the oil content of samples - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/34736/10/10_chapter3.pdf · the oil content of samples ... such as petroleum ether, ... g of Jatropha

CHAPTER 3: Phenotyping: Measuring and recording the oil content of samples

Marker assisted selection for high oil yielding varieties in Jatropha curcas 21

3.1 INTRODUCTION

Phenotypic traits are any observable characteristics of a plant species. Phenotyping

involves measuring and recording the complex as well as directly measured

phenotypic traits of individual accessions in a population. Phenotypic characters are

mainlyquantitative parameters. The complex traits include physiology, ecology,

architecture, resistance, tolerance, yield, growth and development. Directly measured

phenotypic traits include image-based projected leaf area, chlorophyll fluorescence,

stem diameter, plant height/width, compactness, stress pigment concentration, tip

burn, internode length, color, leaf angle, leaf rolling, leaf elongation, seed number,

seed size, tiller number, flowering time, germination time etc.

Most of the phenotypic traits of interest to plant breeders are quantitatively inherited.

Quantitative inheritance constitutes the cumulative action of many genes that interact

with environment to produce continuous distribution of phenotypes over a given

range of individuals (Semagn et al., 2010). QTL Mapping offers the possibility of

direct selection of quantitative traits using DNA–based markers. But the method is

rather time consuming and limited to few crops, as this method requires a biparental

mapping population. Thus, association mapping is captivating which uses natural

populations to identify genes responsible for quantitative variation of complex traits

with agricultural & evolutionary importance. Phenotyping is the most essential

module in association mapping.

Phenotypic scoring can be costly and time consuming, requiring an execution of

feasible and convenient method. Moreover, to increase precision of phenotypic

measurements, the individual accessions from a particular site are duplicated.

Subsequently, the quantitative parameters for each replicative accession are combined

to produce an average estimate of the phenotype of the accession which is less

influenced by environment or measurement errors (Semagn et al., 2010).

Many developed countries are using edible oil-seed crops such as soybean, rapeseed,

groundnut, sunflower for the production of bio-diesel. However, developing countries

like India, that have huge dearth of edible oil (6.31 million tonnes) for the

consumption, cannot afford to use edible oils for bio-diesel production and hence

non-edible oil seeds such as Pongamia (Pongamia pinnata) and Jatropha (Jatropha



curcas) are explored along with fulfilling the additional criteria of greening the waste

lands without compromising the food, fodder security and improved livelihoods

(Hegde et al., 2003; Kaushik et al., 2007). In India, non-edible seed crops like Karanj,

Mahua, Jatropha are used for production of biodiesel depending on locality

(Bhattacharya et al., 2003). However, these activities are at their infancy stages.

Among non-edible plant species, J. curcas, a perennial tree containing high amount of

non-edible oil, is considered best for the formation of biodiesel (Verma et al., 2009).

Jatropha curcas that belongs to the family of Euphorbiaceae and native to South

America has attained significant importance as a petrocrop. Jatropha curcas is a

multipurpose shrub with a variety of applications and enormous economic potentials

for its seed oil, which can be converted into biodiesel and used as an alternative to

petro-diesel. The seeds of J. curcas are considered to be good source of lipids. They

contain approximately 20-39% of oil; due to this it is very enticing in its use as an

energy source for fuel production. The crude oils of Jatropha is composed mainly of

oleic, linoleic, palmitic, and stearic acids (Rodríguez-acosta et al., 2010). The oil

possesses appropriate combustive properties with cetane number for Jatropha oil (23-

41) close to cottonseed (35-40) and better than rapeseed (30-36), groundnut (30-41)

and sunflower (29-37) (Vaitilingom et al., 1997). In addition, the oil from its seeds

has been found useful for the manufacture of candles and soap, in cosmetic industry

and also for medicinal purposes (Akbar et al., 2009; Gubitz et al., 1999).

The fact that Jatropha oil cannot be used for nutritional purposes makes it resourceful

as energy/fuel source. The lipid fraction of Jatropha oil seed can be extracted by

using different techniques. Extraction with organic solvents and water have been the

main approaches (Shah et al., 2004), assuitable mechanical pressure system are not

available for oil extraction from seeds (Openshaw, 2000).

Various methods for recovering the oil from J.curcas seeds have been

investigated.Conventional method such as solvent extraction is most widely used

technique, owing to their high efficiency in oil recovery (90-98%). However, its

major disadvantage is its high energy input and toxicity of solvent. This has led to the

development of enzyme-based techniques (Sharma et al., 2002). The enzyme

supported aqueous extraction offers a non toxic alternative to common extraction

methods using organic solvents.



The present study deals with optimization of oil extraction using solvent and

enzymatic extraction techniques.

3.2 MATERIALS AND METHODS

3.2.1 Materials

A total of 91 J. curcas accessions were collected for the present study,which included

accessions from six different states (10 geographical regions) of India viz. Gujarat,

Rajasthan, Uttar Pradesh, Maharashtra, Tamil Nadu and North India (Himachal

Pradesh, Jammu and Kashmir, Delhi, Uttarakhand and Haryana). In Gujarat, seeds of

J. curcas were collected from Research and Development Center of Forest

Department, Rajkot, Gujarat and Anand Agriculture University, Anand. Damaged

seeds were discarded and healthy and clean seeds were selected. The seeds were dried

in shade prior to use. All chemicals and solvents used were of analytical grade.

3.2.2 Method

3.2.2.1 Oil Extraction

3.2.2.1.1 Solvent Extraction Techniques

Jatropha seeds were cracked, shells carefully removed and the kernels thus obtained

were used for oil extraction. The kernels (1g) were crushed with different organic

solvents (20 ml) such as petroleum ether, hexane and isopropanol. The oil was

extracted by using different techniques such as filtration, centrifugation and separating

funnel. Finally, oil was recovered by evaporating the organic solvents.

3.2.2.1.2 Aqueous enzymatic oil extraction (AEOE) from seed kernels

Jatropha seeds (5g) were soaked in water for 2 hours. The soaked seeds were

grounded (without addition of water to the soaked seeds) to a thick paste by using

mortar and pestle. This paste was then dispersed in distilled water at 1:2 (w/v) paste

to water ratio followed by gentle stirring with a magnetic stirrer. Cellulase (250 mg)

(Himedia 15FTU/ml) was added before the pH of the slurry was adjusted to 4 by

adding appropriate amount of 0.5 N HCl or NaOH. The enzyme mixture was then



incubated overnight at 40°C with constant shaking at 100 rpm. The upper oil phase

was collected by centrifugation at 10,000 x g for 20 minutes. A control (Aqueous oil

extraction, AOE) was also carried out for the above extraction during which no

enzyme was added (Sharma et al., 2002).

3.2.2.1.3 Soxhlet Extraction

An improvisation of procedure described by Akbar et al., 2009 has been implemented

and has shown to be effective in oil extraction. The method involves, homogenizing 3

g of Jatropha curcas seed kernels mechanically using mortar and pestle.

Subsequently, the grounded seeds were defatted in soxhlet apparatus using hexane,

isopropanol and petroleum ether as the solvent. The defatting process ran for 6 hours.

After the oil had dissolved in hexane, isopropanol and petroleum ether, it was

evaporated using a rotary evaporator by exposure to heat in hot air oven at 50˚C. The

flasks with the grounded seeds and solvent were pre-weighed and the same were post

weighed after the solvent evaporation. The amount of oil recovered was calculated as

pre-weight minus the post-weight and represented as percentage of total oil present in

Jatropha curcas seed kernels.

Each extraction was run in triplicate and their average was estimated as final value.

3.3 RESULTS AND DISCUSSION

The amount of oil recovered was calculated as % of total oil present in J.curcas seed

kernels. Three different solvents were used to evaluate their efficiency. The oil

extraction capabilities of petroleum ether, hexane and isopropanol are shown in Table

3.1.

Table 3.1 shows the comparative data of oil extracted by use of various solvents and

extraction method. The extraction yield with petroleum ether was found in range of

10-53%. Similarly, the extraction yield with hexane was found to be in range of 20-

78% and with isopropanol was found to be between 15-59%. The best result was

obtained by using hexane and soxhlet extraction method.



Organic

Solvents

Extraction Methods (% yield)

Petroleum

Ether

Separating

Funnel

Centrifugation

Filtration Soxhlet

Extraction

28.6 ± 4.16

48.3 ± 0.57 10.3 ± 4.0 53.66± 1.05

Isopropanol 19.66 ±

2.3 53.33 ± 1.52

15.66 ± 4.0

59.66 ± 2.5

Hexane 36 ± 9.53 52.66 ± 4.04 20 ± 6.2 78.66 ± 0.9

Table 3.1: Extraction of oil by Use of Various Solvents (n=3)

Use of aqueous oil extraction (AOE), as control, was found to yield 21.4% oil. The

same data i.e. AOE yield in range of 17-21% was reported by others authors (Sharma

et al., 2005). In AEOE method, the enzymes used such as cellulase and pectinase

were unsuccessful in extracting large amount of oil. Their yield was very less (in the

range of 2%) as compared to that described by other workers (Sharma et al., 2005).

A comparison between different techniques of oil extraction and solvents used is

shown in Figure 3.1. The results reveal that extraction by soxhlet method, yields oil in

the range of 53-78%. This value was taken as 100% recovery of oil while calculating

oil recovery by other methods. Soxhlet extraction is better as compared to other

methods because the process is continuous and there is complete oil recovery.

Among the solvents used for oil extraction, hexane is reported as the best solvent

because it yields maximum oil as compared to other solvents (Sepidar et al., 2009).

But, the oil recovered by hexane and isopropanol is slightly yellow in color. This

might pose problems in using oil for further biodiesel production. Thus, it is better to

use petroleum ether for efficient biodiesel production. Kandpal et al., 1995 also used

petroleum ether as a solvent, to extract Jatropha curcas oil from whole seed and

kernel.

In AEOE, enzymes are used to facilitate release of oil from oil bodies enmeshed in

protein and cellulosic/hemicellulosic networks. Use of only cellulase and pectinase

did not improve the yield of oil. For better oil yield, additional enzyme preparation



such as Protizyme, Pectinex Ultra SP-L, Promozyme,etc. are required (Sharma et al.,

2005). This adds to the cost of oil extraction.

Figure 3.1: Effects of solvents and extraction technique in % oil yield from Jatropha

seed kernels

In the present study, soxhlet extraction method gave higher oil yields than other

extraction methods. Petroleum ether is a very efficient solvent though its yield is less

as compared to hexane and Isopropanol. This is because it gives pure and colorless oil

whereas isopropanol and hexane gives light yellow colored oil with white debris.

Furthermore, seed oil content of all 91 collected accessions of J. curcas was

estimated using Soxhlet extraction and petroleum ether as solvent of choice. The oil

content of collected accessions ranged from 18% to 40%.

The following table (Table 3.2) displays the % oil content in all 91 Jatropha curcas

accession representing six different states of India.

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

Separating Funnel Centrifuge Filtration Soxhlet Extraction

Per

cen

tag

e o

il Y

ield

Extraction Techniques

Petroleum ether Hexane Isopropanol



Table 3.2: Percentage oil content in all the selected Jatropha curcas accessions

Sa

mp

le

Oil

content

(%)

Reg

ion

Sa

mp

le

Oil

content

(%)

Reg

ion

Sa

mp

le

Oil

content

(%)

Reg

ion

Sa

mp

le

Reg

ion

Oil

content

(%)

JC-1 22.66

Gu

jara

t

JC-27 32.08

Utt

ar

P

rad

esh

JC-50 26

No

rth

I

nd

ia

JC-73

Ma

ha

rash

tra

36.85

JC-2 28 JC-28 35.24 JC-51 38.73 JC-74 33.06

JC-3 23.33 JC-29 35.92 JC-52 28.91 JC-75 26.42

JC-4 26.66 JC-30 37.2 JC-53 31.38 JC-76 31.74

JC-6 29.33 JC-31 34.76 JC-54 25.88 JC-77 35.64

JC-7 28.33 JC-32 37.02 JC-55 37.77 JC-78 35.69

JC-8 29.33 JC-33 29.5 JC-56 40.29 JC-79 26.87

JC-9 24.33 JC-34 37.28 JC-57 18.33

Ra

jast

han

JC-80 33.17

JC-10 18.33 JC-35 28.45 JC-58 24.66 JC-81

Ta

mil

Na

du

30.7

JC-11 25.66 JC-36 32.67 JC-59 36.4 JC-83 36.07

JC-12 23.33 JC-37 32.78 JC-60 35.5 JC-84 35.2

JC-13 21.66 JC-38 33.98 JC-61 26.4 JC-85 35.6

JC-14 27.66 JC-39 35.57 JC-62 29.57 JC-86 40.5

JC-15 35.33

Ra

jast

han

JC-40 33.06 JC-63 31.7 JC-87 40.8

JC-16 34.33 JC-41 32.21 JC-64 26.7 JC-88

No

rth

In

dia

34.31

JC-19 31 JC-42 36.1 JC-65 32.3 JC-89 30

JC-20 20 JC-43 32.2 JC-66 34.2 JC-90 29.89

JC-22 33.33 JC-44 30.9 JC-67 35.5

JC-91 28.95

JC-23 27.66 JC-45 31 JC-68 35.53 JC-92 38.82

JC-24 28 JC-46 31.3 JC-69 37.05 JC-93 33.28

JC-25 36.66 JC-47 30.7 JC-70 32.65 JC-94 36.31

JC-26 39.23

JC-48 33.92

JC-71 33.05 JC-95 30

JC-49 41.83 JC-72 36.95 JC-96

Raja

st

han

36

Reports are available showing tremendous variability in oil content of seed of J.

curcas accessions from different agro-climatic regions of India. Subramanian et al.,

2005, reported that Jatropha seeds contain 46-58% of oil on kernel weight and 30-

40% on seed weight.Wani et al., 2006, recorded variation in Indian accessions for oil

content (27.8–38.4%) and 100 seed weight (44–77 g). Kaushik et al., 2007, explored

the variability in Haryana-India accessions to find wide variation in 100 seed weight

(49–69 g) and oil content (28–39%). Similarly Rao et al., 2008, found wide variation

in 100 seed weight (57–79 g) and oil content (30–37%) for accessions from Andhra

Pradesh, India.



In the present study, among collected 91 accessions from six states of India

(representing 10 geographical regions), the oil content of provenance collected from

Himachal Pradesh HP-21 showed the highest oil content (41.83%) while Gujarat

GJ/PMS/456 and Rajasthan RJ/UP/010 provenance showed the lowest oil content

(18.33%). Percent oil content of collected accessions varied from 18.33% to 29.33%

in Gujarat, 18.33% to 36.66 % in Rajasthan, 28.45% to 39.23% in Uttar Pradesh,

25.88 % to 41.83% in Himachal Pradesh, 37.77 % to 40.29 % in Jammu Kashmir,

26.42 % to 37.05% in Maharashtra, 30.7% to 40.08 % in Tamil Nadu, 28.95% to

34.31% in Delhi, 33.28 % to 38.82 % in Uttarakhand and Haryana provenance had

22.66% (Fig1.2). In this entire study, highest oil per cent was found in Himachal

Pradesh provenance, whereas lowest in Gujarat and Rajasthan.

(a)

0

20

40

Udaipur Jaipur

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Sample 9

Sample 10

Sample 11



(b)

(c)

0

10

20

30

40

NBRI Biotechpark

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Sample 9

Sample 10

Sample 11

Sample 12

Sample 13

Sample 14

Sample 15

Sample 16

LUCKNOW

0

5

10

15

20

25

30

35

40

Akola

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Sample 9

Sample 10

Sample 11

Sample 12

Sample 13

Sample 14MAHARASHTRA



(d)

(e)

0

5

10

15

20

25

30

35

40

45

Madurai

Sample 1 Sample 2

Sample 3 Sample 4

Sample 5 Sample 6

Sample 7

0

10

20

30

GUJARAT

Banaskatha

Dahod

Chotila

Junagadh

Junagadh2

Junagadh3

Junagadh4

Panchmahal

Panchmahal2

Panchmahal3

Panchmahal4

Panchmahal5

Rajkot

Rajkot2



(f)

Figure 3.2: % Oil content of Jatropha Samples collected from different parts of India (a)

Rajasthan (b) Uttar Pradesh (c) Maharashtra (d) Madurai (e) Gujarat (f) North India

In general, it appears that the environment has a predominant role in the phenotypic

variation among provenances, which could be interpreted as a narrow genetic base of

J. curcas. Studies concerning chemical variations have been focused on the seed oil

content, with minimal attention to other molecules potentially useful as markers for

estimation of genetic diversity. Ovando-Medina et al., 2009b investigated variations

in seed oil content between populations of J. curcas from the coastal zone of Mexican

State of Chiapas. Results showed that seed oil content varied from 12.09 to 44.28%,

apparently related to the aridity of the sites, with the higher contents corresponding to

zones with lower rainfall. Ginwal et al., 2004, reported similar associations between

oil content and rainfall. The variation in oil content can be generated by genetic and

environmental factors, including rainfall and soil fertility (Escobar et al., 2008;

Mishra, 2009), however, several authors have reported high heritability values for this

characteristic, 99% (Kaushik et al., 2007),89.7% (Gohil et al., 2009), and >75%

(Ginwal et al.,2005). There are reports on the composition of J. curcas oil (fatty acids,

sterols and other molecules), but researchers have concentrated on the potential of oil

as food/feed or as biocide (Adebowale et al., 2006; Martínez- Herrera et al., 2006).

0

10

20

30

40

50

Sample1

Sample2

Sample3

Sample4

Sample5

Sample6

Sample7



There are two main postulates for the diversity in oil content; the first, as Ginwal et

al., 2005 and Saikia et al., 2009 suggested, is related to the fact that this species

grows over a wide range of climatic conditions and populations must have

experienced marked differences in selective pressure in their natural habitat. The

problem with this postulate is that Jatropha populations are relatively new in Asia

and Africa. The second explanation is that the variation was introduced with seeds

from tropical America, centuries ago. Whichever could be the reason, it is apparent

from the present study that germplasm used for biodiesel production in various parts

of the country is highly heterogenous.



3.4 REFERENCES

Adebowale, KO. and Adedire, CO. (2006) Chemical composition and insecticidal

properties of the under utilized Jatropha curcas seed oil. African Journal of

Biotechnology 5: 901-06

Akbar, E. Yaakob, Z. Kamarudin, S. and Ismail, M. (2009) Characteristics and

Composition of Jatropha curcas oil seed from Malaysia and its potential as Biodiesel

Feedstock. European Journal of scientific research 29: 396-403

Bhattacharya, P. and B. Joshi (2003) Strategies and institutional mechanism for large

scale cultivation of Jatropha curcas under Agroforestry in the context of proposed

biofuel policy of India. ENVIS Bull Grassland Ecoystem and Agroforestry 1: 58-72

Escobar, JC. Lora, ES. Venturini, OJ. Yáñez, EE. Castillo, EF. Almazán, O. (2008)

Biofuels, Environment, technology and food security. Renew. Sust.Energ. Rev 13:

1275-1287

Ginwal, HS. Phartyali, SS. Rawat, PS. Srivastava, RL. (2005) Seed source variation in

morphology, germination and seedling growth of Jatropha curcas Linn.in Central

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Gohil, RH. Pandya, JB. (2009) Genetic evaluation of Jatropha (Jatropha curcas

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Gubitz, G. M., Mittelbach and M., Trabi (1999) Exploitation of the tropical oil seed

plant Jatropha curcas L. Bioresource Technology 67: 73-82

Hegde, D.M. (2003) Tree oilseeds for effective utilization of wastelands.In

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Kandpal, J. B. and Madan, M. (1995) Jatropha curcas a renewable source of energy

for meeting future energy needs. Renewable Energy 6:159–60



Kaushik, N. Kumar, K. Kumar, S. Kaushik, and N. Roy, S. (2007) Genetic variability

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accessions. Biomass Bioenergy 31: 497–502

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treatments on their levels, in four provenances of Jatropha curcas L. from

Mexico.Food Chemistry 96: 80-89

Mishra, DK. (2009) Selection of candidate plus phenotypes of Jatropha curcas L.

using method of paired comparisons. Biomass Bioenergy 33:542-545

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Ovando-Medina, I. Espinosa-García, F. Núñez-Farfán, J. and Salvador-Figueroa, M.

(2011) Genetic Variation in Mexican Jatropha curcas L. Estimated with Seed Oil

Fatty Acids. Oleo Science 60: 301-311

Rao, G.R., Korwar, GR., Shanker, A.K. and Ramakrishna, Y.S. (2008) Genetic

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Shukla, SK.(2005) Experiences of Chattisgarh biofuel development authority.

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industrial products from Jatropha curcas.Dbv-Verlagfür die Technische Universität

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3.5 APPENDIX 3.5.1 Extraction of Oil

3.5.1.1 OrganicSolvents

All solvents used such as Petroleum ether, hexane and Isopropanol were all of

analytical Grade.

3.5.1.2 Cellulase

250 mg of cellulose was directly added in the mixture. There is no need for

dissolving it in buffer.

3.5.1.3 Chemicals and Reagents Used

Serial No. Reagents/Chemicals Used Company Name

1. Cellulase HiMedia

2. Petroleum Ether Qualigens

3. Hexane Qualigens

4. Isopropanol Qualigens

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the oil content of samples - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/34736/10/10_chapter3.pdf · the oil content of samples ... such as petroleum ether, ... g of Jatropha