Biotechnol Lett (2013) 35:479–490

DOI 10.1007/s10529-012-1116-z

REVIEW

Biodiesel production by transesterification using

immobilized lipase
Sunil Kumar Narwal • Reena Gupta

Received: 18 September 2012 / Accepted: 28 November 2012 / Published online: 18 December 2012
Ó Springer Science+Business Media Dordrecht 2012

Abstract Biodiesel can be produced by transesteri- Introduction

fication of vegetable or waste oil catalysed by lipases.
Biodiesel is an alternative energy source to conven- Sustainability is a key principle in natural resource
tional fuel. It combines environmental friendliness management and it has become increasingly obvious
with biodegradability, low toxicity and renewability. that continued dependence on fossil fuel energy
Biodiesel transesterification reactions can be broadly resources are unsustainable in the long run because of
classified into two categories: chemical and enzy- depleting resources and the contribution of these fuels to
matic. The production of biodiesel using the enzy- environmental and health problems. According to the
matic route eliminates the reactions catalysed under World Energy Forum, fossil-based oil, coal and gas
acid or alkali conditions by yielding product of very reserves will be exhausted in\10 decades.
high purity. The modification of lipases can improve Biodiesel could be a potential alternative to traditional
their stability, activity and tolerance to alcohol. The fossil fuels (Wang et al. 2000). Biodiesel is a monoalkyl
cost of lipases and the relatively slower reaction rate ester of long-chain fatty acids produced by transesteri-
remain the major obstacles for enzymatic production fication reactions of animal fats or vegetable oils such as
of biodiesel. However, this problem can be solved by sunflower oil (Mittelbach 1990; Selmi and Thomas
immobilizing the enzyme on a suitable matrix or 1998), soybean oil (Myniyappa et al. 1996), or palm oil
support, which increases the chances of re-usability. (Basri et al. 1997; Carter et al. 2007). Transesterification
The main factors affecting biodiesel production are is the chemical reaction between triacylglycerols and
composition of fatty acids, catalyst, solvents, molar alcohol in presence of catalyst which results in the
ratio of alcohol and oil, temperature, water content, production of methyl esters along with glycerol. The
type of alcohol and reactor configuration. Optimiza- feedstock used for biodiesel production can be animal fat,
tion of these parameters is necessary to reduce the cost crude, refined or waste oil (Marchetti et al. 2008).
of biodiesel production. The production of biodiesel on a large scale using
vegetable oils promotes plantation of crops resulting in
Keywords Biodiesel
Immobilization
Lipase
recycling of CO2 and minimizing its impact on the
Transesterification
Vegetable oil greenhouse effect (Korbitz 1999; Agarwal and Das 2001).
Biodiesel has a similar engine performance and emits less
harmful emissions as compared to conventional fuel
S. K. Narwal
R. Gupta (&)
(Cardone et al. 2002). Conventional fuel contains more
Department of Biotechnology, Himachal Pradesh
University, Summer Hill, Shimla 171005, India sulphur and thus damages the atmosphere by releasing
e-mail: [email protected] SO2. However, biodiesel has almost zero sulphur content

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480 Biotechnol Lett (2013) 35:479–490

(Vicente et al. 1998; Fukuda et al. 2001). In addition, the reaction generally takes place at the oil–water inter-
biodiesel produced through transesterification using face (Kamini and Iefuji 2001). It helps in lipid
lipases is of high purity and enables easy separation from metabolism, such as fat digestion and absorption.
the by-product, glycerol (Iso et al. 2001; Kose et al. 2002; Lipases occur widely in microorganisms and in higher
Shimada et al. 2002; Ranganathan et al. 2008). The biggest eukaryotes. Microorganisms are the commonly used
advantage of biodiesel as compared to many other source for the production of industrially important
alternative transportation fuels is that it can be used in enzymes as these are one of the potent biocatalysts
diesel engines without any modification, and can be because of their multiple and novel multi-fold appli-
blended at any ratio with petroleum diesel. The animal fats cations in oleo-chemistry, organic synthesis, detergent
are low in free fats and water and also available in limited formulation and nutrition (Pandey et al. 1999).
amount as compared to vegetable oils, so they cannot meet Although lipases have been found in many species of
the fuel needs of the world (Zhu et al. 2008). Therefore, animals, plants, bacteria, yeast and fungi, the enzymes
vegetable oil is preferred for the production of biodiesel. from microorganisms are most interesting because of
The catalysts used for transesterification are acids their small generation time, high yield of product, simple
or bases (Highina et al. 2012) as well as free or cultivation conditions and their potential applications in
immobilized enzymes (Kaieda et al. 1999; Ma and various industries such as food, diary, pharmaceuticals
Hanna 1999; Komers et al. 2001; Suppes et al. 2004). (Rajendran et al. 2009), detergents, textile, bio-diesel
Although biodiesel has been produced using either (Jeong and Park 2008), cosmetic industry, synthesis of
acid or base chemical catalysts there are certain fine chemicals, agrochemical and new polymeric mate-
limitations which restrict the use of chemical catalysts, rials. The reason for the enormous biotechnological
such as glycerol recovery from biodiesel, removal of potential of microbial lipases is that they are stable in
inorganic salt, high temperature and undesirable side organic solvents, do not require cofactors, possess broad
reactions along with transesterification reaction. substrate specificity (Chandrasekeran and Bhartiya 2009)
Because of these advantages, lipase-catalysed biodie- and also exhibit a high enantio-selectivity (Holt and
sel production may become an alternative solution to Hanefeld 2009). The three-dimensional structure of
high gas prices (Vasudevan and Briggs 2008). lipase consists of three domains: ‘‘contact domain’’,
Biodiesel has been produced by lipase-catalysed responsible for distinguishing the surface of substrate,
transesterification (Shaw et al. 1991; Kaied et al. 2001; ‘‘hydrophobic domain’’, responsible for the extraction of
Pizarro and Park 2003; Du et al. 2004, 2006; Nie et al. one substrate molecule and its association with the
2006; Wang et al. 2000; Al-Zuhair et al. 2006; Akoh ‘‘functional’’ domain, and the last domain ‘‘functional
et al. 2007). Thus these are industrially very important domain’’, containing the catalytic triad which includes
enzymes especially in the formation of biodiesel. A Ser, Hys and Asp/Glu (Guncheva and Zhiryakova 2011).
large variety of lipases has been used for transesteri- Lipases cause nucleophilic attack on carbonyl carbon of
fication and esterification (Fjerbaek et al. 2009). A ester. Some lipases are also able to catalyze the processes
recent study showed that the presence of more than 1 % of esterification, interesterification, transesterification,
of water reduces the catalytic power of lipases (Cao acidolysis and aminolysis, and may show enantioselec-
et al. 2008). Lipases can be immobilized on a suitable tive properties (Hasan et al. 2009).
matrix or support. The immobilization of an enzyme is
localization or confinement of the enzyme onto a solid
support or on a carrier matrix. The immobilised lipase Modification of lipases
increases the chances of re-usability due to the solid
particles which can be removed easily from the mixture To improve lipase stability, activity and methanol
and can be reused in future (Xu et al. 2004). tolerance, especially for biodiesel production, the
enzyme can be treated with various reagents such as
substrates or their analogues, organic solvents, salts
Lipase: mode of action and classification and crown ethers (Lu et al. 2010). The most commonly
used method is the usage of substrates or their
Lipases are hydrolases (E.C.3.1.1.3) having both analogues as pre-treatment reagents. Organic solvents
hydrolytic and synthetic activity. The hydrolytic form a complex, i.e., enzyme–imprinter complex,

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which keeps the enzyme in its active conformation and Feed-stocks used for biodiesel production
therefore retains higher activity in the non aqueous
environment as compared to the nonimprinted enzyme Different types of feed-stocks can be used for the
(Rich et al. 2002). Lu et al. (2010) found that treating production of biodiesel. The choice of the source
the enzyme with 1 mM CaCl2 and MgCl2 improved depends largely on the country and its climate (Knothe
enzyme activity, methanol tolerance and operational 2005), and also on economics (Rodrigues et al. 2008).
stability. Methanolysis was achieved faster when Thus the feed-stock can be categorised into three
Candida antarctica was preincubated in methyl oleate groups.
for 0.5 h and subsequently in soybean oil for 12 h
(Samukawa et al. 2000). Virgin oil

It includes different vegetable oils. Biodiesel produc-

Lipase immobilization tion from soybean oil is very popular. Soybean oil has
a high iodine value compared to many other biodiesel
The cost of lipase production is one of the main feedstocks (indicating a relatively low-level of satu-
obstacles for industrial application of lipases. Immo- ration compared to other oils, such as rapeseed and
bilization of lipases decreases cost of production by canola). Different types of oils can be used for
their reusability, which is a necessary to make them biodiesel production such as rapeseed/canola, palm,
more attractive and potent for industrial applications corn, sunflower, cottonseed, peanut, and coconut oils
(Natarajan and Venkatesh 2012). The main purpose of (Knothe 2005; Moser 2009).
immobilization of enzymes is to improve their prop-
erties such as thermo stability, activity in non-aqueous Animal fats
medium, to improve handling, recovery and recycling
of biocatalyst. Recycling of immobilized enzymes The use of animal fats such as beef tallow for the
greatly reduces their cost of production; therefore the production of biodiesel plays an important role in
more effective immobilization matrices and methods pollution control (Krause 2008). Chicken fat obtained
could make the enzymatic biodiesel production better from processing of by-products is a promising alter-
than the chemical processes. The lipase can be native feedstock for biodiesel production because of
immobilized by various methods such as adsorption its easy availability and low cost (Freitas et al. 2009).
(Padmini et al. 1993), ionic and covalent binding Waste fats and vegetable oils are of low-cost such as
(Zaidi et al. 1995), cross-linking (Lima et al. 1995), restaurant greases thus reduces cost of production (Lee
entrapment, encapsulation (Prazer et al. 1993), and et al. 2002).
hydrophobic interactions (Winayanuwattikun et al.
2011). Adsorption is one of the most widely used Aquatic plants
methods for the immobilization of lipases the proce-
dure is simple and easy, conditions are mild and the These include algae and microalgae, that can be grown
method is cheap (Nie et al. 2006; Yagiz et al. 2007). using waste materials such as sewage and without
The selection of matrix depends on several factors displacing land currently used for food production.
such as thermal stability, chemical durability, mechan- Chlorella vulgaris can produce 39 % (w/w) of
ical strength, lipase type and type of reaction system, biomass rich in oleic acid (58 % w/w of total) (Weber
ease of regeneration, loading capacity and its cost and Mukherjee 2008).
(Jegannathan and Abang 2008). Pahujani et al. (2007)
found that the lipase enzyme from Bacillus coagulans
immobilized on nylon-6 had good reusability in Different methods of biodiesel production
performing continuous transesterification (Pahujani
et al. 2007). Also, the higher lipase activity was Vegetable oils cannot be used in diesel engines
obtained using nonpolar resins (Yang et al. 2006). directly because of their high viscosity, which is
There are a number of matrices that can be used for incompatible with engines. Thus to reduce their
immobilization as given in Table 1. viscosity it is essential to break the triacylglycerols

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Table 1 Different techniques of immobilization of lipases used in biodiesel synthesis

Immobilization Matrix Source of lipases Oil Alcohol Yield Reference
method (%)

Adsorption Celite Burkholderia Jatorpha oil Ethanol 98 Shah and Gupta

cepacia (2007)
Adsorption Hydrotalcite and zeolites Thermomyces Waste oil Methanol 92.80 Yagiz et al.
lanuginosus (2007)
Adsorption Macroporous polypropylene Burkholderia Soybean oil Methanol 98 Salis et al. (2008)
fluorescens
Burkholderia
cepacia
Adsorption Cotton membrane Candida sp. Waste oil Methanol 92 Nie et al. (2006)
Entrapment Hydrophobic sol–gel support Burkholderia Soybean oil Methanol 65 Noureddini et al.
cepacia Ethanol (2005)
Entrapment Phyllosilicate sol–gel matrix Burkholderia Restaurant Methanol 87–95 Hsu et al. (2000)
cepacia grease Ethanol
Encapsulation Silica aerogels Burkholderia Sunflower Methanol – Orcaire et al.
cepacia oil (2006)
Candida
antarctica
Encapsulation Sepabeads Candida rugosa Palm oil Methanol – Prazer et al.
(1993)
Covalent bond Acrylic resin Thermomyces Soybean oil Ethanol 75–100 Rodrigues et al.
lanuginosus (2010)
Covalent bond Amino functionalized magnetic Thermomyces Soybean oil Methanol 94 Xie and Ma
nanoparticles lanuginosus (2009)
Covalent bond Silica–PVA composite Burkholderia Palm oil Ethanol 98 Moreira et al.
fluorescens (2007)

bonds of fats which results in synthesis of biodiesel. It diacylglycerols and then diacylglycerols are converted
can be done using four different methods (Ghadge and to monoacylglycerols followed by the conversion of
Raheman 2006). monoacylglycerols to glycerol. The transesterification
of triacylglycerols with methanol produces methyl
(1) Transesterification.
esters along with glycerol. This is a balanced and
(2) Blending.
catalyzed reaction as illustrated in Fig. 1 (Sree et al.
(3) Microemulsions.
2009).
(4) Pyrolysis.
Commonly used alcohols for transesterification
Out of all these methods, tranesterification is one of reactions are short chain alcohols, mainly methanol
the most potent and important method of biodiesel or ethanol (Li and Zongba 2007). In all three steps an
preparation. ester is produced and thus from 1 mol triacylglycerols
3 mol of ester are produced (Sharma and Singh 2008).
Transesterification The chemically-catalysed transesterification using an
acid or alkali-catalyst gives high conversion of tria-
In biodiesel production the transesterification reaction cylglycerols to their corresponding methyl esters in a
occurs among oil (triacylglycerols) which may be short reaction time but the reaction has several
vegetable oil, animal fat or grease and alcohol in the limitations. It is energy intensive using high temper-
presence of a catalyst, either chemical (acid or base) or atures, the acidic or alkaline catalyst has to be removed
enzymatic. It is a sequence of three reversible from the product, the alkaline waste-water requires
reactions in which triacylglycerols are converted to treatment as does separation of glycerol from biodiesel

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Fig. 1 Transesterification CH2 O COR Lipase CH2 OH

reaction between
triacylglycerols and alcohol, CH O COR + 3CH3OH CH OH + 3R COOCH3
yielding biodiesel and
glycerol CH2 O COR CH2 OH

Triacylglycerols Methanol Glycerol Methyl esters

(Biodiesel)

Fig. 2 General process of UPPER BIODIESEL

biodiesel production OIL
PHASE
(Ranganathan et al. 2008)

METHANOL TRANSESTERIFICATION SEPARATION

LOWER GLYCEROL
ENZYME PHASE

and there are undesirable side-reactions. Thus to these can lead to soap formation along with unwanted
overcome this, lipase-catalysed transesterification has side reactions (Ma and Hanna 1999; Shah and Gupta
been developed. The general transesterification pro- 2007). During winter time, oils containing higher
cess used in biodiesel production is shown in Fig. 2 amount of saturated fatty acids can block the fuel lines
(Ranganathan et al. 2008). Biodiesel production leads due to solidification (Pinto et al. 2005; Akoh et al.
to the formation of low-cost by products like glycerol 2007; Demirbas 2008). Biodiesel produced from the
(Yazdani and Gonzalez 2007), which can be used as oils containing a higher amount of oleic acid has
substrate for the synthesis of monoacylglycerols in characteristics that are the most similar to conventional
food industry as suggested by Freitas et al. (2009). biodiesel (Knothe 2005; Robles et al. 2009). Freedman
Lipases from various microorganisms like C. antarc- et al. (1984) compared both crude and refined vege-
tica, Pseudomonas cepacia etc., are suitable for table oils as feed stocks and found that the yield of
biodiesel formation as given in Table 2. methyl esters was increased from 93 to 98 % for the
refined oil and from 67 to 86 % for the crude oil. When
the content of free fatty acid (FFA) was increased from
Factors affecting biodiesel production and its 50 to 85 % in the oil, the yield of biodiesel also
quality increased from 54 to 88 % thus indicating that high
FFA content was beneficial for biodiesel production
Composition of fatty acid (Saifuddin et al. 2009). Gupta et al. (2011) found that
karanja oil was the best source of raw material for bio
The property of biodiesel is greatly affected by the fuel generation in India because it is cheap, non edible
composition of fatty acids and the type of alcohol used and abundant in India (Gupta et al. 2011).
in transesterification reaction. The greater the straight-
chain length of fatty acid the better will be the Catalysts
properties of biodiesel such as heat capacity (Knothe
2010) and cetane number (Dunn 2009). The oil should A catalyst is a substance that increases the rate of
have very low water and free fatty acid content because chemical reaction without being consumed in the

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Table 2 Biodiesel production using lipases extracted from microbes

Source of lipases Oil Alcohol Solvents Time of Yield Reference
incubation (%)
(h)

Candida antarctica Sunflower Methanol tert-Butyl 50 97 Ognjanovic et al.

oil alcohol (2009)
Thermomyces lanuginosa Soybean Ethanol Hexane or 10 70–100 Rodrigues et al.
oil solvent- (2010)
free
Candida rugosa, Pseudomonas Jatropha Ethanol Solvent-free 8 98 Shah and Gupta
fluorescens seed oil (2007)
Candida antarctica Cotton Methanol tert-Butyl 24 97 Royon et al. (2008)
seed oil alcohol
Rhizomucor miehei Soybean Methanol Solvent-free 12 68–95 Guan et al. (2010)
Penicillium cyclopium oil
Candida antarctica Soybean Methyl Solvent-free 14 92 Du et al. (2004)
oil acetate
Candida antarctica Jatropha Methanol Hexane 8 52 Haldar and Nag
oil 27 (2008)
Karanja
oil
Candida antarctica Rapeseed Methanol Solvent-free 24 91 Watanabe et al.
oil (2007)
Penicillium expansum Waste oil Methanol tert-Amyl 24 93 Li et al. (2009)
alcohol
Bacillus subtilis Waste Methanol Solvent-free 72 [90 Ying and Chen
cooking (2007)
oil
Enterobacter aerogenes Jatropha Methanol tert-Butyl 48 94 Kumari et al. (2009)
seed oil alcohol
Burkholderia cepacia Soybean Methanol Solvent-free 90 [80 Du et al. (2004)
oil
Cryptococcus SP-2 Rice bran Methanol Solvent-free 120 80 Kamini and Iefuji
oil (2001)
Rhizomucor miehei, Thermomyces Sunflower Methanol Hexane 24 [80 Soumanou and
lanuginosa, Pseudomonas fluorescens oil Bornscheuer
Sunflower Methanol Solvent-free 24 [90 (2003)
oil
Thermomyces lanuginosa, Candida Rapeseed Methanol tert-Butyl 12 95 Li et al. (2006)
antarctica oil alcohol

reaction. The transesterification reaction can be cata- disadvantage associated with acid is the inhibition
lysed in the presence of chemical (acid or base) or of transesterification reaction due to the formation
enzymatic catalysts. of water along with methyl ester (Canakci and
Van 2001). Also, the yield of biodiesel reduces
Acid-catalysed transesterification due to high temperature and acid concentration
which can damage the oil. Acid is also corrosive
Acid catalysed transesterification reactions have and can thus damage the equipment (Freedman
low rate of reaction (Al-Zuhair 2007). The main et al. 1984).

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Base-catalysed transesterification – It requires lesser amount of alcohol and does not

result in side reactions.
In base-catalysed transesterifications, the raw material
The catalysis of transesterification among methanol
is pre-treated with acid catalyst when it contains large
and palm oil revealed that immobilized lipase from
amount of water and free fatty acids (Freedman et al.
C. rugosa on Sepabeads EC-OD was most promising
1984; Kaied et al. 2001; Zhang et al. 2003). The main
for further development as a biocatalyst for the
aim of pre-treatment is to reduce soap formation and
application of enzyme-catalyzed biodiesel synthesis
also to separate biodiesel and glycerol from the
(Winayanuwattikun et al. 2011).
catalyst and alkaline water (Mittelbach et al. 1983).
Base as a catalyst is very sensitive to oil purity
Solvents
(Freedman et al. 1986). NaOH is the most widely used
alkaline catalyst because it is economical and easy to
The enzymatic transesterification has been performed
prepare (Meka et al. 2007). Besides this, both acid as
in solvent and solvent-free medium by various lipases
well as alkali-catalysed transesterification reactions
immobilized on different matrices (Shah and Gupta
result in production of large amount of waste-water
2007; Royon et al. 2008). The use of organic solvents
which is of environmental concern.
is not suitable for the enzymatic production of
biodiesel due to deleterious effects like toxicity,
Lipase-catalysed transesterification
flammability of solvent, damaging effects on the
environment and consequential requirement for its
The enzymatic catalyst, i.e., lipase, has been used for
removal. Therefore, to make the enzymatic process
the transesterification of vegetable oils due to its high
more efficient, enzymatic solvent-free systems have
efficiency and selectivity, ease of glycerol removal,
been developed (Soumanou and Bornscheuer 2003).
biodiesel purification, toleration of water into the oil,
An organic hydrophilic solvent such as tert-butyl
low energy consumption (reaction requires mild
alcohol has been developed as a novel reaction
conditions) and low waste (Akoh et al. 2007). The
medium for lipase-catalysed transesterification for
main problem associated with enzymatic transesteri-
biodiesel production in which both methanol and
fication is the cost of the enzyme preparation. How-
glycerol are soluble (Li et al. 2006).
ever this can be solved by the process of immobilization
which generally increases the reusability of the enzyme
Type of alcohol
(Roy et al. 2003), therefore the immobilization of
lipases reduces the cost and helps the enzymes to work
The yield of transesterification or esterification reac-
in non-aqueous medium (Shah and Gupta 2007).
tions are better using ethanol as compared to methanol
Enzymatic processes require lower amounts of energy
(Mittelbach 1990; Nelson et al. 1996; Abigor et al.
with fewer steps and a decreased volume of waste-water
2000; Hsu et al. 2000). This can be explained on the
(Fjerbaek et al. 2009). The lipase-catalysed transeste-
basis that the lipases are more tolerant to ethanol as
rification reaction is better than a chemically-catalysed
compared to methanol (Fukuda et al. 2001), or lipases
reaction due to various advantages as given in Table 3
act better on long-chain fatty alcohols than on short-
(Parawira 2010). Thus biodiesel produced by transeste-
chain alcohols (Shimada et al. 1997, 1998). Since
rification reactions using lipase offers following advan-
ethanol can be produced from renewable resources
tages (Haas et al. 2002):
and is less hazardous than methanol, therefore it could
– It requires little or no heating, thus decreases the be another reason for using ethanol instead of meth-
cost of production. anol in transesterification reactions. From the eco-
– No soap production. nomic point of view methanol is most commonly used
– Lipase-catalysed transesterification reactions can in transesterification reactions for the commercial
occur even in the presence of water, whereas the production of biodiesel; however other alcohols such
chemically-catalysed reactions lead to soap and as ethanol, propanol, and butanol can also be used as
froth formation, and is thus difficult to separate. alkyl donors (Moser 2009).

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Table 3 Comparison between chemically and Lipase-catalysed transesterification reactions to produce biodiesel
Variable Alkali catalysed Acid catalysed Lipase-catalysed
transesterification transesterification transesterification

Reaction temperature (°C) 60–70 55–80 30–40

Free fatty acid content in oil Saponified products Esters Methyl esters
Water in transesterification reaction Interferes with reaction Interferes with reaction Less interference
Methyl esters yield Normal Normal Higher
Glycerol recovery Difficult Difficult Easy
Separation of methyl esters By repeated washing By repeated washing No washing
Cost of catalyst Cheap Cheap Relatively expensive

Molar ratio of alcohol and oil alkaline conditions (Rashid and Anwar 2008) and also
deactivates the lipase (Leung and Guo 2006; Eevera
The molar ratio of alcohol and oil plays a vital role in et al. 2009). When the reaction temperature was
biodiesel yield (Freedman et al. 1986; Ma and Hanna controlled at 30–40 °C, the yield of biodiesel was
1999; Canakci and Van 2001; Zhang et al. 2003). A increased from 30 to 56 % with increasing temperature
high molar conversion (80 %) can be obtained using (Pinyaphong et al. 2011). The phenomenon could be
immobilized lipase operated over 30 days without explained on the ground that the viscosity of the reaction
losing catalytic activity (Chen et al. 2011). Higher might be reduced with increasing temperature. When
mass ratio of reactants increases the contact between the temperature was increased from 40 to 60 °C, the
the methanol and oil molecules. So the methyl ester biodiesel yield decreased, however, the content of free
concentration increases with increasing mass ratio of fatty acids increased. This was due to loss in enzymatic
methanol to oil. The yield of biodiesel increases with activity at higher temperature.
increase in the concentration of alcohol up to a certain
concentration. However, further increase of alcohol Water content
content does not increase the yield of biodiesel, but it
also increases the cost of alcohol recovery (Soumanou Water plays a very important role in biodiesel produc-
and Bornscheuer 2003; Leung and Guo 2006). tion. Kaieda et al. (1999) studied the effect of water
Soumanou and Bornscheuer (2003) also investigated content on the production of biodiesel from soybean oil
the influence of methanol on the yield of biodiesel, using lipases obtained from Rhizopus oryzae, C. rugosa,
with the same lipase, and obtained best results with 3:1 Pseudomonas fluorescens, C. antarctica and Burk-
(mol/mol) methanol/oil molar ratio (Knothe 2010). holderia cepacia. In all these cases, the enzyme activity
was low without water, which supports the fact that a
Temperature minimum amount of water is required for the enzyme to
work (Kaieda et al. 1999). On increasing the water
The activity of lipase-catalysed transesterification reac- content in the tranesterification reaction, there was a
tions increases with an increase in temperature, and the considerable increase in biodiesel production, showing
maximum conversion was obtained at 55 °C. Royon the enhancement in the enzyme activity. However
et al. (2007) also carried out the biodiesel production enzyme activity gradually decreased when the water
from cotton seed oil in tert-butyl alcohol at 50 °C. content increased. Similar results were observed in the
Leung and Guo (2006), and Eevera et al. (2009) found production of biodiesel using P. fluorescens lipase,
that the rate of transesterification increased up to the immobilized on Celite. Biodiesel yield was highest
optimal level. However, further increase in the reaction when initial water activity of enzyme was reduced to
temperature beyond the optimal level led to decrease in 0.23; however biodiesel yield decreased with increase in
biodiesel yield because higher reaction temperature initial water activity of enzyme more than 0.23. This
accelerates the saponification of triacylglycerols in was because of the minimal amount of water that

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Biotechnol Lett (2013) 35:479–490 487

associated with enzyme was necessary for the mainte- Conclusion

nance of enzyme’s catalytically active conformation in
methanol (Pinyaphong et al. 2011). Biodiesel is an important new alternative transporta-
tion fuel. It is a form of diesel fuel produced from
Reactor configuration transesterification of vegetable oils, animal fats, or
restaurant greases with alcohol. It is a safe, non toxic,
On a laboratory-scale the most commonly used biodegradable, renewable source and produces less
systems are stirred, batch reactors. The advantage of air pollutants than petroleum-based diesel. Biodiesel
this reactor system is easy handling and simple fuels are attracting increasing attention worldwide as a
configuration of the reactor. There are various direct replacement for diesel in engines and are also
studies on enzymatic transesterification in different for their eco-friendly behaviour. Biodiesel is made
reactor configurations with the emphasis on deter- through a process called transesterification using
mining the best operational parameters, but there is lipase, which is better than chemical catalysts because
not sufficient information on scale up systems (Du of the high efficiency and selectivity, ease of glycerol
et al. 2007; Talukder et al. 2009). However, packed- removal, biodiesel purification, toleration of water
bed reactors are often used for large-scale produc- into oil, low energy consumption (reaction requires
tion because of their high-efficiency and reduced mild conditions) and low waste amounts. Production
shear denaturation of the lipase. They also allow the of biodiesel from waste and non edible vegetable oil
reuse of lipase without separation in comparison to greatly reduces the cost of biodiesel production and
stirred batch reactor. Severac et al. (2011) tested the thus avoids the conflict between food and energy
maximum production of biodiesel using packed bed security and is considered an important step in
reactor. reducing pollution and recycling waste oil. Methanol
is the most commonly used alcohol in biodiesel
Cost analysis for biodiesel production production due to its low cost; however, it has more
hazardous effect on lipase activity than ethanol. One
The primary determinants of profitability are (1) the main problem in production of biodiesel using lipases
cost of the feedstock, (2) the value of and access to is their high cost of production. Therefore, immobi-
markets for biodiesel, (3) the value of and access to lization of lipases, which allows their reusability, is
markets for the co-products (glycerol and oilseed necessary to make them more attractive for industrial
meal), (4) government support policies, and (5) utility biodiesel processes.
costs. The major economic factor to consider for
input costs of biodiesel production is the feedstock,
which is about 80 % of the total operating cost. The References
cost of biodiesel fuel varies depending on the base
Abigor RD, Uadia PO, Foglia TA, Haas MJ, Jones KC, Okpefa
stock, geographic area, variability in crop production E, Obibuzor JU, Bafor ME (2000) Lipase-catalyzed pro-
from season to season, the price of crude petroleum duction of biodiesel fuel from some Nigerian lauric oils.
and other factors. With cooking oils used as raw Biochem Soc Trans 28:979–981
material, the viability of a continuous transesterifica- Agarwal AK, Das LM (2001) Biodiesel development and
characterization for use as a fuel in compression ignition
tion process and recovery of high quality glycerol as engines. J Eng Gas Turbines Power 123:440–447
a biodiesel by product are primary options to be Akoh CC, Chang S, Lee G, Shaw J (2007) Enzymatic approach
considered to lower the cost of biodiesel. The to biodiesel production. J Agric Food Chem 55:8995–9005
competitiveness of biodiesel compared to petrodiesel Al-Zuhair S (2007) Production of biodiesel: possibilities and
challenges. Biofuels Bioprod Biorefin 1:57–66
depends largely on fuel taxation rates and policies Al-Zuhair S, Jayaraman KV, Krishnan S, Chan WH (2006) The
and, generally, the production costs of biodiesel effect of fatty acid concentration and water content on the
remain much higher than those of petrodiesel. Thus, production of biodiesel by lipase. Biochem Eng J
the challenge is to reduce the costs associated with 30:212–217
Basri M, Heng AC, Razak CNA, Wan-Zunus WMZ, Ahmad M,
production of biodiesel and approaches described in Rahman RNA, Ampon K, Salleh AB (1997) Alcoholysis of
this review aim to contribute to lowering costs of the palm oil mid-fraction by lipase from Rhizopus rhizopodi-
process. formis. J Am Oil Chem Soc 74:113–116

123
488 Biotechnol Lett (2013) 35:479–490

Canakci M, Van GJ (2001) Biodiesel production from oil and Guncheva M, Zhiryakova D (2011) Catalytic properties and
fats with high free fatty acids. Trans ASAE 44:1429–1436 potential applications of Bacillus lipases. J Mol Catal B
Cao F, Chen Y, Zhai F, Li J, Wang J, Wang X, Wang S, Zhu W Enzym 68:1–21
(2008) Biodiesel production from high acid value waste Gupta P, Upadhyay LSB, Shrivastava R (2011) Lipase cata-
frying oil catalyzed by superacid heteropolyacid. Bio- lyzed-transesterification of vegetable oils by lipolytic
technol Bioeng 10:93–100 bacteria. Res J Microbiol 6:281–288
Cardone M, Prati M, Rocco V, Seggiani M, Senatore A, Vitolo S Haas MJ, Piazza GJ, Foglia TA (2002) Lipid biotechnol. In: Kuo
(2002) Brassica carinata as an alternative oil crop for the TM, Gardner HW (eds) Enzymatic approaches to the pro-
production of biodiesel in Italy: engine performance and duction of biodiesel fuels. Marcel Dekker, Inc., New York,
regulated and unregulated exhaust emissions. Environ Sci pp 587–598
Technol 36:46–56 Haldar SK, Nag A (2008) Utilization of three non edible vege-
Carter C, Finley W, Fry J, Jackson D, Willis L (2007) Palm oil table oils for the production of biodiesel catalysed by
market and future supply. Eur J Lipid Sci Technol 109: enzyme. Open Chem Eng J 2:79–83
307–314 Hasan F, Shah A, Hameed A (2009) Methods for detection and
Chandrasekeran SM, Bhartiya S (2009) Substrate specificity of characterization of lipases: a comprehensive review. Bio-
lipase in alkoxycarbonylation reaction: QSAR model devel- technol Adv 27:782–798
opment and experimental validation. Biotechnol Bioeng Highina BK, Bugage IM, Umar B (2012) Biodiesel production
30:527–534 using Jatropha caucus oil in a batch reactor using zinc oxide
Chen HC, Ju HY, Wu TT, Liu YC, Lee CC, Chang C, Chung as catalyst. J Appl Hytotechnol Environ Sanit 2:61–66
YL, Shieh CJ (2011) Continuous production of lipase- Holt J, Hanefeld U (2009) Enantioselective enzyme-catalyzed
catalyzed biodiesel in a packed-bed reactor: optimization synthesis of cyanohydrins. Curr Org Synth 6:1005–1018
and enzyme reuse study. J Biomed Biotechnol 2011:1–6 Hsu AF, Jones K, Foglia TA, Marmer WN (2000) Immobilized
Demirbas A (2008) Relationships derived from physical prop- lipase-catalyzed production of alkyl esters of restaurant
erties of vegetable oil and biodiesel fuels. Fuel 87:1743 grease as biodiesel. Biotechnol Appl Biochem 36:181–196
–1748 Iso M, Chen B, Eguchi M, Kudo T, Shrestha S (2001) Produc-
Du W, Xu Y, Liu D, Zeng J (2004) Comparative study on lipase- tion of biodiesel fuel from triacylglycerols and alcohol
catalyzed transformation of soybean oil for biodiesel pro- using immobilized lipase. J Mol Catal B Enzym 16:53–58
duction with different acyl acceptors. J Mol Catal B Enzym Jegannathan KR, Abang S (2008) Production of biodiesel using
30:125–129 immobilized lipase—a critical review. Crit Rev Biotechnol
Du D, Sato M, Mori M, Park EY (2006) Repeated production of 28:253–264
fatty acid methyl ester with activated bleaching earth in Jeong GT, Park DH (2008) Lipase catalyzed transesterification
solvent free system. Process Biochem 41:1849–1853 of rapeseed oil for bio diesel production with tert-butanol.
Du W, Liu DH, Li LL, Dai LM (2007) Mechanism exploration Appl Biochem Biotechnol 148:131–139
during lipase-mediated methanolysis of renewable oils for Kaied M, Samukawa T, Kondo A, Fukuda H (2001) Effect of
biodiesel production in a tert-butanol system. Biotechnol methanol and water contents on production of biodiesel
Prog 23:1087–1090 fuel from plant oil catalyzed by various lipases in a solvent-
Dunn RO (2009) Effects of minor constituents on cold flow free system. J Biosci Bioeng 91:2–15
properties and performance of biodiesel. Prog Energy Kaieda M, Samukawa T, Matsumoto T, Ban K, Kondo A,
Combust Sci 35:481–489 Shimada Y, Noda H, Nomotos F, Ohtsuka K, Izumoto E,
Eevera T, Rajendran K, Saradha S (2009) Biodiesel production Fukuda H (1999) Biodiesel fuel production from plant oil
process optimization and characterization to assess the catalyzed by Rhizopus oryzae lipase in a water-containing
suitability of the product for varied environmental condi- system without an organic solvent. J Biosci Bioeng 88:
tions. Renew Energy 34:762–765 627–631
Fjerbaek L, Christensen KV, Norddahl B (2009) A review of the Kamini NR, Iefuji H (2001) Lipase catalyzed methanolysis of
current state of biodiesel production using enzymatic vegetable oils in aqueous medium by Cryptococcus spp.
transesterification. Biotechnol Bioeng 102:1298–1395 S-2. Process Biochem 37:405–410
Freedman B, Pryde EH, Mounts TL (1984) Variables affecting Knothe G (2005) Dependence of biodiesel fuel properties on the
the yields of fatty esters from transesterified vegetable oils. structure of fatty acid alkyl esters. Fuel Process Technol
J Am Oil Chem Soc 61:1638–1643 86:1059–1070
Freedman B, Butterfield RO, Pryde EH (1986) Transesterification Knothe G (2008) Designer biodiesel: optimizing fatty ester
kinetics of soybean oil. J Am Oil Chem Soc 63:1375–1380 composition to improve fuel properties. Energy Fuels 22:
Freitas L, Da Rós PCM, Santos JC, Castro HF (2009) An inte- 1358–1364
grated approach to produce biodiesel and monoacylglyce- Knothe G (2010) Biodiesel and renewable diesel: a comparison.
rols by enzymatic interestification of babassu oil (Orbinya Prog Energy Combust Sci 36:364–373
sp.). Process Biochem 44:1068–1074 Komers K, Stloukal R, Machek J, Skopal F (2001) Biodiesel
Fukuda H, Kondo A, Noda H (2001) Biodiesel fuel production from rapeseed oil, methanol and KOH 3: analysis of
by transesterification of oils. J Biosci Bioeng 92:405–416 composition of actual reaction mixture. Eur J Lipid Sci
Guan F, Peng P, Wang G, Yin T, Peng Q, Huang J, Guan G, Li Y Technol 103:363–371
(2010) Combination of two lipases more efficiently cata- Korbitz W (1999) Biodiesel production in Europe and North
lyzes methanolysis of soybean oil for biodiesel production America, an encouraging prospect. Renew Energy 16:
in aqueous medium. Process Biochem 45:1667–1682 1078–1083

123
Biotechnol Lett (2013) 35:479–490 489

Kose O, Tuter M, Aksoy AH (2002) Immobilized Candida Noureddini H, Gao X, Philkana RS (2005) Immobilized Pseu-
antarctica lipase-catalyzed alcoholysis of cotton seed oil in domonas cepacia lipase for biodiesel fuel production from
a solvent-free medium. Bioresour Technol 83:125–129 soybean oil. Bioresour Technol 96:769–777
Krause LC (2008) Desenvolvimento do processo de produção de Ognjanovic ND, Saponjic SV, Bezbradica DI, Knezevic DI
biodiesel de origem animal. Dissertation, Federal Univer- (2009) Lipase-catalysed biodiesel synthesis with different
sity of Rio, Brazil acyl acceptors. Bioresour Technol 100:710–716
Kumari A, Mahapatra P, Garlapati VK, Banerjee R (2009) Orcaire O, Buisson P, Pierre A (2006) Application of silica
Enzymatic transesterification of Jatropha oil. Biotechnol aerogel encapsulated lipases in the synthesis of biodiesel
Biofuels 2:1–7 by transesterification reaction. J Mol Catal B Enzym 42:
Leung DYC, Guo Y (2006) Transesterification of neat and used 106–113
frying oil: optimization for biodiesel production. Fuel Padmini P, Raskhit SK, Baradarajan A (1993) Studies on
Process Technol 87:883–890 immobilization of lipase on alumina for hydrolysis of rice
Li B, Zongba Z (2007) Biodiesel production by direct methan- bran oil. Bioprocess Eng 9:43–46
olysis of oleaginous microbial biomass. J Chem Technol Pahujani S, Shukla SK, Bag BP, Kanwar SS, Gupta R (2007)
Biotechnol 82:775–780 Application of lipase immobilized on nylon-6 for the
Li LL, Du W, Liu DH, Wang L, Li ZB (2006) Lipase-catalyzed synthesis of butyl acetate by transesterification reaction in
transesterification of rapeseed oils for biodiesel production n-heptane. J App Polym Sci 106:2724–2729
with a novel organic solvent as the reaction medium. J Mol Pandey A, Benjamin S, Soccol CR, Nigam P, Krieger N, Soccol
Catal 43:58–62 VT (1999) The realm of microbial lipases in biotechnol-
Li N, Zong M, Wu H (2009) Highly efficient transformation of ogy. Biotechnol Appl Biochem 29:119–131
waste oil to biodiesel by immobilized lipase from Peni- Parawira W (2010) Biodiesel production from Jatropha curcas:
cillium expansum. Process Biochem 44:685–688 a review. Sci Res Essays 5:1796–1808
Lima V, Pyle F, Asenjo JA (1995) Factors affecting the esteri- Pinto AC, Lilian LN, Guarieiroa LLN, Rezendea MJC, Ribeiroa
fication of lauric acid using an immobilized biocatalyst: NM (2005) Biodiesel: an overview. J Braz Chem Soc
enzyme characterization and studies in a well mixed 16:1313–1330
reactor. Biotechnol Bioeng 46:69–79 Pinyaphong P, Sribuir P, Phutrakul S (2011) Biodiesel fuel
Lu J, Deng I, Zhao R, Zang R, Wang F, Tan T (2010) Pre- production by methanolysis of fish oil derived from the
treatment of immobilized Candida sp. 99–125 lipases to discarded part of fish catalysed by Carica papaya lipase.
improve its methanol tolerance for biodiesel production. World Acad Sci Eng Technol 76:466–472
J Mol Catal B Enzym 62:15–18 Pizarro AVL, Park EY (2003) Lipase-catalyzed production of
Ma F, Hanna MA (1999) Biodiesel production: a review. Bi- biodiesel fuel from vegetable oils contained in waste
oresour Technol 70:1–15 activated bleaching earth. Process Biochem 38:1077–1082
Marchetti JM, Miguel VU, Errazu AF (2008) Techno-economic Prazer DMF, Garcia FAP, Cabral JMS (1993) An ultrafiltration
study of different alternatives for biodiesel production. membrane bioreactor for the lipolysis of olive oil in
Fuel Process Technol 89:740–748 reversed miceller media. Biotechnol Bioeng 41:761–770
Meka PK, Tripathi V, Singh RP (2007) Synthesis of biodiesel Rajendran A, Palanisamy A, Thangavelu V (2009) Lipase cat-
fuel from safflower oil using various reaction parameters. alyzed ester synthesis for food processing industries. Braz J
J Oleo Sci 56:9–12 Biotechnol 52:207–219
Mittelbach M (1990) Lipase catalyzed alcoholysis of sunflower Ranganathan SV, Narasimhan SL, Muthukumar K (2008) An
oil. J Assoc Off Anal Chem 67:168–170 overview of enzymatic production of biodiesel. Bioresour
Mittelbach M, Worgetter M, Pernkopf J, Junek H (1983) Diesel Technol 99:3975–3981
fuel derived from vegetable oils: preparation and use of Rashid U, Anwar F (2008) Production of biodiesel through
rape oil methyl ester. Energy Agric 2:369–384 optimized alkaline catalyzed transesterification of rapeseed
Moreira ABR, Perez VH, Zanin GM, Castro HF (2007) Biodiesel oil. Fuel 87:265–273
synthesis by enzymatic transesterification of palm oil with Robles MA, Gonzalez PA, Esteban CL, Molina GE (2009)
ethanol using lipases from several sources immobilized on Biocatalysis: towards ever greener biodiesel production.
silica–PVA composite. Energy Fuel 21:3689–3694 Biotechnology 27:398–408
Moser BR (2009) Biodiesel production, properties and feed- Rodrigues RC, Volpato G, Wada K, Ayub MAZ (2008) Enzy-
stocks. In Vitro Cell Dev Biol Plant 45:229–266 matic synthesis of biodiesel from transesterification reac-
Myniyappa PR, Brammer SC, Noureddini H (1996) Improved tions of vegetable oils and short chain alcohols. J Am Oil
conversion of plant oils and animal fats into biodiesel and Chem Soc 85:925–930
co-product. Bioresour Technol 56:19–24 Rodrigues RC, Pessela BCC, Volpato G, Fernandez LR, Guisan
Natarajan E, Venkatesh KP (2012) Production and character- JM, Ayub MAZ (2010) Two step ethanolysis: a simple and
ization of biodiesel using Pongamia oil by immobilized efficient way to improve the enzymatic biodiesel synthesis
Rhizopus oryzae. Adv Biotech 11:32–34 catalyzed by an immobilized-stabilized lipase from Ther-
Nelson LA, Foglia TA, Marmer WN (1996) Lipase-catalyzed momyces lanuginosus. Process Biochem 45:1268–1273
production of biodiesel. J Am Oil Chem Soc 73:1191–1195 Roy I, Gupta A, Khare SK, Bisaria VS, Gupta MN (2003) Immo-
Nie K, Xie F, Wong F, Tan T (2006) Lipase catalyzed meth- bilization of xylan degrading enzymes from Melanocarpus
anolysis to produce biodiesel: optimization of the biodiesel albomyces IIS 68 on the smart polymer eudragit L-100. Appl
production. J Mol Catal B Enzym 43:142–147 Microbiol Biotechnol 61:309–313

123
490 Biotechnol Lett (2013) 35:479–490

Royon D, Daz M, Ellenrieder G, Locatelli S (2008) Enzymatic unrefined oil: comparison of the methanolysis methods.
production of biodiesel from cotton seed oil using t-butanol J Mol Catal B Enzym 60:106–112
as a solvent. APEFE 39:161–169 Vasudevan PT, Briggs M (2008) Biodiesel production-current
Saifuddin N, Raziah AZ, Farah HN (2009) Production of bio- state of the art and challenges. J Ind Microbiol Biotechnol
diesel from high acid value waste cooking oil using an A 35:421–430
optimized lipase enzyme/acid-catalyzed hybrid process. Vicente G, Conteron A, Martinez M, Arcil J (1998) Application
J Chem 6:485–495 of the factorial design of experiments and response surface
Salis A, Pinna M, Monduzzi M, Solina V (2008) Comparison methodology to optimize biodiesel production. Ind Crops
among immobilized lipases on macroporous polypropyl- Prod 8:29–35
ene toward biodiesel synthesis. J Mol Catal B Enzym Wang WG, Lyons DW, Clark NN, Gautam M (2000) Emissions
54:19–26 from nine heavy trucks fuelled by diesel and biodiesel
Samukawa T, Kaieda M, Matsumoto T, Ban K, Kondo A, blend without engine modification. Environ Sci Technol
Shimada Y, Noda H, Fukuda H (2000) Pretreatment of 34:933–939
immobilized Candida antarctica for biodiesel fuel pro- Watanabe Y, Pinsirodom P, Nagao T, Yamauchi A, Kobayashi
duction from plant oil. J Biosci Bioeng 90:180–183 T, Nishida Y, Takagi Y, Shimada Y (2007) Conversion of
Selmi B, Thomas D (1998) Immobilized lipase-catalyzed eth- acid oil by-produced in vegetable oil refining to biodiesel
anolysis of sunflower oil in a solvent-free medium. J Assoc fuel by immobilized Candida antarctica lipase. J Mol
Off Anal Chem 75:691–695 Catal B Enzym 44:99–105
Severac E, Galy O, Turon F, Monsan P, Marty A (2011) Con- Weber N, Mukherjee KD (2008) Lipid biotechnology. In: Akoh
tinuous lipase-catalyzed production of esters from crude CC, Min DB (eds) Food lipids: chemistry, nutrition and
high-oleic sunflower oil. Bioresour Technol 102:4954–4961 biotechnology. CRC Press, Boca Ratón, pp 707–766
Shah S, Gupta MN (2007) Lipase catalyzed preparation of Winayanuwattikun P, Kaewpiboon C, Piriyakananon K, Chu-
biodiesel from Jatropha oil in a solvent free system. Process lalaksananukul W, Yongvanich T, Svasti T (2011) Immo-
Biochem 42:407–414 bilized lipase from potential lipolytic microbes for
Sharma YC, Singh B (2008) Development of biodiesel from catalyzing biodiesel production using palm oil as feed-
karanja, a tree found in rural India. Fuel 67:1740–1742 stock. Afr J Biotechnol 10:1666–1673
Shaw JF, Wang DL, Wang YJ (1991) Lipase-catalyzed ethan- Xie W, Ma N (2009) Immobilized lipase on Fe3O4 nanoparticles
olysis and isopropanolysis of triacylglycerols with long- as biocatalyst for biodiesel production. Energy Fuels
chain fatty acids. Enzyme Microb Technol 13:544–546 23:1347–1353
Shimada Y, Sugihara A, Nakano H, Kuramoto T, Nagao T, Xu Y, Du W, Zeng J, Liu D (2004) Conversion of soybean oil to
Gemba M, Tominaga Y (1997) Purification of decosa- biodiesel fuel using lipozyme TL IM in a solvent-free
hexaenoic acid by selective esterification of fatty acids system. Biocatal Biotransform 22:45–48
from tuna oil with Rhizopus delemar lipase. J Am Oil Chem Yagiz F, Kazan D, Akin AN (2007) Biodiesel production from
Soc 74:97–101 waste oils by using lipase immobilized on hydrotalcite and
Shimada Y, Sugihara A, Minamigawa Y, Higashiyama K, zeolites. Chem Eng J 134:262–267
Akimoto K, Fujikawa S, Komemushi S, Tominaga Y (1998) Yang G, Tian-Wei T, Kai-li N, Fang W (2006) Immobilization
Enzymatic enrichment of arachidonic acid from Mortier- of lipase on macroporous resin and its application in syn-
ella single-cell oil. J Am Oil Chem Soc 75:1213–1217 thesis of biodiesel in low aqueous media. Chin J Biotechnol
Shimada Y, Watanabe Y, Sugihara A, Tominaga Y (2002) 22:114–118
Enzymatic alcoholysis for biodiesel fuel production and Yazdani SS, Gonzalez R (2007) Anaerobic fermentation of
application of the reaction to oil processing. J Mol Catal B glycerol: a path to economic viability for the biofuels
Enzym 17:133–142 industry. Curr Opin Biotechnol 18:213–219
Soumanou MM, Bornscheuer UT (2003) Improvement in Ying M, Chen G (2007) Study on the production of biodiesel by
lipase-catalyzed synthesis of fatty acid methyl esters from magnetic cell biocetalyst based on lipase-producing
sunflower oil. Enzyme Microb Technol 33:97–103 Bacillus subtilis. Appl Biochem Biotechnol 137:793–804
Sree R, Babu S, Prasad SPS, Lingaia N (2009) Transesterifica- Zaidi A, Gainer JL, Carta G (1995) Fatty acids esterification using
tion of edible and non-edible oils over basic solid Mg/Zr nylon-immobilized lipase. Biotechnol Bioeng 48:601–605
catalysts. Fuel Process Technol 90:152–157 Zhang Y, Dube MA, McLean DD, Kates M (2003) Biodiesel
Suppes GJ, Bockwinkel K, Lucas S, Botts JB, Mason MH, production from waste cooking oil: process design and
Heppert JA (2004) Transesterification of soybean oil with technology assessment. Bioresour Technol 89:1–16
zeolite and metal catalysts. Appl Catal A 257:213–223 Zhu LY, Zong MH, Wu H (2008) Efficient lipid production with
Talukder MR, Wu JC, Nguyen TBV, Fen NM, Melissa YLS Trichosporon fermentans and its use for biodiesel prepa-
(2009) Novozyme 435 for production of biodiesel from ration. Bioresour Technol 99:7881–7885

123