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Short Communication

Biodiesel production by a mixture ofCandida rugosa and Rhizopus oryzae

lipases using a supercritical carbon dioxide process

Jong Ho Lee a, SungBong Kim a, Seong Woo Kang a, Yoon Seok Song a, Chulhwan Park b, Sung Ok Han c,Seung Wook Kim a,

a Department of Chemical and Biological Engineering, Korea University, 1, Anam-dong, Sungbuk-ku, Seoul 136-701, South Koreab Department of Chemical Engineering, Kwangwoon University, 447-1, Wolgye-Dong, Nowon-Gu, Seoul 139-701, South Koreac College of Life Science and Biotechnology, Korea University, 1, Anam-dong, Sungbuk-ku, Seoul 136-701, South Korea

a r t i c l e i n f o

Article history:

Received 25 March 2010

Received in revised form 7 August 2010

Accepted 10 August 2010

Available online 13 August 2010

Keywords:

Biodiesel

Lipase

Initial reaction rate

Optimization

Supercritical carbon dioxide process

a b s t r a c t

In thisstudy, variousfactors,such as temperature, pressure, agitation speed, water content, andthe concen-

tration and ratio of immobilized ROL and CRL were investigated for the efficient enzymatic production of

biodiesel using a supercritical carbon dioxide process. Furthermore, a stepwise reaction method for the

maintenance of immobilized lipase activity was optimized. Optimal conditions for biodiesel production

weredetermined tobe asfollows:130 bar pressure,45 C temperature,250 rpmagitation speed, 10%water

content, and20% immobilized ROL and CRL (1:1). When batch process was performed under optimal con-

ditions, thebiodieselconversionyieldwas99.13% at3 h. Biodieselconversion yieldwas 99.99%at 2 h when

90 mmol methanol wasusedin a stepwisereaction.Moreover,the conversion yield ofbiodiesel producedby

the repeated recycling of immobilized lipase in the stepwise reactions was 85% after 20 reuses.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

Biodiesel, one of the most interesting alternatives for renewable

fuel, is produced from renewable sources of feedstock by transeste-

rification (Rosa et al., 2008; Demirba, 2003). Among various pro-

cesses for biodiesel production, enzymatic processes using

various lipases are often selected due to its reasonable energy

intensity and simplicity (Shimada et al., 2002; Tan et al., 2010;

Watanabe et al., 2000). But due to disadvantages such as slowreac-

tion rate, it cannot be acceptable widely for commercialization.

Then many researchers focused on enhancement of reaction rate

via new immobilization process, supercritical carbon dioxide pro-

cess (SCCO2) and stepwise reaction method (Rosa et al., 2008; Shi-

mada et al., 2002; Savage and Pinnarat, 2009; Shieh et al., 2003;

Varma et al., 2010; Watanabe et al., 2000). In our previous work,new processes for biodiesel production were developed using

SCCO2for efficient mass transfer. Moreover, novel reaction mecha-

nisms involving the elimination of the acyl-migration step also

have been developed using an enzyme mixture (Lee et al., 2009).

In this study, to increase the reaction rate and conversion yield,

reaction factors were optimized in SCCO2process contained an en-

zyme mixture systemof hydrolase and esterase. The stepwise reac-

tion method was also investigated to prevent deactivation of

immobilized lipases.

2. Materials and methods

2.1. Lipase immobilization

For the pretreatment of lipase, 1 g ofCandida rugosalipase (CRL,

Sigma) or Rhizopus oryzae lipase (ROL, Fluka) was suspended in

100 ml of 0.25 M MOPs-Sodium phosphate buffer (pH 6.5). The

supernatant was pretreated with 0.1% soybean oil at 40 C for

45 min. For the immobilization of pretreated lipase, 1 g of dry silica

gel was mixed with 10% 3-aminopropyltriethoxysilane in 20 ml of

acetone and incubated at 50 C for 2 h. After incubation, silica gel

was washed and dried. To activate the silica gel, the dried silica

gel was suspended in 20 ml of 0.1 M phosphate buffer solution

(pH 7) containing glutaraldehyde (2 ml) and incubated at 20 C

for 2 h. The activated silica gel was washed and then dried. Acti-

vated silica gel (500 mg) was mixed with 10 ml of pretreated lipase

solution and then immobilized at 20 C. The immobilized lipase

was recovered by filtration.

2.2. Biodiesel production by immobilized C. rugosa and R. oryzae

lipases

Biodiesel was produced in a 100 ml SCCO2 reactor unit (Lee

et al., 2009). In batch reaction, the reactants including 60 mmol

0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2010.08.034

Corresponding author. Tel.: +82 2 3290 3300; fax: +82 2 926 6102.

E-mail addresses: [email protected] (J.H. Lee), [email protected] (C. Park),

[email protected](S.O. Han),[email protected](S.W. Kim).

Bioresource Technology 102 (2011) 21052108

Contents lists available at ScienceDirect

Bioresource Technology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o r t e c h
http://dx.doi.org/10.1016/j.biortech.2010.08.034mailto:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.biortech.2010.08.034http://www.sciencedirect.com/science/journal/09608524http://www.elsevier.com/locate/biortechhttp://www.elsevier.com/locate/biortechhttp://www.sciencedirect.com/science/journal/09608524http://dx.doi.org/10.1016/j.biortech.2010.08.034mailto:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.biortech.2010.08.034


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soybean oil and 240 mmol methanol were mixed with 20% of

immobilized CRL and ROL in the reactor. As temperature of reactor

was increased from30 to 60 C, the pressure was increased from 70

to 160 bar. In the stepwise reaction, the methanol concentration of

the reaction medium containing 60 mmol of soybean oil and 15 g

of immobilized lipases was adjusted to 60120 mmol. Each equiv-

alent amount of methanol was then added with the different time

interval during biodiesel production.

2.3. Analytical method

Biodiesel was analyzed using a GC M600D (Younglin Co. Ltd.,

Korea) with a HP-innowax 1909IN-133 column (30 m 25lm,Agilent, USA). The column temperature was raised from 150 to

180 C at a rate of 15 C min1, and then from 180 to 240 C at a

rate of 5 C min1, after which the temperature was maintained

at 240 C for 1 min. The injector and the detector temperature were

both set at 260 C. Conversion yield and initial reaction rate were

calculated as described inLee et al. (2009).

3. Results and discussion

3.1. Determination of optimal condition for biodiesel production using

SCCO2

In our previous work, the reaction rate of immobilized Candidaantartica lipase with SCCO2 was very low, although the biodiesel

Fig. 1. Effects of temperature (A), pressure (B), agitation speed (C), water content (D), enzyme concentration (E) and enzyme ratio (F) on biodiesel production and initial

reaction rate under supercritical fluid conditions by immobilized CRL and ROL: initial reaction rate (d), conversion yield (bar).

2106 J.H. Lee et al. / Bioresource Technology 102 (2011) 21052108


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conversion yield was increased to 98.92% (Lee et al., 2009). There-

fore, to increase the biodiesel conversion yield and reaction rate,

reaction factors were optimized in the batch reaction. The optimal

conditions determined on the basis of maximum conversion yield

and best initial reaction rate were 45 C of temperature, 130 bar

of pressure, 250 rpm of agitation speed and 10% of water content

(Fig. 1(AD)). As pressure increased at 45 C, observed change of

temperature was less than +1 C. Compared to temperature and

pressure, the increase in efficiency afforded by agitation speed

was relatively low. Accordingly, these results show that the others

are much more effective than mechanical agitation in the mass

transfer of SCCO2. Thus, optimized levels of heat and pressure

can induce modifications in structure of enzyme and changes of

substrate affinity and function, result changes of both in enzyme

activity and mass transfer (Oliveira and Oliveira, 2001).

Immobilized enzyme concentration is also an important factor

because inhibition of diffusion was expected in a high viscosity

system.Gao et al. (2006)reported that inhibition of biodiesel pro-

duction was induced by a high concentration of enzyme. Addition-

ally, if inhibition of mass transfer does not occur, the reaction rate

was increased linearly with enzyme concentration (Rosa et al.,

2008; Gao et al., 2006). Therefore, to decrease the inhibition of

intraparticle diffusion, the optimal concentration of immobilized

lipases should be investigated under the above optimal conditions.

The biodiesel conversion yield and initial reaction rate with 30%

immobilized lipases were 99.91% and 2.97 102 mmol l1 s1,

respectively, as shown inFig. 1(E). Significant differences in con-

version yield were not observed at enzyme concentrations of

20%, 25% and 30%. On the other hand, the biodiesel conversion

yield was decreased markedly when less than 20% immobilized li-

pases was used. Thus, the optimal concentration of immobilized li-

pases was 20% and the conversion yield and initial reaction rate at

the concentration were 99.93% and 2.66 102 mmol l1 s1,

respectively. Transesterification consists of three steps of which

the rate-determining step is acylmigration (Lee et al., 2007). In or-

der to increase reaction rate, the acyl-migration step should be

treated. Therefore, the mixture of 1,3-specific lipase (ROL) andnon-specific (CRL) lipase was used to eliminate the acyl-migration

step, followed by investigation of ratio of CRL and ROL for efficient

biodiesel production. Conversion yield of biodiesel remained al-

most constant, except at a ratio of 1:3 and initial reaction rate

was decreased as the ratio of lipase varied from 1:1 to 3:1 (Fig. 1

(F)). Therefore, the optimal ratio of immobilized ROL and CRL

was 1:1.

3.2. Batch and stepwise reaction of biodiesel production

To prove optimal reaction conditions, biodiesel was produced

using a mixture of immobilized ROL and CRL under optimal condi-

tions. In the batch reaction, biodiesel conversion of this mixture

was 99.13% at 3 h (Fig. 2(A)). Compared with our previous workshowing a conversion rate of over 98% at 4 h, the reaction rate in

the present study is higher (Lee et al., 2009). In this process, the

activity of immobilized lipases was maintained during biodiesel

production. Thus, an SCCO2 process that has the efficient character-

istics of heat and mass transfer is suitable for use in high viscosity

systems such as biodiesel production.

Because lipases were continuously deactivated by initial high

concentration of methanol, a stepwise reaction method was inves-

tigated to decrease the effect of accumulated methanol in the

SCCO2 process. Some researchers reported that lipase can be deac-

tivated using short chain alcohols, due to their intense polarity and

hydrophilic nature (Gao et al., 2006; Shimada et al., 2002). Espe-

cially, in a high heat and mass transfer system such as the SCCO2

process, deactivation is accelerated by rapid methanol transfer.Therefore, prevention of lipase activation can be confirmed if

methanol does not accumulate during biodiesel production. Sev-

eral methanol concentration (60, 90, 120 mmol) was initially

added into the reaction medium and the same equivalent amount

was feed at intervals during biodiesel production. When the initialconcentration of methanol was 90 mmol, the conversion yield of

Fig. 2. Biodiesel production under supercritical fluid conditions (A) and production

using various stepwise reaction methods (B)with a mixture of immobilized CRLand

ROL. Reaction conditions were as follows: pressure 130 bar, temperature 45 C,agitation speed 250 rpm, water content 10% and immobilized enzyme concentra-

tion (CRL:ROL = 1:1) 20%. Arrows indicate the feeding time.

Number of use

0 5 10 15 20

C

onversionyield(%)

0

20

40

60

80

100

Batch process by mixture of immobilized lipases

Stepwise process by mixture of immobilized lipases

Fig. 3. Biodiesel production attained by reusing immobilized CRL and ROL in batch

and stepwise reaction method.

J.H. Lee et al. / Bioresource Technology 102 (2011) 21052108 2107


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biodiesel reached 99.99% at 2 h. At an initial concentration of 60

and 120 mmol, conversion yields of biodiesel at 3 h were 99.16%

and 99.14%, respectively (Fig. 2 (B)). For production of biodiesel

by stepwise reaction, the optimal methanol concentration and

feeding interval was 90 mmol and 0.75 h, respectively.

As acyl-migration, the rate-determining step, is eliminated,

other rate-determining step is regarded. Biodiesel production is

two-substrate reaction therefore a mechanism based on ping-

pong-bi-bi could be used to kinetic study. Because methylation

reaction is relatively slower than esterification reaction and the

methylation reaction might be a rate-determining step when bio-

diesel is produced with a mixture of CRL and ROL under supercrit-

ical condition (Tan et al., 2010; Varma and Madras, 2008; Varma

et al., 2010).

3.3. Reuse of immobilized lipase for repeated biodiesel production

Reuse of immobilized lipase is very important for industrial

applications. After batch and stepwise reactions, immobilized li-

pase was washed with water and isopropyl alcohol, and then re-

used for the next reaction. The biodiesel conversion yield by the

stepwise reaction was about 85% after 20 reuses (Fig. 3). However,

when a batch reaction was performed, the conversion yield of bio-

diesel was decreased to 80% after 13 reuses. These results show

that lipase activity was enhanced as a result of the reduced concen-

tration of accumulated methanol. Thus, the stepwise reaction

method under supercritical fluid conditions is also efficient for bio-

diesel production from vegetable oil and methanol as shown by Lee

et al. (2009).

4. Conclusion

Biodiesel was produced by immobilized ROL and CRL under

supercritical fluid conditions and its production was increased

with optimal reaction factors. When a batch process was per-

formed under optimal conditions, the conversion yield of biodiesel

was 99.13% at 3 h. A stepwise reaction method could prevent the

deactivation of lipases in supercritical fluid condition. When the

initial concentration of methanol in the reaction medium was

90 mmol, and this concentration was feed at 0.75 h intervals, the

biodiesel conversion yield was 99.99% at 2 h. Finally, the conver-

sion yield of stepwise method and batch reaction were about

85% after 20 reuses and 80% after 13 reuses, respectively.

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