Chinese Journal of Chemical Engineering 23 (2015) 1851–1856

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Chinese Journal of Chemical Engineering

journal homepage: www.elsevier.com/locate/CJChE

Biotechnology and Bioengineering

Influences of operating conditions on biocatalytic activity and reusability

of Novozym 435 for esterification of free fatty acids with short-chain
alcohols: A case study of palm fatty acid distillate
Sawittree Mulalee, Pongrumpa Srisuwan, Muenduen Phisalaphong ⁎
Chemical Engineering Research Unit for Value Adding of Bioresources, Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: In the present study, the effects of operating conditions on biocatalytic activity and stability of Novozym 435 for
Received 7 October 2014 repeated-batch biodiesel production from free fatty acid (FFA) were investigated. Thermal deactivation caused
Received in revised form 12 November 2014 by increased operating temperature from 45 to 50 °C could seriously affect the reusability of Novozym 435.
Accepted 12 May 2015
The deactivation of Novozym 435 during the esterification of oleic acid with ethanol tended to be stronger
Available online 20 August 2015
than that in the system with methanol. Under the optimal conditions, considering both biocatalytic activity
Keywords:
and stability of the enzyme, Novozym 435 could be reused for 13 cycles for biodiesel productions from
Biodiesel oleic acid and absolute alcohols (methanol and ethanol) with FFA conversions of at least 90%. The presence of
Oleic acid 4%–5% water in ethanol significantly affected the reusability of Novozym 435. Changes in the surface morphology
Novozym 435 of Novozym 435 during the esterification with various conditions were observed. It was revealed that the reduc-
Reusability tion in catalytic activity was related to the swelling degree of the catalyst surface. Additionally, biodiesel produc-
Palm fatty acid distillate tion from low cost renewable feedstocks, such as palm fatty acid distillate (PFAD) and 95% ethanol was examined.
The esterification of PFAD with 95% ethanol catalyzed by Novozym 435 in 10-repeated batch operation showed
the similar results in FFA conversion as compared to those using oleic acid. Novozym 435 remained active and
could maintain 97.6% of its initial conversion after being used for 10 batches.
© 2015 The Chemical Industry and Engineering Society of China, and Chemical Industry Press. All rights reserved.

1. Introduction process is considered to be a clean and environment friendly technique.

However, the high cost of enzymatic catalysts is considered to be the lim-
The energy supply shortage is currently one of the most important iting factor for their commercialization. Therefore, immobilization tech-
issues that continuously and directly affect humans. In Thailand, biodie- niques are used to enhance the potential for industrial-scale enzymatic
sel is one of the most attractive alternative fuels. Biodiesel is an environ- processes. Immobilization allows for the easy recovery of enzymatic cata-
mentally compatible product that can be produced from renewable lysts and for reuse of the catalyst several times without significant losses
resources, such as vegetable oils, free fatty acids (FFAs), animal fats in activity or stability [10].
[1,2] or waste cooking oil (WCO) [3] with short-chain alcohols, such Immobilized lipase from Candida antarctica lipase B (Novozym 435)
as methanol, ethanol, propanol and butanol [4]. The catalysts used in is an attractive biocatalyst for the production of biodiesel from many
the production of biodiesel can be classified as alkaline catalysts types of oil-containing seed plants in Thailand, such as palm (Elaeis
(NaOH or KOH), acid catalysts (H2SO4) [5], solid acid and base catalysts guineensis), physic nut (Jatropha curcas), papaya (Carica papaya) and
[6] and biocatalysts (lipase enzymes). rambutan (Nephelium lappaceum) [11]. The operating conditions, such
Lipase can selectively and effectively catalyze both transesterification as the temperature, initial molar ratio of FFA to alcohol, mixing rate
and esterification reactions with low energy consumption at mild operat- and enzyme concentration, have important roles in the enzymatic con-
ing temperatures of less than 50 °C [7]. Moreover, this process is more version of FFAs [12–14]. Influence of alcohol structure on the enzymatic
environmentally friendly than chemical processes [8]. Because the use activity was also reported [15].
of enzymatic catalysts does not form soaps, it can esterify both FFA and Oleic acid is a major component of various oils, such as palm oil,
TAG in one step without the need of a subsequent washing step [9]. rapeseed oil and used frying oil [16]. In our previous studies on enzy-
Since there is no discharge of chemicals and wastewater, the enzymatic matic esterification using Novozym 435 in a batch solvent-free system,
the optimal conditions for the conversion of oleic acid with methanol
and ethanol were reported, and the conversion yields were greater
⁎ Corresponding author. than 90% [13]. In this study, we focused on the study of effects of the op-
E-mail address: muenduen.p@chula.ac.th (M. Phisalaphong). erating conditions on the reaction progress and reusability of Novozym

http://dx.doi.org/10.1016/j.cjche.2015.08.016
1004-9541/© 2015 The Chemical Industry and Engineering Society of China, and Chemical Industry Press. All rights reserved.
1852 S. Mulalee et al. / Chinese Journal of Chemical Engineering 23 (2015) 1851–1856

435 for the esterification of oleic acid with short-chain alcohols. More- occur and thereby negatively affect the activity, stability and reus-
over, for further development of “green technology” for a commercial ability of enzymes. Our previous study showed that there was no
biodiesel production from low-cost feedstocks, the esterification of significant difference in the FFA conversion and the reaction rate
palm fatty acid distillate (PFAD), a byproduct from palm oil refining at temperatures between 45 °C and 60 °C [13]. In the present
and 95% ethanol using Novozym 435 as the catalyst was evaluated study, the stability of Novozym 435 during the enzymatic esterifi-
with repeated batch experiments. cation of oleic acid with short-chain alcohols, such as methanol
(99.9%) and ethanol (99.9%) was investigated at the operating tem-
2. Materials and Methods perature of 45 °C and 50 °C. As shown in Fig. 1A, with the use of
methanol, the initial FFA conversion slightly increased as the tem-
2.1. Materials and chemicals perature increased from 45 °C to 50 °C. However, it was found
that the final FFA conversion was not significantly affected by tem-
Novozym 435 (lipase B from C. antarctica, EC 3.1.1.3), a nonspecific perature changes in this temperature range. The effect of the ther-
lipase immobilized on macroporous acrylic resin was purchased from mal deactivation of Novozym 435 at 50 °C was more clearly
S.M. Chemical suppliers Co., Ltd, Bangkok, Thailand. The diameters of observed in the esterification of oleic acid with ethanol as shown
the particle beads are in a range of 0.3–0.9 mm with approximate den- in Fig. 1B. In the first batch with the fresh enzyme, the rates of
sity of 0.4 g·ml−1. The catalytic activity was 10000 PLU·g−1. All other FFA conversion and the final FFA conversions at the operating tem-
chemicals used in this study were purchased from local suppliers in perature of 45 °C and 50 °C were quite similar. However, from the
Thailand. second batch to the fifth batch, the rates of FFA conversion signifi-
cantly decreased as the temperature increased from 45 °C to 50 °C.
2.2. Enzymatic esterification reaction At the operating temperature of 50 °C, the FFA conversion after 8 h
was approximately 91% in the first batch, and it was less than 80%
The batch esterification process using 40 g of oleic acid and short- in the fifth batch, whereas no significant drop in FFA conversion
chain alcohols catalyzed by Novozym 435 was performed in a 250 ml was observed with the operating temperature at 45 °C. The results
Erlenmeyer flask. The optimal operating conditions obtained from indicated that for the long-term use of this biocatalyst, the optimal
our previous work were used, as follows: operating temperature of temperature for the esterification of oleic acid with methanol or ethanol
45 °C, FFA to alcohol molar ratio of 1:2, and enzyme loading of 5% by Novozym 435 should be 45 °C for maintaining high catalytic activity
(w/w of oleic acid) [13]. To minimize the effect of external mass and reusability of the enzyme.
transfer limitations and maintain the enzyme activity, the mixtures
were shaken at a constant rate of 250 r·min− 1 in a shaking incubator 3.1.2. Effect of alcohols
(Innova 4000, New Brunswick Scientific Co., Inc., Germany). Samples The effect of alcohols on the enzyme activity in acyl transfer re-
were collected from the mixtures during the reactions (0–8 h) for the actions includes reversible inhibition and irreversible inactivation
determination of FFA conversion. Water, a reaction byproduct and [17,18]. The type of alcohol might directly affect the reusability of
residual alcohols were removed from the samples via thermal evap- enzymes. In this part, the effects of alcohols, methanol (99.9%) and
oration. The samples were then analyzed using the titration method ethanol (99.9%), on the reusability of Novozym 435 in the esterifica-
to determine the FFA conversion. On the study of the reusability of tion of oleic acid were investigated. In Section 3.1.1, to reduce the ef-
Novozym 435, after each batch, the products and the remaining sub- fects of thermal deactivation on the activity and stability of
strates were removed, and fresh substrates were added for the next Novozym 435, the reactions were performed at a constant operating
cycle. temperature of 45 °C. As shown in Fig. 2, the FFA conversion rates to
produce methyl oleate were higher than those of ethyl oleate. Dur-
2.3. Biodiesel conversion analysis ing the production of methyl oleate, the conversion rate slightly de-
creased as the number of enzyme reuse cycles increased. However, a
The percentage of FFA conversion was determined by the titration gradual decrease in the FFA conversion rate with increasing number
method with 0.1 mol·L−1 KOH solution using phenolphthalein as the of Novozym 435 reuse cycles was more clearly seen during the pro-
indicator. The FFA conversions were calculated from the titration vol- duction of ethyl oleate. Nevertheless, for both reactions, Novozym
umes of the KOH solution. The reported values were the average values 435 could be reused for 13 repeated batches (104 h) with FFA con-
of each duplicate set. versions of at least 90%. Previously, it was reported that among all
enzymes tested, Novozym 435 was the most effective biocatalyst
2.4. Characterization of Novozym 435 for the methanolysis in a continuous process [19]. However, the de-
activation of Novozym 435 in the esterification could occur because
Scanning electron microscope (SEM) was performed to observe of the interaction of alcohols with the surface of Novozym 435
morphology changes of the biocatalyst (Novozym 435) after being through the adsorption of alcohols [20]. The denaturing effect of al-
used in the esterification reaction. Excess oil and solution at the surface cohols on proteins, causing enzyme deactivation is well known. Pro-
of the biocatalysts was blotted out with Kimwipes paper. The samples of gressive deactivation after several reuses of the biocatalyst during
biocatalysts were then sputtered with gold and were examined for mor- the esterification was also observed [20]. In previous reports, 85%
phological structures by the scanning electron microscope JSM-5410LV of the initial Novozym 435 activity was maintained after 9 batches
(Tokyo, Japan). in the transesterification of vegetable oils and ethanol [21], and
90% of the activity of Novozym 435 was maintained over 7 batch re-
3. Results and Discussion actions in the transesterification of vegetable oils and short-chain
alcohols [22].
3.1. Effects of operating conditions on catalytic activity and reusability of
Novozym 435 3.1.3. Effect of initial water content in alcohol
Influence of the initial water content on the esterification and
3.1.1. Effect of operating temperature transesterification reactions is another important issue. It was re-
Higher operating temperature results in higher initial conver- ported that transesterification reactions catalyzed by Candida rugosa,
sion rate and according to the high rates, the reaction reaches equi- Pseudomonas cepacia, and Pseudomonas fluorescens lipases could not
librium sooner. However, thermal deactivation of enzymes might occur in the water-free system [9]. However, Novozym 435 exhibited
 S. Mulalee et al. / Chinese Journal of Chemical Engineering 23 (2015) 1851–1856 1853

Fig. 1. The effect of operating temperature on the reusability of Novozym 435 in the esterification of oleic acid with 99.9% methanol (A) and 99.9% ethanol (B). The cycle time is 8 h and the
operating temperatures are 45 °C ( ) and 50 °C ( ).

Fig. 2. The effect of type of alcohol on the reusability of Novozym 435 in the esterification of oleic acid at 45 °C with 99.9% methanol ( ) and 99.9% ethanol ( ).

the highest activity with a low availability of water content [23,24]. however it exhibited a negative effect on the reusability of Novozym
The presence of water during the reaction could have a negative ef- 435. As shown in Fig. 3, when 95.0% ethanol and 96.0% ethanol were
fect on the conversion of FFAs due to the occurrence of a hydrolysis used, to retain a conversion of at least 90%, the cycles of reuse of
reaction during transesterification [25] and a reversible reaction ef- Novozym 435 should not be greater than 10 and 11 batches, respective-
fect on esterification [4]. Methanol is mostly produced from coal or ly, whereas the number of Novozym 435 reuse cycles in the system
natural gas. It is available commercially in anhydrous form. Ethanol using anhydrous ethanol was about 13 cycles. Previously, in a solvent-
is considerably less toxic and contains more energy than methanol. free system, the deposition of water on the surface of the lipase could re-
Ethanol is mostly produced from renewable materials via fermenta- duce the conversion yield of biodiesel [28]. A fast interaction between
tion processes. During the separation of ethanol by distillation, etha- water and enzymes might deactivate enzymes and the presence of
nol forms an azeotrope with water with a composition of 96.6% water as a byproduct of the reaction could lower the equilibrium con-
ethanol and 3.4% water (by volume). Therefore, hydrated ethanol version [4]. However, it was suggested that the inhibition of the enzyme
(95%–96%, v/v) is more available at a relatively lower price than an- by water is reversible, which could be solved by the removal of water
hydrous ethanol (99.9%, v/v). Thus, in this study, the effect of the ini- [19]. It was reported that the enzyme recovered its catalytic activity
tial water content in ethanol on the reusability of Novozym 435 after being dried to its initial water content [28]. The reusability of en-
during the esterification of oleic acid was investigated. zyme could also be influenced by other factors such as the purity of al-
The initial water content in ethanol was varied by using 99.9% (v/v), cohols and the contaminants of toxic organic compounds.
96.0% (v/v) and 95.0% (v/v) ethanol. During the first hour of the reac-
tion, the initial FFA conversions during the production of ethyl oleate 3.2. Effects of operating conditions on the surface morphology of Novozym
with 96.0% and 95.0% ethanol were slightly higher than that with 435
99.9% ethanol. However, the final conversion reached the same level
of about 90% (Fig. 3). A higher initial rate of esterification catalyzed by Loss of the enzyme activity could also be caused by deactivation of
an enzymatic catalyst in a system containing some amount of water the enzyme from the adsorption of alcohols on the surface. The experi-
was previously reported during the production of biodiesel from soy- mental results in this study showed that in a solvent-free system, the
bean oil catalyzed by various lipases, such as Rhizopus oryzae, deactivation of Novozym 435 during the esterification of oleic acid
C. rugosa, P. fluorescens, C. antarctica and Burkholderia cepacia [26]. It with ethanol tended to be stronger than that observed in the system
was suggested that a certain amount of water was required for many with methanol. Under SEM observation of the surface morphology of
enzymes to work [26,27]. fresh Novozym 435 (Fig. 4A) compared to that after being used in the
The presence of 4%–5% water in ethanol did not considerably affect system using 99.9% methanol at operating temperature of 45 °C for 5 re-
the FFA conversion catalyzed by Novozym 435 in Batch 1 to Batch 9; peated batches (Fig. 4B), a slight change in the surface morphology was
1854 S. Mulalee et al. / Chinese Journal of Chemical Engineering 23 (2015) 1851–1856

Fig. 3. The effect of initial water content in ethanol on the reusability of Novozym 435 in the esterification of oleic acid at 45 °C with 99.9% ethanol ( ), 96.0% ethanol ( ) and 95.0%
ethanol ( ).

Fig. 4. SEM micrographs of Novozym 435 in the esterification of oleic acid with short-chain alcohols at various operating conditions.
 S. Mulalee et al. / Chinese Journal of Chemical Engineering 23 (2015) 1851–1856 1855

noticed. On the other hand, the partial swelling of the catalyst surface 4. Conclusions
was clearly observed after being used for 5 repeated batches in the
system using 99.9% ethanol at 45 °C (Fig. 4D). A significant increase in The reaction progress and reusability of Novozym® 435 for the pro-
the degree of swelling was observed on the surface of Novozym 435 duction of biodiesel from oleic acid with short-chain alcohols, methanol
as the operating temperature was increased to 50 °C. Compared to the and ethanol were studied. Novozym 435 gave the maximum FFA
system using methanol at 50 °C for 5-repeated batches (Fig. 4C), consid- conversion of 94.6% in the production of methyl oleate and 90.5% in
erable increase in the degree of swelling was observed in the system the production of ethyl oleate. The results indicated that temperature,
using ethanol at 50 °C (Fig. 4E). From the experimental results, it was type of alcohol, and water content considerably affected the reusability
noticed that the reduction in catalytic activity or the degree of deactiva- of Novozym 435. It was shown that under the optimal conditions,
tion of Novozym 435 was related to the swelling degree of the catalyst Novozym 435 could be reused in the production of methyl oleate and
surface. From the SEM observation, it was also suggested that the reac- ethyl oleate for 13 cycles with FFA conversions of at least 90%. Changes
tion at a high operating temperature might enhance the adsorption of in the surface morphology related to the degree of deactivation of
alcohols, especially in the system using ethanol, resulting in significant Novozym 435 during the esterification with various conditions were
reduction in the reusability of Novozym 435. observed. Furthermore, the esterification of PFAD and 95% ethanol
Novozym 435 catalyzed the reaction with high activity under highly catalyzed by Novozym 435 in 10-repeated batch operation showed
water-deficient conditions [23,24,29]. Previously, it was suggested that the similar results in FFA conversion as compared to those using oleic
the agglomeration of water might flood the enzyme pores, causing de- acid. It was demonstrated that PFAD and 95.0% ethanol have high
creases in the reaction rates [30]. In this study, it was also shown that potentials as a source of low-cost feedstock for biodiesel production
the presence of water at 4%–5% in ethanol causes some negative effects by enzymatic esterification.
on the reusability of the biocatalyst. Considerable swelling of the cata-
lyst surface was also observed after Novozym 435 was being used for
13 repeated batches in the system using 95% ethanol at 45 °C (Fig. 4F). Acknowledgments

3.3. A case study of palm fatty acid distillate (PFAD) We thank the National Research Council of Thailand (NRCT) for fi-
nancial support. SM also received support from the 90th anniversary
This study also focuses on producing biodiesel from low cost feed- of Chulalongkorn University (Ratchadaphiseksomphot Endowment
stocks such as palm fatty acid distillate (PFAD) (Patum Vegetable Oil Fund).
Co., Ltd., Pathum Thani, Thailand) and 95% ethanol. The esterification
of PFAD and 95% ethanol catalyzed by Novozym 435 in 10-repeated
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