Crc Biodiesel Paper

8/6/2019 Crc Biodiesel Paper

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Production of Biodiesel Using Immobilized LipaseACritical Review

Kenthorai Raman Jegannathan and Sariah Abang Denis Poncelet Eng Seng

Chan and Pogaku Ravindra

QUERY SHEET

Q1: Au: Diatomaceous

Q2: Au: Please cross-check the year in Belafi-bako.

Q3: Au: Please cite reference Hsu

Q4: Au: Please provide the complete page range for reference Reyed (2007).


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Critical Reviews in Biotechnology, xxxx:112, 2008

Copyright c Informa UK Ltd.

ISSN: 0738-8551 print / 1549-7801 online

DOI: 10.1080/07388550802428392

Production of Biodiesel Using Immobilized LipaseACritical Review

Kenthorai Raman Jegannathan and Sariah AbangCentre of Materials and Minerals, School of Engineering and Information Technology, Universiti

Malaysia Sabah, 88999, Kota Kinabalu, Sabah, Malaysia5

Denis PonceletENITIAA: rue de la Geraudiere B.P. 82225 44332 NANTES Cedex 3, France

Eng Seng Chan and Pogaku RavindraCentre of Materials and Minerals, School of Engineering and Information Technology, Universiti

Malaysia Sabah, 88999, Kota Kinabalu, Sabah, Malaysia10

Increase in volume of biodiesel production in the world scenario proves that biodiesel is ac-cepted as an alternative to conventional fuel. Production of biodiesel using alkaline catalyst hasbeen commercially implemented due to its high conversion and low production time. For theproduct and process development of biodiesel, enzymatic transesterification has been suggestedto produce a high purity product with an economic, environment friendly process at mild re-action conditions. The enzyme cost being the main hurdle can be overcome by immobilization.Immobilized enzyme, which has been successfully used in various fields over the soluble coun-terpart, could be employed in biodiesel production with the aim of reducing the production costby reusing the enzyme. This review attempts to provide an updated compilation of the studiesreported on biodiesel production by using lipase immobilized through various techniques andthe parameters, which affect their functionality.

15

20

Keywords adsorption, alcohol, biodiesel, encapsulation, entrapment, enzymatic transesterifica-tion, immobilization, lipase, vegetable oil

INTRODUCTION

Biodiesel, a hot topic in every countrys policy agenda is a

renewable and environment friendly substitute for petroleum-

based diesel fuel. It appears that the Belgian patent granted25

in 1937 constitutes the first report on what is known today as

biodiesel. It describes the use of ethyl esters of palm oil as diesel

fuel (Knothe, 2005). However, it was not until now that the

biodiesel production from plant oil has become commercially

viable due to the soaring price of crude oil.30

Transesterification of triglycerides with alcohol in the pres-

ence of chemical catalyst or biocatalyst leads to the formationof alkyl ester commercially known as the biodiesel. Enzymatic

transesterification using lipase looks attractive and encourag-

ing for reasons of easy product separation, minimal wastewa-35

Address correspondence to Pogaku Ravindra, Department ofChemical Engineering, School of Engineering and IT, UniversitiMalaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia. E-mail:dr [email protected]

ter treatment needs, easy glycerol recovery and the absence of

side reactions, unlike the chemical catalyst (Ravindra, 2006).

Practical use of lipase in pseudohomogenous reaction systems

presents several technical difficulties such as contamination of

the product with residual enzymatic activity, and economic cost

(Al-Zuhair, 2007). In order to overcome this problem, the en-

zyme is usually used in immobilizedform so that it canbe reused

several times to reduce the cost, and also to improve the qual-

ity of the product. Immobilization refers to the localization or

confinement of an enzyme on to a solid support or on a carrier

matrix. The choice of a carrier is dependent upon several fac-tors, namely: mechanical strength, microbial resistance, thermal

stability, chemical durability, chemical functionality hydropho-

bic/hydrophilic character, easeof regeneration, loading capacity,

and cost, which is important in industrial processing applications

(Karube et al., 1977).

Enzyme immobilization is experiencing an important transi-

tion. Combinations of approaches are increasingly applied in the

design of robust immobilized enzyme by rational combination

of basic immobilization techniques i.e., noncovalent adsorption,

1


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2 K.R. JEGANNATHAN ET AL.

covalent binding, entrapment, and encapsulation. The availabil-

ity of a robust immobilized enzyme at an early stage definitely

enables early insight into process development, and saves cost

for process development and production (Cao et al., 2005). The

article provides a review on the various types of immobiliza-60

tion techniques used for enzymatic production of biodiesel, the

achievements and drawbacks, as well as the various factors,which affect the biodiesel production by using the immobilized

enzyme.

VARIOUS IMMOBILIZATION TECHNIQUES USED65FOR BIODIESEL PRODUCTION

Immobilized enzymes are defined as enzymes physically

confined or localized in a certain defined region of space with

retention of their catalytic activities, and which can be used re-

peatedly and continuously. The term immobilized enzyme70

was recommended at the First Enzyme Engineering Conference

in 1971. In general, the methods for enzyme immobilization can

be classified into two basic categories, based on chemical reten-

tion and physical retention (Bommarius and Riebel-Bommarius2000) as shown in Figure 1.75

An immobilized enzyme has to perform two essential func-

tions: namely, the noncatalytic functions that are designed to aid

separation and the catalytic functionsthat are designedto convert

the targeting substrates within a desired time and space (Cao,

2005). The last few years have witnessed the design of high ac-80

tivity biocatalyst preparations for use in nonaqueous systems

(Shah et al., 2004). Various immobilization techniques have

been employed on lipase used for biodiesel production and they

are discussed as follows.

Adsorption85

Adsorption is the attachment of enzymes on the surface of

support particles by weak forces, such as van der Waals or dis-

persion forces. Adsorption is the most straightforward immobi-

lization procedure. The preparation is easy and the associated

costs are small. This immobilization method involves no toxic

chemical and the resulted immobilized enzyme does not experi-

ence internal mass transfer limitations, unlike cross-linking and

entrapment.

Various carrier particles have been explored for immobiliza-tion of lipase for biodiesel production. An extensive list of the

type of carrier used has been summarized in Table 1. Among

them are such as Toyonite 200-M, celite, Accurel, diatonomous

earth, polypropylene EP 100, textile membrane, hydrotalcite,

silica gel, acrylic resin, and anion resin (Iso et al., 2001; Shah

et al., 2004; Yangetal., 2006;Yesiloglu, 2004;Salis etal., 2005; 1

Hsu et al., 2001; Nie et al., 2006; Yagiz et al., 2007; Wang

et al., 2006; Talukder et al., 2006). In general, the conversions

of biodiesel from various vegetables and waste oils by using the

enzyme immobilized in this form ranges from 76 to 100%. The

effect of polar and nonpolar properties of resin as a carrier on 1

the degree of immobilization has also been studied (Yang et al.,

2006). The degree of immobilization was high when lipase wasadsorbed onto a nonpolar resin with a pore diameter of 8.59.5

nm. It was reported that the pore diameter of resin influences

the degree of immobilization where degree of immobilization 1

increases with increasing pore diameter (Yang et al., 2006).

Even though adsorption has various advantages like commer-

cially available and high activity for biodiesel production, the

enzyme could be stripped off the support during the reaction due

to the direct shear involved between the support and the impeller 1

as the adsorption is basically due to weak forces. In such a case

the loss in the activity of enzyme was due to leaching and not

dueto thedeactivation of enzyme(Yadav and Jadhav,2005). De-

pending on the strength of the interaction between the enzyme

and a particular support, the immobilization process can produce 1

distortions of the protein structure. These distortions are more

FIG. 1. Classification of immobilization methods.


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effective at low loadings and cause enzyme erosion (Bosley and

Peilow, 1997). Hence the stability of immobilized enzyme using

adsorption is very low, which makes the reuse limited and unfit

for industrial applications.125

Cross-Linking

The cross-linking method is based on the formation ofintermolecular cross-linkages between the enzyme molecules

by means of bifunctional or multifunctional reagents such as

glutaraldehyde, bisdiazobenzidine, hexamethylene diisocyanate130etc. Cross-linked enzyme aggregates are matrix-free immobi-

lized preparations. Generally, the first step of the immobiliza-

tion process is to precipitate the enzyme using acetone and this

produces physical aggregates of the enzyme. These aggregates

are then cross-linked with glutaraldehyde to form a more robust135structure. The application of this biocatalyst design for the pro-

duction of biodiesel has been explored (Kumari et al., 2007).

The use of cross-linked enzyme aggregates accelerated the rate

of transesterificationand a conversion of 92%has been obtained.

However, one of the intrinsic drawbacks for cross-linkedenzyme140aggregates is that their particle size is usually below 10 m.

Thus, difficulties arise when they are used in heterogeneous re-

action systems, where the substrate particle and the cross-linked

enzyme aggregates particles might be in the same range. This

can create problems in separation of immobilized enzyme from145the product for the continuous use (Cao et al., 2003).

Entrapment

Entrapment of lipaseentails capture of thelipase withina ma-

trix of polymer (Xavier et al., 1990).The lipase immobilized by

entrapment is much more stable than physically adsorbed lipase.150

Unlike the covalent bonding method it uses a relatively simpleprocedure while at the same time the immobilized lipase main-

tains its activity and stability (Kennedy et al., 1990). A novel

procedure for entrapment ofPseudomonas cepacia lipase within

a phyllosilicate sol-gel matrix with tetramethylorthosilicate as155precursor has been developed (Hsu et al., 2001). Lipase from

FIG. 2. Hybrid immoblization methods.

Pseudomonas cepacia was entrapped within a sol-gel polymer

matrix, preparedby polycondensation of hydrolyzed tetramethy-

lorthosilicate and iso-butyltimethoxysilane. The immobilized

enzyme was used in transesterification of soybean oil and a con- 1

version of around 67% was achieved. The low conversion of es-

ter using immobilized lipase by entrapment was due to the poor

diffusion and erosion of enzyme from the surface of the supportduring the processing procedures (Nourenddini et al., 2005).

Encapsulation 1

Encapsulation is the confinement of enzyme with in a porous

membrane forming a bilayer. Encapsulation avoids direct con-

tact between the enzyme and the bulk medium. It also pro-

vides a cage to avoid the enzyme from leaching out (Yadav

and Jadhav,2005). Production of biodiesel using encapsulated 1

lipase in silica aerogel has shown a conversion of 56%. The im-

mobilized enzyme could be recycled several times without any

apparent mechanical deterioration by wear (Orcaire etal., 2006).

On the other hand, a stronger diffusion limitation with encap-

sulated lipase occurred by a high protein concentration in the 1

enzyme, which clogs the pores in the matrices. It is worthwhile

to produce an encapsulated enzyme with smaller size to over-

come the mass transfer problems and to use a purified enzyme

to avoid clogging (Orcaire et al., 2006).

Other Immobilization Techniques 1

The immobilization of enzyme by using a combination of

different immobilization technique are known as hybrid immo-

bilization (Fig. 2). Hybrid immobilization has given promising

results on an industrial scale in fields like food (Reyed, 2007)

and pharmaceuticals (Posorske, 1984; Bonrath et al., 2002). 1

This approach was recently explored for transesterification of p-chlorobenzyl alcohol with vinyl acetate to give p-chlorobenzyl

acetate using lipase adsorbed on hexagonal mesoporous sil-

ica followed by encapsulation on calcium alginate (Yadav and

Jadhav, 2005). The hybrid immobilization system showed 68% 1

conversion and excellent reusability with a decrease of only 4%


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PRODUCTION OF BIODIESEL USING IMMOBILIZED LIPASE 5

in overall conversion after the fourth reuse. The potential of

this technique could be explored for biodiesel production. For

example, it could be used to solve the leakage of enzyme im-

mobilized through adsorption by providing a porous membrane

through encapsulation.195

Similarly, protein-coated microcrystals are becoming popu-

lar for nonaqueous systems. Protein-coated microcrystalshavenegligible mass transfer limitations and high activity compared

to cross-linked enzyme aggregates (Kumari et al., 2007). The

preparation of protein-coated microcrystals involves the mixing200

of enzyme solution with a concentrated solution of salt, sugar,

or amino acid. The combined aqueous mixture is then added

drop wise with rapid mixing to water-miscible organic solvent

like acetone or 1-Propanol (Kreiner etal., 2001). Theproduction

of biodiesel using protein-coated microcrystals has shown high205

conversion (Kumari et al., 2007).

FACTORS AFFECTING THE PRODUCTION

OF BIODIESEL USING IMMOBILIZED LIPASEPretreatment of Immobilized Lipase

Most of the commercial available lipase is in powder form. It210

is necessary to dissolve the lipase enzyme in a coupling media

before immobilization. The type of coupling media used could

influence the activity of the lipase. Both aqueous and nonaque-

ous coupling media have been used for dissolving the lipase

enzyme. Higher ester conversion was obtained when nonaque-215

ous (heptane) coupling media was used compared to aqueous

media (buffer) for immobilization of Candida sp. in a resin

(Yang et al., 2006). The possible reason for higher conversion

with heptane could be that, the treatment of lipase with po-

lar organic solvent changes the lipase conformation from the220

less hydrophobic-closed form (active site is covered by lid) tomore hydrophobic open form (active site is opened), favoring

the binding of hydrophobic substrate to lipase (Chamorro et al.,

1998; Colton et al., 1995). On the other hand the activation of

immobilized enzyme was enhanced by pretreatment of lipase225

in methyl oleate and soybean oil (Samukaw et al., 2000; Dong

et al., 2006). The pretreatment of immobilized Candida antar-

tica lipase in isopropanol has also shown higher methyl ester

conversion. However, the use of nonaqueous coupling media

increases the cost of biodiesel production.230

Feedstock

Most countries have set up policies to use biodiesel alongwith conventional diesel due to environmental concerns and the

raising crude oil price. Various vegetable oils can be used for

the production of biodiesel (Table 1). The majority of studies235

reported the useof soybean oil, sunflower oil, palm oil, rape seed

oil, cottonseed oil, andjatrophaoil (Talukder etal., 2006; Royan

etal.,2007;Lietal., 2006; Shah andGupta, 2006; Mukesh et al.,

2007).

In general, there are no technical restrictions on the use of240

vegetable oils for biodiesel production. However, the main con-

straint for biodiesel production may be the cost of the feedstock.

Al-Zuhair (2007) suggests considering palm oil as a favorable

feedstock for biodiesel production. Palmoil has the highest yield

compared to that of other vegetable oils. The high value of 2

edible vegetable oil as a food product makes production of a

cost-effective fuel very challenging. The demand for vegetable

oils for biodiesel has also raised the use of fertilizers, whichalso contribute to the increase in greenhouse gases. Produc-

ing biodiesel from a fertilizer-heavy crop would generate up to 2

70% percent more greenhouse gases emissions than using reg-

ular diesel ( http://www.oregonlive.com).It is more reasonable

to use jatropha oil, as edible oils are not in surplus supply (Shah

and Gupta, 2006). Jatropha oil has an estimated annual produc-

tion potential of 200 thousand metric tonnes in India (Srivastava 2

and Prasad, 2000). It can be grown in wasteland with minimum

amount of water and fertilizers, unlike sunflower or soybean. It

grows rapidly, takes approximately 2 to 3 years to reach maturity

and it could generate economic yields. It has a productive lifes-

pan in excess of 30 years. The fatty acid composition of Jatropha 2

oil is similar to other edible oils but the presence of some antin-utritional factors such as, toxic phorbol esters renders this oil

unsuitable for cooking purposes (Shah etal., 2004). Jatropha oil

is thus a promising candidate for biodiesel production in terms

of availability and cost. 2

On the other hand, there are large amount of low-cost oils

and fats, such as restaurant waste and animal fats that could

be converted to biodiesel. The problem with processing these

low-cost oils and fats is that they contain large amounts of

free fatty acids that cannot be converted to biodiesel using an 2

alkaline catalyst (Canakci and Gerpen, 2001). Whereas, the

oil with high free fatty acids content can be converted to es-

ters by using immobilized lipase. The use of waste cooking

oil for biodiesel production could reduce production cost and

favors the environment. However, the adequate and sustain- 2

able supply of the waste oils must be ensured for large-scale

production.

In the future there will be an increase demand for land for

the production of vegetable oils Therefore, it is important to

prioritize processes, production systems, and products that are 2

efficient with regard to the land area used and their environmen-

tal impact. Based on these criteria, algal oil represents another

source of renewable raw materials for biodiesel production. Mi-

croalgae oil has currently received more attention as a potential

feedstock owing to their high production of lipids(Chisti, 2007). 2

Algal oil is largely produced through substrate feeding and het-erotrophic fermentation. If algal production could be scaled up

industrially, less than 6 million hectares would be necessary

worldwide to meet current fuel demands, amounting to less than

0.4% of arable land (Sheehan et al., 1998). 2

Lipase Enzyme

The main natural function of lipases (triacylglycerols ester

hydrolases EC 3.1.1.3) is to catalyzethe hydrolysis of long-chain

triacylglycerols (TAG). Contrary to many other enzymes, they


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FIG. 3. Mechanism of lipase in transesterification.

show remarkable levels of activity and stability in nonaqueous295environments, which facilitates the catalysis of several unnatural

reactions such as esterification and transesterification.

Literature reveals the relation between lipase structures and

their catalytic ability (Cecilia et al., 2007). The overall structure

of the triacylglycerol lipases can be described as a structure with300

a central L-sheet with the active serine placed in a loop termed

the catalytic elbow. Above the serine a hydrophobic cleft are

present or formed after activation of the enzyme (Svendsen,

2000). The hydrophobic cleft is an elongated pocket suitable for

acyl moieties to fit into. The activation, which is often necessary305

for the lipase enzyme is the movement of a lid. Thermomyces

lanuginose lipase has an active site and a lid on the surface of

the enzyme. Pseudomonas and Candida antarctica lipase has anactive site and a funnel-like lid. Candida rugosa lipase has an

active site at the end of a tunnel containing the lid in its external310

part (Pleiss et al., 1998). The structural peculiarities of lipase

from different sources might be the reason for showing different

activity in different substrates.

The mechanism of lipase involves the Asp-His-Ser catalytic

triad acting as a charge-relay system (Bommarius and Riebel-315

Bommarius 2000). The carboxylate group on aspartic acid is

hydrogen-bonded to histidine, and the nitrogen on histidine ishydrogen-bonded to the alcohol on serine. The first step in the

reaction is to make the serine alcohol a better nucleophile. This

taskis performed by histidine, whichcompletely pulls the proton 3

off theserine alcohol, forming an oxyanion.The serineoxyanion

then attacks the substrates carbonyl carbon, forming a tetrahe-

dral intermediate1 (Fig. 3).The created oxyanion is stabilized by

nearby amino acids (aspartate and histidine), which hydrogen-

bond to the oxyanion. Next, the electrons on the oxyanion are 3

pushed back to the carbonyl carbon, and the proton currently

on the histidine is transferred to the diglyceride, which is subse-

quently released (Al-Zuhair etal., 2007).The serineester formed

reacts with alcohol to complete the transesterification. The his-

tidine nitrogen removes hydrogen from the alcohol molecule 3forming the alkyl oxide anion. The hydroxide attacks the car-

bonyl carbon, the intermediate oxyanion is stabilized by a hydro-

gen bond (tetrahedral intermediate 2), the electrons are pushed

back to thecarbonyl carbon, andthe free fatty acid is formed. The

serine oxygen then reclaims the hydrogen situated on the histi- 3

dine to re-establish the hydrogen-bonding network. The aspartic

acid serves to pull positive charge from the histidine during the

times it is fully protonated.


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The immobilized lipases from various sources have been used

in biodiesel production (see Table 1). Lipase from Candida an-340

tartica has been selected by the majority of researchers. Other

sources of enzyme used were produced from Pseudomonas flu-

orescens, Pseudomonas cepacia, Burkholderia cepacia, Rhi-

zomucor miehei, Chromobactrium viscosum, Porcine pancreas,

and Thermomyces lanuginosus. On the other hand, an interest-345ing study by Turkan and Kalay (2006) on the elucidating mech-

anism of lipase for methanolysis of sunflower using three dif-

ferent immobilized lipases concludes that immobilized lipase

from Rhizomucor miehei and Thermomyces lanuginosus cat-

alyzethe first step (conversion of triacylglyceride to diglyceride)350

of transesterification faster. Whereas, immobilized lipase from

Candida antarctica catalyzes the second (di acylglyceride to

mono acylglyceride) and third (monoacyl glyceride to acylester)

steps faster. Turkan and Kalay, (2006) suggested using a dual en-

zymatic system rather than the single lipase system in biodiesel355

production.

Acyl AcceptorsAlcohol has been chosen as the acyl acceptors by major-

ity of the researchers (Table 1). Among alcohols, methanol

and ethanol was popular. 2-butanol (Salis et al., 2005) and 2-360

propanol (Mukesh et al., 2006) were also reported as potential

acyl acceptors. On theother hand methylacetate(Du et al., 2005)

and ethyl acetate (Mukesh et al., 2007) werealso used asan acyl

acceptor.

On the other hand, immobilized lipase has been found to be365

deactivated by lower linear alcohols. The degree of deactiva-

tion is inversely proportional to the number of carbon atoms in

the linear lower alcohols (Chen and Wu, 2003). Adding organic

solvents have been adopted to overcome this problem. Nelson

et al., (1996) used hexane to prevent the deactivation by a lower370

alcohol. Other solvents used include: isooctane,petroleum ether,

2-propanol, tetrahydrofuran and t-butanol (Orcaire et al., 2006;

Mittelbach et al., 1990; Iso et al., 2001; Talukder et al., 2006;

Rayon etal., 2007). Sung etal., (2007) recently explored the use

of ionic liquids as solvents. Whereas, Oliveria et al., (2001) and375

Vivek and Giridhar, (2007) have reported the use of supercriti-

cal CO2 as solvent. An advantage of using supercritical fluids,

considering the complete process, was that after the system de-

pressurization, a mixture of products and nonreacted substrate

may be obtained without traces of solvent (Oliveria and Oliveria380

2001). However, the use of solvents is not recommended as it

requires the addition of a solvent recoveryunit and pressure unit,which makes the production process costly.

Stepwise addition of alcohol was also used to get rid of the li-

pase deactivation by lower linear alcohols. The stepwise addition385

of alcohol has been discussed in detail (Shimada et al., 2002).

A list of work reported on biodiesel production by immobilized

lipase using step-wise addition of alcohol is given in (Table 2).

On the other hand, Talukder et al., (2007) used a salt-solution

based controlled release system to control the concentration of390

methanol in the oil phase by dissolving methanol in MgCl2 and

LiCl salt solutions. Methanol was released according to its parti-

tioning coefficient between the oil and the salt-solution phases.

In addition, it was reported that the glycerol produced during

the process dissolves in the salt-solution phase, eliminating the 3

glycerol deposition on the immobilized lipase. However, the use

of salt solutions could generate more wastewater and this could

increase the production cost.In the present scenario where environmental concerns and

production costs are given higher priority, the solvent free trans- 4

esterification could be encouraged. Among the alcohols, using

ethanol as an acyl acceptor for biodiesel production could be

economical. With the increase in the production of ethanol in

the world market, the price of ethanol is getting cheaper, which

makes ethanol the best candidate for biodiesel production. 4

Water Content

Biocatalysts need minimum amount of water to retain their

activity (Bommarius and Riebel-Bommarius, 2000). Lipase pos-

sesses the unique feature of acting at the interface between an

aqueous and an organic phase. Lipase interfacial action is due to 4

the fact that their catalytic activity generally depends on the ag-

gregation of the substrates. Activation of the enzymes involves

unmasking and reconstructing the active site through conforma-

tional changes of the lipase molecule, which requires the pres-

ence of an oil-water interface. Lipase activity generally depends 4

on the available interfacial area. The amount of water available

for oil to form oilwater droplets increases with the addition

of water. It facilitates increase in the available interfacial area

(Al-Zuhair etal., 2006). However, since lipases usually catalyze

hydrolysis in aqueous media, excess water may also stimulate 4

the competing hydrolysis reaction.The optimumwater content is

a compromise between minimizing hydrolysis and maximizing

enzyme activity for the transesterification reaction (Noureddini

et al., 2005).

Several researchers have reported the effect of water on 4

biodiesel production (Table 1). With increased addition of wa-

ter there was an increase in ester production. On the other hand

few researchers have reported that with the addition of water

the ester production decreased (Shimada et al., 1999; Talukder

et al., 2006). The amount of water to be maintained in biodiesel 4

production using immobilized lipase depends on the feedstock

(the water content of feedstock differs for waste oil to that of

refined oil), source of lipase (some commercial lipase are in

powder form which must be dissolved in coupling media before

immobilization process), immobilization technique (some im- 4mobilization technique involves the use of water) and the type

of acyl acceptor (analytical grade or reagent grade). Thus, it is

recommended to optimize the water content depending on the

reaction system used.

OPERATION MODE 4

In general there are three types of operation mode namely

batch, continuous, and fed batchmode. Commonly used reactors

for these operation modes are stirred tank reactor, packed bed


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TABLE2

Productionofbiodieselusingimmobilizedlipasebystepwisead

ditionofalcohol

Oil

Alcohol

Immobilization

technique

Percentageof

immobilized

lipasetothe

weightofoil

Numberof

methanol

addition

steps

Molar

equivalentof

alcoholin

eachstep

Reaction

temperature

C

Reaction

time(h)

Conv

ersion

in

the

1st

step

(%)

Conversion

inthe

2ndstep

(%)

Conversion

inthe

3rdstep

(%)

References

Wasteoil

Methanol

Adsorption

4

3

1/3

30

48

34a

t10h

66at24h

90.4at48

Watanabee

ta

l.,2001

Mixtureofsoya

andrapeseed

Methanol

Adsorption

4

3

1/3

30

48

34a

t10h

66at24h

95.6at48

Watanabee

ta

l.,2001

Soybean

Methanol

Adsorption

4

3

1/3

30

3.5

42.4

at1h

69.8at2.5h

98.7at3.5h

Samukawa

eta

l.,2000

Mixtureofsoybean

andrapeseed

Methanol

Adsorption

4

2

2/3

30

36

32at7h

96.5at36h

Watanabee

ta

l.,2000

Soybean

Methanol

NA

4

3

1/3

NA

NA

32at5h

75at25h

90at45h

Due

ta

l.,2005

Lard

Methanol

Adsorption

20

3

1/3

40

30

28at3h

60at18h

87.4at30h

Lue

ta

l.,2007

8


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TABLE3

Proposedkineticmode

lsforbiodieselproductionusingimmo

bilizedlipase

Triglyceride[TG]

Solvent[A]

Immobilizedlipase

Proposedkineticequation

Vmax

KM[TG]

KM[A]

Ki

References

Soybeanoil

Methylacetate

Can

didaan

tart

ica

Vi=

Vmax(A)(m

ix

MA(A))MTG

Km

TG(A)(1+((A)/Ki))+(Km

A+(A))(m

ix

MA(A))/MTG)

1.9Mol/Min

1Mol/l

16Mol/l

0.0455M

ol/l

Xue

ta

l.,2004

Sunfloweroil

Butanol

Rhizomucorm

iehei

Vi=

Vmax(

TG)

1+K

iTG[TG]+K

TG[TG

](1+[A]K

IA)+K

A[A]

250mMol/min.g

5.3mM

55mM

13mM

Dossateta

l.,2002

Sunfloweroil

Methanol

Rhizomucorm

iehei

V=

Vmax[T

G]

1+K

iTG[TG]+K

TG[TG](1+[A]K

IA)+K

A[A]

0.414Min1

0.16Mol/Cm3

0.98

104Mol/cm3

1.9Mol/c

m3

Al-Zuhair,2005

TG:Triglyceride.

A:Alcohol.

Km[TG]:ApparentMichaelasconstantsfortriglycerides.

Km[A]:ApparentMichaelasconstantsformethylacetateoralcohol.

VMAX:Theinitialmaximumvelocityofreaction.

Ki:InhibitionConstant.

9


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ptember 5, 2008 18:13 802 BBTNA343007


reactor, fluidized bed reactors, and membrane reactors. The type

of reactors to be selected using immobilized lipase depends on445

the nature of the immobilized enzyme. The mechanical stability

of the immobilized enzyme must be considered when using a

stirred tank reactor. Whereas, the pressure drop created across

the reactor packing and the interparticle diffusion limitations

should be considered when using a packed bed reactor (Balcao450et al., 1996). The packed reactor has traditionally been used for

most large-scale catalytic reactors because of its high efficiency,

low cost, and ease of construction and operation (Malcata et al.,

1990). In biodiesel production by immobilized lipase majority

of the researchers selected batchoperation in stirred tank reactor.455

However, Royon et al., (2007) and Watanabe et al., (2000) used

continuous fixed-bed reactors and reported theeffect of flow rate

on biodiesel production. But, in packed bed reactor the glycerol

produced during the reaction decreases the reaction rate of the

immobilized lipase. Conversion could be enhanced if glycerol460

was removed from the reaction mixture as the reaction proceeds

(Watanabe et al., 2000). Glycerol formed during the reaction

was separated continuously using a membrane reactor (ChenandWu, 2003; Belafi-bako et al., 2006). But, one of the limitations

using membrane reactor might be their high cost compared to465

packed bed reactor and stirred tank reactor.

KINETICS OF BIODIESEL PRODUCTION USINGIMMOBILIZED LIPASE

It is essential to understand the kinetics of the reaction to

identify the optimal conditions for lipase-catalyzed transesteri-470

fication. Only a limited number of kinetic studies are found in

the literature for biodiesel production using immobilized lipase.

Most of the kinetic studies are describedby the ping-pong model

with competitive inhibition by alcohol. Xu etal., (2004) reported

the kinetics of lipase-catalyzed interesterification of soybean475

oil with methyl acetate for biodiesel production. A mechanism

was proposed considering the presence of three consecutive re-

versible reactions. The rate constants for the three consecutive

reactions from triacylglyceride to acylester were calculated and

the results indicated that the first step reaction, triacylglyceride480

to diacylglyceride was the rate-limiting step for the overall reac-

tion. The proposed equations and the calculated rate constants

are given in (Table 3). Dossat et al., (2002) proposed a model

basedon the Ping-Pong Bi Bi withalcohol competitive inhibition

mechanism to describe transesterification kinetics, whereas Al-485 Zuhair(2005) proposed a kinetic model for biodieselproduction

by two types of lipase, namely, Rhizomucor miehei lipase im-

mobilized on ion-exchange resins and Thermomyces antarctica

lipase immobilized on silica gel. There was a good agreement

between the experimental results of the initial rate of reaction490

and those predicted by the proposed model equations, for both

enzymes. The proposed model equation (Table 3) can be used

to predict the rate of methanolysis of vegetable oils in a batch

or a continuous reactor.

CONCLUSION 4

The conventional production of biodiesel by chemical cata-

lysts is energy consuming, leads to undesirable side products,

requires expensive wastewater treatment, and makes it difficult

to recover the glycerol. Enzymatic production of biodiesel omits

these difficulties and is workable at milder conditions. It is not 5

economical when an enzyme is used in free form, which makesthe recovery of the enzyme impossible. Immobilization not only

increasesthe stabilityof enzyme, also favors touse ofthe enzyme

several times thereby reducing the production cost. Selection of

suitable immobilization techniques is important to ensure effec- 5

tive usage of enzyme without leakage and detachment from the

support. Use of natural polymers as matrix for lipase immobi-

lization in biodiesel production is yet to be explored, but has

given promising result in other fields like food and pharmaceu-

ticals. Production of biodiesel using nonedible oil can greatly 5

reduce the feedstock cost and is also environmental friendly.

Using ethanol as the acyl acceptors can make the biodiesel pro-

duction green but the amount used should be less than at the

solubility limit, so that it is not present in the separate phase.

Stepwise addition of alcohol can keep the concentration of alco- 5

hol below the critical level. Immobilized lipase which can give

higher ester conversion in a short period and resistant to alcohol

should be used for economic production. Continuouspacked bed

reactor, which has high efficiency, low cost and ease of construc-

tion, operation, and maintenance can be used. Immobilization 5

of lipase using combinatorial techniques must be explored to

achieve a robust catalyst. Production of biodiesel using immo-

bilized lipase shows promising results on the laboratory bench

scale. Working on the pilot scale is essential to bring it to the

commercial scale. 5

ACKNOWLEDGMENTS

This research project is financially supported by E-SCIENCE

fund (Grant no: SCF0012-IND-2006), Ministry of Science,

Technology and Innovation (MOSTI), Malaysia.

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