Kumar et al. 2014. International J Ext Res.

1:36-40
http://www.journalijer.com

International

Journal of Extensive Research

Open Access

Research Article

Efficient Production Methodology For Biodiesel From Non-Edible

Karanja Oil and Its Fuel Characterization Study
Kumar Vinod, Jahan Firdaus and Saxena RK*
Department of Microbiology, University of Delhi South Campus
New Delhi 110021 India

Abstract

Biodiesel production is the transesterification reaction which could be catalyzed either chemically or enzymatically. Enzymatic
transesterification has certain advantages over the chemical catalysis of transesterification, as it is less energy intensive, allows
easy recovery of glycerol and the transesterification of glycerides with high free fatty acid contents. In the present study, transesterification reaction of karanja oil was carried out by using Pseudomonas aeruginosa lipase. Effects of important process parameters were optimized conventionally. Enhanced conversion 85.9% of fatty acid to fatty acid methyl ester within 48 h at 45 C was
achieved by using lipase at concentration of 5% (w/v of oil), molar ratio of 1:4 (oil: methanol), methanol acyl donor, n-hexane
solvent and temperature 45 C. This approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing enzyme, enabling a broader use of biodiesel like fuel in the near future.

Keywords: lipase, biodiesel, transesterification, karanja oil, FAME.

Introduction

Diesel fuel consumption has been continuously increasing over

the past few decades resulting in the depletion of world petroleum
reserves and increased pollution. In recent decades, researches are
being focused on developing renewable raw materials as sources
of sustainable energy (Gog et al. 2012). The biodiesel fuel from
vegetable oil does not produce sulfur oxide and minimize the soot
particulate one-third times in comparison with the existing one
from petroleum (Gog et al. 2012). Because of these environmental
advantages, biodiesel fuel can be expected as a substitute for conventional diesel fuel (Fan et al., 2012).
In this context, the concept of using vegetable oil as a fuel
source in diesel engines is known, however, its development and
utilization as biodiesel fuel has been limited until recently. Technically, biodiesel is defined as the mono alkyl esters of long fatty
acids derived from renewable lipid feedstock such as vegetable oils
or animal fats (Meka et al. 2007). Karanja oil (Millettia pinnata) is
one of such non-edible oils, which has an estimated annual production potential of 200 metric tons in India and it can be grown
in waste land (Shikha and Rita, 2012). The karanja seed kernel
contains 40-60% (w/w) oil, which is constituted of 20 % saturated
and 80 % unsaturated fatty acids. Oleic acid is the most abundant
(44.8 %) followed by linoleic acid (34 %), palmitic acid (12.8 %),
and stearic acid (7.3 %) (Shikha and Rita, 2012). While composition of karanja oil is similar to other oils, which are used for
non-edible purposes, the presence of some anti-nutritional factors
*Corresponding authors email: rksmicro@yahoo.co.in;
vinodudsc@gmail.com
Copyright IJER 2014. All rights reserved.

such as toxic phorbol esters renders this oil unsuitable for use in
cooking (Gbitz et al. 1999).
Although biodiesel is at present produced chemically at industrial level (Kamini & Iefuji, 2001), the disadvantages of using
chemicals of either acid or alkaline catalysts for the transesterification of karanja oil (such as pre-treatment of feedstock, glycerol
recovery, catalyst removal wastewater treatment and high energy
requirement in the process) have drawn many researchers to seek
for more environ- mental friendly approach. Enzymatic approach
is likely to over- come the problems of a chemical-catalyzed process. Enzyme catalyzed reaction is more efficient, highly selective,
involves less energy consumption, produces less waste and it is
recyclable as enzymes can be immobilized onto a support medium
(Robles-Medina et al. 2009). Glycerol recovery in enzymatic process is easier as it would produce high grade glycerol as compared
to an alkaline process (Robles-Medina et al. 2009). It is reported
that the enzymatic reactions are insensitive to FFA and water content in the raw material (Kulkarni & Dalai, 2006).
In this paper karanja oil was studied, but there are no technical
restrictions to the use of other kinds of vegetable oils, although
biodiesels coming from some vegetable oils may not fulfil quality standards (Chang et al. 2005; Garlapati et al. 2013; Kumari et
al. 2007; Soumanou & Bornscheuer, 2003). Our current research
is focussed on the use a cheap lipase preparation from P. aeruginosa was used as the catalyst. The enzymatic transesterification
of karanja oil with methanol in solvent system was studied (Fig.
1). The reaction conditions for were optimized and evaluating the
fuel properties of the produced biodiesel.
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Materials and Methods

Materials
Karanja oil was purchased from Adi biotech Pvt, Ltd. New Delhi. All analytical reagents and media components were purchased
from Sisco Research Laboratories, India and Hi-Media, India. All
other chemicals were also obtained commercially and of analytical
grade.
Microorganism and lipase production conditions
P.aeruginosa, was maintained on nutrient agar slants (g/l): Peptone-1.5, Yeast extract-1.5, NaCl-5, Agar-20). Production of lipase
was carried out in 2 L Erlenmeyer flasks containing 400 ml of
optimized production medium (% w/v) : corn oil (emulsified with
2% gum acacia), 0.5; glucose 0.2; Tryptone 1.5%; Ammonium nitrate 0.2%; MgSO4.7H2O 0.60 mM; pH 8.0) inoculated with 2%
inoculum density and incubated at 30 oC, 200 rpm for 36 h. The
crude enzyme obtained after centrifugation of the culture broth
and subsequent four fold concentration was carried out using 10
kDa cellulose acetate membrane (Millipore). This concentrated
culture filtrate was used as lipase source for further transesterification reactions.
Transesterification Reaction
The reactions for biodiesel synthesis were carried out in a 100 ml
round bottom flask. The reaction mixture consisted of 20.0 g of
oil (karanja oil), 5.0% (w/w) crude lipase and 3.0 molar equivalent
of methanol. This reaction was carried out in presence of n-hexane at 45 C and 200 rpm for 48 h. Samples (2.0 ml) were withdrawn from the reaction mixture at regular intervals of 12 h up to
48 h and centrifuged at 12,000 rpm for 10 min. An aliquot of the
upper layer was precisely taken and biodiesel production was measured by TLC. Spectrophotometric analysis at 715 using copper
pyridine regent for residual fatty acid and finally GC analysis was
carried out to determine fatty acid methyl ester (FAME) content.
Spectrophotometric analysis of fatty acid methyl estyers.
The reaction (Sample size 0.5 ml) was terminated using 0.1 ml
of 6 M HCl. n-hexane (0.5 ml) was added to it and the mixture
was centrifuged at 10,000 rpm for 10 min at 4 C. Now, 0.1 ml
of copper pyridine reagent (50 g copper acetate dissolved in 1.0
L distilled water with pH adjusted to 6.0 using pyridine) was added to 0.5 ml of supernatant. This mixture was homogenized and
absorbance was recorded at 715 nm with a UV/VIS spectrophotometer (Shimada et al. 1999).
The percentage of conversion of fatty acid (FA) at definite
time (t) was determined according to the following formula:
Fatty acid conversion(t ) ( % )

Initial conc. of FA Conc. of FAat time ( t )

100
Intial conc. of FA

GC analysis of fatty acid methyl esters

After the reaction, samples were withdrawn and the product was
analyzed by a gas chromatography. A GC-2014 gas chromatography (Shimadzu, Japan) equipped with a capillary column (Stablewax DA, 30 m x 0.25 mm, 0.2 m film thickness) and flame
ionizing detector (FID) was used. Nitrogen gas was used as the
carrier gas at a flow rate 0f 6.21 mL / min. Injection was done in
split mode (1/5) and the injector and detector temperatures were
at 250 oC and 260 oC, respectively. Samples (1 L) were injected at

an oven temperature of 160 oC, then the oven was heated at 3 oC

/min to 250 oC (holding for 10 min) (Lowry and Tinsley, 1975).
Biodiesel fuel characteristics
Fuel characteristics were checked by American Society for Testing Materials (ASTM) methods. Specific gravities of pure samples were tested (ASTM 1996)with specific gravity bottle. Around
40 ml of sample was taken for viscosity (ASTM 2006), and flash
point testing (ASTM 2002), 80 ml of pure sample was used. Flash
point was not tested for blended sample. The experiments were
repeated two times and the average values were presented.

Fig. 1 Schematic representation of biodiesel produciton via

enzymatic route from non-edible oil

Results and Discussion

Effect of enzyme concentration on transesterification

The transesterification reactions were carried out using different
concentrations (5 15%) of P. aeruginosa lipase to produce maximum FAME production. As presented in (Fig. 2), results show
that the maximum FAME conversion (74.5%) was obtained using
5 % lipase. Use of lipase in lower concentration (1-2.5%) did not
show any significant FAME conversion. However further increase
in the enzyme concentration did not yield in any further enhancement in FAME conversion after 48 h. At a higher enzyme concentration (3%), percentage conversion was very low. The reason
might be due to unavailability of sufficient quantity of substrate
to enzyme for reaction (Nie et al., 2006). The maximum methyl
ester yield was reported by Kose et al., 2002 with the 30% Novozyme based on oil weight in transesterification of cottonseed oil
(Kse et al. 2002). Similar results were in case of methanolysis of
rice bran oil using Cryptococcus sp. S-2 lipase (Kamini & Iefuji,
2001). In another study, Garlapti et al., (2013) also reported the
optimal molar conversion of 62.23% was observed at 10 U of
lipase activity.
Effect of different acyl acceptors on transesterification
The most important factors affecting the transesterification reaction is the different acyl acceptor of solvent. The synthesis of
biodiesel was studied in the methanol, ethanol, propane 2-ol, nbutanol. The result showed that, the maximum conversion (74.8
%) was observed on using methanol as an acyl acceptor in 48 h.
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Effect of molar ratio of acyl acceptors on transesterification

One of the most important factors affecting the transesterification reaction is the molar ratio of triglycerides to methanol. The
synthesis of biodiesel was studied in the range of molar ratio
of 1:3 to 1:12. With 5 % (w/w) lyophilized lipase the maximum
FAME conversion (79.9 %) was obtained at 1:6 molar ratio after
the incubation of 48 h (Fig. 4). However, at higher molar ratio,
there was a decline in the FAME conversion with only 43.3 %
FAME conversion obtained at 1:12 molar ratio.
From the results presented in Fig. 3, it is clear that one mole
of oil is completely esterified by six mole of methanol when crude
lipase is used and approximately similar results were obtained using lipase from Pseudomonas fluorescens. Shimada et al. (1999)
reported the methanolysis of canola oil and methanol for biodiesel production using immobilized lipase from Candida antarctica
(Novozyme 435). Maximum conversion was achieved using subFig. 2 Effect of enzyme concentrations for biodiesel production.
Error bar represents std deviation.

Fig. 3 Effect of acyl acceptor for biodiesel production.

Error bar represents std deviation.

Minimum conversion was observed in case of n-butanol (40.2 %)

(Fig. 3).
In order for the lipase to maintain its activity for repeated reactions, either the enzyme or the entire cell must be immobilized.
One major problem of lipase-catalyzed transesterification is that
glycerol competitively inhibits lipase activity by blocking the active
sites site (Nie et al. 2006). Since glycerol is a byproduct of the
reaction of triglycerides with methanol, new acyl acceptors that
produce other more useful and less detrimental byproducts are
under investigation. Methanol also has a negative impact on the
stability of the enzyme for repeated use because prolonged exposure causes denaturation of lipase. An acyl acceptor functions
as the new attachment of fatty acid chains, in place of glycerol.
The byproduct of this reaction is triacetylglycerol, which has no
negative effects on the reaction, can easily be separated from the
biodiesel product, and has a higher value than glycerol for later use
(Mallory, 2007).

Fig. 4 Effect of molar ratio of oil to methanol on biodiesel

production. Error bar represents std deviation.

Fig. 5 Effect of solvents on biodiesel production.

Error bar represents std deviation.

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strate molar ratio of 3.5:1 and enzyme concentration of 42.3%.

However, in the present investigation, lower concentration of enzyme (5%) was used for biodiesel production.
Effect of organic solvents on transesterification
The transesterification reactions were carried out using different
organic solvents to observe their effect on the FAME conversion.
A suitable solvent system can improve the miscibility of substrates,
which in turn enhance the conversion and rate of the reaction. It
was observed that polar solvents like acetonitrile and acetone did
not favour the reaction, whereas non polar solvents like hexane
(Chang et al. 2005). These values indicate the polarity of the solvents. The higher the value of log P, the lower is the polarity of the
solvent. It is quite obvious that polar solvents cannot solubilize the
non-polar substrates like vegetable oils and hence, result in lower
conversions. In recent years the focus was shifted from non-polar
solvents to a partially polar solvent like tbutanol, which has a log
P value lying between those of polar and non-polar solvents and
hence, it is widely used for transesterification reactions involving
different oils (Kumar et al. 2007; Kumar et al. 2009).
As visible from the results presented in Fig. 5, the maximum
FAME conversion (79.90%) was observed while using n-hexane
as a solvent. Whereas, minimum conversion was observed with
iso-amylalcohol (53.20%). However, n-propanol and n-butanol
showed the average FAME conversion.
Biodiesel Fuel characteristics
The physico-chemical properties of the karanja, based biodiesel
(B100) were evaluated as per the ASTM standards and the experimental procedures for evaluating physico-chemical properties
were followed. The fuel properties data of karanja methyl esters
with diesel are summarized in a tabular form as shown in Table
1. The karanja methyl esters have fuel property values relatively
closer to that of regular petroleum diesel. GC profile of reaction
mixture also presented in the Fig. 6.

Table 1. Fuel properties of biodiesel made from

karanjia oil
Biodiesel
(Enzymatic)

Biodiesel standard
(ASTM D 6751 08)

Density, kg/m3

0.8785

0.85 - 0.95

Kinematic viscosity
at 40 C cSt

8.972

25

Acid value mg
KOH/g

0.82

<0.8

Properties

Pour point C

0 0C

< 9 0C

Water content

<0.05

< 0.1 %

Conclusions

In the present investigation, biodiesel was synthesized using

karanja oil via transesterification reaction using P. aeruginosa lipase.
The optimal conditions determined were as follows: enzyme concentration 5 %, molar ratio 1:6, acyl accepter methanol and temperature 45 C. We report highest yield of biodiesel after transesterification (85.9 %) was achieved after 48 h incubation using
karanja oil. However, in the batch system, the conversion yield of
the biodiesel could not be increased above 85.9% might due to
activity loss of enzyme. Therefore, a stepwise reaction could be
conducted to increase the biodiesel production. This method is
a feasible method for an industrial biodiesel production. However, further research and development on some advanced methods
such as supercritical methanol process could be performed.
Acknowledgement
The authors gratefully acknowledge financial support from the
Ministry of New and Renewable Energy (MNRE) to Vinod
Kumar and from Council of Scientific and Industrial Research
(CSIR) to Firdaus Jahan. The authors would also like to thank
Technology Based Incubator, UDSC New Delhi for providing the
infrastructure support.

Fig. 7 Gas chromatography profile of (a) Fatty acid profile in karanja oil and (b) fatty acid methy esters synthesis during
transesterification reaction

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Conflict of interests
The authors declare that they have no direct financial relation with
any commercial identity above mentioned in this paper that might
lead to a conflict of interests. The authors also declare that they
have no competing interests.
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Article Information:

Received: 08 August 2014

Accepted: 14 September 2014
Published: 19 September 2014

Cite this article as:

Vinod et al. 2014. Efficient production methodology for biodiesel from

non-edible oil and its fuel characterization study. International Journal
of Extensiv Research. Vol. 1:36-40.

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