Bioresource Technology 147 (2013) 395–400

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

Development of a novel integrated continuous reactor system

for biocatalytic production of biodiesel
Soham Chattopadhyay a, Ramkrishna Sen b,⇑
a
Department of Biotechnology, Heritage Institute of Technology, Kolkata, West Bengal 700107, India
b
Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Integrated CSTR-PBR based

continuous biodiesel production
process was developed.
 Biodiesel was produced by
indigenously developed immobilized
enzyme as catalyst.
 Process was perfectly continuous
with no intermittent methanol
addition steps.
 The process was stable up to several
cycles with high productivity.
 Process parameters were
standardized to obtain maximum
percentage conversion.

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

Article history: A novel integrated immobilized enzyme-reactor system involving a continuous stirred tank reactor with
Received 11 June 2013 two packed bed reactors in series was developed for the continuous production of biodiesel. The problem
Received in revised form 31 July 2013 of methanol solubility into oil was solved by introducing a stirred tank reactor to dissolve methanol into
Accepted 5 August 2013
partially converted oil. This step made the process perfectly continuous without requiring any organic
Available online 12 August 2013
solvent and intermittent methanol addition in the process. The substrate feeding rate of 0.74 mL/min
and enzyme loading of 0.75 g per reactor were determined to be optimum for maximum biodiesel yield.
Keywords:
The integrated continuous process was stable up to 45 cycles with biodiesel productivity of 137.2 g/L/h,
Biodiesel
Green continuous process
which was approximately 5 times higher than solvent free batch process. In comparison with the pro-
Integrated CSTR-PBR system cesses reported in literature using expensive Novozyme 435 and hazardous organic solvent, the present
Immobilized lipase process is completely green and perfectly continuous with economic and environmental advantages.
Solvent free system Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction system. This problem can be overcome by stepwise addition of

Biodiesel, the mixture of mono-alkyl esters of long chain fatty
acids, are renewable in nature and can be used as blends with
petroleum diesel. Various lipids have been tried as feedstock for
the production of biodiesel using free and immobilized lipases.
The insoluble methanol droplets present in reaction mixture is
primarily responsible for inactivation of enzyme in solvent free
⇑ Corresponding author. Tel.: +91 3222283752; fax: +91 3222278707.
E-mail addresses: soham.chattopadhyay@heritageit.edu (S. Chattopadhyay),
rksen@hijli.iitkgp.ernet.in (R. Sen).
methanol into the partially converted oil (Lee et al., 2011). Another
approach to reduce the inhibitory effect of methanol is to use a
partially polar solvent like t-butanol, which facilitate the solubility
of methanol into oil (Royon et al., 2007). Though various organic
solvents were studied for biodiesel production, solvent free
systems are still preferred for their environment friendly nature.
At present most of the commercial biodiesel productions are
carried out in batch mode. Recently, the continuous process
employing fixed bed reactor has extensively experimented in
laboratory scale due to its higher productivity than batch process
(Severac et al., 2011). Immobilized C. antarctica lipase, commercially

0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.08.023
396 S. Chattopadhyay, R. Sen / Bioresource Technology 147 (2013) 395–400
known as Novozyme 435, was used for most of the reported liter-
atures (Severac et al., 2011; Sakai et al., 2010; Halim et al., 2009).
This immobilized proparation showed efficient transesterification
property in wide variety of lipid sources. The cost of the Novozyme
435 is significantly high and that makes the overall process expen-
sive. Some relatively inexpensive immobilized system is needed to
be developed to produce biodiesel in economically feasible way. To
avoid methanol inhibition in solvent free process, a three step
methanolysis was described (Lee et al., 2011). In the first step, 1/
3 of the stoichiometric amount of methanol was added into the
oil and the product produced after reaction was kept in separator
for the complete separation of glycerol from partially converted
oil. The next step was performed by adding another 1/3 of the stoi-
chiometric amount of methanol into the partially converted oil and
the same process was continued for the third step. The stepwise
processes described in the literatures were not truly continuous
and sometimes involved environmentally harmful organic solvents
and expensive enzyme Novozyme 435 in their systems.
The present study thus aimed at producing biodiesel in a green,
solvent free and economical way using a less expensive immobi-
lized lipase, IIT-SARKZYME, developed by Chattopadhyay and Sen
(2012). For that purpose, an integrated continuous stirred tank
reactor with two packed bed reactors (CSTR-PBR) in series was
used first time for the simultaneous reaction-cum-solubilization
of the immiscible phases in the solvent free system for the im-
proved conversion in PBR. The stability of the integrated process
was determined and its efficacy for different feedstock with vari-
ous free fatty acids (FFA) was checked to find out the robustness
of the system.
2. Methods
2.1. Materials used
Oils were procured from N.K. Proteins Ltd. (Nagpur, India).
Crude Steapsin (pancreatic lipase Ex-micro organism), and methyl
linoleate standard were purchased from SRL (Mumbai, India), and
the standard is chromatographically pure. HPTLC plates, SDS, and
other chemicals were obtained from MERCK (Germany), and were
of analytical grade. Bradford protein estimation kit was purchased
from Bangalore Genie (Bangalore, India).
2.2. Methods
2.2.1. Preparation of immobilized lipase for transesterification
In our earlier report, development of the immobilized biocata-
lyst system designated as ‘IIT-SARKZYME’ was described in detail
(Chattopadhyay and Sen, 2012). The enzyme loading was
calculated by Bradford protein estimation method using bovine
serum albumin (BSA) as standard and the enzyme activity was
measured by tributyrin method (Chang et al., 2007). The cost of
IIT-SARKZYME was estimated and compared with the most
commonly used commercially available lipase Novozyme 435.
2.2.2. Batch reaction
One and two step solvent free methanolysis was carried out in
batch system. Optimization of different reaction parameters were
described earlier (Chattopadhyay et al., 2011a). In single step pro-
cess, 1:15 molar ratio of oil to methanol was mixed with the
immobilized lipase (1% w/w of oil) and incubated at 37 °C for
24 h with a constant shaking at 150 rpm. In case of two step pro-
cesses, first, methanol was mixed with oil at 1:1 molar ratio and
reaction was carried out for 10 h. Rest of stoichiometric amount
of methanol was added at 10 h and reaction was continued up to
24 h. The product was kept at separating funnel for 2–3 h to
separate glycerol completely. Glycerol layer was collected from
bottom and upper biodiesel layer was analyzed.
2.2.3. Quantitative estimation of the product
The product was analyzed by high performance thin layer chro-
matography (HPTLC) method. The detail description of the method
was described elsewhere (Chattopadhyay et al., 2011b). In brief,
different glycerides were separated by thin layer chromatography
(TLC) using hexane, ethyl acetate and acetic acid (90:10:1) as mo-
bile phase. The TLC plate was scanned at 203 nm under HPTLC
scanner and the percentage yield was calculated from the peak
areas of different glycerides obtained from HPTLC chromatogram
(Chattopadhyay et al., 2011b).
2.2.4. Systematic development of a green continuous process for
biodiesel
2.2.4.1. Continuous process development in a single packed bed
reactor. To eliminate methanol inhibition problem, t-butanol was
used in the continuous system. The IIT-SARKZYME was used in a
single packed bed reactor (PBR) (inner diameter 2 cm, height
45 cm, volume 140 mL) with a bed volume of 100 mL. Reaction
mixture containing oil, t-butanol and methanol was fed continu-
ously into the PBR from bottom through a peristaltic pump with
a flow rate of 1 mL/min. The enzyme loading was kept as one gram.
Reactor temperature was maintained at 37 °C by water circulation
through jacket. The reactor set up comprises single PBR is
presented in Fig. 1a. The product was collected in a separating fun-
nel. The upper biodiesel layer was distilled at 84 °C for 30 min to
separate solvent and unutilized methanol from the product. The
lower layer comprises mostly glycerol was collected from bottom
into the separating funnel. Some important reactor parameters like
void fraction (e) and packing density were determined. The small
void fraction ensured better contact of enzyme to substrate and
useful to calculate the residence time of the reaction. The packing
density determines the flow characteristics of the substrates
through the PBR. The parameter values were determined using fol-
lowing equations (Vikbjerg et al., 2005).
Void fractionðeÞ ¼ VS =V ð1Þ
Packing density ¼ M=VR ð2Þ
Residence time ¼ ðVR  eÞ=F ð3Þ
where, VS is the void volume of the PBR, V is the volume of the IIT-
SARKZYME, VR is the reactor volume, M is the mass of the IIT-SARK-
ZYME, and F is the substrate flow rate.
Since single PBR operation was limited by the reactor design in
terms of enzyme loading and residence time, double PBR system
connected in series was also studied to check whether the product
yield could be enhanced by allowing for more enzyme loading and
reaction time.
2.2.4.2. Continuous process development in double PBRs connected in
series. To increase the yield of the product, two PBRs were con-
nected in series and the substrate mixture was fed continuously
through them (Fig. 1b). The substrate flow rate was varied from
0.12 to 1.78 mL/min by varying the speed of the peristaltic pump.
The enzyme amount was varied from 0.5 to 2 g and distributed in
two columns equally to determine the minimum amount required
for effective transesterification. The product was collected and ana-
lyzed using the methods described in Section 2.2.3.
The biocatalysts based continuous processes are environment
friendly than that of chemical processes. However, use of organic
solvent, t-butanol, being harmful to the environment and human
health, does not make the process completely green. Thus, our next
 S. Chattopadhyay, R. Sen / Bioresource Technology 147 (2013) 395–400 397

(a) (b)
WB WB

PBR PBR PBR D

D

L L L SF
SF BD
BD
G
G
J
J

S
S

P P

Fig. 1. Continuous biodiesel production in (a) single PBR and (b) two PBRs connected in series. S: substrate mixture, P: pump, J: water circulating jacket, L: immobilized lipase,
WB: water bath, PBR: packed bed reactor, D: distillation unit, SF: separating funnel, BD: biodiesel, G: glycerol layer.

focus was to produce biodiesel with higher productivity in solvent
free system.
2.2.4.3. Development of a green continuous process based on an
integrated CSTR-PBR system for solvent free biodiesel produc-
tion. With a view to developing a completely green process, a
CSTR-PBR based integrated enzymatic reactor system employing
our indigenously formulated immobilized enzyme (IIT-SARKZYME)
was designed for continuous biodiesel production in solvent free
system (Patent Application No: 404/KOL/2011). In the integrated
system, oil and methanol was mixed (1:1 molar ratio) and allowed
to react for a small period of time (approximately 4 h) in batch
mode, which partially converted oil to its methyl ester. The en-
zyme loading in the batch process was kept as 1% (w/v of oil).
The reactor was then operated in continuous mode (as CSTR) with
this partially converted oil and fresh feed, where a small fraction of
fresh oil was converted into biodiesel. This initial step involving
partial conversion increased the solubility of methanol in oil and
in turn, eliminated the inhibitory effect of methanol as well as
the requirement of organic solvent. The mixture was then fed into
two PBRs as described previously. The reaction scheme is depicted
WB
PBR
PBR
in Fig. 2. The final product was separated in separating funnel and
analyzed.
Stability of IIT-SARKZYME in continuous process was deter-
mined by calculating the percentage conversion values in each cy-
cle. The total residence time for the two PBRs in series was
calculated and was considered as one cycle.
2.2.5. Mass balance and product productivity
The mass balance of the reactant and the product was calcu-
lated from their volume and specific gravity values. The oil to
methanol molar ratio was determined by considering an average
molecular weight of oil.
The productivity (P) of the entire process at steady state for the
production of 100 mL product was calculated by the following
equations:
Overall dilution rateðDÞ ¼ F=ðn  VÞ ð4Þ
ProductivityðPÞ ¼ C  D  60 ð5Þ
where, D is the dilution rate (per min), F is the volumetric flow rate
(mL/min), n is the number of reactors used, V is the volume of each
reactor (mL), C is the product concentration (g/L) and P is the pro-
ductivity (g/L/h).
Different feedstock of varying FFA compositions like palm, sun-
flower, soybean, rice bran and waste cooking oil along with cotton-
seed oil were transesterified at same conditions using the
integrated continuous process to check the robustness of the
D process.

3. Results and discussion

SF
L L BD
P 3.1. Cost comparison of IIT-SARKZYME with Novozyme 435
P G
Immobilized lipase is the major cost contributing factor in
CSTR
MeOH enzymatic biodiesel production. Therefore, it is very important to
oil J
use a relatively less expensive immobilized enzyme system as an
alternative to the most extensively used and expensive formula-
tion Novozyme 435 for biodiesel production. A comparative cost
P estimate of both IIT-SARKZYME and Novozyme 435 as given in
Table 1. The amount of enzyme that provided same activity was
Fig. 2. Continuous biodiesel production in an integrated CSTR-PBR system. MeOH:
methanol, P: pump, J: water circulating jacket, L: immobilized lipase, WB: water
taken as the basis for the cost comparison. The activity of IIT-SAR-
bath, CSTR: continuous stirred tank reactor, PBR: packed bed reactor, D: distillation KYME was determined to be 3557 U/g immobilized enzyme
unit, SF: separating funnel, BD: biodiesel, G: glycerol layer. (Chattopadhyay and Sen, 2012). From the comparative cost
398 S. Chattopadhyay, R. Sen / Bioresource Technology 147 (2013) 395–400

Table 1 3.4. Standardization of enzyme loading

Comparative analysis of cost between two immobilized enzyme systems.

Immobilized Cost Activity of Amount of
lipase (Rs/g of immobilized immobilized
lipase) lipase (U/g) lipase with
same activity (g)
IIT-SARKZYME 50 3557 1 50
Novozyme 435 984 10,000 0.36
analysis, it is clear that IIT-SARKYME as an immobilized enzyme
system is approximately 7.2 times cheaper than Novozyme 435.
3.2. Batch reaction
As compared to single step process with maximum conversion
of 20% after 48 h, the percentage conversion in two step process
was estimated as 40% after 10 h of reaction (with oil to methanol
molar ratio 1:1). With further methanol addition at 10 h, the con-
version increased and reached to a saturation level of 72% after
24 h (Fig. 3). The inhibitory effect of methanol in solvent free single
step process was eliminated by step wise methanol addition at
10 h, which helped to solubilize methanol into oil and in turn en-
hanced percentage conversion (Hajar et al., 2009).
3.3. Standardization of substrate flow rate
The percentage conversion of oil into biodiesel increased with
increasing flow rate, which may be due to the consequence of high
rate of mass transfer. It was observed that, percentage conversion
reached its maxima at a flow rate of 0.74 mL/min, which was con-
sidered as optimal for further reaction (Fig. 4a). After optimum
flow rate, where mass transfer rate reached its maxima, the per-
centage conversion decreased, which might be due to the small
residence time for the reaction (Lee et al., 2010). Some earlier re-
ports exemplified such trend of drastic reduction of methyl ester
yield at higher flow rates (Chang et al., 2009; Shaw et al., 2008).
The optimum flow rates for maximum percentage conversion, as
reported in literature, are summarized in Table 2. In most of the
cases, very low flow rates ranging from 0.1 to 0.5 mL/min were
used to get maximum conversion (Rosa et al., 2009; Wang et al.,
2011). However, a process with very high flow rate of 6 mL/min
was also reported (Hajar et al., 2009), wherein the productivity
was less as the product was recycled for 72 h before collecting
from the reactor.
80
70
60
% conversion
Total cost (Rs) The total enzyme loading was varied from 0.5 to 2 g and was
equally distributed into two packed bed reactors connected in ser-
ies. It was observed that higher than 1.5 g of enzyme did not able to
improve percentage conversion further (Fig. 4b). With increase in
enzyme amount, the interaction between enzyme and substrates
358
increased. But after that the enzyme-substrate reaction reached
its saturation level and hence, no additional increment in percent-
age conversion was found. Similar enzyme loading of 2 and 1 g and
was reported to be used to achieve maximum percentage conver-
sion (Hajar et al., 2009; Royon et al., 2007). The enzyme loading re-
ported in the various recent literatures is summarized in Table 2.
As cost of the enzyme is the major concern in enzymatic biodiesel
production, minimum amount (1.5 g) that was required for effec-
tive transesterification was used for further experiments.
3.5. Determination of important reactor parameters
From Eqs. (1)–(3), different reactor parameters were calculated.
The void fraction was found to be 0.28 whereas packing density
was 0.88 g/mL. The packing density was calculated and compared
with literature reports (Table 3). From the table it is clear that
the packing density was found to be higher as compared to most
of the literatures but comparable with some (Rosa et al., 2009;
Shaw et al., 2008). Small void fraction with relatively higher pack-
ing density increases enzyme–substrate interaction and reduces
channelling effect inside PBRs. The residence time at flow rate of
0.74 mL/min was calculated as 53 min. As void fraction was not de-
scribed by the available literature reports, comparison of residence
time was not possible with our value.
3.6. Salient features of the integrated continuous process
3.6.1. Productivity
It was observed that oil was partially converted around 25% in
CSTR after 4 h reaction. This partially converted oil was fed into
two PBRs connected in series. After the system achieved steady
state, the overall productivity of the integrated process was
137.2 g/L/h, which was higher than most of the reported literature
values (Wang et al., 2011; Hajar et al., 2009; Shaw et al., 2008). The
comparative analysis of the different continuous processes is sum-
marized in Table 4. Though some processes indicated higher pro-
ductivity than ours (Rosa et al., 2009; Halim et al., 2009), use of
organic solvent and expensive enzyme made those processes eco-
nomically and environmentally unfavourable. Experimental data
indicated that the productivity of the integrated process was
1 step
approximately five times higher than the solvent free batch pro-
2 step cess (26.1 g/L/h). The productivity of the batch processes was less
due to the higher reaction time by stepwise methanol addition into
the system. The integrated process may prove to be economically

50 and environmentally attractive as it excluded the use of organic

solvent and intermittent methanol addition throughout the whole
40 process.

30
3.6.2. Mass balance
The overall mass balance of the entire process was calculated
20
as:
10 100 g oil þ 56:5 g methanol ! 103:5 g BD þ 7:8 g glycerol
0 þ 45:2 g methanol
0 10 20 30 40 50 60
Having considered 100 g oil as the basis, a mass balance of the
Time (h)
transesterification process was carried out by using the actual
Fig. 3. Percentage conversion of oil to biodiesel in single step and two steps batch mass values of the substrates and the products. The average molec-
process. The methanol addition point at 10 h is indicated by an arrow. ular weight of oil was calculated to be 850 g/mole. As indicated by
 S. Chattopadhyay, R. Sen / Bioresource Technology 147 (2013) 395–400 399

80 (a) 80 (b)
70 0.74 70
60 1 60
0.33

% conversion
% conversion
50 1.78 50
40 0.12 40
30 30
20 20
10 10
0 0
0 0.5 1 1.5 2 0.25 0.75 1.25 1.75 2.25
Flow rate (ml/min) Amount of enzyme (g)

Fig. 4. Effect of (a) substrate flow rate and (b) different enzyme loading (in two columns) on transesterification.

Table 2 Table 4
Comparative analysis of optimum flow rates and enzyme loading. Comparative analysis of different continuous processes.

Flow rate (mL/min) Enzyme loading (g) Reference Effective Productivity Remarks on the process Reference
reactor (g/L/h)
0.25 10 Wang et al. (2011)
volume
1.50 3.0 Salum et al. (2010)
(mL)
6.00 2.0 Hajar et al. (2009)
0.50 22.5 Rosa et al. (2009) 5 79.3 Low reaction rate, use of Royon et al. (2007)
0.60 2.8 Halim et al. (2009) organic solvent
0.10 NA Chang et al. (2009) 1.2 40.7 Low reaction rate, use of Shaw et al. (2008)
0.10 1.0 Shaw et al. (2008) organic solvent
0.16 1.0 Royon et al. (2007) 18 9.5 Three step methanolysis, Hajar et al. (2009)
0.74 1.5 This work recycled up to 72 h
21 247.8 High enzyme loading and Rosa et al. (2009)
NA: Data not available use of organic solvent
8 151.5 Use of organic solvent and Halim et al. (2009)
expensive Novozyme 435
23 82.5 Two step methanolysis, Salum et al. (2010)
Table 3 methanol addition at 7 h
Comparison of packing density with different reactor volume. 40 28.7 High reaction time, use of Wang et al. (2011)
organic solvent
Reactor Immobilized Packing Reference 100 137.2 High reaction rate, perfectly This work
volume (mL) enzyme (g) density continuous, no organic solvent,
(g/mL) green, no intermittent methanol
40.2 10 0.25 Wang et al. (2011) addition
22.7 3 0.13 Salum et al. (2010)
17.7 2 0.11 Hajar et al. (2009)
21.0 22.5 1.07 Rosa et al. (2009)
8.3 2.8 0.34 Halim et al. (2009)
1.2 1 0.83 Shaw et al. (2008)
5.1 1 0.20 Royon et al. (2007) 80
140 123 0.88 Our work
70

60
initial optimization experiments (Chattopadhyay et al., 2011a),
50
% conversion

optimum oil to methanol molar ratio of 1:15 was taken. Accord-

ingly, the amount of methanol was calculated using its molecular
40
weight value. The actual masses of biodiesel (103.5 g) and glycerol
(7.8 g) were determined by measuring their respective volumes as 30
collected after distillation and also measuring their respective spe-
cific gravity values. These mass values of biodiesel and glycerol 20
were in exact agreement with their respective masses calculated
by HPTLC quantification method (Chattopadhyay et al., 2011b). 10
From the calculations based on both mass balance and HPTLC anal-
yses, the conversion of oil into biodiesel by the integrated green 0
continuous process was almost 72%. CSO Soybean RBO Palm SFO WCO

Fig. 5. Comparing biodiesel production from different oils as feedstock; CSO:

Cottonseed oil; Soybean: Soybean oil; RBO: Rice Bran oil; Palm: Palm oil, SFO:
3.6.3. Robustness
Sunflower oil, WCO: Waste cooking oil.
The experimental data with different feedstock revealed that
the integrated CSTR-PBR system was able to transesterified all
the six different feedstock with almost equal efficiency (Fig. 5). 3.7. Stability of immobilized lipase in continuous process
The results indicated that the developed integrated continuous
process was robust and could be used for transesterification of dif- It was observed that the biodiesel yield was not significantly re-
ferent feedstock with varying FA compositions. duced up to 45 cycles and the system was considered as stable
400 S. Chattopadhyay, R. Sen / Bioresource Technology 147 (2013) 395–400
120
100
80
% relative yield
60
40
20
0
0 10 20 30 40 50
No. of passes
Fig. 6. Stability of immobilized lipase with respect to number of cycles.
(Fig. 6). This immobilized system was found to be more stable as
compared to the other solvent free systems, in which the stability
of immobilized enzyme was reported up to 6–9 cycles (Salum
et al., 2010; Hajar et al., 2009). It was observed that active confor-
mation of lipase could be maintained by using organic solvents in
the process. Transesterification in presence of organic solvent with
higher operational stability of several hours was reported (Lee
et al., 2010). But the processes involving organic solvents were
not environment friendly. Methanol inhibition and formation of
glycerol layer over the immobilized enzyme during reaction might
be responsible for lower operational stability in case of solvent free
processes (Halim et al., 2009).
3.8. Environment-friendliness of the integrated continuous process
Methanol that was used as a reactant is toxic for both health
and environment. Our process is a close system and no methanol
was disposed to the environment as was evident from the mass
balance. The excess amount of methanol was distilled and reused
for further reactions. No other organic solvent like t-butanol or
hexane etc. were used in the system to facilitate the reaction.
Use of enzyme as biocatalyst, instead of hazardous chemical cata-
lyst, made the process further environment-friendly. Thus the
developed integrated continuous process for biodiesel production
was completely green with zero methanol discharge.
4. Conclusion
The present study has culminated into a patentable solvent free
green process involving an integrated immobilized enzyme-reactor
system with IIT-SARKZYME as the biocatalyst for the continuous
production of biodiesel with zero methanol discharge (Patent Appl.
No.: 404/KOL/2011). Inclusion of CSTR as an inventive step helped
in avoiding enzyme inhibition due to insoluble methanol droplets
and hence, made the process perfectly continuous without requir-
ing any organic solvent and intermittent addition of methanol in
the subsequent steps involving PBRs. This is a perfectly continuous
solvent free process with economic and environmental advantages
over the reported literature.
Acknowledgements
Authors would like to acknowledge Dr. Asoke Deysarkar, CEO,
PfP Technology LLC., Houston, Texas, USA and PfP Technology (In-
dia) Ltd., Mumbai for their moral and financial support. We are
grateful to Prof. Amit Patra, Dean (Alumni Affairs & International
Relations), IIT Kharagpur for his valuable suggestions and encour-
agement. We would like to thank Mr. Ankush, Ms. Priyatama,
and Mr. Vikram for their help during reactor operation. SC is thank-
ful to University Grant Commission (Government of India) for
scholarship.
60 Reference
Chang, S., Chang, S., Yen, Y., Shieh, C., 2007. Optimum immobilization of Candida
rugosa lipase on Celite by RSM. Appl. Clay Sci. 37, 67–73.
Chang, C., Chen, J.H., Chang, C.J., Wu, T.T., Shieh, C.J., 2009. Optimization of lipase-
catalyzed biodiesel by isopropanolysis in a continuous packed-bed reactor
using response surface methodology. New Biotechnol. 26, 187–192.
Chattopadhyay, S., Sen, R., 2012. A comparative performance evaluation of jute and
eggshell matrices to immobilize pancreatic lipase. Process Biochem. 47, 749–
757.
Chattopadhyay, S., Das, S., Sen, R., 2011a. Rapid and precise estimation of
biodiesel by high performance thin layer chromatography. Appl. Energy 88,
5188–5192.
Chattopadhyay, S., Karemore, A., Das, S., Deysarkar, A., Sen, R., 2011b. Biocatalytic
production of biodiesel from cottonseed oil: Standardization of process
parameters and comparison of fuel characteristics. Appl. Energy 88, 1251–1256.
Hajar, M., Shokrollahzadeh, S., Vahabzadeh, F., Monazzamia, A., 2009. Solvent-free
methanolysis of canola oil in a packed-bed reactor with use of Novozym 435
plus loofa. Enzyme Microbiol. Technol. 45, 188–194.
Halim, S.F.A., Kamaruddin, A.H., Fernando, W.J.N., 2009. Continuous biosynthesis of
biodiesel from waste cooking palm oil in a packed bed reactor: Optimization
using response surface methodology (RSM) and mass transfer studies.
Bioresour. Technol. 100, 710–716.
Lee, M., Lee, J., Lee, D., Cho, J., Kim, S., Park, C., 2011. Improvement of enzymatic
biodiesel production by controlled substrate feeding using silica gel in solvent
free system. Enzyme Microb. Technol. 49, 402–406.
Lee, J.H., Kim, S.B., Park, C., Tae, B., Han, S.O., Kim, S.W., 2010. Development of batch
and continuous processes on biodiesel production in a packed-bed reactor by a
mixture of immobilized candida rugosa and rhizopus oryzae lipases. Appl.
Biochem. Biotech. 161, 365–371.
Rosa, C.D., Morandim, M.B., Ninow, J.L., Oliveira, D., Treichel, H., Oliveira, J.V., 2009.
Continuous lipase-catalyzed production of fatty acid ethyl esters from soybean
oil in compressed fluids. Bioresour. Technol. 100, 5818–5826.
Royon, D., Daz, M., Ellenrieder, G., Locatelli, S., 2007. Enzymatic production of
biodiesel from cotton seed oil using t-butanol as a solvent. Bioresour. Technol.
98, 648–653.
Sakai, S., Liu, Y., Yamaguchi, T., Watanabe, R., Kawabe, M., Kawakami, K., 2010.
Production of butyl-biodiesel using lipase physically-adsorbed onto electrospun
polyacrylonitrile fibers. Bioresour. Technol. 101, 7344–7349.
Salum, T.F.C., Villeneuve, P., Barea, P.B., Yamamoto, C.I., Côcco, L.C., Mitchell, D.A.,
Krieger, N., 2010. Synthesis of biodiesel in column fixed-bed bioreactor using
the fermented solid produced by Burkholderia cepacia LTEB11. Proc. Biochem.
45, 1348–1354.
Severac, E., Galy, O., Turon, F., Monsan, P., Marty, A., 2011. Continuous lipase-
catalyzed production of esters from crude high-oleic sunflower oil. Bioresour.
Technol. 102, 4954–4961.
Shaw, J.F., Chang, S.W., Lin, S.C., Wu, T.T., Ju, H.Y., Akoh, C.C., Chang, R.H., Shieh, C.J.,
2008. Continuous enzymatic synthesis of biodiesel with Novozym 435. Energy
Fuel 22, 840–844.
Vikbjerg, A.F., Peng, L., Mu, H., Xu, X., 2005. Continuous production of structured
phospholipids in a packed bed reactor with lipase from Thermomyces
lanuginosa. J. Am. Oil Chem. Soc. 82, 237–242.
Wang, X., Liu, X., Zhao, C., Ding, Y., Xu, P., 2011. Biodiesel production in packed-bed
reactors using lipase–nanoparticle biocomposite. Bioresour. Technol. 102,
6352–6355.