A Report of

INVESTIGATION ON THE POTENTIAL OF SOME

SELECTED WETLAND PLANTS FOR THE

BIODIESEL PRODUCTION

Ms. NEETHU CYRIL

(1936-MRP/14

Assistant Professor

Assumption Autonomous

Changanacherry, Kottayam, Kerala, 686101

University Grants Commission

A Report of Minor Research Project

INVESTIGATION ON THE POTENTIAL OF SOME

SELECTED WETLAND PLANTS FOR THE

BIODIESEL PRODUCTION

Ms. NEETHU CYRIL,

MRP/14-15/KLMG034/UGC-SWRO)

Assistant Professor, Department of Chemistry

Assumption Autonomous College

Changanacherry, Kottayam, Kerala, 686101

Submitted to

University Grants Commission, New Delhi

January 2017

INVESTIGATION ON THE POTENTIAL OF SOME

SELECTED WETLAND PLANTS FOR THE

ACKNOWLEDGEMENTS

The present study was supported by the University grants commission of India

under the grant No:1936-MRP/14-15/KLMG034/UGC-SWRO.

I would like to express my sincere thanks to Dr Sr Amala SH, Principal,

Assumption College, Changanacherry for her inspiration, constant support and for

providing necessary facilities for carrying out this work.

I wish to thank Dr. Jissy Mathew, HOD, Department of Chemistry, Dr. Marykutty

CV, former HOD, Assumption College, Changanacherry and my colleagues for their

constant encouragement and support throughout the period of my work.

I deeply express my gratitude to my parents and family members for their

constant encouragement, valuable support and understanding.

Above all I thank God Almighty for his grace and blessings....

Neethu Cyril

CONTENTS

Abstract 1

1. Introduction 2

1.1 Global energy needs and significance of a sustainable energy resource 2

1.2 Biodiesel – A Clean Energy Source 4

1.3 Transesterification 4

1.3.1 One step transesterification

1.3.2 Two step acid base transesterification

1.3.3 Advanced transesterification process

Ultra sound assisted transesterification

Microwave assisted transesterification

1.4 Oil sources for Biodiesel 9

1.5 Environmental Impact of Biodiesel 10

1.6 Scope of present investigation 12

1.7 Objectives of the present investigation 13

References

2. Review of Literature 16

2.1 Indian Scenerio 16

2.2 Triglyceride as diesel fuels 21

2.3 Transesterification 23

References

3. Materials and Methods 29

3.1 Materials 29

3.2 Methodology 30

3.2.1 Extraction of oil

3.2.2 Determination the oil yield

3.2.3 Determination of Free Fatty Acids

3.2.4 Determination of fatty acid composition of oils

3.2.5 Transesterification of oils

Two step acid- base catalysed transesterification

Acid pre-esterification

Base catalysed transesterification

Transesterification reaction with ultrasound

4. Results and Discussion 34

4.1 Extraction of oil from of selected wetland plants 34

4.2 Estimation of acid value and free fatty acid content (FFA) 34

4.2.1 Procedure for estimation of acid value

4.2.2 Procedure for estimation of FFA

4.3 Fatty acid profile of selected wetland plants 36

4.4 Transesterification Studies 40

4.4.1 Alkali-catalysed transesterification studies of Nymphaea nouchali

4.4.2 Two- step biodiesel production of Pongamia Pinnatta seed oil

4.4.3 Two- step biodiesel production of Thespesia Populnea oil

4.5 Effect of ultrasound on transesterification of oils 48

References

5. Conclusion 51

GC MS results 53

1

Abstract

The present work aims at the sustainable utilisation of our wetland ecosystem for

the production of energy and thus to promote awareness of the importance of wetland

conservation to the general public.In the present work, wetland plants were collected

from the resource based areas of southern Kerala mainly from Kuttanadu region for

screening their potential for biodiesel production. However, taking into account the

magnitude of its abundance, the studies conducted are still negligible. Plants used in the

study were Nymphea nouchali,Thespesia populnea, Pongamia pinnattaL, Derris

trifoliata and Canvalia cathartica.The fatty acid compositions of plants were studied by

Gas Chromatography – Mass Spectroscopy. Amongthe wetland plants,Thespesia and

Pongamia were identified as a cheap and renewable raw material for biodiesel

production.Also, biodiesel yield from conventional method is compared with the

ultrasound assisted transesterification.Thus biodiesel production using ultrasound could

be considered as a potential route for the production of biodiesel, capable of meeting

high demands in short time period, with energy costs that may be less than the expenses

with the conventional method. The identified fatty acids in the hexane extract of

Thespesia populnea, Derris trifoliate and Canavalia cathartica exhibit multifunctional

biological activity. The oil of these seeds contained a significant percentage of

pharmacologically active linoleic and alpha-linolineic fatty acids. The present findings

also proved the traditional use of these plants in the folk medicine.

2

Chapter 1

INTRODUCTION

1.1 Global energy needs and significance of a sustainable energy resource.

Global energy demand is growing at a fast pace. As the world economy expands,

more energy will be needed to fuel the higher levels of activity andliving standards.It

seems clear that significantly more energy will be required over the next twenty years to

enable the world economy to grow and prosper.Fossil fuels remain the principal source

of energy powering the world economy.Oil demand increases by almost 20 Mb/d over

the outlook, with growing use in Asia for both transport and industry. Population and

income are the key drivers behind growing demand for energy.The growth in the world

economy means more energy is required; energy consumption increases by 34% between

2014 and 2035.More than half of the increase in global energy consumption is used for

power generation as the long-run trend towards global electrification continues: the share

of energy used for power generation rises from 42% today to 45% by 2035.If production

and consumption of coal continue at the rate as in 2015, proven and economically

recoverable world reserves of coal would last for about 150 years.This is much more

than needed for an irreversible climate catastrophe. Thus it is important to find out an

alternative energy resources to improve the energy efficiency.Technologies that promote

sustainable energy include renewable energy sources such as hydroelectricity, wind

energy, solar energy, geothermal energy, bioenergy and so on. Biofuel plays an

important role among the renewable energy resources for meeting our global energy

demands.

Biofuels are known as transformative fuels because they can move us towards a

more renewable and sustainable fuel source. Biofuels are: Bioethanol, Biodiesel,

3

Charcoal and Biohydrogen. Plants use a nifty biological mechanism called

photosynthesis to convert sunlight energy into chemical energy. This mechanism is the

single source for all biofuels. A recent survey conducted in 2009 by the US department

of energy found that biodiesel reduces carbon dioxide emissions by 78%, when

compared to petroleum diesel.

According to energy statistics 2016, Ministry of Statistics and Programme

Implementation, the total potential for renewable power generation in the country as on

31.03.15 is estimated at 896603 MW. This includes wind power potential of 102772

MW (11.46%), Small-hydro power potential of 19749 MW (2.20%), Biomass power

potential of 17,538 MW (1.96%), 5000 MW (0.56%) from bagasse-based cogeneration

in sugar mills and solar power potential of 748990 MW (83.54%).

Fig 1: Sourcewise estimated potential of renewable power in India as on 31.03.15 by

the Ministry of Statistics and Programme Implementation.

India’s substantial and sustained economic growth is placing enormous energy

demand in agriculture, industrial, commercial and household sectors has increased

84%

11%

2%

2%1% 0%

Sourcewise Estimated Potential of Renewable Power in

India as on 31.03.15

solar power wind energy small hydro power

Biomass power cogeneration bagasse waste to energy

4

tremendously and placed enormous pressure on its resources. India’s crude oil and

natural gas production has been declining in recent years. This leads to an increase in the

dependency on imports. Presently, almost 83% of India’s crude oil availability is through

imports. To reduce the dependency on imports, Federal and State governments are

encouraging the use of renewable sources of energy.

On account of the increasing need for energy, the government should formulate

policies and programmes for the development of new and renewable sources of energy.

1.2 Biodiesel – AClean Energy Source

Biodiesel is a biofuel produced from an oil product. Biodiesel is a straight chain

of carbon atoms and depending on the source of feedstock oil can either be saturated or

unsaturated. Biodiesel can be defined as mono alkyl esters of free fatty acids from

vegetable oil and animal fats[1]. It can be used either pure, or in blends with petroleum

based diesel fuel. All oils are made up of triacylglycerides, which are three fatty acid

chains connected to a three carbon skeleton known as glyceride.

1.3 Transesterification

Transesterification is a simple process that chemically converts vegetable oil by

treating it with sodium hydroxide and alcohol turning it into a fatty acid methyl ester

(FAME). Obtaining pure esters after transesterification was not easy, since there are

impurities in the esters, such as di and monoglycerides[2]. The examples of alcohol that

can be used in the transesterification of triglycerides are methanol, ethanol, propanol,

butanol and amyl alcohol. Van Gerpen investigated the effect of different alcohol types

on transesterification[3]. The high conversion rates found for long chain alcohols

compared with methyl ester are due to the higher reaction temperatures allowed by their

boiling points. Methanol and ethanol are most frequently used alcohols for

5

transesterification. Catalyst used in the transesterification of triglycerides can be

classified as homogeneous, heterogeneous and enzyme catalyst. In this transesterification

process, there are two types of homogeneous catalyst which is acid catalyst (H2SO4 or

HCl) and alkali catalyst (KOH or NaOH). In heterogeneous catalytic process, usually the

catalyst is a solid and the reactant and product are in liquid or gaseous form. Alkaline

earth metal oxides, anion exchange resins, various alkali metal compounds supported on

alumina and various types of zeolites can be used as heterogeneous catalyst in the

production of biodiesel. Homogeneous basic catalyst provides much faster reaction rates

than heterogeneous catalyst, but it is difficult to separate homogeneous catalyst from the

reaction mixture. Enzymes can also be used as biocatalysts in the reaction.

Fig 2: Transesterification reaction

For an alkali based transesterification, oil and alcohol should be anhydrous [4] as

the presence of water can produce soap. The soap lowers the yield of esters and makes

the separation of ester and glycerol difficult. Also, the free fatty acid content of the

feedstock must be less than 2% [5] for effective transesterification. If more water and

free fatty acids are in the oil, acid catalysed transesterification can be used. The acid

value and hence free fatty acid content of crude oil samples were determined by a

standard titrimetry method. Free fatty acid content in the oil will result in the formation

of soap and water. The soap inhibits the biodiesel- glycerine separation process. Free

fatty acids are a problem because they make the fuel acidic, which will damage the

engine. There are various ways to deal with them. If the level of free fatty acids is less

6

than about 2%, the oil can be still processed with an alkali catalyst. In this case, an

amount of extra catalyst should be added to increase the yield of biodiesel. Above

2%,the amount of soap inhibits the separation of glycerine. The most suitable method is

to esterify the FFA’s using an acid catalyst. The acid catalyzes the esterification reaction

of fatty acids to methyl esters. When the level of free fatty acids has been reduced to

below 0.5%, the mixture can be passed through the transesterification process as if it

were refined oil.

Transesterification is the reaction of a fat or oil triglyceride with an alcohol to

form esters and glycerol. A catalyst is usually added to increase the rate of the reaction.

Since the reaction is reversible, excess alcohol is used to shift the equilibrium to the

product side. The main by-product of the reaction is glycerine. Transesterification is

widely used to reduce vegetable oil viscosity[6].The transesterification reaction proceeds

well in the presence of some homogeneous catalysts such as potassium hydroxide and

sodium hydroxide or heterogeneous catalysts such as metal oxides or carbonates. Even

enzymes like lipases can catalyse transesterification[7]. Sodium hydroxide is preferred

over potassium hydroxide because of its low cost and high product yield. But KOH is

more soluble in alcohol than sodium hydroxide.

1.3.1 One step transesterification

Esters, in the presence of a base, form an anionic intermediate, which can

dissociate back to the new ester or form a new ester. Base catalysed transesterification

proceeds faster than the acid catalysed reaction. In the first step, the base reacts with the

alcohol producing an mediate from which the alkyl ester is formed. Diglycerides and

monoglycerides are converted by the same mechanism into a mixture of alkyl esters and

glycerol. Alkoxides are better catalysts than hydroxides. Alkoxides are prepared by

dissolving the clean metals in analkoxide .Alkoxide ion attacks the carbonyl carbon of

7

the triglyceride generating a tetrahedral interhydrous alcohol. While using NaOH and

KOH, water is formed as a by product which will further result in soap formation. The

unwanted saponification reduces the ester yields and makes the separation of glycerol

difficult. Carbonates can be used as a substitute for NaOH.

Fig 3: Mechanism of alkali catalysed transesterification of vegetable oils.

1.3.2 Two step acid base transesterification

Two-step process was usually used for the production of biodiesel. the free fatty

acid content of the feedstock must be less than 2% [5] for effective transesterification. If

more water and free fatty acids are in the oil, acid catalysed transesterification can be

used. Initially sulphuric acid was used to catalyse the esterification of free fatty acids

present in the oil. After the reduction of free fatty acid content of the oil,

transesterification reaction was carried out.

For acid catalysed esterification, solutions of sulphuric acid in anhydrous alcohol

were prepared at room temperature. The sulphuric acid percentage was based on the

weight of the oil to be reacted. The acid catalyst is dissolved into alcohol by vigorous

8

stirring in a small reactor. The oil is transferred into the biodiesel reactor and then the

catalyst/alcohol mixture is added into the oil. It is already reported in the literature that a

large excess of alcohol (15 to 35 moles per mole of fatty acid) should be used when

sulphuric acid is employed as a catalyst[8]. Freedmann and pryde [9] mentioned that a

30:1 molar ratio of alcohol to oil with 1% sulphuric acid gave good conversion after 44 h

of heating at 60 0C.They highlighted that if oil has more than 1% free fatty acid, the acid

catalyst will be much more fruitful than the alkali catalyst. Acid catalysed esterification

is much slower than alkali-catalysed reaction. The mechanism of acid catalysed

transesterification of vegetable oil is shown below:

Fig 4: Mechanism of acid catalysed transesterification

1.3.3 Advanced transesterification process

1.3.3.1 Ultra sound assisted transesterification.

Transesterification reaction can be accelerated by the application of

ultrasound.Biodiesel production from canola oil withmethanol was studied in the

presence of a base-catalyst by a circulation process at room temperature[10]. It was

found that the conversion of triglycerides to fatty acid methyl esters was greater than

99% within the reaction time of 50 min. The procdure for ultra sound assisted

9

transesterification is similar to simple transesterification process. In the first step, the

reaction mixture is sonicated without adding the base catalyst. In the second step,

catalyst is added and then sonicated. It is already reported in the literature that the

influence of ultrasound on transesterification reaction is of purely physical nature[11].

No radical species are produced during the reaction. Due to microturbulence generated

by cavitation bubbles an emulsion is created between oil and alcohol. The interfacial area

between oil and alcohol increases, which accelerates the reaction. The optimum alcohol

to oil molar ratio for the reactions varies depending on the frequency and intensity of

ultrasound and type of sonicator [11].A study was carried out on the hydrolysis of waste

cooking oil (WCO) under solvent free condition using commercial available

immobilized lipase (Novozyme 435) under the influence of ultrasound irradiation

[12]. The activation energy and thermodynamic study shows that the hydrolysis reaction

is more feasible when ultrasound is combined with mechanical agitation as compared

with the ultrasound alone and simple conventional stirring technique. Application of

ultrasound considerably reduced the reaction time as compared to conventional reaction

[12].

1.3.3.2 Microwave assisted transesterification.

Microwave can be used to increase the efficiency of biodiesel production. Nezihe

Azcanet al studied the efficiency of microwave assisted transesterification for the

biodiesel production and the results shows that that microwave heating has effectively

increased the biodiesel yield and decreased the reaction time[13].

1.4 Oil sources for Biodiesel

The source of biodiesel depends on the crops suitable to the regional climate.

Selection of biodiesel feedstock depends on the oil percentage and the yield per

hectare[14].The most commonly used oils for the production of biodiesel are

10

soyabean[15], cotton seed[16], palm[17], rapeseed[18], canola and

jatropha[19].However, edible oils always compete with food and therefore some species

of plants yielding non-edible oils play a major role in providing resources. But as India

still imports huge quantities of edible oils, the use of edible oils for diesel engine fuel is

not feasible. These plants may be grown on a massive scale on agricultural or waste

lands, so that the primary resourcemay be available to produce biodiesel on commercial

scale. In India, Jatropha and Pongamia pinnata oilseeds were cultivated on wasteland

but the cultivation of these two has been failed.Much effort has been devoted to develop

new biodiesel production processes by using cheaper feedstock.

Edible oils Non edible oils Other sources

Soyabeans[15] Almond Algae

Rapeseed[20] Jatropha Fungi

Coconut[21] Pongamia glabara Waste cooking oil

Wheat[22] Mahua

Cotton seed Jojoba

Rice bran Rubber[23]

Barley Palm

canola Tobacco seed[24]

Table 1: Biodiesel sources

1.5 Environmental Impact of Biodiesel

Vegetable oil has a heating value lower than diesel oil; however vegetable oil has

a high viscosity and low volatility which leads to incomplete combustion and forms

carbon deposits in the fuel injectors. The main advantages of using biodiesel are its

renewability, better-quality exhaust gas emissions, its biodegradability and given that all

the organic carbon present is photosynthetic in origin, it does not contribute to a rise in

the level of carbon dioxide in the atmosphere and consequently to the greenhouse effect.

Transesterification process is most popular than other methods because it is simple and

11

glycerol is obtained as a by-product. Glycerol has a commercial value. Glycerol is

obtained proximately 10% of the oil or fat and proximately one ton of glycerine can be

obtained for every ten tones of biodiesel engenderment. Biodiesel is defined as the mono

alkyl esters of long chain fatty acids derived from renewable feed stocks like vegetable

oils or animal fats. Most of the properties of biodiesel are comparable to petroleum based

diesel fuel.

Property Test

method

ASTM D ASTM D6751

EN14214: 2003 975(Diesel)

(biodiesel,B100)

Limit Limit Test

method Limit

Flash point D 93 325 K min 403 K min ISO/CD3679

374 K min

Water and sediment

D 2709 0.05 max % vol

0.05 max % vol

-

Kinematic viscosity (at 313 K)

D 445 1.3-4.1 mm2/5 1.9-6.0 mm2 /s

EN/ISO310 3.5-5.0

Sulphated ash D 874 - 0.02 max % wt

ISO3987 0.02max % (m/m)

Ash D 482 0.01 max % wt - - -

Sulphur D 5453 0.05 max % wt - - -

Sulphur D 2622 / 129

- 0.05 max % wt

- -

Copper strip corrosion

D130 No.3 max No.3 max Rating No.1 max

Cetane number

D 613 40 min 47 min EN ISO 5165

51 min

Aromaticity D 1319 35 max % vol -

Carbon residue

D 4530 - 0.05 EN ISO1-370

0.3 max

Carbon residue

D 524 0.35 max %mass

- - -

Distillation temp (90% volume recycle)

D 1160 555 K min- 611 K max

- - -

Table 2: Standards for biodiesel

12

1.6 Scope of present investigation

In India, wetlands are distributed in different geographical regions ranging from

Himalayas to Western Ghats. Wetlands provide us with food, fodder, fuel and water for

domestic and industrial purposes. Wetlands support a number of rare and endangered

species of flora and fauna. The services provided by wetland ecosystem are endless and

they are one of the most productive and complex ecosystems on our planet acclimatized

to extraordinary conditions. The biodiversity rich wetland ecosystem of India is

shrinking due to increasing biotic and abiotic pressures. The aim of the present work is

the sustainable utilisation of our wetland ecosystem for the production of energy and thus

to promote awareness of the importance of wetland conservation to the general public.

Plants are the unique biological resources form the basis of life. Due the diverse

ecological conditions of India, we are lucky to have many oil yielding wild plants which

can be utilized for the production of biodiesel. Woefully like other fields, no scientific

investigation has been done on biodiesel prior to this, because the people are unaware to

use these resources for development of this technology due to lack of interactions

between our industries and research institutions. In this contest the present study is a

stepping stone to initiate biodiesel research in India.

More than 100 oil bearing plants have been identified, among which

Calophyllum inophyllum L, Ricinus communis L, Soyabean and Jatropha curcas L are

considered to be potential biodiesel sources. However no systematic studies of wetland

species are available for biodiesel production.

In the present work, wetland plants were collected from the resource based areas

of southern Kerala. Wetlands in the Kuttanadu region are rich in Nymphea species.

However, taking into account the magnitude of its abundance, the studies conducted are

still negligible. In this search, petals and stamens of Nymphea nouchali were collected

13

for oil extraction. Also other plants used in the study were Thespesia populnea,

Pongamia pinnattaL, Derris trifoliata and Canvalia cathartica.

1.7 Objectives of the present investigation

The objectives of the present work were the following:

To identify and shortlist local wetland plants that are viable sources of Biodiesel, in

order to establish a rationale for large scale plantation for the most preferable

Biodiesel producing plants.

Extraction of oil from these selected wetland plants.

To study the fatty acid profile of these selected wetland plants.

Transesterification of oil for biodiesel production.

To study the effect of ultrasound on the biodiesel yield.

14

References

1. Krawczyk, T., Biodiesel. Alternative fuel makes inroads but hurdles remain. 1996.

2. Ma, F. and M.A. Hanna, Biodiesel production: a review. Bioresour Technol, 1999. 70(1):

p. 1-15.

3. Canacki, M. and J.V. Gerpen, Biodiesel production via acid catalysis. Transactions of the

American Society of Agricultural Engineers, 1999. 42: p. 1203-1210.

4. Wright, H., et al., A report on ester interchange. Oil & Soap, 1944. 21(5): p. 145-148.

5. Meher, L.C., V.S.S. Dharmagadda, and S.N. Naik, Optimization of alkali-catalyzed

transesterification of Pongamia pinnata oil for production of biodiesel. Bioresour Technol,

2006. 97(12): p. 1392-1397.

6. Sinha, S., A.K. Agarwal, and S. Garg, Biodiesel development from rice bran oil:

Transesterification process optimization and fuel characterization. Energy Conversion and

Management, 2008. 49(5): p. 1248-1257.

7. Macrae, A., Lipase-catalyzed interesterification of oils and fats. Journal of the American

Oil Chemists’ Society, 1983. 60(2): p. 291-294.

8. Formo, M.W., Ester reactions of fatty materials. Journal of the American Oil Chemists

Society, 1954. 31(11): p. 548-559.

9. Freedman, B. and E. Pryde, Fatty esters from vegetable oils for use as a diesel fuel. 1982,

Dept. of Agriculture, Peoria, IL.

10. Thanh, L.T., et al., Ultrasound-assisted production of biodiesel fuel from vegetable oils in

a small scale circulation process. Bioresour Technol, 2010. 101(2): p. 639-645.

11. Kalva, A., T. Sivasankar, and V.S. Moholkar, Physical mechanism of ultrasound-assisted

synthesis of biodiesel. Industrial & Engineering Chemistry Research, 2008. 48(1): p. 534-

544.

12. Waghmare, G.V. and V.K. Rathod, Ultrasound assisted enzyme catalyzed hydrolysis of

waste cooking oil under solvent free condition. Ultrason Sonochem, 2016. 32: p. 60-67.

13. Azcan, N. and A. Danisman, Microwave assisted transesterification of rapeseed oil. Fuel,

2008. 87(10): p. 1781-1788.

14. Singh, S. and D. Singh, Biodiesel production through the use of different sources and

characterization of oils and their esters as the substitute of diesel: a review. Renewable

and Sustainable Energy Reviews, 2010. 14(1): p. 200-216.

15. Freedman, B., R.O. Butterfield, and E.H. Pryde, Transesterification kinetics of soybean oil

1. Journal of the American Oil Chemists’ Society, 1986. 63(10): p. 1375-1380.

16. Köse, Ö., M. Tüter, and H.A. Aksoy, Immobilized Candida antarctica lipase-catalyzed

alcoholysis of cotton seed oil in a solvent-free medium. Bioresour Technol, 2002. 83(2): p.

125-129.

15

17. Darnoko, D. and M. Cheryan, Kinetics of palm oil transesterification in a batch reactor. J

Am Oil Chem Soc, 2000. 77(12): p. 1263-1267.

18. Saka, S. and D. Kusdiana, Biodiesel fuel from rapeseed oil as prepared in supercritical

methanol. Fuel, 2001. 80(2): p. 225-231.

19. Senthil Kumar, M., A. Ramesh, and B. Nagalingam, Investigations on the use of Jatropha

oil and its methyl ester as a fuel in a compression ignition engine. Journal of the Institute

of Energy, 2001. 74(498): p. 24-28.

20. Billaud, F., et al., Production of hydrocarbons by pyrolysis of methyl esters from rapeseed

oil. Journal of the American Oil Chemists’ Society, 1995. 72(10): p. 1149-1154.

21. Abigor, R., et al., Lipase-catalysed production of biodiesel fuel from some Nigerian lauric

oils. Biochem Soc Trans, 2000. 28(6): p. 979-981.

22. Ugarte, D.G.D.L.T. and D.E. Ray, Biomass and bioenergy applications of the POLYSYS

modeling framework. Biomass and Bioenergy, 2000. 18(4): p. 291-308.

23. Ramadhas, A.S., S. Jayaraj, and C. Muraleedharan, Biodiesel production from high FFA

rubber seed oil. Fuel, 2005. 84(4): p. 335-340.

24. Giannelos, P., et al., Tobacco seed oil as an alternative diesel fuel: physical and chemical

properties. Industrial crops and products, 2002. 16(1): p. 1-9.

16

Chapter 2

REVIEW OF LITERATURE

The biodiesel is an alternative to reduced CO2 and sustainable use of

bioresources. Moreover, the fuel will reduce country over dependence on imported petro-

diesel. Hence, detailed screening process of all available virgin or plant and animal

origin coming up with some restrictions. The cheapest feedstock available is the key

factor for a low cost biodiesel. The recent focus is to find oil-bearing plants that produce

non-edible oils as the feedstock for biodiesel production. Biodiesel obtained from neat

vegetable oil is costly compared to the petroleum diesel fuel. The main advantages of

using biodiesel are its renewability, better-quality exhaust gas emissions, its

biodegradability and given that all the organic carbon present is photosynthetic in origin,

it does not contribute to a rise in the level of carbon dioxide in the atmosphere and

consequently to the greenhouse effect[25]. Recently, a lipase-catalyzed esterification of

various kinds of vegetable oils using an organic solvent or a solvent free system was

studied by environmental friendly process[26]. Research has recently indicated that the

lipids contained in sewage sludge are a potential feedstock for biodiesel[27].Researchers

are also developing microalgae that produce oils, which can be converted to

biodiesel[28]. Much effort has been devoted to develop new biodiesel production

processes by using cheaper feedstock[29].The present study concentrates on screening

untapped natural resources like the lipids present in the selected aquatic plants of

Kuttanad wetland ecosystem of Kerala, India for the production of biodiesel.

2.1 Indian Scenerio

The Planning Commission of India established the National Biodiesel Mission

(NBM) for the development and commercialization of biodiesel from Jatropha and

17

Pongammia seeds. The Cultivation of these two cultivars started in the state of Andhra

Pradesh. Based on extensive research carried out in agricultural research centers, it was

decided to use Jatropha curcas oilseed as the major feedstock for India’s biodiesel

programme. NBM was planned for two phases. Phase I was termed as demonstration

phase and has been carried out from year 2003 to 2007 (Planning Commission, Govt. of

India, 2003). The work done during this phase were development of Jatropha oilseed

nurseries, cultivation of Jatropha on 400,000 hectares (ha) waste land, setting up of seed

collection and Jatropha oil expression centers, and the installation of 80,000 Mt/year

transesterification to produce biodiesel from Jatropha oil. Phase II planned with a self

sustaining expansion of the programme leading to the production of biodiesel to meet

20% of the country’s diesel requirements by 2011–2012. The lack of assured supplies of

vegetable oil feedstock has foiled efforts by the private sector to set up biodiesel plants in

India. Commercial biodiesel production has not yet started in India. So far only two

firms, Naturol Bioenergy Limited (NBL) and Southern Online Biotechnologies, have

embarked on biodiesel projects, both in the southern state of Andhra Pradesh. Naturol

Bioenergy Limited (NBL), a joint venture with the Austrian biodiesel firm Energea

Gmbh and the investment firm Fe Clean Energy (USA), has planned to install a 300

tonnes/day (t/d)/(90,000 tonnes/year) (t/y) biodiesel plant in Kakinada, Andhra

Pradesh[30].The State Government allocated 120,000 ha of land for Jatropha cultivation

to the firm but cultivation has not yet begun or is in initial stage. The farmers were

demanding that the market set the oilseed price, but NBL wants the government to fix a

price to reduce its risks in production. Southern Online Biotechnologies has a 30 t/d

(9000 t/y) project, which would require about 9500 t/y of oil. It was expected to get

about 6000 t/y through cultivation of Jatropha and Pongamia pinnata oilseeds on

wasteland, and plans to make up the balance through animal fats, but the cultivation of

18

these two have been failed. So, there are many constraints for the biodiesel production in

India and phase I of NBM has not given the anticipated results. Azam MM etal studied

thefatty acid profiles of seed oil methyl esters of 75 plant species [31]. Out of these

plants, based on saponification value, iodine number, cetane number and fatty acid

profile, fatty acid methyl esters of 36 species meet the specification of Biodiesel standard

of USA is listed in the (table 1).

Khan et al. reviewed prospects of biodiesel production from micro algae in India

and this paper is an attempt to review the potential of micro algal biodiesel in

comparison to the agricultural crops and its prospects in India[32]. The existing biodiesel

production process is neither completely “green nor renewable’’ because it utilizes

fossil fuels, mainly natural gas as an input for methanol production, also the catalyst

currently in use are highly caustic and toxic . To overcome this process, a new method is

used in which waste vegetable oil and non edible plant oils and biodiesel feed stock and

nontoxic, inexpensive and natural catalyst[33].

19

Table 1 - Fatty acid composition of oils

TABLE 3: a oil from kernel, b oil from seeds, osa: other saturated acids, uk: unknown

20

Table 1 - Fatty acid composition of oils

a oil from kernel, b oil from seeds, osa: other saturated acids, uk: unknown

21

2.2 Triglyceride as diesel fuels

Vegetable oil and animal fats are extensively used as the major source in the

production of biodiesel. The characteristics of biodiesel are close to diesel fuels, and

therefore becomes an alternative source to replace the diesel fuels. Biodiesel has

viscosity close to diesel fuels. Table 2 shows the comparison of fuel properties between

diesel and biodiesel[34].

Table 2 - Comparison of fuel properties between diesel and biodiesel

Depending upon the climate and soil conditions, different countries are

looking for different types of vegetable oils as substitutes for diesel fuels. For

example soyabean oil in US, rapeseed oil in Europe is being considered. However,

edible oils always compete with food and therefore some species of plants yielding

22

non-edible oils play a major role in providing resources. But as India still imports

huge quantities of edible oils, the use of edible oils for diesel engine fuel is not

feasible. These plants may be grown on a massive scale on agricultural or waste

lands, so that the primary resourcemay be available to produce biodiesel on

commercial scale. In India, Jatropha and Pongamia pinnata oilseeds were cultivated

on wasteland but the cultivation of these two has been failed.Much effort has been

devoted to develop new biodiesel production processes by using cheaper

feedstock.The present study aims to screen the suitable wetland plants as energy

crops from a tropical wet-land ecosystem, where the high production of biomass

occurs within a short time span. This biomass is considered as nuisance to the

farmers as well as local people. Moreover it causes economic threat to the farmers.

Hence the study aims to use the biomass for the production of energy. There are

limited studies so far reported from India on the production of biodiesel from wet-

land plants and the present attempt has the significance in the energy sector of the

country especially in the biodiesel production.

Kerala is one of the green States of India and is well known for its wetlands.

The total wetland area in Kerala is 160590 ha[35].In Kerala, wetlands are under

extreme pressure compared to any other state. Partitioning by funds, reclamation and

consequent shrinkage have been implicated as the major reasons for the destruction

of habitat and dwindling of resources. The study aims to find out certain potential

wetland plant species from a tropical wetland system for biodiesel production. The

distribution of plants in the different wetlands is given in the table below[36]:

23

Distribution Trees Shrubs Climbers Herbs Total % to total

species

Coastal wetlands 51 34 40 393 518 71

Inland wetlands 87 47 43 311 488 67

Both in coastal and inland wetlands

35 18 18 210 281 39

Only in coastal wetalnds

16 16 22 183 237 33

Only in inland wetlands

52 29 25 101 207 29

Table 4 - Distribution of plants in the different wetlands

In the present study, some locally available wetland plants are randomly selected

for screening their potential for biodiesel production.There are many unexploited plant

species, which contains oil that can be extracted and used as bio-diesel. Some

unexploited plant species were experimented in this present study for its content. A

preliminary work was done on the transesterification of the crude oil extracted from T.

populnea seed under standard conditions with sodium methoxide as catalyst[37]. Many

research studies had been done on the transesterification of pongamia oil[38,

39].However, the fatty acid profile of plants varies with the external environment. In the

present study, fatty acid profiles of wetland plants in kerala are explored to study their

potential for biodiesel production.

2.3 Transesterification

Different methods have been employed to convert oils and fats to be used in diesel

engines. Among them, transesterification is one of the promising methods for the

production of biodiesel. Transesterification is the process of converting an ester into

another ester by reaction with an alcohol using a suitable catalyst. Transesterification of

TG is a stepwise reaction where the TG reacts first with the alcohol to produce a DG and a

mono-alkyl ester molecule, then the DG reacts similarly to produce a MG and a second

molecule of mono-alkyl ester and finally the MG reacts with another alcohol to produce

24

glycerol and a third molecule of mono-alkyl ester [15]. Transesterification reaction can be

done in the presence of homogeneous catalyst, heterogeneous catalyst and an enzyme.

Homogeneous catalysis can be performed by using base catalyst or an acid catalyst.

Alkaline transesterification is found to be more effective than acid catalysed reaction for

vegetable oil containing FFA level less than 0.5%[40]. Also, it can be performed at lower

temperatures than heterogeneous catalysis. A comparison is made of different basic

catalysts (sodium methoxide, potassium methoxide, sodium hydroxide and potassium

hydroxide) for methanolysis of sunflower oil and their studies shows that although all the

transesterification reactions were quite rapid and the biodiesel layers achieved nearly 100%

methyl ester concentrations, the reactions using sodium hydroxide turned out the

fastest[41].

Efforts have also been made to use heterogeneouscatalysts for the transesterification

of triglycerides. Heterogeneous catalysts have many advantages over homogeneous

catalysts. They can be easily separated from the products after the reaction and thus it can be

reused. However, the heterogeneous catalytic reaction should be done at higher temperature

than homogenous catalysis with an excess of alcohol. Research work has been conducted to

use compounds of calcium and barium, to produce the methyl esters of rapeseed oil.This

research showed that the transesterification of rapeseed oil by methyl alcohol can be

catalysed effectively by basic alkaline-earth metal compounds: calcium oxide, calcium

methoxide and barium hydroxide. Calcium catalysts, due to their weak solubility in the

reaction medium, are less active than sodium hydroxide. However, calcium catalysts are

cheaper and lead to decreases in the number of technological stages and the amount of

unwanted waste products[42]. It was already reported in the literature that mixed oxides of

zinc and aluminium could be used for the transesterification of vegetable oils[43]. Zeolites

can be used as effective catalyst for transesterification reaction. Zhongjun etal studied the

25

transesterification of olive oil with ethanol in the presence of different types of microporous

zeolites (BEA type Beta zeolite and MFI type ZSM-5 zeolite) and micro-mesoporous

zeolites (MFI type ZRP-5 zeolite) with various Si/A1 ratios[44].The results showed that the

zeolites with high Si/Al ratios had better catalytic performance. The catalytic activities of

different metal complexes were compared with classical alkali and acid based

trasesterification[45].It was found that all complexes are active Sn 2+≫Zn 2+>Pb 2+≈Hg 2+.

Transesterification of soybean oil with methanol was carried out at 60, 120, and 150 °C in

the presence of a series NaX faujasite zeolite, ETS-10 zeolite, and metal catalysts[46]. The

ETS-10 catalysts provided higher conversions than the Zeolite-X type catalysts. The

increased conversions were attributed to the higher basicity of ETS-10 zeolites and larger

pore structures that improved intra-particle diffusion.

Research in the field of biological catalysts have also gain momentum recently and

used in the transesterification of different vegetable oils.Dehua Liu et al studied Candida

antarctica lipase catalysed biodiesel production of soyabean oil[47].The optimum

conditions of the transesterification were 30% enzyme based on oil weight; a molar ratio of

methyl acetate/oil of 12:1; temperature 40 °C and reaction time 10 h.

Effect of ultrasonication on the alkaline transesterification of Cynara cardunculus

L. seed oil with methanol for biodiesel production was investigated [48]. In this study it

was reported that the % yield of the extracted methyl esters using mechanical stirring was

lower compared to ultrasonication (50.4 and 85.1% respectively). Microwave irradiation

can also be used for the production of the biodiesel. Shakinaz T.ElSheltawy et al reported

the effect of microwave on the transesterification of jatropha oil[49]. The study showed

that the application of radio frequency microwave energy offers a fast, easy route to this

valuable biofuel with the advantages of enhancing the reaction rate (2 min instead of 150

min) and of improving the separation process.

26

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29

Chapter 3

MATERIALS AND METHODS

3.1 Materials

The petals and stamen of Nymphea nouchali, seeds of Pongamia pinnatta L,

Derris trifoliata and Canvalia cathartica, were collected from Kuttanadu wetland

ecosystem of southern Kerala, India. The seeds of Thespesia populnea were collected

from Koratty, Thrissur. Avicenia marina seeds were collected from Ayiramthengu,

Kollam. All the chemicals used were of analytical grade without further purification.

NaOH and KOH pellets were purchased from Merck India.

Nymphea nouchali Thespesia populnea Derris trifoliate

Pongamia pinnatta LCallophyllum inophyllum Canvalia cathartica

30

3.2 Methodology

3.2.1 Extraction of oil

The plant parts were dried under sunshade for one week. Powdered and extracted

with hexane at 850C for 2 hours in an automatic Soxhlet apparatus, SOX 3, Tulin

Equipments, Chennai. The solvent was removed from the extracts at 450C under vacuum

using a rotary evaporator to obtain crude oil samples. The available oil percentage in the

samples was determined.

Based on the oil yield and availability, Nymphea Nouchali,Calophyllum

Inophylum, Pongamia pinnata and Thespesia Populnea were selectedfor

transesterification studies.The oil samples were then kept in hot air oven at 105 0C for 2

hours in order to remove moisture

3.2.2 Determination the oil yield

The % oil yield was calculated as follows

Oil Content = [ (Weight of oil)/(Weight of sample)] × 100

Fig 5: Automatic soxhlet extractor

31

3.2.3 Determination of Free Fatty Acids

The FFA of oils was determined using the standard procedure. 25 ml of ethanol is

measured into conical flak, neutralised and boiled in a water bath in order to remove

dissolved gases. Approximately 2 g f the oil is transferred into the 25 ml of hot ethanol

with continues heating. A few drops of phenolphthalein indicator were added and titrated

aganist 0.1M NaOH .The end point is the appearance of permanent pink colour.

%��������������������� =� × � × 28.2

�× 100

V = Average volume of NaOH, M= Molarity of NaOH, 28.2g/mol = Molecular weight

of oleic acid, W= Weight of oil

3.2.4 Determination of fatty acid composition of oils

The various vegetable oils are distinguished by their fatty acid compositions. The

fatty acid composition of oils was determined by gas chromatography-mass

spectroscopy. Properties of biodiesel depend on the composition and percentage of fatty

acids in the oil. The fatty acids which are commonly found in vegetable oil and fat are

stearic, palmitic, oleic, linoleic. The other fatty acids which are also present in many of

the oils and fats are mysristic, palmitoleic, arachidic, linolenic and octadectetranoic.

There are many other fatty acids which are also found in oils with the above mentioned

common fatty acids.

The fatty acid composition of the seeds were determined by converting into fatty

acid methyl esters (FAMEs) followed by GC-MS. 0.25g sample was refluxed with 20ml

0.5 M methanolic KOH for 20 min at 55 0C, cooled and esterified with 1.5 ml H2SO4 and

15 ml methanol .The mixture is refluxed for 30 min, cooled to room temperature and 10

ml n-heptane was added, again refluxed for 10 min. The filtrate was transformed to a

32

separating funnel and shaken with 5 ml saturated NaCl. The top layer was collected

through anhydrous sodium sulphate and transferred to 2 ml autosampler vials for GC-MS

analysis. The methyl esters of fatty acid were separated by GC using a DB Wax column

.The compounds were detected by MS and identified by comparison with the NIST mass

spectral database (National Institute of Standards and Technology, Gaithersburg,

Maryland ,USA).

3.2.5 Transesterification of oils

Two step acid- base catalysed transesterification

A two step process, acid catalysed esterification process and followed by base-

catalysed transesterification process, were selected for converting oil samples to ethyl

esters of fatty acids. In order to reduce the FFA content in the oil, the oil was firstly

treated with acid to esterify the free fatty acids. The process was done to convert FFA to

esters using an acid catalyst (1% w/w Sulphuric acid) to reduce the FFA concentration of

the oil below 5%. Second step was base catalysed transesterification.

Acid pre-esterification

Pre-esterification was carried out in a 100 ml three-neck flask. The flask was

immersed in a glass water bath placed on the plate of magnetic stirrer of 400rpm.It was

confirmed that transesterification depends on several factors, namely, alcohol to oil ratio,

catalyst type, reaction time, FFA and water content of oils 8. In the present work, the

optimum condition for the reaction was found to be at 30:1 ethanol to oil molar ratio,

reaction temperature of 500C and reaction time of 2 hrs for Pongamiaoil.

Firstly, in the pre-esterification process, the solution of concentrated sulphuric

acid (1% based on oil weight) in alcohol was heated at 500C and then added into the

reaction flask. Ethanol to oil ratio was kept at 30:1 and reaction temperature at 500C. The

33

reaction was carried out for 2 hrs. The conversion of pre-esterification was calculated by

comparison of the acid values before and after the reaction.

Base catalysed transesterification

It was confirmed that transesterification depends on several factors, namely,

alcohol to oil ratio, catalyst type, reaction time, FFA and water content of oils. The acid

value of the oil was reduced after the acid pre treatment. The oil was preheated in a two

necked round bottomed flask at 500C for reducing its viscosity. The potassium

hydroxide-ethanol solution was prepared. The alkali ethoxide solution was added to

preheated oil while mixing by means of a magnetic stirrer. The stirring was maintained at

350 rpm throughout the reaction. The reaction was stopped after 2h and was poured into

a separating funnel and allowed to settle under gravity. The products of the alkaline

transesterification process result in the formation of two layers viz., an upper layer

containing biodiesel and a lower layer of glycerol. The lower layer was drained off. The

upper layer was washed with hot distilled water until the bottom aqueous layer becomes

clear.

Transesterification reaction with ultrasound

After the pre treatment of the oil, it was then subjected to ultrasound for

transesterification using base catalyst. The tub of ultra sound bath was filled with 300 ml

of distilled water and then the beaker containing the reactants was placed inside. The

temperature was maintained in 500C and the beaker was covered with aluminium foil,

considering that this temperature the evapouration of ethanol is negligible. The

equipment was set to operate at 200 W and 20 kHz for 20 minutes by keeping other

optimum reaction conditions and process parameters was unaltered.

34

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Extraction of oil from of selected wetland plants

The plant parts were sun dried for one week and oven dried at 1200C for 2 hours.

The plant parts are then powdered in a blender and extracted with hexane at 85 0C for 2

hours in an automatic Soxhlet apparatus, SOX 3,Tulin Equipments, Chennai. The solvent

was removed from the extracts at 450C under vacuum using a rotary evaporator to obtain

crude oil samples. The available oil percentage in the samples was determined.

Plant Part used Oil yield (%)

Nymphea nouchali (Blue lotus) Petals and stamen 1.60

Canvalia cathartica Seeds 2.34

Derris trifoliata Seeds 5-7

Pongamia pinnatta Seeds 46.36

Thespesia populnea Seeds 22.59

Calophyllum inophyllum Seeds 36.60

Table 5 :The available oil percentage in the sample

4.2 Estimation of acid value and free fatty acid content (FFA)

4.2.1 Procedure for estimation of acid value

Weigh sufficient quantity of oil in an Erlenmeyer flask. Add 25 ml of freshly

prepared neutralized hot ethanol and 1ml of phenolphthalein indicator. Boil the mixture

for about 5min and titrate while as hot as possible with standard aqueous alkali solution

shaking vigorously during titration.

��������� =��. � × � × �

�

V= Volume in ml of standard KOH

N= Normality of standard KOH,0.1047N

W=weight in gram of oil

35

Table 6 : Acid value of oil

4.2.2 Procedure for estimation of FFA

Same procedure as Acid value

%��������� ������������ =� × � × ��. �

�× ���

%��������� ������������� =� × � × ��

�× ���

Table 7: FFA value of oil

The maximum acid value for alkaline transesterification is 2 mg KOH/g of oil

recommended by Canakci and Van Gerpen. Based on the acid value, the FFA content of

Nymphea nouchali is0.44% thus; it does not need any pre-treatment before

transesterification. Acid value and hence the free fatty acid content of other oils and they

have to be pretreated to reduce FFA.

Oil Acid Value

(mg KOH/ g)

Nymphea nouchali 0.624

Canvalia cathartica 23.64

Derris trifoliata 17.24

Pongamia pinnatta 7.02

Thespesia populnea 8.84

Callophyum inophyllum 15.92

Oil Free fatty acid Value (%)

Nymphea nouchali 0.44

Canvalia cathartica 15.85

Derris trifoliata 12.14

Pongamia pinnatta 4.96

Thespesia populnea 5.21

Callophyum inophyllum 11.22

36

4.3 Fatty acid profile of selected wetland plants

Table 8: Fatty acid composition of selected wetland plants was determined by gas chromatography-mass spectroscopy.

Systemic name Formula Structure Nymphea nouchali

Canvalia cathartica

Derris trifoliata

Pongamia pinnatta

Thespesia populnea

Octanoic C8H16O2 8:0 Trace 0.166

Decanoic C10H20O2 10:0 -----

2-methyl decanoic C11H22O2 10:0 7.6 0.113

Dodecanoic (Lauric acid) C12H24O2 12:0 3.386 3.700 trace 1.698

9(Z)-dodecenoic C12H22O2 12:1 0.161

9(E) – dodecenoic C17H32O2 12:1 0.691

Tetradecanoic(Myristic acid) C14H28O2 14:0 3.5 2.423 2.855 trace 1.536

Hexadecanoic (Palmitic acid) C16H32O2 16:0 4.05 22.04 26.27 7.0 33.315

9(Z)-heptadecenoic C17H32O2 17:1 0.738

9(E),12(E)- octadecadienoic C18H32O2 18:2 2.961 2.416

Octadecanoic(Stearic acid) C18H36O2 18:0 7.0 3.136

9(Z)- octadecenoic (Oleic acid) C18H34O2 18:1 55.7 46.480 42.78 69.0 17.192

9(E)-octadecenoic C18H34O2 18:1 1.273

9(Z),12(Z)-octadecadienoic (Linoleic acid)

C18H32O2 18:2 28.1 12.410 10.87 15.6 33.968

9(Z),15(Z)-octadecadienoic(Mangefiric acid)

C18H32O2 18:2 0.251

Linolenic acid 18:3 13.241 Trace trace Trace

Eicosanoic C20H40O2 20:0 0.652

37

The fatty acid composition of the oils from Thespesia populnea, Derris trifoliate,

Nymphea nouchali, Pongamia pinnatta and Canavalia Cathartica was determined using Gas

Chromatography- Mass Spectroscopy.

The seed oils contain a significant percentage of long chain polyunsaturated fatty acids

especially linoleic acid, oleic acid and α-linolenic acid (ALA). α-linolenic acid undergoes

desaturation and elongation to produce EPA (Eicosapentaenoic acid) and DPA

(Docosapentaenoic acid) in cell membranes is an important factor in determining cell and tissue

function[1]. It prevents inflammation, heart disease[2], stroke, type II diabetes, kidney disease

and certain types of cancers[3]. ALA is essential for the proper working of the nerve system[4].

Edible wild plants provide alpha linolenic acid more than by cultivated plants[5]. Derris trifoliate

contains an unusual fatty acid, cis 11-octadecenoic acid (42.27) as the major fatty acid. Cis-11-

octadecenoic was reported to be present in the seed oil of Doxantha unguis-cati [6]. It was

reported in the literature that some seed oils of Sapindaceae have cis-11-octadecenoic acid as the

principal fatty acid[7].

Thespesia oil contains high level of unsaturated fatty acids (52%), while the saturated

fatty acids accounts for remaining. The dominant unsaturated fatty acid is 9(Z), 12(Z)-

octadecadienoic acid (Linoleic acid) followed by oleic acid. The nutritional value of linoleic acid

was due to its metabolism at tissue levels which produces the hormone-like prostaglandin[8]. The

activity of these prostaglandins includes lowering of blood pressure and contraction of smooth

muscle. High levels of palmitic acid were also detected in the hexane extract of Thespesia

populnea. Lauric acid is an antimicrobial agent useful for infection control in hospitals. It was

already reported in the literature that, linoleic acid and oleic acid possessed potential antibacterial

activity and antifungal activity[9]. This property of Thespesia populnea oil is made use in the

Indian and Chinese folk medicine for the treatment of skin diseases, scabies and ringworm. It

also contains 9(Z), 15(Z)-octadecadienoic acid, a novel fatty acid reported only in the pulp of

mango.

38

Biodiesel fuel properties

Biodiesel properties Thespesia populnea

Pongamia pinnatta

Derris trifoliata

Saturated Fatty Acid 40.610 14.00 32.83

Mono Unsaturated Fatty Acid 18.310 69.01 42.78

Poly Unsaturated Fatty Acid 36.00 15.01 10.870

Degree of Unsaturation 90.310 100.20 64.52

Saponification Value (mg/g) 197.89 197.01 181.236

Iodine Value 81.67 90.30 58.15

Cetane number 55.513 53.685 63.330

Long Chain Saturated Factor 5.548 4.200 2.627

Cold Filter Plugging Point (°C) 0.955 -3.28 -8.224

Cloud Point (°C) 12.532 -1.310 8.826

Pour Point (°C) 6.783 -8.24 2.760

Allylic Position Equivalent 90.310 100.20 64.52

Bis-Allylic Position Equivalent 37.30 15.600 10.870

Oxidation Stability (h) 5.866 10.150 13.440

Higher Heating Value 37.316 39.021 34.001

Kinematic Viscosity (mm2/s) 3.422 3.913 3.133

Density (g/cm3). 0.831 0.864 0.755

Table: 9 - Biodiesel fuel properties are predicted based on fatty acid profile of oil feedstock

determined by gas-chromatography using Biodiesel Analyzer analytical software

39

Biodiesel fuel properties

Biodiesel properties Canvalia

cathartica Nymphea Nouchali

Biodiesel standard (ASTM method)

Saturated Fatty Acid 27.85 15.15

Mono Unsaturated Fatty Acid 46.49 55.70

Poly Unsaturated Fatty Acid 28.24 28.20

Degree of Unsaturation 102.96 112.10

Saponification Value (mg/g) 212.28 208.87

Iodine Value 105.21 101.19 <120

Cetane number 48.33 49.66 48-65

Long Chain Saturated Factor 2.20 0.405

Cold Filter Plugging Point (°C) -9.55 -15.205

Cloud Point (°C) 6.60 -2.86

Pour Point (°C) 0.35 -9.92 -15 to 10

Allylic Position Equivalent 102.97 112.10

Bis-Allylic Position Equivalent 41.48 28.20

Oxidation Stability (h) 6.77 6.77 > 3 hours

Higher Heating Value 40.33 38.82

Kinematic Viscosity (mm2/s) 3.723 3.46 1.9-6.0

Density (g/cm3). 0.900 0.870 0.86-0.90

Table 10: Biodiesel fuel properties are predicted based on fatty acid profile of oil feedstock determined by gas-chromatography using Biodiesel Analyzer analytical software

40

4.4 Transesterification Studies

4.4.1 Alkali-catalysed transesterification studies of Nymphaea nouchali (egyptian blue

lotus) oil

Nymphaea nouchali, often known by its synonym Nymphaea caerulea, or by

common names blue lotus. It is native to southern and eastern parts of Asia, and is the

national flower of Sri Lanka and of Bangladesh. Nymphaea nouchali is widely

distributed along the south-western coast of kerala. However, taking into account the

magnitude of its abundance, the studies conducted are still negligible. In the present

study petals and stamen of Nymphaea nouchali is screened for its potential for biodiesel

production.

Nymphaea nouchali Dried petals and stamen

Two major factors affecting the rate and conversion efficiency of

transesterification are: molar ratio of alcohol to oil and reaction temperature. When

Calophyllum inophyllum oil was processed by base catalysed reaction, the optimum

reaction conditions was 8:1 M ratio of methanol to oil and 60 0C reaction temperature. In

the present experiment, the catalyst concentration was kept at 1% of the lotus oil. KOH

was used as the catalyst.

Experimental Set Up: The oil was preheated in a two necked round bottomed flask at

500C for reducing its viscosity. The potassium hydroxide-ethanol solution was prepared.

The alkali ethoxide solution was added to preheated oil while mixing by means of a

41

magnetic stirrer. The stirring was maintained at 350 rpm throughout the reaction. The

reaction was stopped after 2h and was poured into a separating funnel and allowed to

settle under gravity. The products of the alkaline transesterification process result in the

formation of two layers viz., an upper layer containing biodiesel and a lower layer of

glycerol. The lower layer was drained off. The upper layer was washed with hot distilled

water until the bottom aqueous layer becomes clear.

The density of oil decreased on transesterification. The acid value of the oil was

reduced after transesterification. The alcohol to oil molar ratio is one of the most

important variables affecting the ester yields. In the present work, the ethanol to oil

molar ratio varied from 4:1 to 10:1.Fig.Shows the effect of ethanol to oil molar ratio on

yield of biodiesel at different temperatures. It was observed that the yield of biodiesel

increased with increase in molar ratio. But at molar ratio of 12:1and above, the yield of

biodiesel decreased with increase in the molar ratio. The molar ratio of 6:1 gave the

maximum yield of 89%.

Figure 6: Optimization of reaction conditions of Nymphea nouchali

42

Alcohol: Oil molar ratio Reaction

temperature % (v/v)Yield

6:1

50 78

60 89

70 80

9:1

50 70

60 72

70 70

4:1

50 55

60 60

70 56

Table 11: Optimization of reaction conditions of Nymphea nouchali

Transesterification studies were carried out at different reaction temperature such

as 50, 60 and 70 0C with 1%KOH for a reaction time of 2 h. The yield of biodiesel shows

an increasing trend with increase in reaction temperature up to 60 0C. This is because

higher reaction temperature helps in faster settlement of glycerol. The decrease in the

biodiesel yield beyond 60 0C may be due to saponification of the triglycerides before the

completion of the transesterification.

4.4.2 Two- step biodiesel production ofPongamia Pinnatta Seed Oil

Pongamia pinnata (Derris indica , Indian Beech Tree, Honge Tree, Pongam Tree,

Milletia pinnata) is widely distributed in India. It belongs to mangrove associated plants.

It contains more than 40% oil yield and it varies with different locations.

43

Experimental Set Up:

Acid pre-esterification

Pre-esterification was carried out in a 100 ml three-neck flask. The flask was

immersed in a glass water bath placed on the plate of magnetic stirrer of 400 rpm. It was

confirmed that transesterification depends on several factors, namely, alcohol to oil ratio,

catalyst type, reaction time, FFA and water content of oils .Firstly, in the pre-

esterification process, the solution of concentrated sulphuric acid (1% based on oil

weight) in alcohol was heated at 500C and then added into the reaction flask. Ethanol to

oil ratio was kept at 30:1 and reaction temperature at 500C. The reaction was carried out

for 2 hrs. The conversion of pre-esterification was calculated bycomparison of the acid

values before and after the reaction.

Base catalysed transesterification

It was confirmed that transesterification depends on several factors, namely,

alcohol to oil ratio, catalyst type, reaction time, FFA and water content of oils. The acid

value of the Pongamia oil was reduced to 1.04mg KOH/g after the acid pre treatment.

The oil was preheated in a two necked round bottomed flask at 500C for reducing its

viscosity. The potassium hydroxide-ethanol solution was prepared. The alkali ethoxide

solution was added to preheated oil while mixing by means of a magnetic stirrer. The

stirring was maintained at 350 rpm throughout the reaction. The reaction was stopped

44

after 2h and was poured into a separating funnel and allowed to settle under gravity. The

products of the alkaline transesterification process result in the formation of two layers

viz., an upper layer containing biodiesel and a lower layer of glycerol. The lower layer

was drained off. The upper layer was washed with hot distilled water until the bottom

aqueous layer becomes clear.

In the present work ,the ethanol to oil molar ratio varied from 6:1 to

12:1.Fig..Shows the effect of ethanol to oil molar ratio on yield of biodiesel at different

temperatures. It was observed that the yield of biodiesel increased with increase in molar

ratio. But at molar ratio of 12:1and above, the yield of biodiesel decreased with increase

in the molar ratio. The molar ratio of 9:1 at 50 0C gave the maximum yield of 86%.

Transesterification studies were carried out at different reaction temperature such

as 40, 50 and 60 0C with 1%KOH for a reaction time of 2 h. The yield of biodiesel shows

an increasing trend with increase in reaction temperature up to 50 0C. This is because

higher reaction temperature helps in faster settlement of glycerol. The decrease in the

biodiesel yield beyond 50 0C may be due to saponification of the triglycerides before the

completion of the transesterification. This finally results in the formation of an emulsion

and makes the separation of layers difficult.

Effect of acid pre treatment on the acid value

45

Acid value

Before pre esterification 7.02 mg KOH/g

After esterification 1.04 mg KOH/g

Transesterification of Pongamia pinnatta oil

Alcohol:Oil molar ratio

Reaction temperature % (v/v)Yield

6:1

40 42

50 55

60 32

9:1

40 71

50 86

60 40

12:1

40 42

50 50

60 36

Table 11: Optimization of reaction conditions of Pongamia pinnatta

4.4.3 Two- step biodiesel production ofThespesia Populnea oil

30

40

50

60

70

80

90

40 45 50 55 60

Bio

die

sel y

ield

(%)

Reaction temperature (0C)

6:01

9:01

12:01

Thespesia populnea, commonly known as the Portia tree is species of flowering

plant in the mallow family, Malvaceae.

Experimental set up

Pre-esterification was carried out in a 100 ml three

immersed in a glass water bath placed on the plate of magnetic stirrer of 400 rpm. It was

confirmed that transesterification depends on several factors, name

catalyst type, reaction time, FFA and water content of oils .Firstly, in the pre

esterification process, the solution of concentrated sulphuric acid (1% based on oil

weight) in alcohol was heated at 50

oil ratio was kept at 30:1 and reaction temperature at 50

for 2 hrs. The conversion of pre

values before and after the reaction.

46

Thespesia populnea, commonly known as the Portia tree is species of flowering

plant in the mallow family, Malvaceae.

esterification was carried out in a 100 ml three-neck flask. The flask was

immersed in a glass water bath placed on the plate of magnetic stirrer of 400 rpm. It was

confirmed that transesterification depends on several factors, namely, alcohol to oil ratio,

catalyst type, reaction time, FFA and water content of oils .Firstly, in the pre

esterification process, the solution of concentrated sulphuric acid (1% based on oil

weight) in alcohol was heated at 500C and then added into the reaction flask. Ethanol to

oil ratio was kept at 30:1 and reaction temperature at 500C. The reaction was carried out

for 2 hrs. The conversion of pre-esterification was calculated by comparison of the acid

values before and after the reaction.

Thespesia populnea, commonly known as the Portia tree is species of flowering

neck flask. The flask was

immersed in a glass water bath placed on the plate of magnetic stirrer of 400 rpm. It was

ly, alcohol to oil ratio,

catalyst type, reaction time, FFA and water content of oils .Firstly, in the pre-

esterification process, the solution of concentrated sulphuric acid (1% based on oil

action flask. Ethanol to

C. The reaction was carried out

esterification was calculated by comparison of the acid

47

The oil was preheated in a two necked round bottomed flask at 500C for reducing

its viscosity. The potassium hydroxide-ethanol solution was prepared. The alkali

ethoxide solution was added to preheated oil while mixing by means of a magnetic

stirrer. The stirring was maintained at 350rpm throughout the reaction. The reaction was

stopped after 2h and was poured into a separating funnel and allowed to settle under

gravity. The products of the alkaline transesterification process result in the formation of

two layers viz., an upper layer containing biodiesel and a lower layer of glycerol. The

lower layer was drained off. The upper layer was washed with hot distilled water until

the bottom aqueous layer becomes clear.

Alcohol: Oil molar ratio Reaction temperature % (v/v)Yield

6:1

40 50

50 65

60 87

70 85

9:1

40 52

50 72

60 85

70 80

12:1

40 50

50 66

60 80

70 72

Table 12: Optimization of reaction conditions of Thespesia populnea

48

Figure 8: Optimization of reaction conditions of Thespesia populnea

In the present work, the ethanol to oil molar ratio varied from 6:1 to 9:1.Fig. shows

the effect of ethanol to oil molar ratio on yield of biodiesel at different temperatures. It was

observed that the yield of biodiesel increased with increase in molar ratio. The molar ratio

of 6:1 gave the maximum yield of 87% at 60 0C Transesterification studies were carried

out at different reaction temperature such as 40, 50 and 60 0C with 1%KOH for a reaction

time of 2 h.

4.5 Effect of ultrasound on transesterification of oils

The tub of ultra sound bath was filled with 300 ml of distilled water and then the

beaker containing the reactants was placed inside. The temperature was maintained in 500

C and the beaker was covered with aluminium foil, considering that this temperature the

evapouration of ethanol is negligible. The equipment was set to operate at 200 W and 20

kHz for 10, 20 and 30 minutes by keeping other optimum reaction conditions and process

parameters was unaltered. It was found that transesterification using ultrasound drastically

reduce the reaction time and leads to higher yields of biodiesel greater than 90 %.

30

40

50

60

70

80

90

35 45 55 65 75

%Y

ield

Reaction time

06:01

09:01

12:01

49

Table 13: Effect of ultrasound in transesterification

The percentage yield of biodiesel increases with the application of ultrasound to

the system. At a reaction time of 20 minutes it gave a good yield (>90 %) than the

conventional method of transesterification. Thus it works better than conventional

methods, as it saves time, has high extraction efficiency and a low environmental impact.

In biodiesel production, adequate mixing is required to create su cient contact between

the vegetable oil or animal fat and alcohol, especially at the beginning of the reaction.

Application of ultrasonication provides sufficient mixing. Ultrasonic cavitation and

microbubble formation, which are caused by the ultrasonic energy introduced by the

sonotrode, greatly improve the interfacial contact between the immiscible methanol and

plant oil/ animal fat mixture, thus increasing the reaction rate. Also, the formation and

bursting of microbubbles caused by ultrasonic cavitation intensifies the local energy

transfer and energizes the reactant molecules, thus enhancing the overall reaction rate.

Thus the remarkable decrease in the reaction time can be explained by intense mass

transfer afforded by the unique conditions generated by cavitation noise.

Oil Ultrasound reaction

time (minutes) % (v/v)Yield

Nymphea oil 10 85 20 89 30 >90

Pongamia 10 90 20 94 30 94

Thespesia 10 89 20 90 30 >95

50

References

1. Burdge, G.C., A.E. Jones, and S.A. Wootton, Eicosapentaenoic and docosapentaenoic

acids are the principal products of α-linolenic acid metabolism in young men. British

Journal of Nutrition, 2002. 88(04): p. 355-363.

2. De Lorgeril, M., et al., Mediterranean alpha-linolenic acid-rich diet in secondary

prevention of coronary heart disease. The Lancet, 1994. 343(8911): p. 1454-1459.

3. Abd El-Gleel, W. and M. Hassanien, Antioxidant properties and lipid profile of

Diplotaxis harra, Pulicaria incisa and Avicennia marina. Acta Alimentaria, 2012. 41(2):

p. 143-151.

4. Holman, R., S. Johnson, and T. Hatch, A case of human linolenic acid deficiency

involving neurological abnormalities. The American Journal of Clinical Nutrition, 1982.

35(3): p. 617-623.

5. Simopoulos, A.P., Omega-3 fatty acids and antioxidants in edible wild plants. Biological

Research, 2004. 37(2): p. 263-277.

6. Chisholm, M.J. and C. Hopkins, Fatty acids of Doxantha seed oil. Journal of the

American Oil Chemists’ Society, 1965. 42(1): p. 49-50.

7. Spitzer, V., Fatty acid composition of some seed oils of the Sapindaceae.

Phytochemistry, 1996. 42(5): p. 1357-1360.

8. Belury, M.A., Dietary conjugated linoleic acid in health: Physiological effects and

mechanisms of action 1. Annual review of nutrition, 2002. 22(1): p. 505-531.

9. Huang, W.-C., et al., Anti-bacterial and anti-inflammatory properties of capric acid

against Propionibacterium acnes: A comparative study with lauric acid. Journal of

dermatological science, 2014. 73(3): p. 232-240.

51

CHAPTER 5

CONCLUSION

The present study revealed that Thespesia populnea seeds and Pongamia pinnatta

seeds yield a high content of oil, 22.59% and 46.36% w/w respectively. The biodiesel

derived from these oils meet the international standards. The optimum biodiesel yield

from thespesia oil was 87% at alcohol:oil molar ratio of 6:1, reaction time for 2 hours at

600C and for pongamia pinnatta was 86% at alcohol:oil molar ratio of 9:1, reaction time

of 2 hours at 500C. Pongamia oil with high free fatty acid content affected the catalytic

activity of the alkali process negatively, resulting in saphonification which decreases the

esters yield and causing processing problems. Pongamia oil has to be pre esterified using

acid before subjecting to base catalysed transesterification.

The sonolysis of oil showed considerable gain in the reaction time with respect to

classical transesterification. The remarkable decrease in the reaction time can be

explained by intense mass transfer afforded by the unique conditions generated by

cavitation noise. Thus biodiesel production using ultrasound could be considered as a

potential route for the production of biodiesel, capable of meeting high demands in short

time period, with energy costs that may be less than the expenses with the conventional

method.

The identified fatty acids in the hexane extract of Thespesia populnea, Derris

trifoliate and Canavalia cathartica exhibit multifunctional biological activity. The oil of

these seeds contained a significant percentage of pharmacologically active linoleic and

alpha-linolineic fatty acids. The present findings also proved the traditional use of these

plants in the folk medicine.

52

In the present study, six wetland species of Kuttanad region were selected and

investigated for screening their potential for biodiesel production.The fatty acid

compositions of plants were studied by Gas Chromatography – Mass Spectroscopy.

Among them thespesia and pongamia were identified as a cheap and renewable raw

material for biodiesel production.

53

RESULTS OF GC-MS

54