Kim, T. and Chung, K. 1989. Some characteristics of palm kernel olein hydrolysis by
Rhizopusarrhizus lipase in reversed micelle of AOT in isooctane, and additive
effects. Enzyme and Microbial Technology. 11(8): 528-532.
Klibanov, A. M. 1983. Immobilized enzymes and cells as practical catalysts. Science.

219(4585): 722-727.
Knothe, G. 2010. Biodiesel and renewable diesel: a comparison. Progress in Energy

and Combustion Science. 36(3): 364-373.
Koh, M. Y. and Ghazi, T. I. M. 2011. A review of biodiesel production from
Jatrophacurcas L. oil. Renewable and Sustainable Energy Reviews. 15(5):
2240-2251.
Kouzu, M., Nakagaito, A. and Hidaka, J. S. 2011. Pre-esterification of FFA in plant

oil transesterified into biodiesel with the help of solid acid catalysis of
sulfonatedcation-exchange resin. Applied Catalysis A: General. 405(1): 36-44.
Kumari, V., Shah, S. and Gupta, M. N. 2007. Preparation of biodiesel by lipase-

catalyzed transesterification of high free fatty acid containing oil from Madhuca
indica. Energy and Fuels. 21(1): 368-372.
Kureel, R.S., Singh, C.B., Gupta, A.K. and Pandey, A. 2007. A Report on Wild
Apricot by National Oilseeds and Vegetable Oils Development Board, Ministry
of Agriculture, Govt. of India. 94p
Leung, D. Y. C. and Guo, Y. 2006. Transesterification of neat and used frying oil:
optimization for biodiesel production. Fuel Processing Technology, 87(10):
883-890.
Leung, D. Y., Wu, X. and Leung, M. K. H. 2010. A review on biodiesel production

using catalyzed transesterification. Applied Energy, 87(4): 1083-1095.
Li, C., Jiang, S., Zhao, X. and Liang, H. 2017. Co-immobilization of enzymes and
magnetic nanoparticles by metal-nucleotide hydrogelnanofibers for improving
stability and recycling. Molecules. 22(1): 179-189.
Linsha, V., AbooShuhailath, K., Mahesh, K. V., Mohamed, A. A. P. and

Ananthakumar, S. 2016. Biocatalytic Conversion Efficiency of Steapsin
Lipase Immobilized on Hierarchically Porous Biomorphic Aerogel Supports.
ACS Sustainable Chemistry and Engineering. 4(9): 4692-4703.
Lotero, E., Liu, Y., Lopez, D. E., Suwannakarn, K., Bruce, D. A. and Goodwin, J.
G. 2005. Synthesis of biodiesel via acid catalysis. Industrial and Engineering
Chemistry Research. 44(14): 5353-5363.
Lotti, M., Pleiss, J., Valero, F. and Ferrer, P. 2015. Effects of methanol on lipases:
Molecular, kinetic and process issues in the production of biodiesel.
Biotechnology Journal, 10(1): 22-30.
Lu, H., Liu, Y., Zhou, H., Yang, Y., Chen, M. and Liang, B. 2009. Production of
biodiesel from Jatropha curcas L. oil. Computers and Chemical Engineering,
33: 1091–1096.
Lunagariya, J., Dhar, A. and Vekariya, R. L. 2017. Efficient esterification of n-
butanol with acetic acid catalyzed by the Brönsted acidic ionic liquids:
influence of acidity. RSC Advances, 7(9): 5412-5420.
Luo, H., Yin, H., Wang, R., Fan, W. and Nan, G. 2017. Caprolactam-Based Brønsted
Acidic Ionic Liquids for Biodiesel Production from Jatropha Oil. Catalysts,
7(4): 102-111.
Ma, F. and Hanna, M. A. 1999. Biodiesel production: a review. Bioresource

Technology, 70(1): 1-15.
Makareviciene, V., Gumbyte, M., Skorupskaite, V. and Sendzikiene, E.

2017.Biodiesel fuel production by enzymatic microalgae oil transesterification
with ethanol. Journal of Renewable and Sustainable Energy, 9(2): 023101.
Maleki, B., Shirvan, H. K., Taimazi, F. and Akbarzadeh, E. 2012. Sulfuric acid
immobilized on silica gel as highly efficient and heterogeneous catalyst for the
one-pot synthesis of 2, 4, 5-triaryl-1H-imidazoles. International Journal of
Organic Chemistry, 2(1): 93-99.
Mane, M. A. 2013. Karanja oil as an alternative fuel for direct injection CI engine-A
Review. International Journal of Science and Research, 2(10): 203-206.
Manjare, M. S., Deorukhkar, O. A. and Sathe, V. S. Esterification Reaction of

Acetic Acid and Ethanol with H2SO4: Batch Kinetics and Simulation Approach.
3(1): 73-79.
Mansourpoor, M. and Shariati, A. 2012. Optimization of biodiesel production from

sunflower oil using response surface methodology. Journal of Chemical
Engineering and Process Technology. 3(4).
Marchetti, J. M., Miguel, V. U., and Errazu, A. F. 2007. Possible methods

for biodiesel production. Renewable and sustainable energy reviews. 11(6):
1300-1311.
Meher, L. C., Sagar, D. V. and Naik, S. N. 2006. Technical aspects of biodiesel

production by transesterification—a review. Renewable and Sustainable Energy
Reviews. 10(3): 248-268.
Melero, J. A., Iglesias, J. and Morales, G. 2009. Heterogeneous acid catalysts for
biodiesel production: current status and future challenges. Green Chemistry.
11(9): 1285-1308.
Moser, B. R. 2009. Biodiesel production, properties, and feedstocks. In Vitro Cellular
and Developmental Biology-Plant. 45(3): 229-266.
Narasimharao, K., Lee, A. and Wilson, K. 2007. Catalysts in production of biodiesel:

a review. Journal of Biobased Materials and Bioenergy. 1(1): 19-30.
Narwal, S. K., Saun, N. K., Dogra, P., Chauhan, G. and Gupta, R. 2015.
Production and characterization of biodiesel using nonedible castor oil by
immobilized lipase from Bacillus aerius. Bio.Med. Research International.
2015(281934): 1-6.
Nasaruddin, R. R., Alam, M. Z. and Jami, M. S. 2013. Enzymatic biodiesel

production from sludge palm oil (SPO) using locally produced Candida
cylindracea lipase. African Journal of Biotechnology. 12(31) 4966-4974.
Nawani, N., Singh, R. and Kaur, J. 2006. Immobilization and stability studies of a
lipase from thermophilic Bacillus sp. The effect of process parameters on
immobilization of enzyme. Electronic Journal of Biotechnology. 9(5): 1-7.
Nelson, L. A., Foglia, T. A., and Marmer, W. N. 1996. Lipase-catalyzed production

of biodiesel. Journal of the American Oil Chemists' Society. 73(9): 1191-1195.
Osório, N. M., Dubreucq, E., da Fonseca, M. M. R. and Ferreira-Dias, S. 2009.

Operational stability of immobilised lipase/acyltransferase during
interesterification of fat blends. European Journal of Lipid Science and
Technology. 111(4): 358-367.
Patel, R. L. and Sankhavara, C. D. 2016. Biodiesel production from Karanja oil and
its use in diesel engine: A review. Renewable and Sustainable Energy Reviews.
(71): 464-474.
Patil, K. D. and Kulkarni, B. D. 2014. Kinetics studies on esterification reaction of

acetic acid with iso-amyl alcohol over ion exchange resin as catalysts. Inter. J.
Engine. Res. 3(8): 488-493.
Patil, V., Tran, K. Q. and Giselrød, H. R. 2008. Towards sustainable production of

biofuels from microalgae. International Journal Of Molecular Sciences. 9(7):
1188-1195.
Patni, N., Pillai, S. G. and Dwivedi, A. H. 2011. Analysis of current scenario of

biofuels in India specifically bio-diesel and bio-ethanol. In International
Conference on Current Trends in Technology. 8(10): 382-386
Pokharel, S. 2007. An econometric analysis of energy consumption in Nepal. Energy
Policy, 35(1): 350-361.
Qin, H. E., Yan, X. U., Yun, T. E. N. G. and Dong, W. A. N. G. 2008. Biodiesel

production catalyzed by whole-cell lipase from Rhizopus chinensis. Chinese
Journal of Catalysis. 29(1): 41-46.
Ranganathan, S. V., Narasimhan, S. L. and Muthukumar, K. (2008). An overview of

enzymatic production of biodiesel. Bioresource Technology. 99(10): 3975-3981.
Reşitoğlu, İ. A., Keskin, A. and Gürü, M. 2012. The optimization of the esterification
reaction in biodiesel production from trap grease. Energy Sources, Part A:
Recovery, Utilization, and Environmental Effects. 34(13): 1238-1248.
Ribeiro, A., Carvalho, J., Castro, J., Araújo, J., Vilarinho, C. and Castro, F. 2013.
Alternative feedstocks for biodiesel production. Materials Science Forum. 730:
623-629.
Robles-Medina, A., González-Moreno, P. A., Esteban-Cerdán, L. and Molina-

Grima, E. 2009. Biocatalysis: towards ever greener biodiesel production.
Biotechnology advances. 27(4): 398-408.
Romanski, J., Nowak, P., Kosinski, K. and Jurczak, J. 2012. High-pressure

transesterification of sterically hindered esters. Tetrahedron Letters. 53 (39):
5287–5289.
Sadaka, S. and Boateng, A. A. 2009. Pyrolysis and bio-oil.Cooperative Extension

Service, University of Arkansas, US Department of Agriculture and County
Governments Cooperating. pp 1-6.
Sales, A. 2011. Production of biodiesel from sunflower oil and ethanol by base
catalyzed transesterification. Thesis, M.Sc. Royal Institute of Technology
(KTH) Stockholm, Sweden 69p.
Salis, A., Pinna, M., Monduzzi, M. and Solinas, V. 2008. Comparison among
immobilised lipases on macroporous polypropylene toward biodiesel synthesis.
Journal of Molecular Catalysis B: Enzymatic. 54(1): 19-26.
Samukawa, T., Kaieda, M., Matsumoto, T., Ban, K., Kondo, A., Shimada, Y. and
Fukuda, H. 2000. Pretreatment of immobilized Candida antarctica lipase for
biodiesel fuel production from plant oil. Journal of Bioscience and
Bioengineering. 90(2): 180-183.
Samukawa, T., Kaieda, M., Matsumoto, T., Ban, K., Kondo, A., Shimada, Y., and
Fukuda, H. 2000. Pretreatment of immobilized Candida antarctica lipase for
biodiesel fuel production from plant oil. Journal of Bioscience and
Sánchez, M., Avhad, M. R., Marchetti, J. M., Martínez, M. and Aracil, J. 2016.
Enhancement of the jojobyl alcohols and biodiesel production using a
renewable catalyst in a pressurized reactor. Energy Conversion and
Management. 126: 1047-1053.
Shahid, E. M. and Jamal, Y. 2011. Production of biodiesel: a technical review.

Renewable and Sustainable Energy Reviews, 15(9): 4732-4745.
Shaw, S. Y., Chen, Y. J., Ou, J. J. and Ho, L. 2006. Preparation and characterization
of Pseudomonas putida esterase immobilized on magnetic nanoparticles.
Enzyme and Microbial Technology, 39(5): 1089-1095.
Shay, E. G. 1993. Diesel fuel from vegetable oils: status and opportunities. Biomass
and Bioenergy. 4(4): 227-242.
Shimada, Y., Watanabe, Y., Samukawa, T., Sugihara, A., Noda, H., Fukuda, H.
and Tominaga, Y. 1999. Conversion of vegetable oil to biodiesel using
immobilized Candida antarctica lipase. Journal of the American Oil Chemists'
Society. 76(7): 789-793.
Shimada, Y., Watanabe, Y., Sugihara, A., and Tominaga, Y. 2002. Enzymatic
alcoholysis for biodiesel fuel production and application of the reaction to oil
processing. Journal of Molecular Catalysis B: Enzymatic, 17(3): 133-142.
Sims, R. E., Mabee, W., Saddler, J. N. and Taylor, M. 2010. An overview of second
generation biofuel technologies. Bioresource Technology. 101(6): 1570-1580.
Singh, D., Kumar, V., Sandhu, S. S. and Sarma, A. K. 2016. Process optimization
for biodiesel production from indigenous non-edible Prunusarmeniaca
oil. Advances in Energy Research. 4(3): 189-202.
Singh, K., Singh, B., Verma, S. K. and Patra, D. D. 2014. Jatrophacurcas: a ten year
story from hope to despair. Renewable and Sustainable Energy Reviews. 35:
356-360.
Srivastava, A and Prasad, R. 2000. Triglycerides-based diesel fuels, Renewable

and Sustainable Energy Reviews. 4(2): 111–133.
Stamenkovic, O.S., Lazic, M.L., Todorovic, Z.B., Veljkovic, V.B. and Skala, D.U.
2007. The effect of agitation intensity on alkali catalyzed methanolysis of
sunflower oil. Bioresour Technol. 98(14): 2688–2699.
Subramanian, K. A., Singal, S. K., Saxena, M., and Singhal, S. 2005. Utilization of
liquid biofuels in automotive diesel engines: an Indian perspective. Biomass and
Bioenergy. 29(1): 65-72.
Sun, J., Chen, Y., Sheng, J. and Sun, M. 2015. Immobilization of Yarrowia lipolytica
lipase on macroporous resin using different methods: characterization of the
biocatalysts in hydrolysis reaction. Biomed Research International. (139179):
1-7.
Sun, Y. P., Li, X. Q., Cao, J., Zhang, W. X. and Wang, H. P. 2006. Characterization
of zero-valent iron nanoparticles. Advances in Colloid and Interface Science.
120(1): 47-56.
Sundqvist, T., Oasmaa, A. and Koskinen, A. 2015. Upgrading fast pyrolysis bio-oil
quality by esterification and azeotropic water removal. Energy and Fuels. 29(4):
2527-2534.
Suryawanshi, M. A., Shinde, N. H. and Nagotkar, R. V. 2014. Kinetic study of

esterification reaction for the synthesis of butyl acetate. International Journal of
Advanced Researach in Science, Engineering and Technology. 1(1): 2350 – 0328.
Swain, K. C. 2014. Biofuel Production in India: Potential, Prospectus and Technology.

Journal of Fundamentals of Renewable Energy and Applications. 4(1):
2090-4541.
Tan, T., Lu, J., Nie, K., Deng, L. and Wang, F. 2010. Biodiesel production with
immobilized lipase: a review. Biotechnology Advances. 28(5): 628-634.
Tapanes, N. C. O., Aranda, D. A. G. and Antunes, O. A. C. 2008. Transesterification of

Jatropha curcas oil glycerides: theoretical and experimental studies of biodiesel
reaction. Fuel. 87(10): 2286-2295.
Tavano, O. L., Fernandez-Lafuente, R., Goulart, A. J. and Monti, R. 2013.

Optimization of the immobilization of sweet potato amylase using
glutaraldehyde-agarose support. Characterization of the immobilized enzyme.
Process Biochemistry. 48(7): 1054-1058.
Tewari, A., Shah, S., Singh, N. and Tamta, K. K. 2015. Diploknema butyracea
(Roxb.) Lamb.: A Viable Livelihood Option For Hill Communities of Central
Himalayan Region. International Journal of Recent Scientific Research. 6( 5):
3937-3940.
Thangaraj, B., Jia, Z., Dai, L., Liu, D. and Du, W. 2016. Lipase NS81006
immobilized on Fe3O4 magnetic nanoparticles for biodiesel production. Ovidius
University Annals of Chemistry. 27(1): 13-21.
Thirumarimurugan, M., Sivakumar, V. M., Xavier, A. M., Prabhakaran, D. and

Kannadasan, T. 2012. Preparation of biodiesel from sunflower oil by
transesterification. International Journal of Bioscience, Biochemistry and
Bioinformatics. 2(6): 441.
Tsai, S. W. and Chang, C. S. 1993. Kinetics of lipase catalyzed hydrolysis of lipids

in biphasic organic-aqueous systems. Journal of Chemical Technology and
Tsai, S. W., Wu, G. H. and Chiang, C. L. 1991. Kinetics of enzymatic hydrolysis of

olive oil in biphasic organic aqueous systems. Biotechnology and
Tweddell, R. J., Kermasha, S., Combes, D. and Marty, A. 1997. Optimization of the
reaction medium for esterification catalyzed by A lipase from Pseudomonas
fluorescens. Biotechnology Letters. 19(9): 939-942.
US Energy Information Admisttation. 2010. Annual energy outlook 2010. 338p.
US Energy Information Admisttation. 2016. Annual energy outlook 2017. 64p.
Van-Tol, J. B. A., Jongejan, J. A., Duine, J. A., Kierkels, H. G., Geladé, E. F.,
Mosterd, F. and Kamphuis, J. 1995. Thermodynamic and kinetic parameters
of lipase-catalyzed ester hydrolysis in biphasic systems with varying organic
solvents. Biotechnology and Bioengineering. 48(3): 179-189.
Vimala, M. 2016. Energy consumption in india-recent trends. Asia Pacific Journal of

Research. 1(36).
Wang, L. and Yu, H. 2012. Biodiesel from Siberian apricot (Prunus sibirica L.) seed
kernel oil. Bioresource Technology. 112: 355–358.
Wu, L. Q., Wu, Y. F., Yang, C. G., Yang, L. M. and Yang, L. J. 2010. Silica
supported perchloric acid: an efficient catalyst for the synthesis of 14-aryl-14H-
dibenzo [a, i] xanthene-8, 13-diones. Journal of the Brazilian Chemical Society.
21(5): 941-945.
Xiu, S. and Shahbazi, A. 2012. Bio-oil production and upgrading research: A
review. Renewable and Sustainable Energy Reviews. 16(7): 4406-4414.
Zabeti, M., Daud, W. M. A. W. and Aroua, M. K. 2009. Activity of solid catalysts for
biodiesel production: a review. Fuel Processing Technology. 90(6): 770-777.
Zhang, D. H., Yuwen, L. X., and Peng, L. J. 2013. Parameters affecting the
performance of immobilized enzyme. Journal of Chemistry. (946248): 1-7
Zhang, X., Zhao, Y., Xu, S., Yang, Y., Liu, J., Wei, Y., and Yang, Q. 2014. Polystyrene
sulphonic acid resins with enhanced acid strength via macromolecular self-
assembly within confined nanospace. Nature Communications. 5(3170): 1-9.
Zheng, J. L. and Wei, Q. 2011. Improving the quality of fast pyrolysis bio-oil by
reduced pressure distillation. Biomass and Bioenergy. 35(5): 1804-1810.
Appendices
ANNEXURE
Annexure 1. The list of equipment used during the experimentation

S.N. Instruments Manufacturer
1 Electronic balance Adair Dutt Ltd., India
2 Magnetic Stirrer Tarsons Pvt. Ltd., India
3 Sonilcator Sonics Pvt. Ltd., India
4 Ice Flaker machine Harrison Scientific instruments
5 Lyophilizer Freeze Drying System
6 Hot air oven Sanco Pvt. Ltd., India
7 Micropipette HiMedia Pvt. Ltd.
8 Refrigerator LG Pvt. Ltd., India
9 Spectrophotometer Thermoscientific Pvt. Ltd., India
10 Incubator shaker Unilab Pvt. Ltd., India
11 pH meter Eutech Instruments Pvt. Ltd.
12 Zeta - sizer Malvern Instruments Ltd.
13 GC-MS RTX-5 MS GCMS-QP2010 Plus
Annexure ............
Annexure 2. Tabulated data of Different experiments.
Annexure 2.1 HClO4-SiO2 Catalyzed Esterification of Lauric Acid

and Propanol (Section 3.4)

Time
(Hours)
Control reaction HClO4-SiO2 catalyzed Bare SiO2 catalyzed
0.00 0.00 ±0.00 0.00 ±0.00 0.00 ± 0.00
0.5 6.57 ±0.41 29.11 ±0.41 8.22± 0.41
1.00 8.22 ±0.41 39.20 ±0.41 13.62 ± 0.41
1.50 -6.57 ±0.41 54.69 ±0.41 -0.23 ± 0.41
2.00 -11.74 ±0.41 65.73 ±0.41 -9.86 ± 0.41
2.50 -9.15 ±0.0.00 67.61 ±0.00 -9.15 ± 0.00
3.00 -7.28 ±0.41 71.36 ±0.41 -6.81 ± 0.41
3.50 -7.04 ±0.00 75.35 ±0.41 -5.16 ± 0.00
4.00 -6.81 ±0.41 75.35 ±0.00 -4.93 ± 0.00
Annexure ............
Annexure 2.2 Effect of Acid to Alcohol Molar ratio on Esterification
Reaction (Section 3.5.1)

S/N MOLAR RATIO
I II III Mean Standard Deviation
1 1:3 83.26 85.59 85.07 84.64 ±1.00
2 1:4 90.00 89.37 88.67 89.35 ±0.54
3 1:5 89.22 89.82 89.94 89.66 ±0.31
4 1:6 92.00 92.31 92.62 92.31 ±0.25
5 1:7 91.67 90.77 91.60 91.35 ±0.41
6 1:8 91.80 91.87 90.98 91.55 ±0.40
7 1:9 90.91 90.99 91.07 90.99 ±0.07
8 1:10 90.10 87.62 90.10 89.27 ±1.17
Annexure 2.3 Effect of HClO4-SiO2 Amount on Esterification Reaction

(Section 3.5.2)

Catalyst Amount
S/N Standard
(mol%) I II III Mean
Deviation
1 1.00 66.00 66.67 65.77 66.15 ±0.46
2 0.50 69.13 68.46 70.00 69.19 ±0.77
3 0.25 89.26 88.67 89.93 89.29 ±0.63
4 0.125 86.58 85.91 85.91 86.13 ±0.33
5 0.1 65.33 65.77 65.77 65.63 ±0.25
Annexure ............
Annexure 2.4 Effect of Reaction Temperature on Esterification Reaction
(Section 3.5.3)

Reaction Temperature
S/N
(˚C) Standard
I II III Mean
Deviation
1 70 81.88 80.54 80.41 80.9405 ± 0.82
2 80 88.51 89.26 88.51 88.76292 ± 0.43
3 90 91.28 91.95 92.00 91.74049 ± 0.40
4 100 95.27 94.59 94.63 94.83191 ± 0.38
Annexure 2.5 Effect of Reaction Temperature on Esterification Reaction

(Section 3.5.3)
Time Reaction Progress (%)

(Hours) 70 80 90 100
0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
0.5 51.45 ± 0.20 58.52 ± 0.23 73.21 ± 0.68 83.41 ± 0.32
1.00 72.61 ± 0.58 75.34 ± 0.44 83.93 ± 0.62 93.27 ± 0.03
1.50 76.84 ± 0.44 82.51 ± 0.67 90.18 ± 0.37 95.07 ± 0.41
2.00 83.07 ± 0.45 87.89 ± 0.67 92.19 ± 0.37 93.50 ± 0.40
Annexure ............
Annexure 2.6 Reusability of HClO4-SiO2 Catalyst System (Section 3.5.4)

S/N Reaction Cycle
1 1 91.95 92.67 91.95 92.19 ± 0.42
2 2 83.33 83.89 82.55 83.26 ± 0.67
3 3 76.67 77.70 77.48 77.28 ± 0.55
4 4 69.13 68.46 70.67 69.42 ± 1.13
5 5 62.16 62.91 63.09 62.72 ± 0.49
Annexure 2.7 Protein Estimation (Section 3.10.5)
A595
S.N. BSA(mL) BSA (µg)
I II Mean
1 0.00 0 0.026 0.027 0.0265
2 0.10 10 0.06 0.058 0.059
3 0.20 20 0.105 0.115 0.11
4 0.40 40 0.158 0.16 0.159
5 0.60 60 0.201 0.21 0.2055
6 0.80 80 0.25 0.261 0.2555
7 1.00 100 0.026 0.027 0.0265
Enzyme (before immobilization) 0.026 0.032 0.029
Enzyme (after immobilization) 0.198 0.209 0.2035
Annexure ............
Annexure 2.8 Lipase Assay (Section 3.10.5)
S.N. ENZYME KOH (titer volume) Lipase Activity

I II III I II III Mean
1 Free Enzyme 7.8 8.2 8.1 6.50 6.83 6.75 6.69±0.17

(before immobilization)
2 Residual Enzyme 6.4 6.3 6.5 5.33 5.25 5.42 5.33±0.8

(after immobilization)
3 Immobilized Enzyme 0.8 0.7 0.9 0.67 0.58 0.75 0.67±0.8
Annexure 2.9 Effect of Oil to Aqueous Phase Volume ratio on Lipase

Activity (Section 3.11.1)
S.N. Volume ratio Lipase Activity

(oil/methanol)
I II III Mean SD
1 1:6 2.58 2.25 2.08 2.31 0.25
2 1:4 2.92 3.17 2.83 2.97 0.17
3 1:2 3.50 3.75 3.75 3.67 0.14
4 1:1 4.17 4.50 4.25 4.31 0.17
5 1:0.8 2.67 2.58 2.50 2.58 0.08
6 1:0.6 2.17 2.33 2.17 2.22 0.10
7 1:0.4 1.67 1.58 1.42 1.56 0.13
8 1:0.2 1.25 1.17 1.25 1.22 0.05
Annexure ............
Annexure 2.10 Effect of Substrate Concentration on Lipase Activity
(Section 3.11.2)
Free Lipase Activity Immobilized Lipase

Oil concentration (µmol/min) Activity(µmol/min)
S.N.
(mM)
I II III Mean SD I II III Mean SD
1 0 0.00 0.00 0.00 0.00 ±0.00 0.00 0.00 0.00 0.00 ±0.00
2 200 1.17 1.33 1.17 1.22 ±0.10 0.75 0.83 0.75 0.78 ±0.05
3 400 3.00 2.83 2.50 2.78 ±0.25 1.50 1.42 1.33 1.42 ±0.08
4 600 4.50 4.17 4.17 4.28 ±0.19 2.08 1.92 2.25 2.08 ±0.17
5 800 5.17 5.08 4.92 5.06 ±0.13 2.50 2.58 2.75 2.61 ±0.13
6 1000 5.33 5.33 5.50 5.39 ±0.10 2.75 2.92 2.92 2.86 ±0.10
7 1200 5.67 5.92 5.83 5.81 ±0.13 3.17 3.25 3.17 3.19 ±0.05
8 1400 5.67 5.83 6.00 5.83 ±0.17 3.42 3.17 3.08 3.22 ±0.17
Annexure 2.11 Effect of Reaction Temperature on Lipase Activity

(Section 3.11.3)
Lipase Activity (µmol/min)

S/N Temperature (˚C)
1 25 2.08 2.08 2.00 2.06 ±0.05
2 30 2.50 2.58 2.50 2.53 ±0.05
3 35 3.00 3.17 3.08 3.08 ±0.08
4 40 3.33 3.58 3.67 3.53 ±0.17
5 45 1.67 1.75 1.67 1.69 ±0.05
Annexure ............
Annexure 2.12 Effect of pH on Lipase Activity (Section 3.11.4)

S/N pH
1 5.00 0.92 0.83 0.83 0.86 ±0.05
2 5.50 1.17 1.25 1.17 1.19 ±0.05
3 6.00 1.17 1.25 1.42 1.28 ±0.13
4 6.50 2.42 2.25 2.33 2.33 ±0.08
5 7.00 2.58 2.67 2.75 2.67 ±0.08
6 7.40 3.67 3.58 3.67 3.64 ±0.05
7 8.00 2.50 2.67 2.83 2.67 ±0.17
8 8.50 1.33 1.33 1.50 1.39 ±0.10
9 9.00 1.58 1.25 1.33 1.39 ±0.17
Annexure 2.13 Storage Stability (Section 3.12.1)

S/N Time (hours)
1 12 6.50 6.83 6.75 6.69 ±0.17
2 36 3.58 3.83 4.33 3.92 ±0.38
3 60 1.00 1.33 1.25 1.19 ±0.17
4 72 1.25 0.92 1.00 1.06 ±0.17
Annexure ............
Annexure 2.14 Organic Solvent Stability (Section 3.12.2)

S/N Organic Solvent
1 Methanol 1.00 1.00 0.92 0.97 ±0.05
2 Ethanol 1.67 1.50 1.67 1.61 ±0.10
3 Isopropanol 1.50 1.42 1.42 1.44 ±0.05
4 Hexane 3.00 3.08 3.08 3.06 ±0.05
Annexure 2.15 Reusability (Section 3.12.2)
Lipase Residual Activity (µmol/min)

S/N Reaction Cycles
1 1 3.33 3.92 4.25 3.83 ±0.46
2 2 3.50 3.17 3.00 3.22 ±0.25
3 3 2.75 2.58 2.50 2.61 ±0.13
4 4 2.00 2.08 1.83 1.97 ±0.13
5 5 1.17 1.25 1.25 1.22 ±0.05
Annexure ............
Annexure 3. GC-MS Analysis
Annexure 3.1 GC-MS analysis of bio-oil esterified by HClO4-SiO2
Annexure ............
List of chemical detected
Peak No. Retention Area% Name of compound

Time
1 4.527 2.25 2-furanmethanol, tetrahydro-
2 7.542 9.67 .Beta.-d-glucopyranose, 1,6-anhydro-
3 8.597 0.95 1,3-di-o-acetylpentopyranose
4 9.977 0.31 .Alpha.-.beta.-d-ribopyranose, 1,3-di-o-acet
5 18.381 0.25 Hexadecanoic acid, methyl ester
6 19.522 7.37 Hexadecanoic acid
7 22.336 0.27 Methyl octadeca-9,12-dienoate
8 22.509 1.11 9-octadecenoic acid (z)-, methyl ester
9 23.920 54.17 Octadec-9-enoic acid
10 24.246 0.33 Octadecanoic acid
11 26.860 2.20 Glycidyl palmitate
12 29.489 0.24 Oleic acid, 3-hydroxypropyl ester
13 30.326 1.06 1,8,11-heptadecatriene, (z,z)-
14 30.446 9.51 2-hydroxy-3-[(9e)-9-octadecenoyloxy] PROPYL

(9E)-9-octadecenoate
16 31.521 0.60 1,2-benzenedicarboxylic acid
17 33.778 0.64 Octadecanoic acid, 4-methoxy-, methyl ester
18 38.972 0.52 1,7-dimethyl-4-(1-methylethyl)cyclodecan
19 44.296 7.08 9-octadecenal, (z)-
20 45.727 1.05 2-heptadecyloxirane
100.00
Annexure ............
Annexure 3.2 GC-MS analysis of Cheura oil Biodiesel prepared by alkali
catalyzed transesterification.
Annexure ............
List of compound detected.
Peak Retention Area Compound Name

No. Time %
1 13.155 0.11 Tridecanoic acid, 12-methyl- ester
2 15.972 0.03 Pentadecanoic acid, methyl ester
3 17.812 0.26 9-hexadecenoic acid, methyl ester, (z)-
5 20.192 0.02 7-Hexadecanoic acid methyl ester, (z)-
6 20.796 0.07 Heptadecanoic acid, methyl ester
7 22.382 1.48 9,12-octadecadienoic acid (z,z)-, methyl est
8 22.868 39.53 9-octadecenoic acid methyl ester, (E)-,
9 23.283 3.56 Methyl stearate
11 27.491 0.15 Eicosanoic acid, methyl ester
12 28.929 0.09 Tetracosanoic acid, methyl ester
13 30.408 0.11 2-hydroxy-3-[(9e)-9-octadecenoyloxy] propyl (9e)-

9-octadecenoate
14 31.515 0.03 Docosanoic acid, methyl ester
15 31.763 0.05 2,6,10,14,18,22-tetracosahexaene, 2,6,10, 15,19,23

hexamethyl
100
Annexure ............
Annexure 3.3 GC-MS analysis of Wild Apricot oil Biodiesel prepared by alkali
Annexure ............
List of compounds detected
Peak Retention Area Compound Name

No. Time %
1 13.224 0.07 Tetradecanoic acid, methyl ester
2 17.858 2.78 9-Hexadecenoic acid, methyl ester, (z)-
6 20.237 0.53 Cyclopropaneoctanoic acid, 2-hexyl-, methyl ester
8 22.449 2.02 9,12-octadecadienoic acid (z,z)-, methyl est
10 23.522 2.93 Octadecanoic acid, methyl ester
12 24.171 0.06 Ethyl 9-octadecenoate
14 26.319 0.40 Hexadecadienoic acid, methyl ester
15 26.788 0.28 Oxiraneoctanoic acid, 3-octyl-, methyl ester
16 26.860 0.25 0.25 Glycidyl palmitate
17 26.972 0.46 11-eicosenoic acid, methyl ester
20 30.462 3.05 Glycidyl oleate
21 30.983 0.16 Dotriacontane
23 32.892 0.05 Hexacosane
24 33.430 0.06 Tricosanoic acid, methyl ester
25 33.780 0.15 Octadecanoic acid, 4-methoxy-, methyl es
hexamethyl
29 40.587 0.14 2h-1-benzopyran-6-ol, 3,4-dihydro-2,7,8-trim
30 46.093 0.29 Stigmast-5-en-3-ol, (3.beta.)-
100
Annexure ............
Annexure 3.4 GC-MS analysis of Karanj Biodiesel prepared by alkali catalyzed
transesterification.
Annexure ............
List of compound detected
Peak Retention Area% Name of compound
No. Time
1 10.146 0.16 6-methoxy-1h-indole-3-carbaldehyde
7 22.408 5.30 9,12-octadecadienoic acid (z,z)-, methyl ester
8 23.129 38.66 9-octadecenoic acid, methyl ester, (e)-
12 25.533 0.06 6,9,12-octadecatrienoic acid, methyl ester
14 26.014 0.17 Hexadecatrienoic acid, methyl ester
16 26.361 0.36 9,12,15-octadecatrienoic acid, methyl este
18 26.773 2.03 9-octadecensaeure, 12-hydroxy-, methyles
21 27.812 0.87 Oxiraneoctanoic acid, 3-octyl-, methyl es
22 28.312 0.16 13,21-cyclo-18-norpregnan-20-ol, 20-methyl-
23 28.512 0.07 Naphthalene, decahydro-1,6-dimethyl-
24 28.665 0.25 Tricyclo[20.8.0.0e7,16]triacontan, 1(22),7(16)-d
25 29.549 0.18 Heneicosanoic acid, methyl ester
27 30.419 0.57 Glycidyl oleate
28 30.996 0.37 13-docosenoic acid, methyl ester, (z)-
30 32.425 0.51 2,2-dimethyl-7-hydroxy-6-[(2'-phenyletheny
31 32.841 1.60 2-phenyl-furo[b]benzopyran-4(4h)-one
32 33.150 1.27 2-[5-(2-methyl-1,3-benzoxazol-7-yl)-1h-pyra
37 37.455 0.78 4-(1,1-dimethylethyl)-3-(2,6-dimethylphenyl
38 37.833 0.28 1h-benzimidazol-2-ylmethyl 4-quinazoliny
39 38.715 0.84 Hexacosanoic acid, methyl ester
40 39.769 0.70 Quino[2,3-b]acridine-7,14-dione, 12,13a-dihydro-2-methyl-
41 40.762 0.36 4-(3-methylphenyl)-6,7,8,9-tetrahydro[1] benzothieno[3,2-e]
[1,2,4]triazolo[4,3-A]pyrimidin-5(4H)-one
100.00
Annexure ............
Annexure 3.5 GC-MS analysis of Sunflower oil Biodiesel prepared by alkali
Annexure ............
Peak R. Time Area% Name of compound
No.
3 17.692 0.04 Methyl (7e)-7-hexadecenoate #
9 23.350 74.64 9-octadecenoic acid, methyl ester, (e)-
14 24.157 0.03 9-octadecenoic acid (z)-, ethyl ester
17 25.772 0.11 Cis,cis,cis-6,9,12-Octadecatrienoic acid, propyl ester
18 26.031 0.11 Hexadecatrienoic acid, methyl ester
21 26.638 0.07 Cis,cis,cis-6,9,12-Octadecatrienoic acid, propyl ester
25 28.311 0.07 13,21-cyclo-18-norpregnan-20-ol, 20-methyl-
26 28.668 0.14 Tricyclo[20.8.0.0e7,16]triacontan, 1(22),7(16)-d
27 29.916 0.14
28 30.984 0.15
30 32.865 0.05 Tetratetracontane
32 34.697 0.04 Hexacosane
hexamethyl
36 36.996 0.04 Heneicosanoic acid, methyl ester
38 38.699 0.11 Hexacosanoic acid, methyl ester
100.00
Annexure ............
Annexure 3.6 GC-MS analysis of Cheura oil Biodiesel prepared by Lipase
Annexure ............
Peak Retention Area% Name of Compound

No. Time
8 22.618 16.52 9-octadecenoic acid, methyl ester
15 30.460 1.96 2-hydroxy-3-[(9e)-9-octadecenoyloxy]propyl ester
17 33.787 0.23 3-(3-Methylbutyl)thiophene-1,1-dioxide
18 36.658 0.35 2,6,10,14,18,22-TETRACOSAHEXAENE,

2,6,10,15,19, 23 hexamethyl
19 40.604 0.24 2h-1-benzopyran-6-ol, 3,4-dihydro-2,7,8-trimethyl-2-

(4,8,12-trimethyltridecyl)-
100.00
Annexure ............
Annexure 3.7 GC-MS analysis of Wild Apricot Biodiesel prepared by Lipase
Annexure ............
Peak# R.Time Area% Name
10 24.065 0.36 9-octadecenoic acid (z)-, ethyl ester
13 26.967 0.88
16 30.480 3.44 2-hydroxy-3-[(9e)-9-octadecenoyloxy]propyl
18 33.806 0.26 Octadecanoic acid, 4-methoxy-, methyl est
19 34.725 7.68 9-octadecenoic acid (z)-, 2-hydroxy-1-(hydr
20 35.755 0.34 Benzyl (9z)-9-hexadecenoate
21 36.679 0.25 2,6,10,14,18,22-tetracosahexaene, 2,6,10,15,19, 23 hexamethyl
22 39.708 0.18 5-(1-hexynyl)-2-furohydrazide
23 40.626 0.50 2h-1-benzopyran-6-ol, 3,4-dihydro-2,7,8- trimethyl-2-(4,8,12-

trimethyltridecyl)-
100.00
Annexure ............
Annexure 3.8 GC-MS analysis of Karanj Biodiesel prepared by Lipase catalyzed
transesterification.
Annexure ............
No. Time
2 17.828 0.04 9-hexadecenoic acid, methyl ester, (Z)-
6 22.416 12.85 9,12-octadecadienoic acid (Z,Z)-, methyl ester
12 26.751 1.03 10-undecenoic acid, methyl ester
15 27.814 0.32 (9e,12e)-9,12-octadecadienoyl chloride
16 30.325 0.24 1,8,11-heptadecatriene, (Z,Z)-
17 30.451 1.85 2-hydroxy-3-[(9E)-9-octadecenoyloxy]propyl (9E)-9-
octadecenoate
18 30.998 0.25 Methyl dihydromalvalate
21 32.687 0.15
22 32.888 2.74 2-(2-phenyl-2-propenyl)-1h-isoindole-1,3(2H)- dione
23 33.147 1.72 2-[5-(2-methyl-1,3-benzoxazol-7-yl)-1H-pyrazol-3-
yl]phenol
25 33.683 0.06 1,1-dichloro-2,2,3,3-tetramethylcyclopropane
26 33.788 0.36 Octadecanoic acid, 4-methoxy-, methyl ester
27 34.717 7.30 9-octadecenoic acid (Z)-, 2-hydroxy-1-
(hydroxymethyl)ethyl ester
29 35.614 0.29 2,2-dimethyl-7-hydroxy-6-[(2'-phenylethenyyl)carbonyl]-
(benzo-2h-pyran)
30 37.473 1.08 4-(1,1-dimethylethyl)-3-(2,6-dimethylphenyl)-1-methyl-5-
phenylimidazol-2(1H)-one
31 37.859 0.35 N-methyl-4-[(4-methyl-1-phthalazinyl)amin
32 38.701 0.56 4h,8h-benzo(1,2-b:3,4-b')dipyran-4-one, 2,3-dih
33 39.785 0.96 Quino[2,3-b]acridine-7,14-dione, 12,13a-dihydro-2-
methyl-
34 40.798 0.67 Diazene, bis[2,6-bis(1-methylethyl)phenyl]-
35 41.537 0.19 1H-indene-1,3(2H)-dione, 2-[[4-(diethylamino)-2,6
dimethylphenyl]imino]-
100.00
Annexure ............
Annexure 3.8 GC-MS analysis of Sunflower Biodiesel prepared by Lipase
Annexure ............

No. Time
8 23.559 0.66 Heptadecene-(8)-carbonic acid-(1)
9 26.924 1.68 Hexadecanoic acid, 1-(hydroxymethyl)-1,2-1,2-ethanediyl

ester
12 30.464 8.00 9-octadecenal, (z)-
13 31.545 1.43 Triacontanoic acid, methyl ester
14 33.788 0.20 Heptanoic acid, docosyl ester
16 36.664 0.30 2,6,10,14,18,22-tetracosahexaene, 2,6,10,15,19,23

hexamethyl
17 42.029 1.52 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltride
100.00
Annexure ............
VITA
The author of this manuscript Manendra Singh Negi was born on 16th August,
1996 in Villege Timali Chhoti, Yamkeshwar Block, Pauri district of Uttarakhand,
India. He has successfully completed his High school from S.V.M. Inter College Avas
Vikas Rishikesh in 2010 and Intermediate from S.V.M. Inter College Avas Vikas
Rishikesh in 2012. The author passed his B.Sc. from, Pt. L.M.S. Govt.(Autonomous)
Post Graduate College Rishikesh in 2015. He was admitted to the College of Post
Graduate Studies in Department of Biochemistry, CBSH, in 2015-16 in G.B. Pant
University of Agriculture and Technology, Pantnagar, Uttarakhand, India, for the
degree of M.Sc. with major in Biochemistry and Minor in Molecular Biology and
Biotechnology. During Graduation Post Graduation, he was also awarded with DST-
INSPIRE Scholarship for Higher Education.
Permanent Address
Manendra Singh Negi
S/o Mr. Rajendra Singh Negi
Street No. 15 Shivaji Nagar, P/O Pashulok
Rishikesh (Dehradun)
Uttarakhand
Pin code: 249203
Email: m97negi@gmail.com
Name : Manendra Singh Negi Id. No. : 49364
Sem. & year : 1st semester, 2015-2016 Degree : M.Sc.
of admission Deptt. : Biochemistry
Major : Biochemistry
Minor : Molecular biology and Biotechnology
Thesis Title : “EFFICIENT PRODUCTION OF BIODIESEL BY ESTERIFICATION/
ENZYME AND CHEMICAL CATALYST”
Advisor : Dr. A. K. Verma
ABSTRACT
Renewable energy sources such as biofuels (biodiesel and bioethanol) are

sustainable and eco-friendly. A variety of non-edible oil plant species from agro-climatic
zones of India is promising feedstock for biodiesel production. In the present
investigation, biodiesel was produced by chemical catalyzed or lipase-mediated
esterification/ transesterification reactions. Biodiesel produced by perchloric acid
(HClO4) immobilized on silica gel (SiO2) and lipase immobilized on the iron
nanoparticle. Three tree-seed-oil such as Cheura (Diploknema butyracea), Wild Apricot
(Prunus armeniaca), Karanj (Pongammia pinnata) and one refined Sunflower oil
(Helianthus annus) was transesterified with alcohol and the product was analyzed by
GC-MS. Further bio-oil obtained from sugarcane residues were also esterified with
propanol and analyzed by GC-MS. A comparative study of kinetic parameters of HClO4-
SiO2 and immobilized lipase for esterification/transesterification was carried with alkali-
catalyzed transesterification. HClO4-SiO2 was efficient and reusable esterifying
dodecanoic acid with propanol at optimum parameters molar ratio 1:6, temperature 90˚C,
catalyst amount 0.25mol% and produce greater than 92% yield of ester in 2 hours.
However, HClO4-SiO2 could not transesterify tree-seed-oils. Alkali catalyst was found
fast and an efficient transesterification in tree–seed-oils with a high yield of 90-99.71%
FAME in 3 hours at 70 ˚C. Immobilized lipase showed a decrease in enzyme efficiency
(Kcat/Km 0.341 mM-1s-1) but increase in thermostability from 37 ˚C to 40 ˚C with 5
times reusability. Immobilized lipase was found effective in the transesterification of
tree-seed-oils and gave 75-85% FAME yield, with different oils in 24 hours. Lesser yield
and longer reaction time observed in lipase-catalyzed transesterification but it was
compensating the high energy cost of alkali-catalyzed transesterification. Highest
biodiesel yield 99.71% achieved by alkali-catalyzed transesterification of Cheura oil.
uke % eusUnz flag usxh ifjp;kad % 49364
l= ,oa izo"ks"k o"kZ
o"kZ % izFke] 2015&2016 mikf/k % LukrdksÙkj
izeq[k % tSo jlk;u foKku foHkkx % tSo jlk;u foKku
xkS.k fo"k; % vk.kfod tho foKku ,oa tSo rduhdh
'kks/k dk 'kh"kZd % “fLfFkjhd`r ,Utkbe rFkk jlk;fud mRizsjd ds mi;ksx ls bZLVjhdj.k@
VªkalbZLVjhdj.k }kjk tSo Mhty dk dq’ky mRiknu”
lykgdkj % MWk0 ,0 ds0 oekZ
lkjka'k
uohuhd`r mtkZ lks= tSls tSo b/kZu¼tSo Mhty tSo bSFkuky ½ LFk;h rFkk iz;kZoj.k fgrS”kh gSA
Hkkjr dh d`f”k tyok;q esa mxus okys fofHkUu izdkj ds xSj [kk| rsy ikni tSo Mhty ds vfHkUu lzksr
gSA orZeku v/;;u esa j;k;fud mRizsjd ij ijDyksfjd vEy (HClO4) dks flfydk tSy (SiO2) ij
rFkk ykbist dks ykSg uSuks d.kksa ij fLfFkjhd`r fd;k x;k rFkk bldk mi;ksx fpmjk ¼fMIyksuhek
C;qVk;hjdk½] txayh [kqekuh ¼izuql vesfZ u;k½] dajt ¼iksxa eh;k fiUUkkVk½ rFkk lqjteq[kh ¼gSyh,aFkl ,sul½ ds
chtks dks VªkalbZLVjhdj.k dj GC-MS }kjk v/;;u fd;k x;k rFkk xUusa ds ikS/ksa ds vo’ks”k ls cus
tSo rsy dk bZLVjhdj.k dj GC-MS }kjk v/;;u fd;k x;kA HClO4-SiO2 rFkk fLfFkjhd`r
ykbist mRizfs jr bZLVjhdj.k@VªkalbZLVjhdj.k vfHkfdz;k ds cyxfrdh; ekudksa dk rqyukRed v/;;u
{kkj mRizfs jr VªkalbZLVjhdj.k ds lkFk fd;k x;kA vuqdy
q re ekudksa tSls ekSyj vuqikr 1%6] rki 90°C
rFkk vfHkdkjd 0-25 ekSy izfr’kr ij HClO4-SiO2 }kjk 92 izfr’kr ls vf/kd bZLVj mRiknu ds lkFk
MksMus kbZd vEy ¼ykfjd vEy½ dk dq’ky bZLVjhdj.k ik;k x;k ijUrq ;g mRizsjd o`{k chtksa ds rsy ds
VªkalbZLVjhdj.k esa vlQy ik;k x;kA {kkj mRizsjd 3 ?k.Vs esa 90&99-7 izfr’kr FAME mRiknu ds lkFk
o`{k chtksa ds rsy ds VªkalbZLVjhdj.k esa dq’ky rFkk rhoz ik;k x;kA fLfFkjhdj.k ls ykbist ds xfrdh;
ekudksa esa ifjoZru vk;k ¼ Kcat/Km 0.341 mM-1s-1½ rFkk lEiw.kZ ,atkbe n{krk esa fxjkoV gqbZ ijUrq
,atkbe dh fLFkjrk rFkk iqu% izlkstu {kerk ¼ikWp ckj½ c<+ x;hA fLfFkjhd`r ykbist 75&86 izfr’kr
FAME mRiknu 24 ?k.Vsa esa nsrs gq, o~{k chtksa ds VªkalbZLVjhdj.k esa izHkko’kkyh ik;k x;kA ykbist
mRizfs jr VªkalbZLVjhdj.k esa nh?kZ vfHkfØ;k dky rFkk derj mRiknu ik;k x;k] ijUrq ;g {kkj mRizfs jr
bZLVjhdj.kesa yxus okyh mPp mtkZ ykxr dh {kfr iwfrZ dj jgk FkkA fpmjk rst ds {kkj mRizfs jr
VªkalbZLVjhdj.k ls vf/kdre tSoMhty mRiknu 99-7 izfr’kr ik;k x;kA

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EFFICIENT PRODUCTION OF BIODIESEL BY

G.B. Pant University of Agriculture & Technology

Manendra Singh Negi

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

On the occasion of accomplishment of the present study I would like to express my

With deep sense of gratitude I extend my warmest thanks to the members of my

I express my sincere thanks to Head Department of Biochemistry, Dr. Sanjeev

This acknowledgment will be incomplete my love and gratitude toward my Idol my

Pantnagar (Manendra Singh Negi)

Pantnagar (A. K. Verma)

(T. K. Bhattacharya) (B. R. Singh) (Ashutosh Dubey)

3. Materials and Methods

4. Results and Discussion

5. Summary and Conclusion

Table No Table Title Page No

2.1 Fuel property of petroleum diesel and biodiesel.

2.2 Oil seed plants as potential alternative feedstock of biodiesel.

2.3 Summary of various acid catalyzed transesterification

3.1 Fat components of different Tree Oilseed

3.2 Reaction mixtures of various Acid to Acohol molar ratio

3.3 Catalyst amount for various mol% of catalyst

3.4 Reaction mixtures for transesterification of various tree seed

3.5 Reaction mixtures of different volume ratio of Oil to Aqueous

3.6 Reaction mixtures of different Substrate concentration for

3.7 Reaction mixtures of different molar ratio Substrate

4.1 Summery of GC-MS analysis obtained from esterified

4.2 Summery of GC-MS analysis of biodiesel obtained by alkali

4.3 Change in kinetic parameters of lipase after immobilization

4.4 Summery of GC-MS analysis of biodiesel obtained by lipase

Table Figure Title Page

2.1 Schemes of esterification andTranesterification

3.1 Bovine Serum Albumin (BSA) standards for estimation of protein

4.1 HClO4-SiO2 catalyzed esterification of Lauric Acid with Propanol

4.2 Esterification of lauric acid with propanol at different molar ratio

4.3 Effect of HClO4-SiO2 amount on esterification reaction

4.4 Effect of Temperature on Esterification of Lauric Acid with Propanol

4.5 Reaction progress with time at different Temperatures.

4.6 Reusability of HClO4-SiO2 up to five cycles.

4.8 Hydrodynamic property of Iron Nanoparticles: (A), (B) Zeta size of

4.10 Effect of Oil to Aqueous Phase Volume ratio on Lipase Activity

4.11 Michaelis-Menten curves for free and immobilized lipase

4.12 Lineweaver-Burk plot for free and immobilized lipase

4.13 Effect of Temperature on Lipase Activity

4.14 Effect of pH on Lipase Activity

4.15 Storage stability of immobilized lipase

4.16 Organic solvent stability of immobilized Lipase

4.17 Reusability of immobilized Lipase

4.18 Immobilized lipase catalyzed esterification of lauric acid with

4.19 Comparative analysis of transesterification and biodiesel yield in

AIRF Advanced Research Instrumentation Facility

BSA Bovine Serum Albumin

CBB Coomassie Brilliant Blue

DAG Di Acyl glycerol

FFA Free Fatty Acid

GC-MS Gas Chromatography and Mass Spectroscopy

MAG Mono Acyl gycerol

TAG Tri Acyl Glycerol

TAN Total Acid Number

Current global energy consumption is heavily dominated by fossil fuels derived

1. Immobilization of perchloric acid on Silica gel and lipase and on Iron

2. Optimization of esterification/transesterification reaction with both the

3. Comparative analysis of esterification/transesterification of oil of seeds of

Energy is a fundamental necessity for fulfilling social economic and