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Bioresource Technology 99 (2008) 39753981


An overview of enzymatic production of biodiesel

Srivathsan Vembanur Ranganathan, Srinivasan Lakshmi Narasimhan,
Karuppan Muthukumar *
Department of Chemical Engineering, A.C. College of Technology, Anna University, Chennai 600 025, India
Received 5 March 2007; received in revised form 29 April 2007; accepted 29 April 2007
Available online 25 June 2007

Biodiesel production has received considerable attention in the recent past as a biodegradable and nonpolluting fuel. The production
of biodiesel by transesterication process employing alkali catalyst has been industrially accepted for its high conversion and reaction
rates. Recently, enzymatic transesterication has attracted much attention for biodiesel production as it produces high purity product
and enables easy separation from the byproduct, glycerol. But the cost of enzyme remains a barrier for its industrial implementation.
In order to increase the cost eectiveness of the process, the enzyme (both intracellular and extracellular) is reused by immobilizing
in a suitable biomass support particle and that has resulted in considerable increase in eciency. But the activity of immobilized enzyme
is inhibited by methanol and glycerol which are present in the reacting mixture. The use of tert-butanol as solvent, continuous removal of
glycerol, stepwise addition of methanol are found to reduce the inhibitory eects thereby increasing the cost eectiveness of the process.
The present review analyzes these methods reported in literature and also suggests a suitable method for commercialization of the enzymatic process.
 2007 Elsevier Ltd. All rights reserved.
Keywords: Lipase; Biodiesel; Transesterication; Immobilization; Whole cell

1. Introduction
Biodiesel has gained importance in the recent past for its
ability to replace fossil fuels which are likely to run out
within a century. The environmental issues concerned with
the exhaust gases emission by the usage of fossil fuels also
encourage the usage of biodiesel which has proved to be
eco-friendly far more than fossil fuels. Biodiesel is known
as a carbon neutral fuel because the carbon present in the
exhaust was originally xed from the atmosphere. Biodiesel
is a mixture of mono-alkyl esters obtained from vegetable
oils like soyabean oil, jatropha oil, rapeseed oil, palm oil,
sunower oil, corn oil, peanut oil, canola oil and cottonseed oil (Peterson, 1986). Apart from vegetable oils, biodiesel can also be produced from other sources like animal fat

Corresponding author. Tel.: +91 44 22203500.

E-mail address: m_kumar77@yahoo.co.in (K. Muthukumar).

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.

(beef tallow, lard), waste cooking oil, greases (trap grease,

oat grease) and algae (Pearl, 2002). A method utilizing all
the above mentioned sources was patented by Foglia et al.
(1998) claiming the process to be a cost eective one as it
uses inexpensive feedstocks.
The direct usage of vegetable oils as biodiesel is possible
by blending it with conventional diesel fuels in a suitable
ratio and these ester blends are stable for short term usages.
The blending process is simple which involves mixing alone
and hence the equipment cost is low. But direct usage of
these triglyceric esters (oils) is unsatisfactory and impractical for long term usages in the available diesel engines due
to high viscosity, acid contamination, free fatty acid formation resulting in gum formation by oxidation and polymerization and carbon deposition. Hence vegetables oils are
processed so as to acquire properties (viscosity and volatility) similar to that of fossil fuels and the processed fuel can
be directly used in the diesel engines available. Three processing techniques are mainly used to convert vegetable oils


S.V. Ranganathan et al. / Bioresource Technology 99 (2008) 39753981

catalyst, heterogeneous catalyst or using alcohols in their

supercritical state. The general reaction is shown below



Vegetable oil Methanol ! Biodiesel Glycerol





Fig. 1. Biodiesel production sequence by transesterication.

to fuel form (Ma and Hanna, 1999) and they are pyrolysis,
microemulsication and transesterication. Pyrolysis refers
to chemical change caused by application of heat to get
simpler compounds from a complex compound. The process is also known as cracking. Vegetable oils can be
cracked to reduce viscosity and improve cetane number.
The products of cracking include alkanes, alkenes, and carboxylic acids. Soyabean oil, cottonseed oil, rapeseed oil
and other oils are successfully cracked with appropriate
catalysts to get biodiesel (Ma and Hanna, 1999). By using
this technique good ow characteristics were achieved due
to reduction in viscosity. Disadvantages of this process
include high equipment cost and need for separate distillation equipment for separation of various fractions. Also
the product obtained was similar to gasoline containing
sulfur which makes it less eco-friendly (Ma and Hanna,
1999). Microemulsication is another technique that has
been reported to produce biodiesel and the components
of a biodiesel microemulsion include diesel fuel, vegetable
oil, alcohol, surfactant and cetane improver in suitable proportions (Ma and Hanna, 1999). Alcohols such as methanol, ethanol and propanol are used as viscosity lowering
additives, higher alcohols are used as surfactants and alkyl
nitrates are used as cetane improvers. Viscosity reduction,
increase in cetane number and good spray characters
encourage the usage of microemulsions but prolong usage
causes problems like injector needle sticking, carbon
deposit formation and incomplete combustion (Ma and
Hanna, 1999). The most popular method of producing biodiesel is the transesterication of vegetable oils. Biodiesel
obtained by transesterication process is a mixture of
mono-alkyl esters of higher fatty acids. Transesterication
is the alcoholysis of triglyceric esters resulting in a mixture
of mono-alkyl esters and glycerol and the sequence of processes is shown in Fig. 1. The high viscosity component,
glycerol, is removed and hence the product has low viscosity like the fossil fuels. The mixture of these mono-alkyl
esters can hence be used as a substitute for fossil fuels.

2. Transesterication process
The transesterication process can be done in a number
of ways such as using an alkali catalyst, acid catalyst, bio-

In the alkali process sodium hydroxide (NaOH) or potassium hydroxide (KOH) is used as a catalyst along with
methanol or ethanol. Initially, during the process, alcoxy
is formed by reaction of the catalyst with alcohol and the
alcoxy is then reacted with any vegetable oil to form biodiesel and glycerol. Glycerol being denser settles at the bottom
and biodiesel can be decanted. This process is the most ecient and least corrosive of all the processes and the reaction rate is reasonably high even at a low temperature of
60 C. There may be risk of free acid or water contamination and soap formation is likely to take place which makes
the separation process dicult (Ma and Hanna, 1999;
Fukuda et al., 2001; Barnwal and Sharma, 2005).
The second conventional way of producing biodiesel is
using an acid catalyst instead of a base. Any mineral acid
can be used to catalyze the process; the most commonly
used acids are sulfuric acid and sulfonic acid. Although
yield is high, the acids, being corrosive, may cause damage
to the equipment and the reaction rate was also observed to
be low (Freedman et al., 1984).
It has been recently found that enzymes such as lipase
can be used to catalyze transesterication process by
immobilizing them in a suitable support. The advantage
of immobilization is that the enzyme can be reused without
separation. Also, the operating temperature of the process
is low (50 C) compared to other techniques. Disadvantages include inhibition eects which was observed when
methanol was used and the fact that enzymes are expensive
(Nelson et al., 1996; Shimada et al., 2002). Heterogeneous
catalysts such as amorphous zirconia, titanium-, aluminium-, and potassium-doped zirconias have also become
popular for catalyzing the transesterication of vegetable
oils. Research is still in progress in order to solve the problems encountered in this process such as exhaustion of catalyst and to achieve higher conversions (Furuta et al.,
The transesterication process can be carried out even
without catalyst but with considerable increase in temperature. Yield is very low at temperatures below 350 C and
therefore higher temperatures were required. However at
temperatures greater than 400 C thermal degradation of
esters occurred (Demirbas, 2006). Recently it has been
found that alcohols in their supercritical state produce better yield and researchers have experimented this process
with methanol in its supercritical state. The process eciency can be increased by using calcium oxide as a catalyst
(Demirbas, 2006).
Of all the methods mentioned above for production of
biodiesel, only the alkali process is carried out in an
industrial scale. It is cost eective and highly ecient.
But problems arise in the downstream operations including separation of catalyst and unreacted methanol from

S.V. Ranganathan et al. / Bioresource Technology 99 (2008) 39753981











Fig. 2. Production of biodiesel by alkali process.





this review presents critical analysis of the methods

reported which can contribute to the global eort of industrial implementation of the enzymatic production of biodiesel in the near future. The enzyme that was found to
be capable of catalyzing methanolysis is lipase and lipase
is obtained from micro-organisms like Mucor miehei, Rhizopus oryzae, Candida antarctica, Pseudomonas cepacia.
Various alcohols are tried for the transesterication process including methanol, ethanol, iso-propanol and butanol
but methanol is considered for industrial production
because of its low cost and availability.
3. Transesterication by enzymatic technique







Fig. 3. Enzymatic production of biodiesel.

biodiesel. The removal of the catalyst involves many complications and biodiesel requires repeated washing for
attaining the necessary purity. Figs. 2 and 3 compare the
dierence in downstream operation required for alkali
and enzymatic production.
The production of biodiesel using a biocatalyst eliminates the disadvantages of the alkali process by producing
product of very high purity with less or no downstream
operations (Fukuda et al., 2001). This method of production of biodiesel using a biocatalyst was also patented by
Haas (1997). But the process has not yet been implemented
in an industrial scale due to certain constraints like enzyme
inhibition by methanol, exhaustion of enzyme activity and
high cost of enzymes. Research works have been reported
in the literature in order to overcome these problems and

Enzymatic production is possible using both extracellular and intracellular lipases. In both the cases the enzyme is
immobilized and used which eliminates downstream operations like separation and recycling. Hence in all the works
reported in the literature either immobilized (extracellular)
enzymes or immobilized whole cells (Intracellular enzymes)
are used for catalysis. Both the processes are reported to be
highly ecient compared to using free enzymes.
4. Extracellular lipase
Mittelbach (1990) reported transesterication of sunower oil with primary alcohols like methanol, ethanol
and butanol using M. miehei and C. antarctica (Novozym
435) in the presence and absence of the solvent, petroleum
ether. Yields obtained for ethanol and butanol were high
even without the solvent but methanol was found to produce only traces of methyl esters without the solvent. Nelson et al. (1996) conducted batch experiments and found
that C. antarctica was suitable for secondary alcohols
(80% conversion) like iso-propanol and 2-butanol and
M. miehei was ecient for primary short chain alcohols
(95% conversion) like methanol, ethanol, propanol and
butanol in the presence of hexane as a solvent. However
in the absence of the solvent, methanol was the least ecient with a methyl ester yield of 19.4%. The low yield
was attributed to the inhibitory eects caused by methanol
on the immobilized enzyme. This was again conrmed by
Abigor et al. (2000) who reported the conversion of palm
kernel oil using methanol and ethanol as 15% and 72%,
respectively. Noureddini et al. (2001) used methanol and
ethanol for the tranesterication of soyabean oil in the
presence of immobilized enzyme obtained from Pseudomonas ourescens and reported conversions 67% and 65% for
methanol and ethanol respectively. But conversions as high
as 97% is possible which was demonstrated by Linko et al.
(1998) using 2-ethyl-1-hexanol for the transesterication of
rapeseed oil. Similarly, usage of other alcohols as alternate
acyl acceptors instead of methanol have been experimented
and conversions as high as 90% were constantly obtained.
Iso et al. (2001) reported 90% conversion of vegetable oil
using P. uorescens enzyme with butanol as the acyl acceptor. The reaction was carried out in a solvent free medium


S.V. Ranganathan et al. / Bioresource Technology 99 (2008) 39753981

under an optimum condition of 0.3% water and 60 C. Propane-2-ol was used as an acyl acceptor by Modi et al.
(2006) for the transesterication of Jatropha, Karanj and
sunower oil achieving a maximum conversion of 92.8%,
91.7% and 93.4%, respectively. With propane-2-ol, the reusability of lipase was maintained over 12 cycles while it
dropped to zero after 7 cycles when methanol was used.
In order to achieve high conversions with methanol as
the acyl acceptor several attempts have been made to minimize the inhibitory eects of methanol. Using a common
solvent for methanol and the oil was found to be eective
by many researchers since insoluble methanol is responsible for inhibition. Iso et al. (2001) reported high conversion
with methanol using 1,4 dioxane as a solvent. Rather than
using butanol as an acyl acceptor, it was suggested to use
tert-butanol as a solvent for methanolysis of oil. Li et al.
(2006) reported the usage of tert-butanol as a solvent for
the transesterication of rapeseed oil. A conversion of
95% was obtained using Lipozyme TI LM and Novozyme
435 in a suitable ratio (3:1) under optimum conditions. tertButanol as a novel solvent for the enzymatic production
was again demonstrated by Royon et al. (2007) who used
Novozyme 435 for the transesterication of cotton seed
oil and yield as high as 97% was reported within 24 h at
55 C. With a continuous xed bed reactor 95% conversion
was reported under optimum ow conditions. Usage of
tert-butanol as a solvent could hence be recognized as
one possible solution for reducing the inhibitory eects of
methanol and for the industrial implementation of the
Pretreatment of the immobilized enzyme was believed to
minimize the deactivation of the enzyme and Samukawa
et al. (2000) illustrated this by preincubation of the enzyme
in methyl oleate for 0.5 h and subsequently in soyabean oil
for 12 h. Inhibitory eects were considerably reduced and
high conversions were obtained. Chen and Wu (2003) suggested the usage of tert-butanol and 2-butanol for regenerating the activity of deactivated enzymes. They conducted
experiments by completely deactivating Novozyme 435
with methanol and regenerating by washing the enzyme
with tert-butanol and 2-butanol. The activity of the enzyme
increased by 10 times compared to the untreated enzyme
and the completely deactivated enzyme was restored to
56% and 75% of its original value when washed with
2-butanol and tert-butanol, respectively. The same process
was also patented by Chen and Wu (2002).
Recently, acyl acceptors other than alcohols are experimented to improve the eciency of transesterication process. A novel acyl acceptor was developed by Du et al.
(2004) who used methyl acetate for transesterication of
soyabean oil with Novozyme 435. While a 1:1 molal ratio
of methanol to oil caused serious inactivation of enzyme,
even a 12:1 molal ratio of methyl acetate to oil did not have
any noticeable negative eect on the enzyme. The activity
of the enzyme was found to be unaltered even after 100
batches. Conversion, as high as 92%, was reported for
soyabean oil. Crude soyabean oil was found to have equal

conversion incontrast to methanolysis which gave only

traces of methyl ester with crude soyabean oil. Since the
by product triacetylglycerol is a valuable compound, this
process was recommended for industrial production. Ethyl
acetate was tried as an acyl acceptor by Modi et al. (2007)
with Novozyme 435 and they reported conversions above
90% for oils such as jatropha, karanj and sunower oil.
Comparison was made with ethanolysis in which activity
could not be maintained for more than 6 cycles whereas
with ethyl acetate, even after 12 cycles there was no appreciable decrease in activity. Ethyl acetate to oil ratio of 11:1
was recommended for the process and even in such high
concentrations no deactivation occurred.
5. Eective methanolysis using extracellular lipase
Methanolysis yield was found to be low in the absence
of a suitable solvent and even in the presence of such a solvent high conversions attained by other alcohols are not
possible. Since the deactivation of the enzyme was due to
insolubility of methanol, any methanol to oil ratio above
1.5:1 caused serious inhibition. To overcome this diculty,
Shimada et al. (1999) suggested stepwise addition of methanol through which 95% conversion was attained even after
50 cycles of operation. Similar to their work Watanabe
et al. (2000) conducted experiments on two step batch wise
addition of methanol and three step continuous addition of
methanol. They reported high conversions of above 90%
even after 100 cycles of repeated operation. Stepwise addition was also demonstrated by Samukawa et al. (2000) who
preincubated the enzyme before usage, were successful in
obtaining a conversion of 97% with three stepwise addition
of 0.33 molar equivalents of methanol at 0.250.4 h interval. For stepwise addition of methanol Kaieda et al.
(2000) studied the dierence in the usage of regiospecic
and non-regiospecic lipases. Non-regiospecic lipases like
Candida rugosa, P. cepacia and P. ourescens are tolerant
to the inhibition of methanol. P. cepacia gave especially
high conversion even with two to three molar equivalents
of methanol. With regiospecic lipases like R. oryzae,
8090% conversion was possible with stepwise addition
of methanol in the presence of 430% water. Shimada
et al. (2002) obtained conversions above 90% with waste
cooking oil using stepwise addition of methanol. They also
conducted experiments on continuous systems and
obtained equally high conversions. Thus it was satisfactorily proved that the inhibitory eects of methanol can be
minimized by stepwise addition of methanol and high conversions are possible even in a solvent free medium.
Bako et al. (2002) suggested that the inhibitory eects
are partly due to glycerol that is formed during the reaction
and to reduce the inhibitory eects, they recommended
in situ glycerol removal by dialysis. Experiments were conducted with two and three stepwise addition of methanol
and glycerol was removed continuously by dialysis. This
method was found to increase the eectiveness of the process it was reported that a minimum ow rate of 85 ml of

S.V. Ranganathan et al. / Bioresource Technology 99 (2008) 39753981

glycerol/liter of reacting mixture was required for eective

separation to produce a conversion of 97% at 50 C. Xu
et al. (2004) suggested that glycerol removal was also possible using iso-propanol in a process employing stepwise
addition of methanol. Thermomyces lanuginosus (Lipozyme
TL IM) was used to catalyze the transesterication of soyabean oil and a maximum yield of 98% was reported at
40 C and a conversion of 94% was maintained even after
15 repeated cycles. Nie et al. (2006) conducted optimization
experiments on batch and continuous transesterication
process and reported a maximum conversion of 96% for
a batch process under optimal conditions with three stepwise addition of methanol using immobilized Novozyme
435. The lipase was found to retain its activity for more
than 20 days of continuous operations. In the continuous
process conversions of 92% and 93% were obtained for vegetable oil and waste cooking oil respectively and the process was recommended for industrial production.
6. Intracellular lipase
The eciency of the process can be highly increased by
using intracellular lipase (whole cell immobilization) in the
place extracellular lipase which requires complex purication steps before immobilization. A comparison of the
immobilization process of extracellular and intracellular
lipases is shown in Fig. 4. It can be clearly seen that considerable reduction in cost can be achieved with intracellular
Whole cell biocatalyst was developed by Matsumoto
et al. (2001) by immobilizing R. oryzae cells and permeabilizing them by air drying. It was then used for the production of methyl esters by three stepwise methanolysis of
plant oil in solvent free and water containing system. The
methyl ester content in the reaction mixture was reported







Fig. 4. Comparison of steps involved in the immobilization of extracellular and intracellular enzymes: (a) extarcellular lipase and (b) intracellular


to be 71 wt% after a 165-h reaction at 37 C with stepwise

addition of methanol.
Ban et al. (2001) utilized immobilized whole cell R. oryzae for the transesterication of vegetable oils and investigated the culture conditions, cell pretreatment eects and
eect of water content on the production process. To
enhance the methanolysis activity of the immobilized cells,
several substrate related compounds were added to the
medium, of which olive oil and oleic acid were found to
be eective. It was reported that, with stepwise addition
of methanol and with 15% water content, a high conversion
of 90% was obtained which was comparative with the
extracellular process.
Inorder to stabilize R. oryzae cells, cross-linking treatment with 0.1% glutaraldehyhde was examined by Ban
et al. (2002). It was reported that without glutaraldehyde
treatment, in the stepwise methanol addition process, conversion level dropped to 50% after 6th batch cycle whereas
with glutaraldehyde treatment conversion can be maintained at 7283% even after 6 batch cycles. Fukuda and
Kondo (2003) patented the process of producing biodiesel
utilizing whole cell biocatalyst by pretreating the cells with
lower alcohols. They claimed a 350600 times increase in
the reaction rate using cells treated with lower alcohols
compared to untreated cells.
Whole cell immobilization was then experimented by
many researchers in order to increase the eciency of enzymatic transesterication. Hama et al. (2004) studied the
increase in enzyme stability with the variation of the fatty
acid composition of the cell membrane. The fatty acid composition of the cell membrane can be controlled by the
addition of various fatty acids to the culture medium. It
was reported that oleic acid and linoleic acid enriched cells
showed higher enzymatic activity than saturated fatty acid
enriched cells and palmitic acid enriched cells showed
higher stability than unsaturated fatty acid enriched cells.
Therefore an optimum ratio of unsaturated to total fatty
acids was determined as a compensation for both activity
and stability and it was found to be 0.67. It was reported
that the methanolysis yield was consistently high and even
after 10 repeated cycles it was above 55%. Hama et al.
(2006) reported the existence of two types of lipases, one
bound to the cell wall (ROL 34) and the other to the cell
membrane (ROL 31). They reported that the increase in
enzyme activity with addition of olive oil or oleic acid
was due to the increase in the production of membrane
bound lipase (ROL 31) suggesting that ROL 31 plays a crucial role in the methanolysis activity.
Hama et al. (2007) studied the production of biodiesel
using a packed bed reactor utilizing R. oryzae whole cell
biocatalyst by plant oil methanolysis. Compared with
methanolysis reaction in a shaken bottle, the packed bed
reactor enhanced repeated batch methanolysis by protecting immobilized cells from physical damage and excess
amounts of methanol. Lipase-producing R. oryzae cells
were immobilized within 6 mm 6 mm 3 mm cuboidal
polyurethane foam biomass support particles (BSPs)


S.V. Ranganathan et al. / Bioresource Technology 99 (2008) 39753981

Table 1
Comparison of various works on enzymatic production of biodiesel





Technique employed

Cost of

Vegetable oil, Novozyme

Soyabean oil, Novozyme
Vegetable oil, R. oryzae



Stepwise addition of methanol






Iso et al. (2001)a

Triolein, P. ourescens






Xu et al. (2004)a




Li et al. (2006)a

Waste cooking oil,

Novozyme 435
Sunower oil, Novozyme
Soyabean oil, Novozyme
Soyabean oil, Novozyme
Rapeseed oil, Novozyme
435 & Lipozyme TL IM
Cotton seed oil, Novozyme
Jatropha oil, Novozyme
Soyabean oil, R. oryzae


Shimada et al.
Bako et al.
Du et al. (2004)a

Stepwise addition methanol and preincubation of

enzyme in methyl oleate and soyabean oil
Stepwise addition of methanol and application of
glutaraldehyde for stability of enzyme
Butanol was used as an acyl acceptor and no
solvent was used
Stepwise addition of methanol


Watanabe et al.
Samukawa et al.
Ban et al. (2001)b





Royon et al.
Modi et al.
Hama et al.



Stepwise addition of methanol and removal of

glycerol by dialysis
A novel acyl acceptor, methyl acetate which had no
inhibitory eects was used
Stepwise addition of methanol and removal of
glycerol using the solvent, iso-propanol
Combined use of Lipozyme TL IM and Novozyme
435 along with tert-butanol as solvent
tert-Butanol was used as a solvent




Ethyl acetate having no inhibitory eects was used



Stepwise addition of methanol in a packed bed




Extracellular lipase.
Intracellular lipase.

during batch cultivation in a 20-l air-lift bioreactor. Hama

et al. (2007) reported that the emulsication of the reaction
mixture resulted in increased yield (75.5%) due the increase
in interfacial surface area whereas without emulsication a
reasonable high conversion of 63% was obtained. During
the investigation the ow rate of reaction mixture was varied between 5 and 55 l/h and higher ow rates caused exfoliation of immobilized enzyme whereas low ow rates
resulted in decreased enzyme activity due to inecient mixing. An optimum of 25 l/h was suggested to give a maximum conversion of 90%.
Fukuda and Noda (2006) patented the process of using
whole cell biocatalyst with waste oil containing water
claiming no decrease in the eciency of the process. This
encourages the process employing intracellular lipase for
commercialization. Table 1 summarizes the signicant
works reported in the literature with respect to enzymatic
production of biodiesel.
7. Conclusions and future prospects
The biodiesel fuel production has gained importance for
its ability to replace fossil fuels, its environmental benets
and the fact that it is a renewable source of energy. Since
the direct usage of vegetable oils as biodiesel is impractical,
many processes have been developed to convert them into a
suitable form. Pyrolysis and microemulsication are not
satisfactory and hence only the transesterication process
is accepted for large scale production of biodiesel. The

alkali transesterication process has already been implemented on an industrial scale and it gives high conversion.
However it has several drawbacks including the diculty in
recycling catalyst and the need for removal of glycerol. To
overcome these drawbacks, which may limit the availability
of biodiesel fuel, enzymatic processes using lipases have
recently been developed. The enzymatic process is much
simpler since recovery of unreacted methanol and glycerol
requires less downstream operations. As the cost of lipase
production is the main hurdle for commercialization of
the lipase-catalyzed process, several attempts have been
made to develop cost eective systems. The cost eective
system can be achieved by reusing the enzyme which is possible with immobilization of enzyme in suitable support
particles. Both extracellular and intracellular lipase can
be immobilized and used for catalyzing the transesterication reaction. The choice of method is based on a balance
between simplied upstream operations as in whole cell
immobilization and high conversions as in the usage of
extracellular lipases. However when methanol was used
as the acyl acceptor the immobilized enzyme suered serious inactivation. This is due to the inhibitory eect of
undissolved methanol and glycerol present in the reaction
medium. Although other acyl acceptors, with no inhibitory
eects have been developed, usage of methanol has been
extensively researched for its low cost and availability. Several methods have been developed by researchers to minimize the inhibitory eects of methnol and glycerol
thereby increasing the overall yield of the process. The

S.V. Ranganathan et al. / Bioresource Technology 99 (2008) 39753981

use of t-butanol as a common solvent for oil and methanol

eliminates the inhibitory eects of methanol. Continuous
glycerol removal by dialysis or solvent extraction reduces
the inhibitory eects of glycerol on the enzyme activity.
Highest conversion was obtained for the stepwise addition
of methanol to oil by which methanol concentration in the
medium was always kept low and thus eliminating enzyme
deactivation. The conversion of whole cell biocatalyst process was enhanced to match the extracellular process by
adding certain promoters and maintaining suitable reaction
conditions. Combining the whole cell biocatalyst process
with stepwise addition of methanol, signicant reduction
in the cost the production of biodiesel could be expected.
Such novel system is promising for the industrial scale
enzymatic production of biodiesel.
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