Biogas Technology in Sub Saharan Africa

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Critical Reviews in Biotechnology

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Biotechnological production of biodiesel fuel using biocatalysed

transesterification: A review
Wilson Parawira a
a
Institute of Food Science, Nutrition and Family Sciences, University of Zimbabwe, Harare, Zimbabwe
First Published on: 01 May 2009
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Critical Reviews in Biotechnology, 2009, 1–12, iFirst
REVIEW ARTICLE
Biotechnological production of biodiesel fuel using

biocatalysed transesteriication: A review
Wilson Parawira
Institute of Food Science, Nutrition and Family Sciences, University of Zimbabwe, Harare, Zimbabwe
Abstract
Biotechnological production of biodiesel has attracted considerable attention during the past decade com-
pared to chemical-catalysed production since biocatalysis-mediated transesteriication has many advantages.
Currently, there are extensive reports on enzyme-catalysed transesteriication for biodiesel production; the
related research can be classiied into immobilised-extracellular and immobilised-intracellular biocatalysis and
this review focusses on these forms of biocatalyst for biodiesel production. The optimisation of the most impor-
Downloaded By: [Parawira, Wilson] At: 12:09 2 May 2009
tant operating conditions afecting lipase-catalysed transesteriication and the yield of alkyl esters, such as the
type and form of lipase, the type of alcohol, the presence of organic solvents, the content of water in the oil,
temperature and the presence of glycerol, are discussed. However, there is still a need to optimise lipase-cata-
lysed transesteriication and reduce the cost of lipase production before it is applied commercially. Optimisation
research of lipase-catalysed transesteriication could include development of new reactor systems with immobi-
lised biocatalysts, the use of lipases tolerant to organic solvents, intracellular lipases (whole microbial cells) and
genetically modiied microorganisms (intelligent yeasts). Biodiesel fuel is expensive in comparison with petrole-
um-based fuel and 60–70% of the cost is associated with feedstock oil and enzyme. Therefore ways of reducing
the cost of biodiesel with respect to enzyme and substrate oils reported in literature are also presented.
Keywords: Biodiesel; transesteriication; lipase; biocatalysis; vegetable oils; biotechnology; renewable energy
Introduction
Biofuels are considered in part a solution to such issues as rape seed oil, canola oil, peanut oil, cottonseed oil, Jatropha
sustainable development, energy security and a reduction of oil and others; animal fats (beef tallow, lard) and ish oil; or
greenhouse gas emissions. Biodiesel fuel for use in motors from waste cooking oil and greases. he source for biodiesel
with internal combustion and compression ignition is an production is chosen according to the availability in each
environmentally friendly fuel similar to petro-diesel in com- of the producing countries. he prices of edible vegetable
bustion properties. Biodiesel is biodegradable, non-toxic oils such as soybean oil, rapeseed oil and sunlower oil are
and has a low emission proile. he environmental issues higher than those of diesel fuel, therefore waste vegetable
concerned with the exhaust gases emission by using fossil oils and non-edible crude vegetable oils should be preferred
fuels also encourages the use of biodiesel which has proved as potential low-priced biodiesel sources. Since biodiesel
to be far more eco-friendly than fossil fuels (Ranganathan comes from renewable sources, it does not contribute to the
et al., 2008). he use of biodiesel fuel is becoming increas- emission of new carbon dioxide (one of the gases responsi-
ingly important due to diminishing petroleum reserves and ble for the greenhouse efect), unlike fossil diesel.
environmental regulations. Biodiesel is a methyl or ethyl Vegetable oils are a very promising alternative to diesel
ester of a fatty acid made from renewable biological resources oil since they are renewable and have similar properties to
such as vegetable oils (both edible and non-edible), recycled fossil diesel. he use of vegetable oils as alternative fuels
waste vegetable oils and animal fats (Demirbas, 2000; Kinney started around 1900, when the inventor of the diesel engine
and Clemente, 2005; Modi et al., 2007; Recep et al., 2000). (Rudolph Diesel) irst tested peanut oil in his compression-
hus biodiesel is a renewable fuel derived from: vegetable ignition engine (Shay, 1993). However, due to the avail-
oils such as sunlower oil, palm oil, castor oil, soybeans oil, ability of inexpensive petroleum products, the use of such
Address for correspondence: [email protected]

( Accepted 20 January 2009)
ISSN 0738-8551 print/ISSN 1549-7801 online © 2009 Informa UK Ltd
DOI: 10.1080/07388550902823674 http://www.informapharmascience.com/btn
2 Wilson Parawira
non-conventional fuels has only recently become a practi- CH2–OOC–R1 R1–COO–R′ CH2–OH
cal proposition, and has received considerable attention
recently worldwide, because of its many advantages and CH–OOC–R2 + 3R′OH Catalyst R2–COO–R′ + CH–OH
the need to replace fossil fuels which are likely to run out
within the present century. Biodiesel derived from surplus CH2–OOC–R3 R3–COO–R′ CH2–OH
edible oils, such as soybean oil, sunlower oil and rapeseed Triglyceride Alcohol Mixture of alkyl Glycerol
oil, is already being used in the USA and Europe to reduce (vegetable oil) esters
air pollution, reduce dependency on already-depleting
Figure 1. Transesteriication reaction of triglycerides.
fossil fuels localised in speciic regions of the world and
to counteract increases in crude oil prices (Agarwal, 2007;
Berchmans and Hirata, 2008; Foidl et al., 1996; Ma and he reaction is reversible and therefore excess alcohol is used
Hanna, 1999; Meher et al., 2006; Openshaw, 2000; Sarin to shift the equilibrium to the products’ side (Barnwal and
et al., 2007). he use of edible oil to produce biodiesel in Sharma, 2005; Meher et al., 2006).
Africa and other developing continents is not feasible he alcohols that can be used in the transesteriication
because of the huge gap between the demand and the sup- process are methanol, ethanol, propanol, butanol and amyl
ply of such oils in the developing world. here is therefore alcohol, with methanol and ethanol being frequently used.
a need to explore alternative non-edible oils for use in the Transesteriication as an industrial process is usually car-
production of biodiesel. To this end, several non-edible ried out by heating an excess of the alcohol with vegetable
oils have already been found to be suitable for biodiesel oils under diferent reaction conditions in the presence of
production (Azam et al., 2005). an inorganic catalyst. he reactions are often catalysed by
he direct use of vegetable oil (triglyceric esters) as a homogeneous catalysts such as an acid or a base, by het-
biodiesel is possible but is unsatisfactory for long-term erogeneous catalysts such as metal oxides or carbonates, or
usage in today’s direct and indirect diesel engines (Ma and by lipase, so as to improve the reaction rate and yield.
Hanna, 1999). his is because they have high viscosity (about he process of transesteriication brings about drastic
10 times that of No. 2 diesel fuel), are contaminated by acid, changes in viscosity of the vegetable oil. he high-viscosity
phospholipids and water; and form gum due to oxidation component (glycerol) is removed and hence the product
and polymerisation of free fatty acids during storage and has a low viscosity, like the fossil fuels. he biodiesel pro-
combustion; they deposit carbon on engines and thicken duced is totally miscible with mineral diesel in any propor-
lubricating oil (Banapurmath et al., 2008; Crabbe et al., 2001; tion. he lash point of the biodiesel is lowered after trans-
Foglia et al., 1996; Openshaw, 2000; Srivastava and Prasad, esteriication and the cetane number is improved. he yield
2000). Consequently vegetable oils are processed so as to of biodiesel in the process of transesteriication is afected
acquire viscosity and volatility characteristics similar to No. by several process parameters which include the presence
2 diesel fuel (Agarwal, 2007; Demirbas, 2008). he main of moisture and free fatty acids (FFAs), reaction time, reac-
processing techniques used to convert vegetable oils into the tion temperature, catalyst type, molar ratio of alcohol and
fuel form are blending, pyrolysis, micro-emulsiication and oil, and organic co-solvent addition (Sharma et al., 2008).
transesteriication (Demirbas, 2000; Ma and Hanna, 1999).
Among these, transesteriication is by far the most important Variables afecting the transesteriication
step in the production of a cleaner and environmentally safe reaction
fuel from vegetable oils and animal fats.
In the conventional transesteriication of vegetable oils and
fats for biodiesel production, the gylceride should have an
he transesteriication (alcoholysis) process acid value of < 1 and all materials should be substantially
he transesteriication (an alternative term that is frequently anhydrous. An acid value > 1 requires that the process uses
used is alcoholysis) of vegetable oils is the most popular more sodium hydroxide to neutralise the free fatty acids.
method of producing biodiesel and it yields the best products Transesteriication yields are signiicantly reduced if the
(Antolin et al., 2002). It is the reaction of a fat or oil (a trigyl- reactants do not meet this requirement (Dorado et al., 2002;
ceride) with an alcohol to form fatty acid alkyl esters (valuable Freedman et al., 1986; Goodrum, 2002; Ma et al., 1998).
intermediates in oleo chemistry), methyl and ethyl esters he presence of water causes the transesteriication reac-
(which are excellent substitutes for biodiesel) and glycerol tion to partially change to saponiication, which produces
(Figure 1). To obtain the biodiesel, the esters (biodiesel) in the soap, thus lowering the yield of esters. Saponiication also
vegetable oil are separated from glycerine. Transesteriication renders the separation of ester and glycerol diicult since
is the process of exchanging the alk-oxy group of an ester com- it increases the viscosity and forms gels (Berchmans and
pound with another alcohol. he overall process is a sequence Hirata, 2008). Free fatty acids and water always produce
of three consecutive and reversible reactions, in which diglyc- negative efects, since their presence causes soap formation,
erides and monoglycerides are formed as intermediate com- consumes catalyst and reduces catalyst efectiveness, all of
pounds (Ma and Hanna, 1999). he stoichiometric reaction which result in a low conversion rate (Kasudiana and Saka,
requires 1 mol of triglycerides and 3 mol of alcohol (Figure 1). 2004). Most of the biodiesel is currently made from edible
Biotechnological production of biodiesel fuel using biocatalysed transesteriication 3
oils by using methanol and alkaline catalyst. However, there reactive, while acids can catalyse the reaction by donating
are large amounts of low-cost oils and fats that cannot be a proton to the carbonyl group, thus also making it more
converted to biodiesel using methanol and alkaline cata- reactive.
lyst because they contain high amounts of free fatty acids
and water. In some instances, crude vegetable oil quality Alkali catalyst
gradually deteriorates due to improper handling and inap- he commercial production methodology frequently uses
propriate storage conditions, which cause various chemical alkaline media for the transesteriication of the oil or fats,
reactions such as hydrolysis, polymerisation and oxidation in the presence of an alcohol, producing methyl esters of
to occur. Two-step processes, such as acid-catalysed esteri- fatty acids and glycerol. he detailed reaction mechanism
ication followed by base-catalysed transesteriication, have of alkali-catalysed transesteriication has been described
been developed for these oils in which initially the free fatty as a three-step process (Ma and Hanna, 1999; Meher et al.
acids are converted to fatty acid methyl esters by an acid- 2006). Sodium hydroxide or potassium hydroxide is used as
catalysed pretreatment and then transesteriied using an the basic catalyst, with either methanol or ethanol as well as
alkaline catalyst in the second step (Ghadge and Raheman, the vegetable oil. Sodium hydroxide is less expensive and
2005; Velkovic et al., 2006). A two-stage transesteriication produces a high product yield and is therefore the one that
process for crude Jatropha curcas L. seed oil with a high is widely used in large-scale processing (Demirbas, 2003).
content of free fatty acids was studied by Berchmans and he alkaline catalyst concentrations in the range of 0.5–1%
Hirata (2008). he irst stage was an acid pretreatment proc- by weight yield a 94–99% conversion rate of most vegetable
ess which reduced the free fatty level to < 1%. he second oils into esters (Barnwal and Sharma, 2005; Srivastava and
stage was an alkali-base catalysed transesteriication proc- Prasad, 2000).
ess which gave a 90% methyl ester yield. Alkali-catalysed transesteriication is much faster than
he conversion rate increases with reaction time and acid-catalysed transesteriication and is also less corrosive
temperature, and therefore these factors are important in the to industrial equipment. Consequently it is the most often
transesteriication process, which can occur at diferent tem- used process commercially (Agarwal, 2007; Ma and Hanna,
peratures depending on the oil used. Generally the reaction is 1999; Marchetti et al. 2007). here are several disadvantages
carried out close to the boiling point of methanol (60–70°C) at in using an alkaline catalysis process, although it does ofer
atmospheric pressure and at a molar ratio (alcohol to oil) of high conversion levels of triglycerides to their corresponding
6:1 (Huaping et al., 2006; Pramanik, 2003; Ranganathan et al., methyl esters with short reaction times. he process is energy
2008; Srivastava and Prasad, 2000). Freedman et al. (1986) intensive, recovery of glycerol is diicult, the alkaline cata-
observed that temperature clearly inluenced the reaction rate lyst has to be removed from the product, alkaline wastewater
and yield of esters when they investigated transesteriication generated requires treatment and the level of free fatty acids
of soybean oil with methanol (6:1) at 32°C, 45°C and 60°C. and water greatly interfere with the reaction. he content of
he stoichiometric ratio for transesteriication requires free fatty acids present in oils should be lower than 0.5% wt%
3 mol of alcohol per mole of triglyceride to yield 3 mol of (Ma and Hanna, 1999) and the water concentration should
fatty esters and 1 mol of glycerol. he transesteriication be limited to 0.1 wt% or less (Hass, 2004). he risk of free
reaction is shifted to the right by using excess alcohol or acid or water contamination results in soap formation, mak-
continuously removing one of the products from the reac- ing downstream recovery and puriication very diicult and
tion mixture. A molar ratio of 6:1 (with alkali as the catalysts) expensive (Barnwal and Sharma, 2005; Fukuda et al. 2001).
is normally used in industrial processes to obtain yields of For substrates such as waste cooking oil with a 2% (w/w)
methyl esters higher than 98% by weight. Ratios greater than normal concentration of free fatty acid, pretreatment of the
6:1 do not increase the yield, but tend to interfere with the oil by esteriication with alcohol using sulphuric acid is rec-
separation of glycerol because there is an increase in glyc- ommended, continuing afterwards with the normal alkali-
erol solubility. When glycerine remains in solution, it helps catalysed transesteriication process (Canakci, 2007). For
drive the equilibrium back to the left, lowering the yield of producing diesel, Tiwari et al. (2007) found that the optimum
esters (Tomasevic and Marinkovic, 2003). When using acid combinations for reducing the free fatty acids of Jatropha
catalysts the desirable product is obtained with 1 mol% of curcas oil from 14% to < 1% were 1.43% (v/v) sulphuric acid
sulphuric acid with a molar ratio of 30:1 at 65°C, and a con- catalyst, 0.28 (v/v) methanol-to-oil ratio and 88 min reaction
version of 99% is achieved in 50 h. time at 60°C compared to 0.16 (v/v) methanol-to-pretreated
oil ratio and a 24 min reaction time at 60°C. his process gave
an average yield of biodiesel of > 99%.
he efects of catalysts
To make the transesteriication process possible, a catalyst Acid catalyst
in the form of an alkali or acid, or a heterogeneous catalyst Biodiesel can also be produced conventionally by using an
such as an enzyme, etc is required (Du et al., 2004; Hama acid to catalyse the transesteriication process. Sulphuric
et al., 2004; Noureddini et al., 2005; Oda et al., 2005; Shieh acid, sulphonic acids and hydrochloric acids in methanol
et al. 2003; Zhang et al., 2003a). Bases catalyse the reaction are the usual acid catalysts but the most commonly used is
by removing a proton from the alcohol, thus making it more sulphuric acid (Demirbas, 2005; Freedman et al. 1986; Ma
4 Wilson Parawira
and Hanna, 1999). he mechanism of acid-catalysed trans- transesteriication of the free fatty acids and the use of mild
esteriication of vegetable oil proceeds via protonation of conditions in the process, with yields of at least 90%. he
the carbonyl group of the ester. his leads to the carbonation enzymatic process is therefore a sustainable biotechnologi-
which, after a nucleophilic attack of the alcohol, produces cal alternative to chemical catalysis (Devanesan et al., 2007;
a tetrahedral intermediate. his intermediate eliminates Mamoru et al., 2001; Oznur and Melek, 2002). he advan-
glycerol to form a new ester and to regenerate the catalyst tages and disadvantages of the methods of catalysing the
(Meher et al. 2006). Acid catalysts are used if the trigyl- transesteriication for biodiesel production are summarised
ceride has a higher free fatty acid content and more water. in Table 1.
he amount of acid catalyst that is required to be added to The enzymatic catalysed transesterification can be car-
the reactor varies from 0.5 mol% to 1 mol%, although some ried out by extracellular and intracellular lipases. Lipase-
authors have used 3.5% (Zhang et al., 2003b). In general, a catalysed transesterification is more appropriate for
1 mol% of sulphuric acid is a reasonable amount for a inal the production of biodiesel from feedstocks containing
conversion rate of 99% in a time of around 50 h. Although high free fatty acids and water, such as waste or recycled
the yields can be high, the corrosiveness of the acids may oils and greases, because the free fatty acids are directly
cause damage to the equipment and the reaction rate can esterified into biodiesel (Canakci, 2007; Hsu et al., 2003;
also be low, sometimes taking more than a day to inish Nelson et al., 1996; Rivera et al., 2007). Biocompatibility,
(Freedman et al. 1986). According to some authors, the biodegradability and environmental acceptability of
reactions are also slow, requiring typically temperatures the biotechnological procedures when using lipase as
above 100°C and more than 3 h to complete the conversion a catalyst are the desired properties in this alternative
(Meher et al. 2006). For example, Freedman et al. (1986) biodiesel production method (Devanesan et al., 2007;
studied the transesteriication of soybean oil in the pres- Marchetti et al., 2007). Two major problems in using
ence of 1% sulphuric acid with an alcohol/oil molar ratio of lipases to catalyse the transesterification process for
30:1 at 65°C, and the conversion was completed in 20 h. As biodiesel production are that the lipase activity is lower
in the alkali-catalysed transesteriication process, if excess than that of chemical catalysts and the enzyme is liable
alcohol is used in the experiment then a better conversion to deactivation by lower alcohols. Harding et al. (2007)
of the triglycerides is obtained, but recovering glycerol recently made a life-cycle comparative study between
becomes more diicult and that is why the optimal rela- the alkali and the enzyme catalysis for the production of
tionship between the alcohol and the raw material should biodiesel using five flow-sheet options from rapeseed oil.
be determined experimentally, taking into account each The life-cycle assessment showed that the enzymatic-
process as an individual problem. Recently, Guan et al. catalysed biodiesel production route was environmen-
(2009) investigated the transesteriication of corn oil with tally more favourable with improvements seen in all
methanol using ptoluene sulphonic acid in the presence of impact categories such as global warming, acidification
dimethyl ether. hey obtained a 100% oil conversion using and photochemical oxidation. The lower pressures and
a methanol/oil ratio of 6:1 at 80ºC for 2 h in the presence of temperatures obtained in biological catalysis help give
dimethyl ether. it the more favourable life-cycle assessment results. The
advantages and disadvantages of using lipase as catalysts
Heterogeneous catalysts for the transesterification for biodiesel production are
Heterogeneous catalysts, such as enzymes, amorphous given in Table 2.
zirconia, titanium and potassium zirconias, heterogenised
on organic polymers have also been used for catalysing the
transesteriication of vegetable oils. Huaping et al. (2006)
Table 1. Comparison of the diferent transesteriication methods for
demonstrated the potential of preparing biodiesel from the production of biodiesel (Crabbe et al., 2001; Chen and Wu, 2003;
Jatropha curcas oil catalysed by a solid super base of cal- Watanabe et al., 2000).
cium oxide with its excellent reining process. When treated Variable Alkali catalysis Acid catalysis Lipase catalysis
with ammonium carbonate solution and calcinated at high Reaction 60–70 55–80 30–40
temperature, calcium oxide becomes a solid super base, temperature (°C)
which shows high catalytic activity in transesteriication. Free fatty acid in Saponiied Esters Methyl esters
raw materials products
Under optimum conditions, the conversion of Jatropha cur-
Water in raw Interference with Interference No inluence
cas oil can reach a level of 93%. he heterogeneous catalyst
materials reaction with reaction
eliminates the additional costs associated with the homoge- Yields of methyl Normal Normal Higher
neous sodium hydroxide used to remove the catalyst after esters
transesteriication. Recovery of glycerol Diicult Diicult Easy
Puriication of Repeated washingRepeated None
Lipase catalyst methyl esters washing
Recently, it has been found that enzymatic catalysis spe- Production cost of Inexpensive Inexpensive Relatively
ciically synthesise alkyl esters resulting in a high purity catalyst inexpensive
product, allows simple recovery of the glycerol, the total Reaction time Short Short (9 h) Long (36 h)
Table 2. he advantages and disadvantages of using lipases in reduce processing costs (Hsu et al., 2004a). Immobilisation
transesteriication (Chen and Wu, 2003; Fukuda et al., 2001, 2007; is also used to increase enzyme stability. However, the cost
Marchetti et al., 2007) of enzymes remains a barrier to their industrial implemen-
Advantages Disadvantages tation (Nelson et al., 1996; Shimada et al., 2002).
Biocompatible, biodegradable Loss of some initial activity due to
and environmental acceptability the volume of the oil molecule
Lipases catalyse more speciic he immobilised lipase is Immobilised-extracellular-lipase catalysed
reactions than chemical catalysts, deactivated by lower alcohols such transesteriication for biodiesel production
and hence produce purer as methanol and ethanol
products Most of the researches on the transesteriication of vegeta-
here is the possibility of he number of support enzymes is ble oils for biodiesel production use the pure commercial
regeneration and reuse of the not uniform enzymes in several reaction media (solvents, the presence of
immobilised residue, because it
additives, polar ions solutions, supercritical luids or using
can be left in the reactor if the
reactive low is maintained the enzyme immobilised on celite and polymers). Several
he use of enzymes in the he production of commercial reports using immobilised extracellular lipases have dem-
reactors allows the use of high enzymes is still prohibitively costly, onstrated the high transesteriication activity of various fat
concentrations of them and although the potential costs are and oil substrates including low-value feedstocks such as
that allows for longer activation being reduced greases. hese immobilised lipases catalyse the alcoholysis
of the lipases
of animal fats and vegetable oils with primary and second-
Immobilisation of the lipase could he activity of the lipase is
protect it from the solvent that relatively lower than that of
ary alcohols. For example, Nelson et al. (1996) demonstrated
the lipase-catalysed production of biodiesel from soybean
could be used in the reaction and chemical catalysts

that will prevent all enzyme oil, rapeseed oil, and tallow and recycled restaurant grease.
particles getting together hey established conditions for converting tallow and recy-
Separation of the product will be – cled grease to alkyl esters with more than a 95% yield using
easier using this catalyst,
various commercial lipases. hey also developed conditions
producing a product of very
high purity with less or no efective for transesterifying feedstocks high in free fatty con-
downstream operations tent to their respective alkyl esters. Simple alkyl esters for use
A greater thermal stability of – as a biodiesel were readily produced from tallow and greases
the enzyme due to the native state using phyllosilicate-sol-gel-immobilised Pseudomonas cepa-
in which they are used cia lipase (Hsu et al., 2001, 2002, 2004a). he inal conversion
of grease or tallow to alkyl esters was aided by the addition of
molecular sieves (0.4 wt% of substrates) to the reaction mix-
ture. he phyllosilicate sol-gel-matrix-immobilised lipase
Biocatalysed transesteriication for biodiesel efectively converted grease and tallow to ethyl esters with
production a > 95% yield using ethanol. he immobilised enzyme could
he enzyme that was found to be capable of catalysing be reused at least ive times without losing its activity.
transesteriication is lipase (glycerol ester hydrolase EC Wanatabe et al. (2000) demonstrated the conversion of oils
3.1.1.3). his enzyme is produced intracellularly and extra- to biodiesel using Candida antarctica lipase immobilised on
cellularly in several microorganisms, such as Mucor miehei, a macroporous acrylic resin. Shimada et al. (2002) produced
Rhizopus oryzae, Candida antarctica, hermomyces lanugi- esters from vegetable oils using immobilised Candida antarc-
nous, Pseudomonas luorescens, Pseudomonas alcaligenes, tica lipase in a continuous reaction process, which is neces-
Pseudomonas mendocina and Pseudomonas cepacia (Du sary for the economic production of these esters as biodiesel.
et al., 2004; Hama et al., 2004; Noureddini et al., 2005; Oda Lipases from C. antarctica and hermomyces lanuginosa
et al., 2005; Shieh et al., 2003). Enzymatic biodiesel produc- supported on granulated silica also have high esteriication
tion is possible using both intracellular and extracellular activity for ester synthesis (Christensen et al., 2003). Hsu et al.
lipases. (2004b) compared the production of alkyl esters from fats and
In all of the work in the literature on lipases, the enzymes oils using lipase from Pseudomonas cepacia and hermomyces
or whole cells are immobilised and used for catalysis since lanuginosa immobilised in a phyllosilicate sol-gel matrix.
they are usually more stable than free lipases and whole cells. At 50°C and 48 h reaction time, immobilised hermomyces
he advantage of immobilisation is that the enzyme can be lanuginose lipase gave higher alkyl ester yields (70–100%)
reused without separation and the immobilised process is from fats and oils regardless of chain length or the degree of
highly eicient compared to the use of free enzymes and unsaturation of the acyl groups in the triacylglycerols than did
whole cells (Mateo et al., 2007). Immobilising the enzyme immobilised Pseudomonas cepacia lipase (20–90%), which
(both intracellular and extracellular) in a suitable biomass preferred unsaturated oils. Both immobilised lipases catalysed
support particle (BSP) increases the cost-efectiveness of the ester formation (80–90%) from greases containing a range of
enzymatic process. he immobilisation of enzymes extends free fatty acids (2.6–36%). However, the addition of molecu-
their utility since immobilisation promotes recovery and lar sieves had no efect on ester yields in the hermomyces
reuse, and the development of continuous processes which lanuginosa lipase-catalysed transesteriication of greases but
6 Wilson Parawira
did improve yields (5–10%) in the immobilised Pseudomonas regarded as immobilised and can be separated easily. his
cepacia lipase-catalysed reactions. can clearly reduce the cost of the transesteriication produc-
Shah and Gupta (2007) reported transesteriication of tion as reported by Ban et al. (2001) and Hama et al. (2004;
Jatropha oil with ethanol using Pseudomonas cepacia lipase 2007) where they used R. oryzae for the transesteriication
immobilised on celite. hey optimised the process with process of vegetable oils. Yeast cells containing high lipase
regard to pH tuning, immobilisation, varying water content activity were developed, permeated by air drying and used
in the reaction media, varying enzyme concentration and as whole-cell biocatalysts for methanolysis in a solvent-free
varying temperature of the reaction. he best yield (98% and water-containing system (Matsumoto et al., 2001). he
w/w) was obtained at 50°C in the presence of 4–5% (w/w) methyl ester content in the reaction mixture reached 71%
water at 8 h. he immobilised biocatalyst could be used four (wt) after 165 h reaction at 37°C with the stepwise addition
times without any loss of activity. of methanol. hese results indicated that an eicient whole-
From the literature, it seems that diferent enzymes have cell biocatalyst can be prepared by intracellular overproduc-
diferent requirements for biodiesel synthesis. Hernandez- tion of lipase in yeast cells and then permeabilised.
Martin and Otero (2008) carried out a comparative study of Processes using lipase-producing microorganisms immo-
diferent immobilised lipase preparations for the synthesis bilised within BSPs as whole-cell biocatalysts are much more
of biodiesel from diferent vegetable oils (sunlower, borage, straightforward since no puriication of lipase is necessary
olive and soybean) in methanol and ethanol. hey observed and cell immobilisation can be achieved naturally during
that loss of lipase activity induced by the nucleophile was batch cultivation. Ban et al. (2001; 2002) found that dried
greater with methanol than with ethanol, and was greater Rhizopus oryzae cells immobilised within BSPs eiciently
for the sn1(3)regio-speciic lipase Lipozyme® (a registered catalysed methanolysis in the presence of 4–20% water and
trademark of hermomyces. lanuginosus lipase from Novo that the lipase activity of Rhizopus oryzae cells treated with
Nordisk, Bargsvaerd, Denmark) TL IM than for the non- 0.1% glutaraldehyde solution was maintained during the six
speciic lipase Novozyme® 435 (a registered trademark repeated-batch reaction cycles, with the methyl ester con-
of Candida antarctica type B lipase, from Novo Nordisk, tent in each cycle reaching 70–80% within 72 h.
Bargsvaerd, Denmark). Maximum conversion rates were Tamalampundi et al. (2008) recently reported that whole-
obtained with Lipozyme® TL IM with a molar ratio of alco- cell R. oryzae immobilised on to BSPs catalysed the meth-
hol to fatty acid residues of 0.33. In contrast, Novozyme® 435 anolysis of Jatropha oil more eiciently than Novozym 435
required at least a 2:1 ratio. Alcoholysis of the vegetable oils in their comparative study of immobilised whole-cell and
was faster with Lipozyme® TL IM than with Novozyme® 435. commercial lipases as biocatalysts. he presence of water in
he use of a high loading of Novozyme® 435 (50% w/w) and Jatropha oil had a signiicant efect on the rate of methanol-
a large molar excess of ethanol were required to obtain an ysis, and Rhizopus oryzae cells exhibited their highest activ-
initial rate similar to that obtained with Lipozyme® TL IM at ity in the presence of 5% (v/v) of added water. In contrast
a lower enzyme loading (10% w/w) and an equimolar ratio to Rhizopus oryzae cells, Novozym 435 activity was inhib-
of ethanol and free fatty acid residues. Novozyme® 435 pro- ited by the presence of added water and it needed almost
duced quantitative conversions in only 7 h at 25°C, but com- anhydrous media for eicient catalysis. Several researchers
plete conversions were not obtained with Lipozyme® TL IM (Du et al., 2004; Shimada et al., 1999) have reported that the
in a single step process. he three-stage stepwise addition of commercially available Novozym 435 (Candida antarctica
ethanol yielded 84% conversion to ethyl esters for Lipozyme® lipase B immobilised on acrylic resin) was the most efec-
TL IM. Hence the choice of Novozyme® 435 to catalyse the tive catalyst among the lipases tested for the production of
transesteriication process should always be made. biodiesel fuel. However, the laborious and expensive puri-
ication processes of this lipase from culture broth restrict
its application in biodiesel fuel production on an industrial
Immobilised whole-cell-catalysed scale. he direct use of lipase-producing Rhizopus oryzae
transesteriication for biodiesel production cells is thus an efective way to reduce the cost of lipase
he use of extracellular lipase as a catalyst requires compli- production, which is the main hurdle to the commercialisa-
cated recovery, puriication and immobilisation processes for tion of the enzymatic process (Ban et al., 2002; Li et al., 2008;
industrial applications (Ban et al., 2001). Consequently, the Zeng et al., 2006).
direct use of a whole-cell biocatalyst of intracellular lipases he facilitatory efect of immobilised lipase-producing
has received considerable research attention (Devanesan Rhizopus oryzae cells on acyl migration in biodiesel fuel pro-
et al., 2007; Kaieda et al., 1999; Li et al., 2007; Matsumoto duction was reported by Oda et al. (2005). Rhizopus oryzae
et al., 2001; Tamalampundi et al., 2008; Fukuda et al., 2008). cells producing 1,3positional speciicity were cultured with
he cost of the transesteriication process using lipase can be polyurethane-foam BSPs in a scaled-up 20 L air-lift bioreactor.
signiicantly decreased by using immobilised intracellular he cells, immobilised within BSPs, were used as a whole-cell
lipase (whole-cell immobilisation) instead of extracellular biocatalyst in a repeated batch-cycle methanolysis reaction
lipase which demands complex puriication stages before of soybean oil. his repeated batch methanolysis reaction
immobilisation. Whole-cell biocatalysts are prepared simply using the dried BSP-immobilised cells maintained a methyl
by cultivation, and the enzymes trapped inside the cells are ester content of 65–80% during 20 batch reaction cycles. he
hydrolytic activity of the whole-cell biocatalyst, on other Yong and Shiyi, 2007). Devanesan et al. (2007) reported a
hand, was stable regardless of the number of reaction cycles. maximum yield (72%) of biodiesel from the transesterii-
Fukuda et al. (2007) investigated the methanolysis reaction in cation of Jatropha oil and a short-chain alcohol (methanol
a packed-bed reactor (PBR) using dried Rhizopus oryzae cells on hexane) using a commercial immobilised Pseudomonas
immobilised within BSPs in preparation for a large-scale prac- luorescens MTCC 103 in sodium alginate gel as a whole-cell
tical biodiesel fuel production. he reaction was carried out biocatalyst. he optimum conditions for the transesterii-
in a glass column (of 25 mm internal diameter and 400 mm cation were a temperature of 40°C, pH of 7.0, molar ratio of
in height). he reaction conditions were investigated in an 1:4, amount of beads of 3 g and reaction time of 48 h.
attempt to increase the reaction rate and the lipase stability. Biodiesel is usually produced from food-grade vegeta-
Comparison with the methanolysis reaction in a shaken bot- ble oils that are more expensive than diesel fuel (Canakci,
tle suggested that the PBR enhances repeated-batch meth- 2007). herefore, biodiesel produced from food-grade vege-
anolysis by protecting immobilised cells from physical dam- table oil is currently economically infeasible. he feedstock
age and excess amounts of methanol. Emulsiication of the in many studies for biodiesel production was focussed on
reaction mixture containing soybean oils and water improved edible vegetable oils such as sunlower oil, soybean oil or
the methanolysis reaction rate. When the low rate was varied cottonseed oil, but the researches on animal fats are few.
between 5 L/h and 55 L/h, they obtained the highest methyl Waste cooking oils, restaurant grease and animal fats are
ester content of over 90% at a low rate of 25 L/h in the irst potential low-cost feedstocks for biodiesel production and
cycle, and over 80% was still being maintained after the tenth are available in large amounts in many countries. Edible and
cycle. hey concluded that this was a promising process for non-edible lard can be used as biodiesel feedstocks owing
industrial biodiesel fuel production. to their highly centralised generation in slaughter/process-
Ban et al. (2002) also reported the stable repeated use ing facilities and their historically low prices. Immobilised
of whole-cell biocatalysts immobilised within BSPs for Candida sp. 99–125 whole cells were successfully used in
biodiesel fuel production. he lipase activity of Rhizopus enzymatic esteriication for biodiesel production from lard
oryzae cells immobilised on BSPs without glutaraldehyde by Lu et al. (2007). here the optimal conditions for process-
treatment decreased considerably (to 50% after six batch ing 1 g of lard were as follows: 0.2 g of immobilised whole
cycles) in the presence of methyl esters produced by meth- cells, 8 mL of nhexane as solvent, 20% of water based on the
anolysis, but the activity of cells treated with 0.1% glutaral- fat weight, a temperature of 40°C and the three-step addi-
dehyde solution showed no signiicant decrease during six tion of methanol. he fatty acid methyl esters yield was 87%
batch cycles and the methyl ester in the reaction mixture and the immobilised Candida sp. 99–125 whole cell proved
reached 70–83% in each cycle. Also, good methanolysis of to be stable when used repeatedly for 180 h.
90% methyl ester yield was reported by Ban et al. (2001) he display of novel enzymes on the yeast cell surface
when using Rhizopus oryzae cells immobilised within is a very powerful method for the development of eicient
BSPs as whole-cell biocatalysts for biodiesel production. whole-cell biocatalysts, because the difusion problem of
To enhance the methanolysis activity of the immobilised substrate and product is circumvented (Kondo and Ueda,
cells under the culture conditions used, various substrate- 2004). Kondo and Fukuda (2007) developed a new method
related compounds were added to the culture medium, of for displaying novel enzymes and their applicability to
which olive oil and oleic acid were signiicantly efective. the production of biofuels from biomass. Plasmids were
he cost of oil sources is another inluential factor on constructed of various lengths for the cell surface display
biodiesel cost; producing biodiesel from reined vegeta- of lipases by fusion with the cell-wall anchoring proteins
ble oils is obviously not competitive and therefore there is -agglutinin and locculin lo 1p, and introduced into yeast
a need to explore the use of relatively low-cost crude and cells. he biodiesel production from plant oil and methanol
acidiied oils for biodiesel production. Diferent feedstocks catalysed by yeast cells displaying Rhizopus oryzae lipase
(reined, crude and acidiied rapeseed oil) were adopted was investigated in a water-containing system without an
by Li et al. (2007) for biodiesel production in a tert-butanol organic solvent. It was found the methyl ester yield reached
system using whole-cell Rhizopus oryzae (IFO 4697) cells 80% by the stepwise addition of methanol to the reaction
immobilised within BSPs as a catalyst. hey found that when mixture (Matsumoto et al., 2001). he displayed enzymes
acidiied rapeseed oil was used as a feedstock, the reaction are regarded as a type of self-immobilised enzyme on the
rate and inal methyl ester yield were signiicantly higher cell surfaces. Cell-surface engineered Saccharomyces cerevi-
than that of reined and crude rapeseed oil. he reaction siae exhibiting the expression of Rhizopus oryzae lipase was
rate increased with the increase of free fatty acid content in successfully used by Ueda et al. (2002) to produce biodie-
oils, the water content had a varied inluence on reaction sel. Matsumoto et al. (2002) constructed a yeast whole-cell
rate and biodiesel yield, and using adsorbent to remove biocatalyst overproducing lipase with a pro-sequence from
excess water increased biodiesel yield signiicantly. Rhizopus oryzae IFO 4697 and successfully used it for biodie-
For the industrial transesteriication of fats and oils, sel production. Such whole-cell biocatalysts overproducing
Pseudomonas sp. immobilised with sodium alginate gel intracellular lipase can be used to reduce the cost of lipase
can be used directly as a whole-cell biocatalyst (Devanesan preparation signiicantly and ofer a promising prospect for
et al. 2007; Foidl et al., 1996; Mohamed and Uwe, 2003; industrial biodiesel production.
8 Wilson Parawira
Other attempts to improve lipase-catalysed continuous reaction process without the use of any organic
transesteriication solvent is needed for the industrial production of biodiesel.
A better solution is to regenerate immobilised lipase for
Various alcohols are being investigated for the transesteri- transesteriication when its activity becomes lower than a set
ication process using lipase including methanol, ethanol, value. Immobilised lipase is frequently deactivated by lower
isopropanol and butanol. However, immobilised lipase is alcohols with the deactivation being caused by the immis-
frequently deactivated by lower alcohols such as methanol cibility between triglycerides and methanol and ethanol.
and ethanol. Pretreatment of immobilised Candida ant- When these lower alcohols are adsorbed to the immobilised
arctica lipase enzyme (Novozyme® 435) for biodiesel fuel enzyme, the entry of triglycerides is blocked, which causes
production from plant oil was demonstrated by Samukawa the reaction to stop. An alcohol with three or more carbon
et al. (2000) to reduce the deactivation of the lipase. he atoms, preferably 2-butanol or tert-butanol, can regener-
methanolysis reaction was observed to be much faster when ate the deactivated immobilised enzyme. Chen reported
Novozyme® 435 was preincubated in methyl oleate for 0.5 h that the activity of immobilised lipase could be signiicantly
and subsequently in soybean oil for 12 h. When preincubated increased when such alcohols were used for an immersion
Novozyme® 435 was used, the methyl ester content reached pretreatment of the enzyme. he activity of Novozyme® 435
over 97% within 3.5 h by the stepwise addition of 0.33 mol increased about ten-fold in comparison to the enzyme not
equivalent of methanol at 0.25–0.4 h intervals. Improvement being subjected to any pretreatment. Following the complete
in the lipase-catalysed synthesis of fatty acid methyl esters deactivation of lipase by methanol, washing with 2-butanol
from sunlower oil was achieved by carrying out the alco- and tert-butanol successfully regenerated the enzyme and
holysis in organic solvents (Soumanou and Bornscheuer, restored it to about 56% and 75% of its original activity level,
2003). he highest conversion (80%) was found in nhexane respectively.

and petroleum ether. Apart from substrate concentrations, temperature has
Jackson and King (1996) used immobilised lipases as also been reported to afect the rate of the transesteriication
biocatalysts for the transesteriication of corn oil in lowing reaction considerably. he trend observed in all studies that
supercritical carbon dioxide and reported an ester conver- investigated the efect of temperature on the production of
sion of more than 98%. But the activity of the immobilised biodiesel by lipase was that initially the reaction increased
enzyme is inhibited by the methanol and glycerol present as the temperature increased. However, the reaction rate
in the mixture. he use of tert-butanol as a solvent, the con- decreased sharply at the onset of denaturation of the enzyme
tinuous removal of glycerol and the stepwise addition of in all the studies that investigated the efect of temperature
methanol are found to reduce the inhibitory efects, thereby on the production of biodiesel by lipase (Al-Zuhair et al.
increasing the cost-efectiveness of the process (Li et al., 2003). he optimum operating temperature for lipases from
2006; Royon et al., 2007; Samukawa et al., 2000). diferent sources is reported to be approximately 40°C.
he efect of alcohol on the enzymatic production of Biodiesel production can also be improved by using a
biodiesel from vegetable oils was also investigated. In the mixture of lipases. Kim et al. (2007) optimised biodiesel pro-
absence of organic solvents, Samukawa et al. (2000) and duction by using a mixture of immobilised Rhizopus oryzae
Shimada et al. (2002) have both reported that biodiesel and Candida rugosa lipases. he ratio of Rhizopus oryzae
production increased when the methanol concentration and Candida rugosa lipases in the mixture was optimised
increased up to an oil-to-methanol ratio of 3:1 equivalents, to 3:1 (w/w). It was found that when 3 mmol of methanol
and decreased when methanol concentration was further was initially added to the reaction medium and 3 mmol of
increased. Efective methanolysis using extracellular lipase methanol was added every 1.5 h during biodiesel produc-
has been reported to improve with the stepwise addition tion, biodiesel conversion reached over 98% at 4 h. In addi-
of methanol through which a 90–95% conversion can be tion, when the mixture of immobilised lipases was reused,
achieved even after 50 and 100 cycles of repeated operation biodiesel conversion was maintained at levels exceeding
(Samukawa et al., 2000; Shimada et al., 2002; Soumanou and 80% after ive reuses.
Bornscheuer, 2003; Watanabe et al., 2000). Room-temperature ionic liquids (organic salts entirely
here have been several further attempts to overcome the composed of ions) are recently emerging as desirable sub-
problem of deactivation of immobilised enzyme by lower stituents for volatile, toxic and lammable organic solvents,
alcohols such as methanol and ethanol, and to improve which are major causes of environmental pollution. Ha et al.
the lipase activity. For example, Nelson et al. (1996) used (2007) reported improved lipase-catalysed biodiesel pro-
hexane as a diluent to prevent the deactivation by lower duction from soybean oil in ionic liquids. Among tested 23
alcohols. Samukawa et al. (2000) attempted to maintain ionic liquids, the highest fatty acid methyl esters production
a very low concentration of methanol during the reaction. after 12 h at 50°C was achieved in [Emim] [TfO].” [Emim]
However, these methods have some inherent problems. [TfO] from C-TRI (Suwon, Korea) was one of the 23 ionic
Using a diluent decreases the reaction rate and the precise liquids used for production of fatty acid methyl esters and
control necessary to maintain the methanol concentration compared with tert-butanol, a commonly used solvent. he
at a very low level is not appropriate for the production of production yield of 80% was eight times higher compared to
biodiesel on a large scale. From an economic point of view, a the conventional solvent-free system, and it was 15% higher
than the fatty acid methyl esters production system using cells during biodiesel production in a solvent-free and tert-
tert-butanol as an additive. he efect of substrate molar ratio butanol system. During repeated Rhizopus oryzae whole-
(methanol: soybean oil) fatty acid methyl esters production cell catalysed biodiesel production, whole-cell stability
was also investigated. he optimum substrate molar ratio of was still poor even with the stepwise addition of methanol,
methanol to soybean oil for fatty acid methyl esters produc- while it improved considerably in a tert-butanol system
tion in [Emim] [TfO] was found to be 4:1. Methanolysis in compared to that in a solvent-free system. he diference in
[Emim] [TfO] was signiicantly decreased when molar ratio whole-cell stability was found to be due to the diferences
of methanol to soybean oil was increased to 8:1 and greater. of product accumulation between the solvent-free and the
his might be explained by the inactivation of Novozym 435 tert-butanol systems. After a 144 h reaction, glycerol and
caused by high methanol concentration. hese high produc- methyl ester accumulated in the cell in the solvent-free sys-
tion yield results in ionic liquids show that they are potential tem to an extent of about 1000 mg/g and 350 mg/g dry mass,
reaction media for biodiesel production. However, ionic respectively, while in the tert-butanol system, the glycerol
liquids are considered to be expensive solvents and their use and methyl ester were maintained at a relatively low level of
would increase the cost of biodiesel production. approximately 100 mg/g and 2 mg/g dry mass, respectively.
Optimisation of the reaction parameters involved in hey concluded that the accumulated glycerol inluenced
lipase-catalysed biodiesel production is commonly made by the whole-cell stability through mass transfer only, while
varying one factor at a time and keeping the others constant the accumulated methyl ester inluenced whole-cell sta-
(Hsu et al., 2003; Iso et al., 2001; Salis et al., 2005, 2008). But bility through both mass transfer limitations and product
this classical method is ineicient as it fails to explain the inhibition.
relationships between the variables and the responses when
there are interactions between the variables. Response sur-

Research gaps
face methodology (RSM) is an efective statistical technique
for the investigation of complex processes like lipase-cata- he studies on biodiesel production from vegetable oil using
lysed biodiesel production because it has many advantages lipase in the literature are purely experimental. Kinetic
(Rodrigues et al., 2008). he use of RSM for optimising enzy- information on the rate of product formation and the efects
matic biodiesel production has been reported by various of changes in operating conditions are essential for the
authors. Chang et al. (2005) investigated the alcoholysis of designing of suitable reactors, control systems and process
canola oil by Novozym 435 using RSM and central compos- optimisation. here are only limited reports on the kinetics
ite design (CCD), and obtained a methyl ester yield of 99% of transesteriication reactions in the literature (Al-Zuhair
under optimum conditions. Shieh et al. (2003) studied the et al., 2006, 2007; Jansen et al., 1999). Experimental deter-
optimisation of lipase-catalysed synthesis for biodiesel using mination of the separate efects of palm oil and methanol
RSM in combination with CCD (soybean oil methyl ester) by concentrations on the rate of their enzymatic transesterii-
Lipozyme IM77 and obtained a maximum molar conver- cation was used to propose the Ping Pong Bi Bi mechanism
sion of 92%. Dermirkol et al. (2006) reported the optimisa- as the suitable kinetic model for the production of biodiesel
tion of enzymatic methanolysis of soybean oil by RSM using (Al-Zuhair et al., 2007). he reactions were carried out in an
Lipozyme RM IM. Rodrigues et al. (2008) optimised lipase- n-hexane organic medium with lipase from Mucor miehei.
catalysed ethanolysis of soybean oil in a solvent-free system Kinetic studies reported in the literature investigated the
using CCD and RSM and obtained a yield conversion of 96% esteriication of free fatty acids rather than the transesterii-
under the optimum conditions with a relatively low enzyme cation of triglycerides (oil) which is the substrate of industrial
content and in a short time. he improvement of biodiesel interest (Jansen et al., 1999; Watanabe et al., 2002). here is a
production by lipozyme TL IM-catalysed methanolysis need for more kinetic studies on the production of biodiesel
using RSM and an acyl migration enhancer was reported by from vegetable oils using lipase in single- and multi-step
Wang et al. (2008). hey optimised the process of biodiesel processes.
production from corn oil catalysed by lipozyme TL IM with here is also a need to carry out experiments on lipase
regard to the efect of enzyme dosage, the ratio of tbutanol catalysed in pilot-scale or full-scale reactors to conirm the
to oil (v/v) and the ratio of methanol to oil (mol/mol) on the beneits of using lipase-catalysed transesteriication. Most of
methyl esters yield of the methanolysis. Waste oil was found the experimental data in the literature were obtained using
to be a suitable feedstock and could yield 93.7% methyl esters reactors ranging from 2 mL (Eppendorf tubes) to 50 mL (Ban
under the optimum conditions that they created. Adding et al., 2001; Iso et al., 2001; Shah et al., 2004; Watanabe et al.,
triethylamine, an acyl migration enhancer, could efectively 2002) with the exception of the excellent work in a 20l packed-
improve the methyl esters yield of the methanolysis of corn bed reactor by Hama et al. (2007). he mixing was eiciently
oil from 85% to 92%. done using orbital shakers or magnetic stirrers and therefore
Using immobilised whole cells such as Rhizopus oryzae cannot replicate the large-scale industrial reactors.
as catalysts for biodiesel production has many advan- Studies in the literature have shown that diferent enzyme
tages; however, the stability of whole-cell biocatalysts was preparations have diferent requirements for the synthe-
reported to be poor in solvent-free systems. Li et al. (2008) sis of biodiesel from diferent vegetable oils and therefore
investigated the factors inluencing the stability of whole transesteriication reactions need to be optimised for each
10 Wilson Parawira
application. Several studies have shown the possibility of varying one factor at a time and keeping the others constant.
recycling the biocatalysts when immobilised, but the reac- RSM and CCD analyses were successfully used by many
tion time is still unfavourable when compared to the alkali- authors to further optimise enzyme-catalysed biodiesel pro-
catalysed transesteriication process. Some critical enzyme duction in solvent-free systems.
properties always must be improved before their implemen- Both immobilised extracellular lipase and whole-cell
tation at an industrial scale, such as stability, activity, inhibi- intracellular lipase can be used to catalyse the transesteri-
tion by reaction products and selectivity. ication process. Several researches have demonstrated that
Biodiesel is usually produced from food-grade vegetable immobilised Rhizopus oryzae whole cells could eiciently
oils that are more expensive than diesel fuel. With the cur- catalyse the methanolysis of reined vegetable oils for biodie-
rent shortage of food and the high prices worldwide, there is sel production in solvent-free systems. he stepwise addi-
a need to focus research on non-food-grade oils, restaurant tion of methanol was recommended so as to minimise the
waste oils and on rendered animal fat as low-cost feedstocks negative efect of methanol on Rhizopus oryzae whole cells.
for biodiesel production. here are large amounts of restau- However, the stability of the whole cell during repeated uses
rant waste oils and rendered animal fats in many countries was poor. In many studies, tert-butanol was demonstrated
that are potentially available for biodiesel production. Waste as an ideal media for biodiesel production, in which the
restaurant oils and animal fats have relatively high levels of stability of the biocatalysts could be enhanced signiicantly.
saturation, are highly contaminated with free fatty acids and A large percentage of the biodiesel fuel cost is associated
moisture in varying amounts, and the technique for biodie- with feedstock oil and enzyme. he cost of enzyme remains
sel production from them needs to be improved. Levels of a barrier to its industrial implementation and much efort
free fatty acids were reported to vary from 0.7% to 41%, and is being made to improve the cost-efectiveness of the
moisture from 0.01% to 55% in waste cooking oils, restaurant process. Several research programmes have been reported
grease and animal fat by Canakci (2007). hese wide ranges which are searching for a lipase which is readily available as
indicate that an eicient process for converting waste grease an industrial enzyme and which works well for catalysing
and animal fats which tolerate a wide range of feedstock the transesteriication. he high cost of the enzymes that
properties needs to be developed. are currently being investigated should not be allowed to
thwart the attempts of carrying out more extensive research
to identify the most promising ones and to determine the
Conclusions optimal conditions for their application. In fact, the results
Developing alternative renewable energy sources to replace of such research should provide the incentive for the com-
traditional fossil fuel has recently become more and more mercial development aimed at inally producing the lipase
attractive due to the high energy demand, the limited enzymes economically on a large scale. he costs of new
resources of fossil fuel and environmental concerns. Of the lipase development are expected to decrease as technol-
several methods available for producing biodiesel, trans- ogy and techniques advance, as fermentation becomes
esteriication of vegetable oils is currently the method of optimised and as less-expensive growth substrates are
choice. Generally, biodiesel is produced by alkali-catalysed explored for the cultivation of the parent microorganisms
transesteriication of the oils. here are several diiculties and as improvements are made in downstream process-
associated with the chemical methods currently used to ing. Utilising immobilised whole-cell biocatalysts instead
produce biodiesel. herefore the enzymatic method is pre- of immobilised lipase is a feasible way to reduce the costs
ferred over chemical methods because, among other advan- of the enzymatic process since it can avoid the complex
tages, it produces a high-purity product and enables easy procedures of isolation, puriication and immobilisation.
separation from the by-product, glycerol. Lipases of various Several attempts are being made to explore ways of reduc-
origins have been widely employed to catalyse the transes- ing the cost of raw material including using waste or used
teriication of vegetable oils under diferent conditions. he oil and the use of non-edible oils. In general, biocatalysts
present paper presents an overview of the biotechnological ofer promising prospects for industrial biodiesel produc-
production of biodiesel that has been reported in the litera- tion, although much further work, such as scaling up and
ture. Since biocatalysts are more expensive than traditional optimization, require further investigation.
chemical catalysts, it is essential to be able to recycle them
so as to overcome economical constraints. his is possible
Acknowledgements
through enzyme immobilisation on solid support, which
allows continuous processes. Methanol, the most frequently Declaration of interest: he author reports no conlicts of
used alcohol, can be enzyme denaturing and mixes poorly interest.
with the oil or the fat. In order to overcome these drawbacks,
a multi-step addition of methanol to the reaction medium
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could be used in the reaction and chemical catalysts

2003). he highest conversion (80%) was found in nhexane respectively.

there are interactions between the variables. Response sur-

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