Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications

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USE OF LIPASES FOR THE PRODUCTION OF BIODIESEL

329

and Pseudomonas fluorescens lipase (about 57%). Using secondary alcohols as

substrates for ester production, conversion by 2-propanol was found to be lower

than that by isobutanol. In all cases, better conversion levels were obtained when

cottonseed oil was transesterified by 1-propanol and isobutanol (Soumanou and

Bornscheuer, 2003b).

4. REACTION CONDITIONS FOR BIODIESEL PRODUCTION

4.1. Solvent Versus Solvent-free Processes

Miscibility between methanol and triglycerides is very poor, thus the presence of

a solvent helps to form a monophasic system. However, the use of a solvent has

some disadvantages such as its storage before use and its removal and disposal

after the process both of which generate environmental problems. Kamini and Iefuji

(2001) found that addition of organic solvents (10% w/v) increased the content of

methyl esters from 80.2% (no solvent) to 85% (DMSO), 86.1% (n-hexane) and

87.2% (petroleum ether), whereas the addition of diethylether decreased methyl ester

content to 70.4%. The same authors stated that although the addition of solvents

gives favourable results in some cases, their use is not recommended for reasons of

economy and safety. The same conclusions were arrived at by Iso et al. (2001). They

reported that the methanolysis and ethanolysis of triolein and safflower oil were

better carried out in the presence of 1,4-dioxane as solvent. Conversely, activity in

other solvents such as benzene, chloroform and tetrahydrofuran was extremely low.

As an alternative they proposed the use of 1-propanol and 1-butanol which allow

full miscibility with the oil and yield high reaction rates.

Soumanou and Bornscheuer (2003b) found that n-hexane was necessary to

perform methanolysis of various vegetable oils. The highest conversion (97%) was

obtained with Thermomyces lanuginosa lipase after 24 h. By contrast, this lipase was

found to be almost inactive in solvent-free reaction medium where methanol or 2-

propanol were the alcohol substrates. In successive work these authors investigated

the effect of other solvents, namely cyclohexane, n-heptane, isooctane, acetone and

petroleum ether. All gave conversions in the range 60–80% with three immobi-

lized lipases (from Mucor miehei, Thermomyces lanuginosa and Pseudomonas

fluorescens). Acetone, by comparison, gave a conversion in the range 5–20%.

This polar solvent may alter the native conformation of the enzyme by disrupting

hydrogen bonding and hydrophobic interactions, thereby leading to a very low

alcoholysis rate (Soumanou and Bornscheuer, 2003b).

An interesting solvent process was proposed by Kojima et al. (2004). The

enzymatic methanolysis of waste oil from activated bleaching earth (ABE) was

performed by using diesel fuel as solvent (0.6 mL/g of waste ABE). In this system,

the lipase from Candida cylindracea showed high stability and activity reaching

approximately 100% yield of FAME in 3 h. Fuel analysis showed that the FAME

produced in this way complied with the Japanese diesel standard. The use of diesel

oil as solvent in FAME production from the waste ABE simplified the process since

there is no need to separate the organic solvent from the FAME-solvent mixture.

330 SALIS ET AL.

4.2. Water Content

Water content in biocatalysis in non-conventional media is a very important

parameter since it strongly affects enzymatic activity (Adlercreutz, 2000; Salis et al.,

2005a). The reaction rates of methanolysis catalysed by the Candida rugosa and

Pseudomonas fluorescens lipases decreased significantly when water content was

low since water prevents the inactivation of these lipases by methanol (Kaieda et al.,

2001). Conversely, the rate of the same reaction catalysed by Pseudomonas cepacia

lipase remained high even under low water content conditions. Shah et al. (2004)

found that the addition of water to Chromobacterium viscosum lipase increased

ethyl ester yield from 62 to 73% for the free preparation (1% of water) and from 71

to 92% for the immobilized (on celite) preparation (0.5% of water). Cryptococcus

spp. S-2 lipase was able to catalyse methanolysis of rice bran oil in the presence

of high water content (80% by weight of substrate), thus obtaining a methyl ester

yield of 80.2% after 120 h (Kamini and Iefuji, 2001).

There are conflicting reports on the effect of water content in the synthesis

of biodiesel. Some authors state that ester yield increases if high amounts of

water are present in the system (Kamini and Iefuji, 2001), whilst others claim the

contrary (Hsu et al., 2002). This apparent contradiction might be explained by

considering that the effect of water in these systems depends on the enzyme, the

support and the medium (solvent or solvent-free). As a general consideration, high

water content should decrease ester yield since undesirable triglyceride hydrolysis

occurs. Although water is involved in several enzyme denaturing processes, a certain

amount of water must be present to ‘lubricate’ polypeptide chains and keep an

enzyme in its active conformation. In this regard, water content is better expressed

in terms of water activity (Halling, 1994) since only the use of this thermodynamic

parameter allows a real comparison of the effects of water among different enzymes

in the same medium. Unfortunately, the majority of papers reporting studies on the

effect of water cite water concentration instead of water activity.

4.3. Biocatalyst Recycling and Continuous Processes

One of the main drawbacks to the biocatalytic production of biodiesel is the cost

of the enzyme. Enzyme recycling might be the solution to this problem.

Pseudomonas fluorescens lipase immobilized on kaolinite lost one third of its

activity when used for the second time, but no further decrease was observed in

successive applications. The initial decrease in activity was put down to enzyme

desorption from the solid support that was not observed after repeated (10 times)

use (Iso et al., 2001). Repeated batch reactions showed that Mucor miehei lipase

showed high stability, retaining about 70% of its initial conversion after 8 cycles

(24 h each cycle), whereas Thermomyces lanuginosa retained only 35% of the initial

conversion under the same experimental conditions. This difference was ascribed

to various factors such as inactivation of the biocatalyst in the oil phase, the type

of carrier used for the immobilisation or enzyme sensitivity to long-term methanol

exposure (Soumanou and Bornscheuer, 2003b).

USE OF LIPASES FOR THE PRODUCTION OF BIODIESEL

331

A two-step batch process employing Novozym 435 was proposed in which 1/3

equivalent of methanol is added initially and the remaining 2/3 is added in the second

step. More than 95 % conversion was observed. When the reaction was repeated

by transferring the immobilized lipase to a fresh substrate mixture, it was observed

that the enzyme could be reused for 70 cycles (105 days) without any decrease in

the conversion. However, when the two-step reaction was conducted using a reactor

equipped with an impeller, the support was destroyed thus limiting reusability to just

a few cycles (Watanabe et al., 2000). As an alternative to recycling the biocatalyst,

continuous processes might help to overcome the economic drawbacks of enzymatic

biodiesel production.

Du et al. (2003) studied the effects of temperature, the oil/alcohol molar ratio and

the presence of the by-product glycerol during Lipozyme TL IM-catalysed continuous

batch operation when short-chain alcohols were used as the acyl acceptor. In non-

continuous batch operation the oil/alcohol ratio and temperature optima were 1:4

and 40–50



C, respectively; however, during continuous batch operation the optimal

conditions were found to be 1:1 and 30



C. Enzymatic activity was noted to be 95%

after 10 cycles when iso-propanol was used to remove glycerol during repeated use

of the lipase. It was also found that the enzyme lost its activity rapidly at 40



C 30



was found to be the optimal temperature at which lipase retains high activity,

even after many batches. Continuous and discontinuous processes have different

optimal temperatures. Presumably, lipases exhibit relatively high activity during

short-term operation in non-continuous reactions, but during long-term operation

they may lose their activity rapidly, particularly at relatively high temperatures.

The lipase from Burkholderia cepacia, immobilized on a phyllosilicate sol-gel

matrix, was used for the continuous production of ethyl esters of grease (Hsu et al.,

2004a). An ester yield>96% was obtained.

4.4. Lipase Inhibition and Regeneration

Both reagents and products of the alcoholysis reaction and some substances

contained in the feedstock can inhibit lipases. Below are described the different

kinds of inhibition and their possible remediation. A schematic summary is also

shown in Table 4.

4.4.1. Methanol inhibition

Methanol is poorly miscible with oil/fat and tends to inactivate enzymes. Candida

antarctica lipase B, an enzyme that is very stable in organic media (Salis et al.,

2003b), was found to be progressively inactivated by methanol in amounts above

1/2 molar equivalent. To overcome this problem, multi-step addition of methanol

was proposed (Shimada et al., 1999): only 1/3 of the stoichiometric amount is used

in the first step. After this first addition, a mixture of methanol, FAME, mono- and

di-glycerides is formed. The system thus composed is less aggressive toward the

enzyme compared to the initial conditions when only the oil and alcohol are present

(Shimada et al., 2002). It has also been shown that pre-incubation of Novozym 435

332 SALIS ET AL.

Table 4. Type of inhibition and possible remediation for discontinous and continuous enzymatic production of biodiesel.

Type of process Lipase Inhibitor Remediation Reference

Discontinuous

(Batch)

Candida antarctica Methanol Three step addition of

methanol

Shimada et al., 1999

Candida antarctica Methanol Addition of 10 wt% of silica

gel

Lee et al., 2002

Candida antarctica Methanol Lipase immersion

pre-treatment in 2-butanol

or tert-butanol

Chen and Wu, 2003

Candida antarctica Phospholipids Oil degumming Watanabe et al.,

2002

Candida antarctica Phospholipids Lipase immersion

pre-treatment

Du et al., 2004b

Candida antarctica Phospholipids Simultaneous dewaxing and

degumming

Lai et al., 2005

Thermomyces

lanuginosa

Methanol Three step addition of

methanol

Xu et al., 2004

Glycerol Enzyme washing with

isopropanol

Continuous

(Packed-bed

reactor)

Rizhomucor miehei Glycerol Addition of silica gel;

Use of hexane amended

with acetone;

Rinsing of the catalyst

Dossat et al., 1999

Candida antarctica Methanol Three-step flow reaction Watanabe et al.,

2002

Candida antarctica Glycerol Use of a dialysis membrane Bélafi-Bakó et al.,

2002

USE OF LIPASES FOR THE PRODUCTION OF BIODIESEL

333

in methyl oleate for 0.5 h followed by soybean oil for 12 h resulted in a methyl

ester yield of 97% within 3.5 h (Samukawa et al., 2000).

Chirazyme L-2 (Candida antarctica lipase B) activity was found to be inhibited

in the presence of more than one mol of methanol. Therefore methanol was added to

the reaction in three consecutive steps. It was also found that addition of 10% silica

gel to the reaction mixture increased the conversion rate. It has been postulated that

the excess methanol forms a barrier around the lipase structure that may involve the

active site, thereby hindering contact with the acyl donor and leading to inactivation

of the lipase. Silica gel may act as a methanol depot thus preventing direct exposure

of the lipase to high concentrations of methanol and resulting in an increased

reaction rate (Lee et al., 2002).

Chen (2003) proposed a procedure for enzyme regeneration that solves the

problem caused by methanol. They found that an alcohol with three or more carbon

atoms, preferably 2-butanol or tert-butanol, can regenerate deactivated immobilized

enzyme. The procedure consists of an immersion pre-treatment of the enzyme in the

alcohol. By this means the activity of Novozym 435 was increased about ten-fold

compared to the untreated enzyme. Following complete deactivation of the enzyme

by methanol, washing with 2-butanol or tert-butanol successfully regenerated the

enzyme restoring about 56 % and 75 % of its original activity, respectively.

Another way to overcome the methanol inhibition of Candida antarctica lipase

was developed by Xu et al. (2003). They proposed the use of methyl acetate as a

novel acyl acceptor for biodiesel production. When used for soybean oil transes-

terification it gave 92% methyl ester yield with a molar ratio methyl acetate:oil =

12:1. Unlike the case for methanol (inhibition at ratios greater than 1:1) no adverse

effects were observed. In addition, the use of methyl acetate enabled crude soybean

oil to be used, attaining the same yield as in the case of refined oil. No activity

loss was observed with methyl acetate as the acyl acceptor upon repeated use (100

batches) of the lipase. Moreover, the by-product triacetylglycerol is an important

chemical of greater economic value than glycerol (Du et al., 2004a). The kinetics

of the lipase catalysed inter-esterification of triglycerides with methyl acetate was

studied by Xu et al. (2005). Three consecutive and reversible reactions occurred

in the inter-esterification. The main finding was that the first reaction step was

limiting for the overall process.

Distinctly from other microbial lipases, Pseudomonas cepacia lipase was found

to give high methyl ester contents in reaction mixtures containing methanol:oil

equivalent ratios of up to 2–3:1. This enzyme seems to be substantially resistant to

methanol (Kaieda et al., 2001). Also Rhizopus oryzae lipase was not inactivated by

methanol (Lara and Park, 2003).

4.4.2. Glycerol inhibition

Glycerol has a negative effect on FAME production by Thermomyces lanuginosa

lipase (Selmi and Thomas, 1998). Enzyme washing with iso-propanol was found to

be effective at removing glycerol. In the presence of iso-propanol the lipase showed

334 SALIS ET AL.

relatively high activity and a methyl ester yield of over 94% was retained after

repeated use over 15 batches (Xu et al., 2004).

4.4.3. Inhibition by phospholipids

Watanabe et al. (2002) found that gum present in crude soybean oil inhibits Candida

antarctica lipase since this negative effect was not observed with degummed oil.

The main components of gum are phospholipids (PLs). The inhibition observed

may be due to PLs bound to the immobilized preparation that interfere in the lipase-

substrate interaction. When degummed oil was used it was successfully converted

(93.8%) into its corresponding methyl esters, and the lipase could be reused for

25 cycles without any loss of activity. Similarly, Du et al. (2004b) found that the

methyl ester yield was significantly lower with crude compared to refined soya-bean

oil. The major difference between refined and crude oils was found to be in their

contents of PLs, free acid and water, which have various influences on biodiesel

production. PL content was found to be the most important parameter: the higher

the PL content in the oil, the lower the methyl ester yield. Water and free fatty acid

content did not affect ester yield to the same extent as PL content. The inhibitory

effect of PLs was overcome by an immersion pre-treatment of the lipase (from

Candida antarctica) in crude oil for 120 h. This resulted in a methyl ester yield of

94% that obtained with refined oils (Du et al., 2004b). Lai et al. (2005) observed

inactivation of lipase by PLs and other minor components during the methanolysis

of crude rice bran oil. To avoid this, simultaneous dewaxing/degumming was found

to be efficient.

4.4.4. Inhibition during continuous operation

Glycerol is formed during the alcoholysis of triglycerides. If this reaction is

performed in a Packed-bed reactor glycerol tends to adsorb onto the hydrophilic

supports instead of being withdrawn by the flow. This adsorption inhibits enzyme

activity. Two possible mechanisms of inhibition have been proposed: the adsorption

of glycerol may cause a decrease in the (thermodynamic) water activity of the

enzyme, or glycerol may form a layer around the enzyme that inhibits diffusion of

the hydrophobic substrate. Studies on the butanolysis of high oleic sunflower oil

(> 80% oleic acid content) catalysed by Lipozyme (the lipase from Rhizomucor

miehei) favoured the second hypothesis. Various procedures were tested for their

ability to retain the high initial enzymatic activity: addition of silica gel to the

reactor bed in order to address the preferential adsorption of the glycerol; use

of n-hexane amended with acetone as a reaction medium; and a semi-continuous

process consisting of a transesterification reaction and rinsing of the catalyst in order

to evacuate the adsorbed glycerol out of the reactor. This latter procedure permitted

the restoration of initial enzymatic activity by using a rinsing solution amended

with water with the thermodynamic water activity adjusted to an optimal value

(Dossat et al., 1999).

When immobilized Candida antarctica lipase was used for the continuous

methanolysis of a mixture of soybean and rapeseed oils in a Packed-bed reactor,

USE OF LIPASES FOR THE PRODUCTION OF BIODIESEL 335

two kinds of enzyme inhibition were identified (Watanabe et al., 2000). One

was due to the substrate (methanol) and the other to the by-product (glycerol)

(Bélafi-Bakó et al., 2002). The first problem was overcome by performing a

three-step flow reaction. As regards the second, it was proposed that glycerol

inhibition could be eliminated by continuous operation in a membrane biore-

actor using a suitable dialysis membrane. The reaction takes place in the

primary side of the module, and the glycerol produced during the reaction passes

through the membrane and is accumulated in the secondary (aqueous phase) side

(Bélafi-Bakó et al., 2002).

4.5. Statistical Optimisation of Reaction Parameters for Biodiesel

Production

The parameters – temperature, reaction time, amount of lipase, mole ratio of

reactants – of the enzymatic (Pseudomonas cepacia lipase) ethanolysis of restaurant

grease were optimised by response surface methodology (RSM). The regression

equation obtained by RSM predicted optimal reaction conditions under which the

predicted yield of ethyl ester was 85.4%. Subsequent experiments, using combi-

nations of the theoretical parameters, revealed a trend in which experimental

percentage yields of ethyl esters were consistently lower than the predicted values.

This problem was overcome by adding 5% SP435 (Candida antarctica lipase) one

hour after the beginning of the reaction. This resulted in an ester yield greater than

96% (Wu et al., 1999).

The ability of commercial immobilized Mucor miehei lipase (Lipozyme IM-77)

to catalyse the methanolysis of soybean oil was investigated. RSM and 5-level-

5-factor central composite rotatable design were used to evaluate the effects on

reaction time, temperature, enzyme amount, molar ratio between reagents, and

added water content on the percentage weight conversion to soybean methyl ester

by transesterification. Experiments performed under these experimental conditions

gave a molar conversion of 90.9% that should be compared with the theoretical

value of 92.2% (Shieh et al., 2003). The same method was used to optimise the

methanolysis of canola oil using Novozym 435 as catalyst (Chang et al., 2005).

As in previous cases, the investigated parameters were reaction time, temperature,

enzyme concentration, substrate molar ratio, and added water. Reaction temperature

and enzyme concentration were the most important variables. High temperature

and excess methanol inhibited the ability of Novozym 435 to catalyse the synthesis

of biodiesel. Based on analysis of ridge max, the optimum synthesis conditions

predicted a weight conversion value of 99.4%, very close to the actual experimental

value (97.9% weight conversion).

De Oliveira et al. (2004) adopted a ‘Taguchi experimental design’ considering the

following variables: temperature (35–65



C), water (0–10 wt%), enzyme (5–20 wt%)

concentrations, and oil to alcohol molar ratio (1:3 to 1:10) for the enzymatic

ethanolysis of castor oil. Novozym 435 and Lipozyme IM were the catalysts.

336 SALIS ET AL.

Experimental conversions of 81.4% and 98% were observed for the two enzymes

respectively, in agreement with the theoretical values (82% and 99.6%).

5. CONCLUSIONS

Lipases are suitable catalysts for biodiesel production. They catalyse both the

alcoholysis of triglycerides and the esterification of free fatty acids. This is especially

important for low value oil/fat of high acidity. Indeed, such substrates would

otherwise require pre-treatment or an esterification step in the presence of an

acid catalyst followed by the alkaline transesterification. Lipases can catalyse both

reactions in the same reactor. These enzymes can work in the presence or in the

absence of a solvent, but the second option is preferred in order to avoid the

environmental and safety problems associated with the use of solvents. Various

substances can inhibit lipase activity (methanol, glycerol, phosphlipids); however

several ways have been proposed to resolve these inconveniences. Lipases work

under mild operating conditions at room temperature and at atmospheric pressure.

Consequently, energy-saving processes can be carried out. The biocatalytic process

does not produce soaps or other by-products. If the reaction reaches completeness,

only esters and glycerol are produced. This simplifies purification steps and conse-

quentially reduces plant costs. A possible flow diagram for biodiesel production by

lipases is outlined in Fig. 3. Once immobilized, lipases can be used several times

or even in continuous processes. This overcomes the main disadvantage related to

their high cost. In conclusion, biodiesel is an environmentally friendly fuel which,

if obtained by biocatalysis, will contribute to reducing negative impacts on the

environment. It may thus be justifiable to rename it ‘bio-biodiesel’.

Immobilised

lipase

Triglyceride

alcohol

mixture

Reactor

(alcoholysis)

Separation of

reaction

mixture

Purification

of glycerol

Biodiesel

Glycerol

(Upper phase)

(Lower phase)

Figure 3. Flow diagram of biodiesel production through lipases

ACKNOWLEDGEMENTS

MIUR and CSGI (Firenze) are acknowledged for their financial support.

USE OF LIPASES FOR THE PRODUCTION OF BIODIESEL

337

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