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Review
Abstract
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
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.04.060
3976
VEGETABLE OILS
Catalyst
BIODIESEL
METHANOL
GLYCEROL
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.,
2006).
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
TRANSESTERIFICATION
ALKALI
+
MeOH
SEPARATION
EVAPORATION OF MeOH
UPPER
PHASE
WASTE
WATERALKALINE
WASHING
LOWER
PHASE
BIODIESEL
PURIFICATION
SAPONIFIED
PRODUCTS
GLYCEROL
OILS
TRANSESTERIFICATION
ENZYME
SEPARATION
UPPER
PHASE
EVAPORATION OF MeOH
MeOH
3977
LOWER
PHASE
BIODIESEL
GLYCEROL
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
3978
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
process.
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
CULTIVATION
CULTIVATION
+
IMMOBILIZATION
SEPARATION
SEPARATION
PURIFICATION
METHANOLYSIS
IMMOBILIZATION
METHANOLYSIS
Fig. 4. Comparison of steps involved in the immobilization of extracellular and intracellular enzymes: (a) extarcellular lipase and (b) intracellular
lipase.
3979
3980
Table 1
Comparison of various works on enzymatic production of biodiesel
S.No
Authors/year
Oil/enzyme
Acyl
acceptor
Conversion
(%)
Technique employed
Cost of
production
Methanol
9093
Moderate
Methanol
97
Methanol
90
Triolein, P. ourescens
Butanol
90
90
Methanol
97
Xu et al. (2004)a
Methyl
acetate
Methanol
92
98
Li et al. (2006)a
Methanol
Shimada et al.
(2002)a
Bako et al.
(2002)a
Du et al. (2004)a
High
Watanabe et al.
(2000)a
Samukawa et al.
(2000)a
Ban et al. (2001)b
Methanol
95
Methanol
Ethyl
acetate
Methanol
10
11
12
a
b
Royon et al.
(2007)a
Modi et al.
(2007)a
Hama et al.
(2007)b
Low
Moderate
Low
97
High
High
91.3
High
90
Low
High
High
High
Extracellular lipase.
Intracellular lipase.
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
3981