Bioresource Technology 102 (2011) 63526355

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Biodiesel production in packed-bed reactors using lipasenanoparticle biocomposite

Xia Wang a, Xueying Liu a, Chuanming Zhao a, Yi Ding b,, Ping Xu c,a,
a

State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, PR China
School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China
c
MOE Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, PR China
b

a r t i c l e

i n f o

Article history:
Received 5 January 2011
Received in revised form 27 February 2011
Accepted 1 March 2011
Available online 5 March 2011
Keywords:
Biodiesel
Fe3O4
Lipase
Nanoparticle
Packed-bed reactor

a b s t r a c t
The development of appropriate reactors is crucial for the production of biodiesel. In this study, a packedbed reactor system using lipaseFe3O4 nanoparticle biocomposite catalyst was successfully developed for
biodiesel production based on soybean oil methanolysis. Emulsication before methanolysis improved
the reaction rate. The lipasenanoparticle biocomposite showed high activity and stability in the single-packed-bed reactor at an optimal ow rate (0.25 mL min 1). After 240 h of reaction, the conversion
rate was sustained as high as 45%. The conversion rate and stability achieved using the four-packedbed reactor were much higher than those achieved using the single-packed-bed reactor. The conversion
of biodiesel was maintained at a high rate of over 88% for 192 h, and it only slightly declined to approximately 75% after 240 h of reaction. The packed-bed reactor system, therefore, has a great potential for
achieving the design and operation of enzymatic biodiesel production on the industrial scale.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Biodiesel produced from vegetable oil does not produce sulfur
oxide and can reduce soot production by one-third compared
with existing petroleum-based products (Ma and Hanna, 1999;
Al-Zuhair, 2007; Ranganathan et al., 2008). Biodiesel is, therefore,
expected to be a good substitute for fossil fuel (Baroutian et al.,
2011; Iso et al., 2001; Sivasamy et al., 2009). However, owing to
the high cost of enzyme catalysts and the relatively slow reaction
rate, the enzymatic production of biodiesel has not yet been applied on the industrial scale (Robles-Medina et al., 2009). For
highly efcient applications of enzyme catalysts, they have to be
immobilized onto appropriate substrates that allow repeated cycling in a rather simple manner. However, the immobilization of
an enzyme alone is not sufcient to ensure a high biodiesel yield.
After successful enzyme immobilization, the development of
appropriate reactors is of critical importance if enzymatic biodiesel
production on an industrial scale is to be realized.
The enzymatic transesterication of vegetable oils can be carried out substantially faster and more economically in continuous
reactors than in batch reactors (Komers et al., 2010; Lee et al.,
2010; Rosa et al., 2009). Batch operation is considered slow, tedious, labor intensive, and not well suited for automation (Chen
et al., 2010). In comparison with a batch process, a continuous pro Corresponding authors at: School of Chemistry and Chemical Engineering,
Shandong University, Jinan 250100, PR China. Tel.: +86 531 88369463; fax: +86 531
88567250 (P. Xu), Tel.: +86 531 88366513; fax: +86 531 88369463 (Y. Ding).
E-mail addresses: yding@sdu.edu.cn (Y. Ding), pingxu@sdu.edu.cn (P. Xu).
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.03.003

cess has evident advantages: the production cost and time can be
adjusted according to the work level, a great amount of biodiesel
per labor unit can be obtained, and the equipment design is exible
in terms of optimizing of biodiesel quality (Halim et al., 2009).
In our previous work, a lipasenanoparticle biocomposite was
prepared by a simple and effective immobilization procedure using
Fe3O4 as a support (Wang et al., 2009), and batch production of biodiesel by using this biocomposite was achieved. In the present
work, we focused on the development of lipasenanoparticle biocomposite-based packed-bed reactors that allow effective and continuous biodiesel production.
2. Methods
2.1. Chemicals
Pseudomonas cepacia lipase was purchased from SigmaAldrich
(St. Louis, MO, USA). Rened soybean oil was purchased from Jinlongyu Company (China).
2.2. Instrumentation
A glass column was used as the packed-bed reactor, in which
the lipasenanoparticle biocomposite (including 40 mg of lipase)
loaded with cotton (approximately10 g) served as a support. The
specication of the packed-bed reactor was as follows. The inner
radius and axial height of the packed-bed reactor were 1.6 and
20 cm, respectively. The volume of the packed-bed reactor was

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6353

160 mL. The weight of the packing material in the packed-bed

reactor was 40 g, and the working volume was 136 mL. The lipasenanoparticle biocomposite loading was easily adjustable because the lipasenanoparticle biocomposite particles were evenly
distributed in the cotton network.
2.3. Enzymatic transesterication reaction
Magnetic Fe3O4 nanoparticles and the lipasenanoparticle biocomposite were prepared as previously described (Wang et al.,
2009). The transesterication reaction was carried out in a single-packed-bed reactor (Fig. 1A), or a four-packed-bed reactor
(Fig. 2A), using a mixture of soybean oil, distilled water, methanol,

Fig. 2. (A) A schematic diagram of biodiesel production in the four-packed-bed

reactor. a, inlet close tank; b, peristaltic pump; c, four-packed-bed reactor; d, outlet
open tank. (B) Biodiesel production in the four-packed-bed reactor.

and n-hexane. The ratio of the volume of soybean oil:distilled

water:methanol:n-hexane is 6:3:1:0.2. The temperature was controlled at 40 C. In the single-packed-bed process, the mixture
was added to a 500 mL round-bottom ask, and then transferred
from the round-bottom ask into the packed-bed for the reaction.
After 24 h of reaction, the mixture was removed such that the concentration of methyl esters could be determined, and a new mixture was added for the reaction. For optimization of the ow
rate, 50 mL of the mixture was added to a 500 mL round-bottom
ask, and then fed into a single-packed-bed at different ow rates.
When all of the mixture owed out of the single-packed-bed, the
efuent liquid was removed to determine the concentration of
methyl esters. In the four-packed-bed process, the mixture of rened soybean oil, distilled water, methanol, and n-hexane was
incubated in a 500 mL round-bottom ask with an agitation system. Subsequently, the mixture was fed to a four-packed-bed reactor from the inlet. The efuent liquid from the outlet was removed
to determine the concentration of methyl esters.
2.4. Analytical method
The methyl esters were extracted with n-hexane for gas chromatography analysis. Methyl heptadecanoate served as the internal standard. The content of methyl esters in the reaction
mixture was analyzed by gas chromatography using a Varian
CP3380 (CP3380, Varian Associates, Inc.) equipped with a SPB-5
column (0.32 mm  0.25 lm  30 m, Supelco) and a ame ionization detector. The column temperature was maintained at 150 C,
and then raised to 170 C at a rate of 0.5 C min 1. The injector
and detector temperature were set at 300 C.
3. Results and discussion
Fig. 1. (A) A schematic diagram of biodiesel production in the single-packed-bed
reactor. a, inlet close tank; b, peristaltic pump; c, single-packed-bed reactor. (B)
Effect of ow rate on the content of methyl esters content. 1, 0.125 mL min 1; 2,
0.25 mL min 1; 3, 0.5 mL min 1; 4, 1.0 mL min 1; 5, 2.0 mL min 1. (C) Biodiesel
production in the single-packed-bed reactor.

3.1. Optimization of ow rate

The key operational variables in a packed-bed reactor are the
amount of enzyme and the ow rate (Robles-Medina et al.,

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X. Wang et al. / Bioresource Technology 102 (2011) 63526355

2009). Therefore, the effect of ow rate on the conversion rate of

biodiesel was investigated at different ow rates, ranging from
0.125 to 2 mL min 1 in a single-packed-bed reactor (Fig. 1A). The
conversion rate increased as the ow rate increased, and the maximum conversion rate was obtained at a ow rate of 0.25 mL min 1
(Fig. 1B). After this point, the conversion rate decreased as the ow
rate continued to increase indicating that the shear stress of the
uid ow at the highest ow rates damaged the lipasesnanoparticle biocomposite, leading to the loss of lipase activity. This phenomenon was also observed by other researchers (Hama et al.,
2007). On the basis of the above results, a ow rate of
0.25 mL min 1 was used in the subsequent experiments.
3.2. Production of biodiesel in a single-packed-bed reactor
In our previous study, a batch process for biodiesel production
using the lipasenanoparticle biocomposite was developed (Wang
et al., 2009). However, deactivation of lipase as a result of the high
methanol concentration and shearing force was a technical concern preventing its application to the large-scale production of biodiesel. Therefore, in the present study, we attempted a prolonged
continuous ow reaction with packed-bed reactors. The production of biodiesel in the single-packed-bed reactor was carried out
with a substrate ow rate of 0.25 mL min 1 (Fig. 1A). Before methanolysis, the reaction mixture was emulsied by an agitation system with an impeller. As expected, the lipasenanoparticle
biocomposite showed high activity and stability in the singlepacked-bed reactor. The conversion rate increased with the reaction time, as shown in Fig. 1C. The maximum conversion rate
(approximately 75%) was observed at 12 h, followed by a stationary phase lasting another 132 h approximately. After 240 h, the
conversion rate remained as high as 45%. In contrast, after nine cycles, the conversion rate remained at only 18.5% in the batch process in our previous study (Wang et al., 2009). This noticeable
increase that in the conversion rate from that of the batch process
was probably caused by a decrease in the deactivation of the lipasenanoparticle biocomposite resulting from the high methanol
concentration and shearing force. Furthermore, the emulsication
of the reaction mixture has a benecial effect on methanolysis
through formation of oilwater droplets, thereby increasing the
available interfacial area. In addition, because the lipase catalysis
occurs in the interfacial layer between the hydrophobic and hydrophilic phases (Al-Zuhair et al., 2003; Rostrup-Nielsen et al., 1990),
the much larger surface area of the water/oil interface seems to result in an increased accessibility of the substrates to the lipase. Finally, the packed-bed reactor may be operated with no need for
separation of the biocatalyst from the reaction products as is the
case with stirred-tank reactors (Robles-Medina et al., 2009).
3.3. Production of biodiesel in a four-packed-bed reactor
For repeated and highly efcient use of lipase, we developed a
four-packed-bed reactor as shown in Fig. 2A. Biodiesel was continuously produced at a feed ow rate of 0.25 mL min 1. The conversion rate of biodiesel was maintained over 88% for 192 h (Fig. 2B).
After 240 h of reaction, the conversion rate slightly dropped to
approximately 75%. Compared with the single-packed-bed reactor,
an evidently high conversion rate and good stability were obtained
with the four-packed-bed reactor. The reasons for this may be that
the four-packed-bed reactor provided a longer residence time of
the reaction mixture in the reactor and reduced the inhibition of
the lipasenanoparticle biocomposite by products, which may
have further improved the reaction efciency (Hama et al., 2007;
Thanh et al., 2010). Furthermore, the effective recycling of the enzyme catalysts contributes to lowering of the cost of biodiesel production (Fjerbaek et al., 2009). Interestingly, our results differ from

those of other studied in which only 67% conversion was observed

at the optimal conditions in the biodiesel production from soybean
oil (Noureddini et al., 2005), and 80% conversion was maintained in
a packed-bed reactor for 120 h (Halim et al., 2009). It is important
to note that, from the economic point of view, the cost involved in
lipase immobilization would also result in an overall increase in
the fabrication cost of biodiesel, the main obstacle preventing complete reuse of lipase (Al-Zuhair, 2007). In this study, the lipase
nanoparticle biocomposite was prepared using a simple and effective immobilization procedure in which Fe3O4 served as a support,
as shown in our previous work (Wang et al., 2009). Thus, the
advantages of using an immobilized enzyme could outweigh the
increased cost of immobilization. On the laboratory scale, these results have great potential for improving the design and operation
of large-scale enzymatic systems.
4. Conclusions
In this study, a method for the continuous production of biodiesel in a packed-bed reactor system was successfully developed
using lipaseFe3O4 nanoparticle biocomposite catalyst. Compared
with the single-packed-bed reactor, a high conversion rate and
great stability were achieved with the four-packed-bed reactor.
The efcient reuse of the enzyme was realized via a simple and
effective immobilization procedure that resulted in a high initial
activity without inactivation or inhibition. The packed-bed reactor
system presented here highlights the possibility of designing and
operating even larger-scale enzymatic systems.
Acknowledgements
This work was partially supported by grants from the National
Natural Science Foundation of China (20706035), New Teacher
Foundation of the Ministry of Education of China
(20090131120005), and Excellent Middle-Aged and Youth Scientist Award Foundation of Shandong Province (BS2010SW016).
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