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Lipase-catalyzed process for biodiesel

production: Enzyme immobilization, process
simulation and optimization
ARTICLE in RENEWABLE AND SUSTAINABLE ENERGY REVIEWS APRIL 2015
Impact Factor: 5.9 DOI: 10.1016/j.rser.2014.12.021
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Retrieved on: 10 February 2016
Renewable and Sustainable Energy Reviews 44 (2015) 182197
Contents lists available at ScienceDirect
Renewable and Sustainable Energy Reviews

journal homepage: www.elsevier.com/locate/rser
Lipase-catalyzed process for biodiesel production: Enzyme

immobilization, process simulation and optimization
Xuebing Zhao a,n,1, Feng Qi b,1, Chongli Yuan c, Wei Du a, Dehua Liu a,nn
a
Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
College of Life Sciences/Engineering Research Center of Industrial Microbiology, Fujian Normal University, Fuzhou 350108, China
c
School of Chemical Engineering, Purdue University, 480 Stadium Mall Drive, West Lafayette, IN 47907, USA
b
art ic l e i nf o
a b s t r a c t
Article history:
Received 1 May 2014
Received in revised form
12 November 2014
Accepted 12 December 2014
Transesterication of oil feedstocks using immobilized lipase (IL) is a promising process for biodiesel
production. However, the running cost of this process is still higher than that of conversional chemicalcatalyzed approaches. To address this challenge, both upstream and downstream processes have to be
optimized. This review provides an overview of recent progresses in improving IL-catalyzed biodiesel
production, focusing on mid- and down-stream processing such as immobilization of lipase, bioreactors
development, process optimization, simulation and techno-economic evaluation. The immobilization of
lipase is a costly process. Most of the commercial ILs are prepared by adsorption of free lipase on
polymeric materials. However, to further reduce cost, works should be focused on developing cheap
carriers and strengthening the interaction between enzyme and carrier but without signicant loss of
lipase activity. Running cost of lipase also can be reduced by improving its lifetime during transesterication. To achieve this goal, solvents can be used to prevent lipase leaching and eliminate the inhibitive
effects of alcohol (usually methanol) and glycerol. Downstream processing includes important units to
purify biodiesel products. In this part, works should be focused on minimizing energy consumption and
waste efuents. A global process integration and optimization with economic evaluation also should be
gured out to improve the economic feasibility of Il-catalyzed production of biodiesel.
& 2014 Elsevier Ltd. All rights reserved.
Keywords:
Biodiesel
Immobilized lipase
Transesterication
Process optimization
Techno-economic evaluation
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Immobilization of lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Techniques for immobilization of lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1.
Overview of lipase immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
Immobilization of lipase by physical adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.3.
Immobilization of lipase by ionic bonding or covalent bonding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.4.
Immobilization of lipase by entrapment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.5.
Immobilization of lipase by cross-linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.6.
Commercialization of immobilized lipase for biodiesel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Process optimization and reactors for IL-catalyzed transesterication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Parameter optimization for transesterication process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Reactors for IL-catalyzed transesterication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.
Stirred tank reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2.
Packed-bed reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3.
Airlift loop reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.4.
Other heterogeneous reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Process simulation and optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel./fax: 86 10 62772130.

Corresponding author. Tel./fax: 86 10 62792128.
E-mail addresses: zhaoxb@mail.tsinghua.edu.cn (X. Zhao), dhliu@mail.tsinghua.edu.cn (D. Liu).
1
Both authors contributed equally to this work.
nn
http://dx.doi.org/10.1016/j.rser.2014.12.021
1364-0321/& 2014 Elsevier Ltd. All rights reserved.
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X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197
4.1.
4.2.
Kinetic modeling of IL-catalyzed transesterication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Process simulation and product purication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1.
Process simulation and unit optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.2.
Biodiesel purication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Techno-economic evaluation of IL-catalyzed production of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Concluding remark and recommendation for future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Lipase-catalyzed transesterication of oil feedstocks has been
considered as one of the most promising techniques for producing
biodiesel, a mixture of fatty acid alkyl esters (FAAE). This process
has become a research hot-spot in academic community during
last 10 years. An increasing number of scientic publications
including articles, review papers, book chapters, patents and
conference abstracts have been published (Fig. 1). However, conventional biodiesel plants adopt transesterication processes
using chemical catalysts such as alkalis and acids. Only a few
plants have employed enzymatic process for industrial productions of biodiesel [1,2]. This is mainly because that the catalyst
(lipase) used in the enzymatic process is much more expensive
Fig. 1. Publications in years of 20002013 found in Web of Science

the term biodiesel and lipase separated by document types.
TM
database by
183
189
190
190
191
192
193
194
194
than chemical catalysts, such as NaOH and H2SO4. Various lipases

have been used for biodiesel production, among which
immobilized-lipase (IL) shows great potential for industrial application since ILs are more tolerant to organic solvents, heat and
shearing force, and much easier to recover than free lipases (FLs).
To reduce the production cost of enzymatic transesterication,
strategies can be made in up-, mid- and down-stream processings
(Fig. 2). Specically, in up-stream processing the catalytic stability
and activity of lipase can be improved by protein engineering,
strain optimization and metabolic engineering techniques as
reviewed in our previous paper [3]. In addition, further reduction
of running cost of the enzyme-catalyzed biodiesel production can
be achieved by process intensication strategies, for example by
improving the immobilization as well as process design and
optimization. Immobilization of lipase enzymes has been studied
for many years, and various carriers have been used. However,
only a few types of carrier and immobilization process have been
commercialized. Nevertheless, these commercialized ILs are still
too expensive to be used for biodiesel production. Some newly
developed immobilization technologies by using magnetic and
nano-particles have been reported, but they are still far away from
industrial application. One of the solutions to the high cost of
lipase for biodiesel production is to increase its lifetime in
transesterication. At this point, reaction media, operation parameters as well as reactor development should be considered.
For example, the stability of ILs in conventional aqueous system
is usually poor due to the leaching of enzyme from carriers and the
inhibitive effects of methanol and glycerol. However, by introducing novel solvent, e.g. tert-butanol, as reaction media, the stability
of lipase can be greatly improved [1]. Reactor design is important
for scale-up of IL-catalyzed production of biodiesel, but the
development of high-efciency reactor for IL-catalyzed production
of biodiesel goes slowly. Commonly used reactors are stirred tank
reactor (STR), packed-bed reactor (PBR) or their combination.
However, much improvement is still needed for intensifying mass
Fig. 2. Unit operations and corresponding works that can be done to reduce the cost of lipase-catalyzed biodiesel production.
184
Fig. 3. Various techniques for enzyme immobilization (adapted from [5,7,12]).
transfer with minimizing mechanic shearing force to damage

carriers and enzymes. Down-stream processing is crucial to obtain
biodiesel product that meets corresponding quality standards.
Simulation is usually used to obtain mass and energy balance data
and process optimization. Dozens of review papers have been
published on lipase-catalyzed biodiesel production as summarized
in Fig. 1. However, most of them are mainly focused on lipase
production and parameter optimization for transesterication
reaction, and lack of recently reported data. In this article, we
have comprehensively reviewed the mid-and down-stream processing of IL-catalyzed production of biodiesel, with focusing on
immobilization techniques, reactor designs, process optimization,
simulation and techno-economic evaluation. Critical perspectives
were provided for unit operations to reduce the operational cost of
biodiesel production.
2. Immobilization of lipase
The rst immobilized enzyme was reported by Nilson and
Grifin more than a century ago [4], while Chibata and co-workers
developed the rst industrial immobilized enzyme, i.e., Aspergillus
oryzae aminoacylase for synthetizing racemic DL amino acids [5].
In recent years, production of biodiesel using immobilized lipases
has attracted great interest. Signicant progresses have been made
on both of the immobilization techniques and process development for IL-mediated biodiesel production.
An immobilized enzyme is dened as the enzyme physically
conned to a certain dened region while retaining its most
catalytic activity [6]. Similar to other immobilized enzymes, ILs
show many advantages over FLs for the large-scale application in
biodiesel production [7], such as easy recovery and reuse, higher
adaptability for continuous operation, less efuent problems,
greater pH and thermal stability, and higher tolerance to reactants
and products. However, the current ILs still show several drawbacks for industrial applications, including: (1) loss of enzymatic
activity during immobilization; (2) high cost of the carriers;
(3) low stability in oilwater systems; and (4) requirement of
novel reactors for well mixing and maximizing oil-to-biodiesel
conversion.
Many materials have been explored in literatures to immobilize

lipases, including various polymer resins, celite, silica, ceramics [8],
carbon nanotubes [9], magnetic particles [10] and microspheres [11].
However, for industrial applications, the carrier material must be of
low cost. In addition, the immobilization procedure should be easy to
perform with a high active lipase recovery rate, and the IL activity
must be maintained for a long running time. Generally, these goals
can be achieved by: (1) improving the immobilization technologies;
(2) optimizing the transesterication process; (3) developing novel
bioreactors; and (4) intensifying the process integration to reduce the
operation cost.
2.1. Techniques for immobilization of lipase
2.1.1. Overview of lipase immobilization
Various techniques have been developed for lipase immobilization (Fig. 3) as intensively reviewed in some recently published
papers [2,12]. Generally, these techniques can be classied into
three types: carrier bonding, cross-linking and entrapment.
Depending on the type of interactions between enzymes and
carriers, these techniques can be further classied into irreversible
and reversible immobilization techniques [5]. In irreversible
immobilization, once enzymes are attached to supporting materials, they cannot be detached without destroying either the
biological activity of the enzyme or the support. In reversible
immobilization, enzymes can be detached from the support under
gentle conditions. Covalent bonding, entrapment and cross-linking
are the most commonly used procedures for irreversible immobilization of lipases. Physical adsorption and various non-covalent
bondings, such as afnity bonding and chelation bonding, are
well-known reversible immobilization procedures. Each immobilization technique has its own merits and inevitably some disadvantages for lipase immobilization.
2.1.2. Immobilization of lipase by physical adsorption
Adsorption is a commonly-used method to immobilize lipase.
Several non-covalent interactions are involved in this immobilization, including non-specic physical adsorption, bio-specic
adsorption, afnity adsorption, electrostatic interaction (also
185
Table 1
Some reported immobilizations of lipase by different techniques for biodiesel preparation in recent years.
Carrier
Lipase source
Immobilization techniques
References
Accurel EP-100
Activated carbon
Aldehyderesin
Carbon cloth
Celite
Celite supported sol-gel
Ceramics
Chitosan
EpoxySiO2PVA
Hydrophilic resins
Hydrotalcite
Lewatit
Magnetic particles
Penicillium expansum
Pseudomonas sp.
Candida antarctica
Thermomyces lanuginosus
Pseudomonas cepacia
Yarrowia lipolytica
Candida antarctica
Pseudomonas cepacia
Candida rugosa
Penicillium camembertii
Rhizomucor miehei
Candida rugosa
Penicillium camembertii
Candida rugosa
Saccharomyces cerevisiae
Burkholderia cepacia
Pseudomonas uorescens
Staphylococcus haemolyticus
Steapsin lipase
Pseudomonas cepacia
Candida rugosa
Pseudomonas cepacia
Candida sp.
Lipolases (Aspergillus oryzae)
Penicillium expansum
Candida antarctica
Rhizopus orizae and Candida rugosa
Enterobacter aerogenes
Candida antarctica
Cross-linking
Physical adsorption
Physical adsorption
Covalent bonding
Physical adsorption
Physical adsorption
Entrapment
Physical adsorption
Covalent bonding
Covalent bonding
Physical adsorption
Physical adsorption
Covalent bonding
Covalent bonding
Covalent bonding
Ionic adsorption
Physical adsorption
Physical adsorption
Covalent bonding
Covalent bonding
Physical adsorption
Physical adsorption /entrapment
Covalent bonding
Physical adsorption
Physical adsorption
Physical adsorption
Physical adsorption
Physical adsorption
Covalent bonding
Physical adsorption
Entrapment
Physical adsorption
Physical adsorption
Entrapment
Entrapment
Covalent bonding
Covalent bonding
Covalent bonding
Physical adsorption
Entrapment
[38]
[90]
[108]
[109]
[108]
[111]
[36]
[110]
[112]
[31]
[113]
[114]
[115]
[82]
[116]
[31]
[117]
[118]
[119]
[120]
[121]
[40]
[53]
[122]
[123]
[121]
[121,123]
[124]
[125]
[126]
[127]
[128]
[123]
[129]
[35]
[130]
[131]
[132]
[114]
[33]
MANAE-agarose
MCM-41 materials
Mg-Al hydrotalcites
Nb2O5 and SiO2-PVA
Olive pomace
Organosilicate
Poly (methacrylate-co-divinyl benzene)
Polyacrylic bead
Polyacrylonitrile
Polymethacrylate
Polypropylene
Polystyrene
Polyurethane foam
Pretreated textile
PVA/chitosan lm
Resin D4020
Silica
Silica aerogel
Silica gel
Styrenedivinylbenzene copolymer
Zeolites
-carrageenan
ionic binding), and hydrophobic interaction [13]. Compared with

other immobilization techniques, adsorption immobilization is
advantageous in the following aspects [12]: (1) mild conditions
and easy operation; (2) relatively low cost of carrier materials and
immobilization procedure; (3) no requirement of chemical additives during adsorption; (4) easy regeneration of carriers for
recycling; and (5) high lipase activity recovery.
Generally, physical adsorption, in which the enzyme is
adsorbed via non-specic forces such as van der Waals forces,
hydrogen bonds and hydrophobic interactions, is frequently used
for preparing ILs. Various carriers have been used to adsorb lipase
as summarized in Table 1. The commonly used support materials
are polymer resins such as polystyrene, polypropylene, polyacrylate etc. These polymers are cheap to obtain. One of the most
common commercial ILs, Novozyms 435, is prepared by immobilizing lipases on acrylic resin by adsorption [14]. This IL has been
widely tested to catalyze the transesterication of various vegetable oil feedstocks, animal fats, yeast lipids and algae oils [1520].
Both carrier properties and immobilization conditions have signicant inuences on the immobilization efciency (enzyme and
activity recoveries). The chemical and physical characteristics of the
carrier, such as polarity, the molar ratio of hydrophilic to hydrophobic
groups, particle size and surface area, porosity and pore size can
determine the amount of lipase bound and lipase activity after
immobilization [8]. Typically, porous support is advantageous since
lipases can be adsorbed at both the outer surface and within the
pores of supporting material [7]. The hydrophobicity of the carrier

has been found to show signicant inuence on the lipase activity
after immobilization. Relative activity of lipase is increased when the
enzyme is selectively adsorbed on hydrophobic supports, because
lipases can recognize the surfaces similarly to those of their natural
substrates and they suffer interfacial activation during immobilization [21]. However, the hydrophobicity of carrier is not an independent parameter dominating lipase immobilization efciency.
Polarity also showed important impact on the microenvironment
of lipase after immobilization. It is reported that native and hydrophobic lipase derivatives showed higher specic activities when
immobilized on polar polymers compared with non-polar polymers
[22]. More specically, the carriers with well established hydrophobicity and medium polarity seem to create an appropriate
microenvironment for lipase after immobilization [23]. It is also
reported that the activity and stability of immobilized lipases could
be enhanced by treatment with polar solvents prior to lyophilization, which was mainly ascribed to the conversion of the inactive
closed form of the enzymes to the active open form as a result of
the treatment [24].
The immobilization parameters such as enzyme concentration,
carrier-to-enzyme ratio, pH, ionic strength also impact the immobilization efciency. The recent work by Zhao et al. [15] has shown that
the kinetics of lipase adsorption on cross-linked polystyrene resin
beads can be signicantly affected by resin-to-lipase ratios. Increase
of resin-to-lipase ratio can enhance the adsorption rate constant but
186
decrease the equilibrium adsorption capacity [15,25]. The pH of

buffer at which the adsorption is conducted is also equally important
since ionic interactions are crucial for immobilization. Typically,
maximum adsorption is observed at pH values close to the isoelectric
point of the enzyme [26,27].
The driving force for lipase adsorption onto carrier surface is
mainly electrostatic interaction and hydrophobic interaction [28].
Particularly, the hydrophobic interaction, which is forced on the
non-polar compounds by the polar environment such as water, is
found to be mainly responsible for enzyme adsorption. Therefore,
once the structure of water is changed by dissolving salts or
organic solvents in water, the hydrophobic interactions would be
affected. Generally, the amount of lipase adsorbed on carrier
surface increases with ionic strength [29] because of enhanced
hydrophobic interactions.
Although immobilization of lipase by physical adsorption is
commercially advantageous due to its easy operation and high
enzyme activity for biodiesel preparation, the stability of IL still
needs to be further improved. In particular, the emulsion of water
oil system could accelerate the leaching of lipase from carriers. As a
result, the activity of IL decreased continually with recycling batches
[15,30]. This is mainly because that hydrophobic interaction is
relatively weak and the lipase can be stripped off from the carrier
during the transesterication process [6]. Increasing the interaction
between lipase and carrier without decreasing enzyme activity is
important for further improvement of this immobilization technique. Generally, the interaction can be intensied by increasing either
hydrophobic or electrostatic forces. Strong hydrophobic interactions
can be achieved by using hydrophobic supports or hydrophobic
enzymes, while strong electrostatic interaction can be achieved by
using highly charged supports or charged enzymes [22]. Therefore,
by attaching hydrophobic groups, such as monomethoxypolyethylene glycol, acetyl-dehyde, dodecyl-dehyde, methyl acetonimidate,
methyl 4-phenylbutyrimidate on lipase molecule, the hydrophobicity of lipase can be increased, and thus the modied lipase can be
more strongly adsorbed on the carriers [22].
2.1.3. Immobilization of lipase by ionic bonding or covalent bonding

In the immobilization process by ionic bonding, the enzymes
are bound through salt linkages [5]. The carriers typically
contain ion-exchange residues such as polysaccharides and
synthetic polymers [7]. Mendes et al. [31] used anionic
exchange resin MANAE-agarose to immobilize Penicillium
camembertii Lipase G. They found that the procedure was quite
fast, and the immobilization step was completed within 60 min
resulting in protein immobilization up to 87% corresponding to
4.52 7 0.18 mg protein g 1 carrier. However, the activity of the
enzyme is slightly decreased during this immobilization procedure. The ionic bonding process can be easily performed, but the
interactions between lipase and carrier are much stronger than
physical adsorption. Compared with covalent bonding method,
ionic bonding can be conducted under much milder condition;
therefore, the ionic binding method causes little changes in the
conformation and the active site of the lipase, retaining lipase
activity in most cases. However, the binding forces between
enzymes and carriers are less strong than that of covalent
binding, and leakage of enzyme from the carrier may occur in
substrate solutions of high ionic strength or upon variation of
pH [7].
Covalent binding refers to the immobilization process of
enzymes via the chemical reaction between the active amino acid
residues outside the active catalytic and binding site of the
enzyme towards the active groups of the carrier [8]. The groups
usually used in the covalent binding are thiol and amine groups of
enzymes [5]. In Table 1, some recent progresses in immobilizations
of lipase by covalent binding are summarized. Various carriers

have been employed for covalent binding, including polymers,
silica-gel, chitosan, and magnetic particles etc. Since the binding
force between lipase and carrier is strong, the IL obtained using
this approach shows high stability during transesterication with
almost no lipase leaching. This type of IL is also resistant to
extreme conditions (pH range, temperature) [7]. Mendes et al.
[31] compared the immobilizations of lipase by different strategies, and found that covalent immobilization on epoxy-SiO2-PVA
in organic medium seemed be the most promising protocol for
immobilizing the lipase and rendered the IL the highest hydrolytic
activity. However, as summarized by Zhang et al. [12] the preparation condition for covalent immobilization is rigorous with
use of some toxic coupling reagents, and the enzyme might lose its
activity during the immobilized process. Therefore, the cost of this
immobilization is high.
2.1.4. Immobilization of lipase by entrapment
Entrapment immobilization refers to the capture of enzymes
within a polymeric network or microcapsules of polymers that allows
the substrate and products to pass through but retains the enzyme
(Fig. 3) [5,12]. After entrapment, lipase proteins are not attached to
the polymeric matrix or capsule, but their diffusion is constrained.
Compared with physically adsorbed lipases, entrapment-mobilized
lipases are more stable. Entrapment immobilization is relatively
simple to perform than covalent bonding while the activity of lipases
is maintained. However when entrapped lipases are used for
biodiesel production, the conversion rate is relatively low. In addition,
the entrapped lipases also show relatively low stability. The recent
work by Jegannathan et al. [32] showed that Burkholderia cepacia
lipase can be encapsulated into -carrageenan with an encapsulation
efciency of 42.6%. The encapsulated lipase retained 72.3% of its
original activity after 6 cycles of hydrolysis of p-NPP. However, when
the same type of lipase was used for transesterication reactions, the
biodiesel yield decreased to only 40% after 10 cycles [33]. Macario
et al. [34] compared the catalytic stabilities of lipase immobilized by
physical adsorption and encapsulation. They found that the adsorbed
lipase retained only about 34% of initial activity after two cycles, but
the encapsulated lipase kept 60% of its initial activity after 5 cycles.
Similar results have been reported by other groups [35,36]. The major
challenge for using entrapped lipases in biodiesel production is the
mass transfer limitation. In addition, the produced glycerol can
increase the viscosity of the system and adhere to the outside surface
of the carrier. This further limits the free diffusion of reactants and
products. Therefore, improving the mass transfer is critical for
utilizing this type of immobilized enzymes in industrial biodiesel
productions.
2.1.5. Immobilization of lipase by cross-linking
Immobilization of lipase by cross-linking refers to the process
to immobilize the enzyme via the formation of intermolecular
cross-linkages. It can be achieved by the addition bi- or multifunctional crosslinking reagents such as glutaraldehyde [7]. This
immobilization technique is usually support-free and involves
joining enzymes to each other to form a three-dimensional
structure [37]. Lipase can be directly immobilized from fermentation broth and recovered as cross-linked enzyme aggregates
(CLEAs). The formed CLEAs demonstrate signicantly high stability
in aqueous solutions within a broad range of pH and temperature
values [38]. The work by Gupta et al. [39] showed that CLEAs can
be formed when Thermomyces lanuginosa lipases were treated
with glutaraldehyde. The aggregates were larger in size than free
lipases, and showed more than 90% residual activity after 10
repeating cycles. Similar observations were made by Kim [40] that
the stability of cross-linked enzyme aggregates of Photobacterium
lipolyticum lipase M37 increased signicantly compared with free

lipases, particularly in the presence of high concentrations of
alcohols, e.g., methanol, ethanol, 1-propanol, and n-butanol.
In spite of all the advantages, cross-linking reactions are usually
performed under relatively harsh conditions, such as using crosslinking reagents that can change the conformation of lipases and
potentially lead to signicant loss of activity. Other disadvantages
associated with cross-linking immobilization are low immobilization yields and absence of desirable mechanical properties. To
address these two concerns, cross-linking is always coupled with
other immobilization techniques, e.g., adsorption. It has been
reported that lipase can be immobilized by a hybrid process
starting with cross-linking of the enzymes and followed by
adsorption on supporting materials, such as polymer membrane
[41,42], macroporous resin [43] and inorganic salt micro-crystals
[44]. These hybrid approaches can improve the stability as well as
mechanical properties of ILs.
2.1.6. Commercialization of immobilized lipase for biodiesel
production
Although many processes have been developed for immobilization of lipases in lab, only a few techniques have been successfully
commercialized. The major roadblock for the technical transfer is the
high cost of immobilization steps. For example, the market price of
Novozyms 435 is $1000/kg [8]. To reduce cost, the carrier must be
easy to synthesize or commercially available at low prices. The
immobilization process should be efcient for recovering proteins
and retaining their enzymatic activities. The ILs should have high
stability to avoid enzyme leaching or activity loss. Some commercial
ILs are listed in Table 2. Among these commercial ILs, Novozyms
435 is the most widely-reported in literatures for biodiesel production. In 2006, the rst industrial plant for enzymatic production of
biodiesel was built in China, with a capacity of 20,000 t/yr. Lipozymes TL IM and Novozyms 435 were both used in this plant. tertbutanol was selected as the reaction medium. The highest biodiesel
yield of the plant can reach 95% using rapeseed oil as feedstock [1].
Another biodiesel production line was established in 2007 with a
capacity of 10,000 t/yr. In this process, lipase of Candida sp. 99125
immobilized on textile membranes was used as catalyst. The cost of
lipase was estimated to only 32$/t biodiesel (200 CNY/t biodiesel)
[2], which is much lower than that of Novozyms 435 if the enzyme
is only used for one batch.
3. Process optimization and reactors for IL-catalyzed

transesterication
3.1. Parameter optimization for transesterication process
Parameter optimizations for IL-catalyzed transesterication of oil
feedstocks have been extensively studied in recent years. Hundreds of
papers have been published on optimization of parameters for
transesterication process with various lipases. Although the optimal
operation conditions vary depending the type of lipases and immobilization techniques, the type of parameters that needs to be
187
considered remain the same, including acyl acceptor types and

concentration, water content, enzyme loading, alcohol to oil ratio
and reaction temperature. Various short-chain alcohols have been
used as acyl acceptors including methanol, ethanol, isopropanol,
2-propanol, n-butanol, and isobutanol [45,46]. Methanol is the most
commonly used for biodiesel production due to its relatively low price.
In addition, some short-chain alkyl acetates such as methyl acetate
[47,48] and ethyl acetate [49] also have been employed as acyl
acceptors. The highest reaction rate is typically obtained when
methanol is used. Increasing acyl acceptor concentration generally
can enhance reaction rate and nal oil-to-biodiesel conversion.
Theoretically, 3 mol of alcohol is needed to completely convert 1 mol
of glyceride, but in practice excess alcohol is used since the transesterication is a reversible reaction. However, high concentrations of
alcohol (especially methanol) signicantly reduce the activity of
lipases, and stepwise addition of methanol is, therefore, performed
to minimize the deactivation of lipase [2].
The transesterication reaction can be performed in either
organic solvent or solvent-free systems. Because oil and methanol is not complete miscible in a solvent-free system, the water
content of the system can signicantly affect the reaction rate
and yield. Water can affect the catalytic activity and stability of
lipases. Thus the minimum water content is necessary in the
system to keep the enzyme active in the non-aqueous reaction
[50]. This is mainly because that the available interfacial area
generally determines lipase activity. In solvent-free system, with
the increase of water content, more oilwater droplets can be
formed and thereby increasing the available interfacial area [51].
However, too high water content may lead to decrease of acyl
acceptor concentration in the system and increase of glyceride
hydrolysis to form fatty acid. Consequently, the apparent transesterication rate and biodiesel yield become lower. It has been
reported that in solvent-free system, the optimum water content
varies between trace amount to 20%, depending on the oil
feedstock and ILs [52,53]. Another issue involved in a solventfree system is the negative effect of glycerol on lipase activity.
Since glycerol is highly hydrophilic and insoluble in oils, it can be
easily adsorbed onto the surface of the ILs causing decrease of
the activity and stability of lipase [52,54]. It is also possible that
the high viscosity of glycerol-contained system decreases the
diffusion of reactant and product. Some strategies have been
developed to remove glycerol, such as addition of silica gel to
absorb glycerol and washing lipases with certain organic solvents periodically [48]. However, these strategies cannot be
easily adapted to large scale operations. Alternatively, introducing organic solvents to the reaction can covert the liquidliquid
bi-phase into a homogenous phase. Hydrophobic organic solvents such as n-hexane and petroleum ether [54], and tertbutanol, have been used as reaction media for IL-catalyzed
transesterication. For example, the stability of Novozyms 435
in tert-butanol was greatly enhanced [5557]. With the introduction of tert-butanol solvent, ILs can be reused for more than
200 cycles with yield of 95% in a batch operation [55] or be
used for over 500 h with biodiesel yield of 97% in a continuous
operation [58]. However, there is an optimum dosage of tert-
Table 2
Some commercial immobilized lipases used for biodiesel production [2].
Commercial name
Enzyme origin
Support
Hydrophobicity/philicity
Producer or inventor
Novozyms 435
Lipozymes RM IM
Lipozymes TL IM
Lipase PS Amano IM
Candida antarctica form B

Rhizomucor miehei
Thermomyces lanuginosa
Candida sp. 99125
Lewatit VP OC 1600
Duolite A568
Silica granules
Diatomaceous earth
Textile membrane
Medium hydrophobic
Hydrophilic
Hydrophilic
Hydrophilic
Hydrophobic
Novozymes (Denmark)
Novozymes (Denmark)
Novozymes (Denmark)
Amano (Japan)
Beijing University of Chemical Technology (China)
188
Fig. 4. Window of operation for STR (left) and PBR (right) (A, B and C illustrate different expansions of the window) (Adapted with permission from [71]).
butanol in order to achieve a high reaction rate and biodiesel

yield. According to Li et al. [55] the optimum dosage of tertbutanol is 75% (v/v) of oil, while further increasing tert-butanol
led to a decrease of reaction rate due to dilution of the reactant.
3.2. Reactors for IL-catalyzed transesterication
Most of the IL-catalyzed biodiesel productions in lab are
performed in shaking asks, but for a larger-scale operation, the
reactor must be carefully designed. Several types of reactors have
been used for biodiesel production, such as stirred tank reactor
(STR) [59,60], packed-bed reactor (PBR) [61,62], uidized bed
reactor (FBR) [63] and bubble column reactor (BCR) [64]. However,
only a few of these rectors are potentially suitable for industrial
production.
3.2.1. Stirred tank reactor
STR is a well-mixed reactor. It is the most often-used reactor for
bioprocesses at different scales because of the ease of construction,
operation and maintenance [65]. STR can be operated in both
batch (BSTR) and continuous modes (CSTR). High oil-to-biodiesel
conversion is usually achieved using this type of reactor. BSTR is
usually used on small scale, especially in laboratory. Ognjanovic
et al. [66] performed the enzymatic conversion of sunower oil to
biodiesel in a solvent-free system using BSTR equipped with
six-bladed turbine impeller. The biodiesel yield could be as high
as 98% when 12:1 M ratio of methyl acetate/oil was used at
100 rpm stirring rate. They found that agitation intensity and the
mode of agitation appear to be of a particular importance for the
transesterication process. Good mixing could improve contact
between substrate and biocatalyst and provide a good dispersion
of the biocatalyst in the reaction mixture, and thus it reduces mass
transfer resistance and increases overall reaction rate. However,
the relative activity of the ILs decreased with reuse. Sanches and
Vasudevan [67] found that Novozym 435 retained 95% of its
activity after ve batches and about 70% after eight batches, but
as low as 41% after 11 batches.
CSTR seems to be more applicable on large scales because of
high productivity. Continuous operation with multi-stage STRs
also allows that the tanks can be operated with enzymes of
different ages/activities and units can be installed between the
reactors to separate out the glycerol formed during the reaction
[68]. According to the work of Chen and Wu [69], 70% biodiesel
yield could be obtained in a CSTR. The reactor can run continuously for over 70 days with regeneration of the lipase by tertbutanol washing. Keng et al. [59] found that a STR with Rushton
turbine impellers at a relatively lower agitation speed could obtain
a biodiesel yield of 95.8%. The process was successfully scaled up
to 75 L with 50 L working volume based on a constant impeller tip
speed approach, and the yield reached 97.2% after 5 h reaction.

Bassheer et al. [70] invented a process to produce biodiesel by
enzymatic transesterication using two CSTRs in series, with a
bottom sintered glass lter to remove the by-product glycerol and
excess water formed during the reaction. High conversions (98%)
could be obtained over a short space of time (4 h), and the
enzymes were reused in over 100 consecutive batch cycles.
One of the major disadvantages of STR refers to the high
shearing force caused by the impeller which can damage the
carrier and thus limiting the reusability of the catalyst [2]. The
shearing force is greatly dependant on the stirring speed and
the impeller types. Therefore, to minimize the damage of IL, the
stirring speed must be optimized and impeller must be improved.
Probably axial ow impellers with down-pumping are more
appropriate for IL-catalyzed production of biodiesel [71], but this
needs further investigation. The stirring speed is important but
easy to control. Too low stirring speed may lead to low productivity because of mass transfer limitation. Too high stirring speed,
however, may cause damage of carrier and denaturation of
enzyme. Therefore, there exists a safe zone in which the process
can be robustly performed regarding to lipase loading and power
input into the system. Xu [71] has proposed using window of
operation (Fig. 4), which is an operating space determined by
constraints and correlations of a process, to guide the process
design and optimization of IL-catalyzed production of biodiesel in
STR. The transesterication rate is mostly dependent on the lipase
activity and mass transfer of substrates by mixing in STR, which
relates to the lipase loading and power input, respectively. The
glycerol associated mass transfer problem sets another constraint
of minimum stirring speed/power input. The damage to immobilized enzyme particles is the correlated effect from shear stress
from the impeller, which are also related to the lipase loading and
power input. The operational window is thus formed by constraints for these variables and their correlation. As rstly proposed by Woodley and Titchener-Hooker [72], windows of
operation thus may be used to help understand and optimize
the process.
3.2.2. Packed-bed reactor

PBR is usually used for a continuous operation. It is the most
promising reactor for industrial-scale production of biodiesel. PBR
is basically composed of a column in which ILs are packed, and
auxiliary equipments such as a water bath for maintaining
required reaction temperature and pumps for transferring reactants [71]. Compared with STR, PBR can obtain a larger reacting
surface area per unit volume with associated higher volumetric
productivity in continuous industrial processes [65]. The parameters that should be considered for optimizing biodiesel production with PBR mainly include the ow rate, reaction temperature,
molar ratio of methanol to oil [73] and solvent dosage if used.

Flow rate is the most important operational variable. A low ow
rate is usually used in order to obtain an enough long retention
time for a high biodiesel yield. However, long-term operation of a
PBR is still a challenge. It has been reported that in a solvent-free
system, the packed-bed reactor can be operated for 37 days
without decreasing ester yield [30,74]. A longer operation could
reduce biodiesel yield, since the accumulated glycerol might
deposit on the surface of the ILs and decrease the catalytic
efciency [2]. To solve this issue, process with stepwise methanolysis and removal of glycerol has been developed by Shimada et al.
[75]. The oil conversion could maintain over 95% during 108 days
of operation. Another solution for decreasing the negative effects
caused by glycerol is to introduce solvent such as tert-butanol as a
reaction medium. According to the results of Sverac et al. [76],
when Novozyms 435 was used for conversion of crude high-oleic
sunower oil to biodiesel with butanol as an actyl acceptor, a high
productivity of 13.8 t yr 1 kg enzyme 1 with a butyl-ester purity
of 96.5% was obtained, and the reactor could be performed for over
50 days without any loss of enzyme activity. Other researchers
reported that the biodiesel yields with PBR for transesterication
were in the range of 7096% with operation time of 24 h to 100
days [77]. However, one of the major disadvantages for PBR is the
high pressure drop associated with small carrier size, high ow
velocity or obstruction of the catalyst bed by accumulation of
insoluble components from the reaction mixture [7]. In solventfree system, pressure drop becomes more signicant due to the
higher viscosity than that of solvent system. Commercial ILs
usually have carrier diameters of less than 4 mm. However, when
particle size is in this range the pressure drop is 41 bar/m reactor
at a rapeseed oil ow velocity of 0.01 m/s at 25 1C [78]. Another
drawback of PBR refers to the mass transfer limitations, which is
due to the restricted ow pattern inside PBR because of the limited
ow velocity relative to the pressure drop allowance [7]. According
to the study of Halim et al. [62], ow velocity signicantly affected
the mass transfer and thus affecting biodiesel yield. At low velocity
of substrate the mass transfer dominated the biodiesel yield,
whereas at high velocities the reaction dominated the yield.
Particularly, the internal mass transfer showed an important
inuence on the apparent kinetics of biodiesel formation in PBR.
As analyzed by Fjerbaek et al. [78], the effectiveness factor, ,
dened as the ratio of rate of reaction with pore diffusion
resistance and rate of reaction with surface conditions, decreased
rapidly from 1 towards 0.66 in the range of particle diameters of
existing commercial biocatalysts. It indicates that intensication of
the internal mass transfer is an important aspect regarding to
further optimizing biodiesel production with RBR.
Similarly, the window of operation for PBR can be set up by
selection of the constraint parameters as shown in Fig. 4 (right).
Supercial velocity and cross-section area of the reactor are two
important operational variables, and conversion per pass, mass
transfer and pressure drop are important constrains for PBR.
The boundaries of the operational zone can be expanded by
different strategies. For example, the boundary can be moved
towards the lower velocity region by using organic solvents to
reduce the viscosity of the reaction mixture and improving the
mutual solubility of the substrates; increasing the rigidity and
particle size of the carriers can decrease pressure drop [71].
However, the interactions of these parameters are complicated.
To obtain high biodiesel yield sometimes has to sacrice other
aspects such as high pressure drop or loss of lipase activity.
3.2.3. Airlift loop reactor

Airlift loop reactor (ALR) also has been tested for IL-catalyzed
transesterication of oils in solvent system. ALR has a channel for
189
gasliquid upowthe riser, and a separate channel for the

downow [79]. The gas is injected near the bottom of the riser,
making the ILs and reactant mixed. A novel airlift loop reactor
with no need of external gases (ALR-NEG) was developed and
patented [80]. In this reactor, the internal gas at the top (methanol
and tert-butanol vapor) is recycled by a pump to the bottom, and
the liquidsolid (ILs) can be well mixed. By using this reactor,
production cost as well as loss of reactants and solvent can be
reduced. Compared with STR, the energy consumption of ALR can
be dramatically reduced, and the carriers cannot be damaged.
However, since the mixing is not so severe, the mass transfer of
ALR should be further intensied. For solvent-free system, ALR
probably does not work well for mixing.
3.2.4. Other heterogeneous reactors

Some other heterogeneous reactors have been also reported for
IL-catalyzed transesterication of oil feedstocks. A biodiesel yield
as high as 98.95% can be obtained by transesterication of babassu
oil with ethanol under the catalysis of Novozyms 435 in a
continuous uidized bed reactor (FBR) combined with a packed
column to remove glycerol [81]. Ricca et al. [63] employed FBR for
continuous transesterication of olive husk oil catalyzed by ILs and
found FBR had a productivity of 30% higher than that of batch
operation. They also found that the recycle ratios (R/F) dened as
the ratio of fresh feed ow rate (F) and the recycle rate (R) had an
important inuence on the productivity and oil conversion.
Increasing R may cause more dilution of the stream owing within
the reactor thus decreasing reaction rate (productivity), but higher
R may increase retention time thus yielding higher conversion.
Therefore, the R/F should be optimized with integrated consideration of productivity, biodiesel yield and catalyst lifetime. Compared with PBR, FBR showed more effective for mixing the solid
catalyst with the viscous oil, leading to a good contact of IL with its
substrate. Mass and heat transfer are also well improved by FBR.
However, the shearing force caused by uidization may cause
damage of carrier as well as leaching of lipase from the carriers.
Productivity and lifetime of ILs can be improved through
modication of conventional heterogenous reactor by combining
with separation system to simultaneously remove byproduct
glycerol or excess water. Extraction [82,83], adsorption [81] or
membranes [84] have been applied as separation unit integrated
with STR or PBR. By removing glycerol simultaneously, the mass
transfer resistance is reduced and lipase lifetime is expanded.
However, these modications also increase the complexity of
equipments as well as operation.
4. Process simulation and optimization

4.1. Kinetic modeling of IL-catalyzed transesterication
Understanding the kinetics of IL-catalyzed transesterication is
important, which can serve as a tool for further optimization of the
process, investigation of the reaction mechanisms and reactor
design. In most of the reported kinetic studies, the reaction system
is considered as a pseudo-homogenous phase and the effect of
mass transfer is usually neglected. Similar to the lipase-catalyzed
hydrolysis and etherication processes [85], pseudo-homogenous
transesterication is usually modeled by ping-pong bibi mechanism, by which a product is released before the addition of the other
substrate [8689]. For the simplest mechanisms in which the oil
transesterication is considered to consist of three consecutive
reversible reactions, the initial reaction rate can be described as the
190
CH3OH
CH2OOCR
CHOOCR
CH3OOCR
k1
k2
CH2OOCR
CH2OH
CH3OH
CHOOCR
CH2OOCR
Glycerol
RCOOH
CH2OH
k7
CHOH
k10
CH2OOCR
CH2OOCR
k15
CH3OH
k16
k8
CH3OH CH3OOCR
CH3OH CH3OOCR
CH2OH
CH2OH
k11
k9
CHOH
k14
CHOOCR
k6
H2O
k13
CH2OH
k4
CH2OOCR
k5
CH3OOCR
k3
CH3OOCR
k12
CHOH
CH2OH
H2O
Fig. 5. A comprehensive kinetic model for lipase-mediated methanolysis of glyceride for formation of biodiesel and glycerol with consideration of esterication,
transesterication and acyl migration (adapted from with permission [93]).
following equation [86,87]:

vi
V max TGA
K m;TG A1 A=K i;A K m;A TG TGA
where vi is the initial reaction rate; Vmax, Km,TG, Ki,A, and Km,A are
corresponding kinetic constants; and [TG] and [A] are concentrations
of triglycerides and acyl acceptor, respectively. The kinetic constants
thus can be tted by experimental data. However, the values for these
kinetic constants may be varied with the oil feedstock, lipase, acyl
acceptor and reaction medium used in the experiments. According to
Dossat et al. [86], when the enzymatic transesterication of high oleic
sunower oil was performed with butanol as an acyl acceptor under
the catalysis of immobilized Lipozymes in n-hexane system, the
determined values for Vmax, Km,TG, Ki,A, and Km,A were 250 mole
min 1 g 1, 5.3 mM, 13 mM and 55 mM, respectively. According to the
study of Xu et al. [87], when rened soybean oil was inter-esteried by
Novozyms 435 with methyl acetate as the acyl acceptor in solventfree system, the kinetic constants were determined as Vmax 1.9
mol min 1 L 1; Km,TG 1 mol/L; Km,A 16 mol/L, and Ki,A 0.0455
mol/L, and the rst step reaction, triacylglyceride to diacylglyceride
was the rate-limiting step for the overall reaction. Nevertheless, Eq. (1)
is just a simplied model, in which the effects of water and hydrolysis
of glycerides are not considered. However, hydrolysis of glyceride
happens inevitably especially when the system has a high water
contents. Therefore, another possible reaction mechanism for fatty
acid ester formation is the two-step reactions: hydrolysis of the ester
bond and release of the fatty acid moiety followed by an esterication
with the alcohols [78]. Theoretically, both of the above two reaction
mechanisms are co-present in the system, but which one is dominant
still needs further investigation. Cheirsilp et al. compared the impact of
three transesterication mechanisms on the kinetic modeling of
biodiesel production by immobilized lipase [90]. They found that
hydrolysis and transestercation occur simultaneously, and the reaction mechanism regarding to stepwise hydrolysis followed by etherication seems not to well describe the actual kinetics. However, the
rate constants for alcoholysis are much higher than those for the
hydrolysis reaction. It should be noted that the reactions involved in
enzymatic preparation of biodiesel are complex, which include direct
transesterication, hydrolysis, esterication, as well as acyl migration
[91]. Acyl migration has been often observed during enzymatic
conversion of triglycerides to biodiesel using 1,3-positional specic
lipase. The acyl migration can happen between 1,2-diglyceride (1,2DG) and 1,3-diglyceride (1,3-DG), or 2-monoglyceride (2-MG) and 1monoglyceride (1-MG) [92]. A more comprehensive kinetic model has
been developed by Li et al. [93] as shown in Fig. 5. According to their
experimental results, acyl migration between 2-MG and 1-MG as well
as between 1,2-DG and 1,3-DG took place independent of enzymatic
catalysis. The acyl migration can be described as rst-order

reversible reactions, and temperature and water activity were
two crucial factors inuencing acyl migration kinetics [92].
However, the mechanism of acyl migration still needs further
investigation. Particularly, the thermodynamic and kinetic driving forces of this phenomenon should be gured out. Moreover,
it seems that the apparent kinetics of lipase-catalyzed production of biodiesel may be greatly dependent on the reaction
medium, acyl acceptor type and concentration, water content,
activity of lipase, and origin of the oil feedstocks.
Another problem associated with most of the current researches
on kinetic modeling of IL-catalyzed transesterication is the negligence of mass transfer. In these researches reaction mixture is
regarded as a homogenous phase, where all reactants and enzymes
are considered as soluble in the solvent. However, this assumption is
probably not true particularly in solvent-free system, because the
mass diffusion through liquid lm and inside the carrier pores might
become limiting-steps. The kinetic modeling with consideration of
mass transfer thus should be developed.
4.2. Process simulation and product purication
4.2.1. Process simulation and unit optimization
Process simulation can obtain general mass and energy balances, which are prerequisites for economic evaluation of biodiesel
production. Simulation tools such as ASPEN PLUS, HYSYS, VMGsim
etc. have been employed to simulate the biodiesel production by
supercritical transesterication [94,95], alkali catalysis [96] and
acid catalysis [97]. To well calculate the energy consumption of the
process, the physical properties of the reactants and products must
be accurately estimated or determined, and the thermodynamic
model should be carefully selected. For most of the simulation,
triolein is routinely used as a model compound of vegetable oils,
because it composes around 4080% of fatty acids in a variety of
vegetable oils such as rapeseed, canola, olive, palm and peanut oils
[95]. This compound also can be directly found in the databases of
simulation softwares. However, the values for normal boiling point
(Tb) of triolein, which is used to predict other properties such as
critical temperature and pressure, are discrepant in the databases
or literatures, varying from 541.7 to 879.9 1C. Lee et al. [95]
determined the Tb of triolein as 412.8 1C by thermogravimetric
analysis (TGA) method; however, this data is much lower than
those mentioned above. Another important aspect for accurate
simulation refers to the selection of thermodynamic model. When
ASPEN PLUS is used for simulation, non-random two-liquid (NRTL)
model is usually selected because the transesterication reactions
occur in a highly non-ideal chemical system. However, sometimes

P-101
Crude Oil
P-102
Tert-Butanol
102
P-201
R-101
M-101
M-102
P-203
201-A
P-104
105
102-A
104
203
201
202-A
P-103
Methanol
101-A
101
191
103
103-A
202
Sumpmentary Methanol
P-202
303-A
M-103
CDS3
P-302
GAS
501
CDS4
T-501
502
Waste Oil
QB2
QB3
QB1
S-401
P-401
403
STEAM
CDS1
H-201
FAME
204
CDS2
303
401
P-301
EA3
EA1
EA2
302
Q1
301
503
Glycerol
402
OUT3
OUT2
OUT1
R-201
triple effect evaporator

Fig. 6. A process simulation for IL-catalyzed production of biodiesel with ASPEN PLUS [98].
the binary NRTL coefcients are not included in the database,

which should be estimated by corresponding thermodynamic methods. For example, Zheng [98] estimated the binary
NRTL coefcients of triolein/methanol and triolein/tert-butanol
by UNIFAC method, which enabled the simulation running
successfully.
By process simulation, the unit operation condition can be optimized, and the parameter sensitivities can be analyzed. The economic
feasibility of the process thus can be further determined. Zheng [98]
performed the process simulation and optimization of Novozyms
435-catlyzed production of biodiesel in tert-butanol system using
ASPEN PLUS as shown in Fig. 6. He found that triple-effect evaporation
was more feasible than column distillation to recover methanol and
solvent, and distillation column with partial condensation was recommended for the purication of fatty acid methyl ester (FAME). Based
on an overall energy balance, Zheng showed that IL-catalyzed process
consumed less energy compared with the acid/alkali and supercritical
processes [98]. Sotoft et al. compared the enzymatic production of
biodiesel from high quality rapeseed oil and methanol in solvent-free
and cosolvent processes based on process simulations and economical
evaluations [97]. Their results indicated that the cosolvent process was
the most expensive and not a viable choice, while the solvent-free
process was viable for a scale of 200,000 t biodiesel/yr with the
enzyme price of 1000 US$/kg. Enzyme cost amounted for 50% of the
total raw material costs in solvent-free process, while only for 22% in
tert-butanol system due to the improved enzyme performance. Being
similar to other biodiesel production processes with chemical catalysts, production capacity is very important. Too small scale usually is
not economically feasible.
4.2.2. Biodiesel purication

Biodiesel products need purication to meet the corresponding
product standards as shown in Table 3. The impurities contained
in the crude biodiesel include free fatty acids (FFA), water, alcohol,
glycerides, metals (soap or catalyst when alkali is used for
catalysis) and glycerol [99]. In a conventional separation process
for solvent-free system, glycerol is usually rst removed out after
transesterication. Phase separation can be well observed since
biodiesel and glycerol are not miscible. Glycerol has a higher
density (1050 kg/m3, or higher) than that of biodiesel (880 kg/m3);
therefore simple techniques such as gravitational settling or
centrifugation are effective for the separation of biodiesel and

glycerol phases [100]. For a cosolvent system, phase separation can
be conducted after the solvent is separated. The solvent recovery
can be simply achieved by conventional evaporation, distillation or
rectication. Compared with those of alkali or acid-catalyzed
processes, the purication of biodiesel produced by IL-catalysis is
less complex. As reviewed by Atadashi et al. the catalyst used in
transesterication, oil to alcohol ratio, water and free fatty acids
contents are found to have signicant effects on the complexity of
downstream processing and purication [100]. Conventionally,
biodiesel is puried by washing with distilled water. For the
alkali or acid-catalyzed transesterication, a large amount of
water is usually consumed in order to remove soap and other
contaminants, and reduce the alkaline metal (Na, K) concentrations less than 5 ppm or acid value less than 0.5 mg KOH/g.
However, for IL-catalyzed transesterication these requirements
are easy to achieve since no alkaline metals and mineral acid are
introduced into the reaction system. Nevertheless, another problem associated with lipase-catalyzed production of biodiesel is
the formation of free fatty acid (FFA) because the reaction system
must contain a certain amount of water to keep the lipase's
activity. Higher amount of FFA can cause more signicant emulsion
of the system, resulting to the difcult of phase separation.
Therefore, FFA should be removed by any chemical or enzymatic
treatment. Preferably, FFA can be converted to biodiesel by
esterication with alcohols. This treatment not only increases
the conversion of oil feedstock to biodiesel, but also reduces the
acid value of the product.
Another purication technique refers to treatment of the
biodiesel product with solid adsorbent or ion-exchange resins
[101]. This technique is also termed as dry wash process.
Absorbents such as Magnesol or bleaching earth have been found
to selectively absorb hydrophilic materials such as glycerol and
mono- and diglycerides [100]. In this process, the solid absorbent
is added to a stirring solution of biodiesel. After stirring the slurry
for a requisite time, the biodiesel in puried form can be obtained
by separation of mixture with a drum lter or related ltration
techniques [102]. When ion-exchange resins are used, the process
is usually conducted by a packing bed column lled with the
resins, where the crude biodiesel passes through resin bed in a
continuous ow until the impurities are removed. According to the
results of Berrios et al. adsorption and resin treatment can remove
192
Table 3
Biodiesel standard in different countries or area.
Specication
Density 15 1C
Viscosity 40 1C
Distillation
Flashpoint (Fp)
CFPP
Cloud point
Sulfur
CCR 100%
Carbon residue (10%dist.residue)
Sulphated ash
Water
Total contamination
Cu corrosion max
Oxidation stability
Cetane number
Acid value
Methanol
Ester content
Monoglyceride
Diglyceride
Triglyceride
Free glycerol
Total glycerol
Iodine value
Linolenic acid ME
C(x:4) & greater unsaturated esters
Phosphorus
Alkalinity
Gp I metals (Na, K)
GpII metals (Ca, Mg)
g/cm
mm/s
%@1C
1C
1C
1C
mg/kg
%mass
%mass
%mass
mg/kg
mg/kg
3 h/50 1C
hrs;110 1C
mgKOH/g
%mass
%mass
%mass
%mass
%mass
%mass
%mass
%mass
%mass
mg/kg
mg/kg
mg/kg
mg/kg
Europe
Germany
USA
China
EN 14214:2003
0.860.90
3.55.0
DIN V 51606
0.8750.90
3.55.0
ASTM D 6751-12
Z 120
country specic
Z 110
Summer 0, spr/aut 10, winter 20
GB/T20828-2007
0.820.90
1.96.0
90%, r 360 1C
Z 130
n
Report
r 10
r 10
r 0.05
r 0.3
r 0.03
r 300
r 20
No. 1
r 0.3
r 0.02
r 500
r 24
No. 1
Z6
r 51
r 0.5
r 0.20
Z 96.5
r 0.8
r 0.2
r 0.2
r 0.02
r 0.25
r 120
r 12
r1
r 10
1.96.0
90%, r 360 1C
Z 130
n
Report
r 15
r 0.05
r 0.05
r 0.02
r 500
r 50
No. 3
Z3
Z 47
r 0.5
r 0.2 or FpZ 130 1C
Z 49
r 0.5
r 0.3
r 0.8
r 0.4
r 0.4
r 0.02
r 0.25
r 115
r 0.4
r 10
r5
r 10
r 0.02
r 0.24
r5
r5
r 0.3
r 0.02
r 500
Trace
No. 1
Z6
Z 49
r 0.5
r 0.02
r 0.24
r5
r5
Table 4
Reported economic evaluations of IL- catalyzed production of biodiesel.
Capacity (t/yr)
Reaction media
Oil feedstock
Glycerol credit included
Enzyme price ($/kg)
Product cost ($/kg)
References
8000
200,000
8000
200,000
8000
200,000
8000
200,000
1000
8500a
8000
8000
Solvent-free
Solvent-free
tert-butanol
tert-butanol
Solvent-free
Solvent-free
tert-butanol
tert-butanol
Solvent-free
tert-butanol
Supercritical CO2
Supercritical CO2
Rapeseed oil
Rapeseed oil
Rapeseed oil
Rapeseed oil
Rapeseed oil
Rapeseed oil
Rapeseed oil
Rapeseed oil
Palm oil
Waste oil
Waste cooking sunower oil
Waste cooking sunower oil
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
1000
1000
1000
1000
10
10
10
10
1200
2000 $/klU
800/kg
8/kg
1.95
0.96
3.12
2.23
0.98
0.065
2.87
1.97
2.414
0.9b
1.64/L
0.75/L
[97]
[97]
[97]
[97]
[97]
[97]
[97]
[97]
[105]
[133]
[134]
[134]
a
b
Calculated according to authors data.

Raw materials cost.
soap, methanol and glycerol effectively, while have no effect on

density, kinematic viscosity, FAME content or glyceride content of
the biodiesel [84].
Membranes also have been used for biodiesel purication. This
process exhibits several advantages over the conventional ones,
such as minimization of capital cost and other related production
costs, and high specic area of mass transfer [100]. Membrane
separation can be conducted simultaneously with the transesterication using membrane reactors [84,103], or after the transesterication process [104]. It has been found that membrane
separation was able to reduce the amount of soap in crude
biodiesel, and ultraltration membrane of 10 kDa could reduce
glycerol effectively to be less than 0.02 wt% [104]. Although these
purication processes are considered promising on industrial
scale, the process still needs optimization with systematic consideration of water and energy consumptions as well as capital
and operation costs. The overall process also should be further

optimized in order to reduce running cost.
5. Techno-economic evaluation of IL-catalyzed production of

biodiesel
Techno-economic evaluation is important to estimating production cost and determining the costliest units for further
optimization. Works on techno-economic evaluation of chemical
production of biodiesel have been frequently reported, but only a
few publications refer to the economic estimation of IL-catalyzed
production of biodiesel (Table 4). Economic evaluation usually
consists of several steps: development of process ow sheets, time
charts, equipment lists followed by estimations of equipment cost,
plant cost and manufacturing cost [104]. Most of the techno-
193
100 $/kg, respectively. Another promising solution is to increase

the specic activity of IL to decrease enzyme loading used for
transesterication, which can be done by lipase modication with
protein engineering approaches. However, process design with
consideration of engineering aspects such as heat integration is
also important.
6. Concluding remark and recommendation for future work
Fig. 7. Effect of IL reused time on the estimated lipase cost under different enzyme
prices. IL loading: 2% based on raw oil feedstock; oil-to-biodiesel conversion: 95%.
economic evaluations are performed based on process simulation

results. Once the owsheet, mass balance and energy consumption
are determined, equipment size and cost estimation of the process
equipments and total capital investment (TCI) can be carried out.
This can be done by using commercial software such as Aspen
Icarus Process Evaluator [97]. Economic evaluation can become
easier when the cost of a plant with the same technology has been
known (base case). Generally, the capital cost of a plant with new
size can be estimated by the following exponential scaling expression:

New size n
New cost Base cost
2
Base size
where n is a characteristic scaling exponent depending on some
characteristic of the equipment related to production capacity.
For complete process plants, n is usually taken as 0.60.7. However
in cases of process plants where capacity is increased by duplicating a number of pieces of equipment (scale-out), n can be higher
and may even approach a value of 1. In the economic analysis of
pilot plant scale, n was set to be 0.9 for technologies requiring
scale-out and 1 for those not requiring scale-out. In greater scales,
smaller values of n (from 0.7 to 0.9) were applied in the technoeconomic model [105,106].
The economic feasibility of enzymatic production of biodiesel
depends on a series of factors. These factors mainly include the
raw material costs such as the prices of oil feedstock, alcohol and
enzyme; the process parameters, such as oil-to-biodiesel conversion ratio, retention time for transesterication, biodiesel recovery
yield, lipase lifetime and solvent loss (if used); process design
regarding water recycle and heat integration; and by-product
credit. It has been found that lipase cost contributes a great part
of the total production cost. The extensively used IL, Novozym 435,
has a high price per kilogram, indicating that a very high
productivity is required for the process to be cost effective [107].
As studied by Jegannathan et al. [105], when the IL is reused for
5 batches the manufacturing cost of IL-catalyzed process is twice
that of alkali process, while 30.9% that of free-lipase catalyzed
process. Corresponding lipase cost accounts for 49.7% of the
manufacturing cost. Therefore, the reusability of ILs is important
to reduce biodiesel production cost. As shown in Fig. 7, reuse time
of IL has a signicant inuence on enzyme cost for IL-catalyzed
production of biodiesel. It can be estimated that to make the
enzyme cost less than 0.1$/kg of biodiesel, the IL should be reused
for more than 320, 210, 160, 50 and 20 batches without loss of
enzyme activity when lipase price are 1500, 1000, 750, 200 and
Lipase-catalyzed production of biodiesel has attracted great

attention recently, due to the merits such as mild reaction
conditions, environmental friendliness and wide adaptability for
feedstocks. Immobilization of lipase facilitates enzyme recovery
and increases the stability of the enzyme. This technique shows
great potential for industrial-scale production of biodiesel. Various
approaches have been developed for lipase immobilization, mainly
including physical adsorption, ionic bonding, covalent bonding,
entrapment and cross-linking. Nevertheless, only a few of them
seem to be economically feasible. Each immobilization technique
has its own advantage and disadvantage, and lipase immobilization is usually performed by a combination of two or more of these
approaches. Most of the commercial ILs are prepared by adsorption of free lipase on polymeric materials, because that this process
is simple and the carrier is relatively easy to make at cheap cost.
However, the stability of ILs still should be enhanced, especially to
strengthen the interaction between lipase and carriers to prevent
enzyme leaching.
Several operation parameters have been found to affect the
biodiesel yield and stability of ILs. These parameters mainly
include acyl acceptor types and concentration, water content,
enzyme loading, alcohol to oil ratio, temperature and reaction
media. Parameter optimization is important to obtain high biodiesel yield and maximize reuse of the enzyme. However, the
optimum condition is greatly dependent on oil feedstock and IL
employed. In solvent-free system, IL is easy to loss activity, which
is mainly ascribed to the leaching of lipase protein from the carrier.
This problem can be solved by introducing inert solvent such as
tert-butanol as reaction media. Mass transfer resistance is reduced
and the inhibitive effects of glycerol and alcohol on IL can be
decreased in a solvent system. Nevertheless, use of solvents may
incur additional expense caused by solvent loss and energy
consumption for solvent recovery.
Reactor is important for scaling-up of IL-catalyzed biodiesel
production. Commonly used reactors are STR and PBR or their
combination. STR is a well-mixed reactor, and can be operated in
both batch and continuous modes. High oil-to-biodiesel conversion usually can be obtained by STR, because that good mixing can
improve contact between substrate and lipase and provide a good
dispersion of the biocatalyst in the reaction mixture. However, the
major disadvantage of STR refers to the high shearing force caused
by the impeller which can damage the carrier and thus limiting
the reusability of the catalyst. PBR is usually used for a continuous
operation. Compared with STR, PBR can obtain a larger reacting
surface area per unit volume with associated higher volumetric
productivity in continuous industrial processes. However, poor
mass transfer with glycerol accumulation and high pressure drop
are the main shortcomings for PBR. STR or PBR can be coupled
with separation system to remove glycerol by centrifugation,
adsorption or membrane. By this integration, lipase reusability
and biodiesel yield can be improved.
Kinetic modeling can provide as a tool for further process
optimization and understanding reaction mechanisms of ILcatalyzed transesterication. In most of reported kinetic studies,
the reaction system is considered as a pseudo-homogenous phase,
and the model is developed based on ping-pong bibi mechanism.
194
Two possible reaction mechanisms have been proposed for oil-tobiodiesel conversion. One refers to the direct transesterication of
glyceride with alcohol to form biodiesel, and the other refers to
hydrolysis of the ester bond to release fatty acid moiety followed
by an esterication with the alcohols. Both hydrolysis and transestercation take place simultaneously during IL-catalyzed biodiesel production; however, the rate constants for alcoholysis are
much higher than those for the hydrolysis reaction. Acyl migration
has been often observed during enzymatic conversion of triglyceride, and thus it should be considered for a comprehensive
kinetic study.
Process simulation based on experimental results can obtain
general mass and energy balances for economic evaluation.
This can be done by using simulation tools such as ASPEN PLUS.
However, in order to obtain a relatively accurate simulation
results, the thermodynamic properties of the reactants and products should be well established. By process simulation, unit
operation can be optimized to minimize consumptions of water
and energy.
Biodiesel needs to be puried in order to meet corresponding
standard. Compared with those of alkali or acid-catalyzed processes, the purication of biodiesel produced by IL-catalysis is less
complex. The impurities contained in the crude biodiesel include
glycerol, free fatty acids (FFA), water, alcohol and glycerides.
Generally, these impurities can be respectively removed by phase
separation, water washing, evaporation and distillation. Adsorption and membrane separation also may be used for biodiesel
purication.
Techno-economic evaluation is important for IL-catalyzed production of biodiesel. Production cost can be estimated and the unit
with the highest cost can be determined by this evaluation. Lipase
cost contributes a great part of the total production cost. This part
of expenditure can be decreased by reducing lipase loading
(increasing lipase specic activity) or increasing reusability of IL.
However, to further reduce production cost, the whole process
optimization with consideration of water and heat integrations
should be performed.
For future works, it is recommended that the following aspects
should be considered to improve the economic competiveness of
IL-catalyzed production of biodiesel.
(1) Increase the stability of ILs during trasesterciation: This can be
done by preventing lipase from leaching off the carriers and
denaturing to loss activity caused by accumulation of alcohol
and glycerol or shearing force of stirring. Increasing the
interaction between lipase and carrier should be performed
but the lipase activity should not be notably decreased.
The multi-step addition of alcohol and simultaneous removal
of glycerol can increase the IL stability, but the process still
needs optimization to make it less complex and more efcient.
To improve the tolerance of IL towards alcohol and glycerol,
biological or chemical modication of the lipase by protein
engineering may be a promising pathway.
(2) Develop novel reactors and gure out the window of operation:
Since mass transfer is important to IL-catalyzed production of
biodiesel, the reactor used for the reaction must have good
mixing; however, it should not cause damage to carriers and
activity loss of lipase. Impeller can be modied to reduce
shearing force in STR, while mass transfer in PBR can be
improved by intensifying radial diffusion. It is recommended
that the window of operation of reactors used for the process
should be gured out to guide the reactor improvement.
(3) Comprehensively and intensively study the kinetics of ILcatalyzed conversion of oil to biodiesel: In order to understand
the mechanisms of oil-to-biodiesel conversion under the
catalysis of ILs, the intermediate products such as fatty acid,
monoglyceride and diglyceride should be determined. For

macro-kinetic studying, transesterication, esterication,
hydrolysis acyl migration and lipase activity loss should be
considered in the models. However, for micro-kinetic studying, mass transfer limitation caused by the internal and
external diffusions should be considered.
(4) Rigorous simulation should be performed to obtain more
accurate data. In most of the reported process simulation
works, the oil feedstocks are simply modeled using glyceryl
trioleate. However, the oil feedstock actually contained fatty
acid chains primarily with 1418 carbon atoms. Therefore,
for accurate simulation, these glycerides should be considered.
The thermodynamic properties of corresponding glycerides,
fatty acids, monoglycerides and diglycerides should be well
estimated or experimentally determined. These properties
parameters are important to obtain an accurate estimation
on energy consumption for biodiesel purication.
(5) Process integration and optimization should be further investigated. Since the total production cost is dependent on the
whole process rather than one or two units, the process
integration with consideration of water and heat recycle
should be conducted. Optimization of the whole process
should be done with the production cost as the nal objective
function. Sensitivity analysis for the main variables such as IL
price, feedstock price, oil-to-biodiesel conversion and solvent
loss (if used) should be performed.
Acknowledgments
Authors are grateful to the nancial support of National Natural
Science Foundation of China (No. 21406130) and Tsinghua Scientic Research Funding (Nos. 2012Z02295 and 2012Z98148) to
this work.
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Lipase-catalyzed process for biodiesel

65 PUBLICATIONS 1,332 CITATIONS

64 PUBLICATIONS 2,423 CITATIONS

Available from: Xuebing Zhao

Renewable and Sustainable Energy Reviews 44 (2015) 182197

Contents lists available at ScienceDirect

Renewable and Sustainable Energy Reviews

Lipase-catalyzed process for biodiesel production: Enzyme

Corresponding author. Tel./fax: 86 10 62772130.

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

Kinetic modeling of IL-catalyzed transesterication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fig. 1. Publications in years of 20002013 found in Web of Science

than chemical catalysts, such as NaOH and H2SO4. Various lipases

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

Fig. 3. Various techniques for enzyme immobilization (adapted from [5,7,12]).

transfer with minimizing mechanic shearing force to damage

Many materials have been explored in literatures to immobilize

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

ionic binding), and hydrophobic interaction [13]. Compared with

pores of supporting material [7]. The hydrophobicity of the carrier

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

decrease the equilibrium adsorption capacity [15,25]. The pH of

2.1.3. Immobilization of lipase by ionic bonding or covalent bonding

of lipase by covalent binding are summarized. Various carriers

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

lipolyticum lipase M37 increased signicantly compared with free

3. Process optimization and reactors for IL-catalyzed

considered remain the same, including acyl acceptor types and

Candida antarctica form B

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

butanol in order to achieve a high reaction rate and biodiesel

speed approach, and the yield reached 97.2% after 5 h reaction.

3.2.2. Packed-bed reactor

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

molar ratio of methanol to oil [73] and solvent dosage if used.

3.2.3. Airlift loop reactor

gasliquid upowthe riser, and a separate channel for the

3.2.4. Other heterogeneous reactors

4. Process simulation and optimization

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

following equation [86,87]:

catalysis. The acyl migration can be described as rst-order

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

triple effect evaporator

the binary NRTL coefcients are not included in the database,

4.2.2. Biodiesel purication

centrifugation are effective for the separation of biodiesel and

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

Glycerol credit included

Enzyme price ($/kg)

Product cost ($/kg)

Calculated according to authors data.

soap, methanol and glycerol effectively, while have no effect on

and operation costs. The overall process also should be further

5. Techno-economic evaluation of IL-catalyzed production of

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

100 $/kg, respectively. Another promising solution is to increase

6. Concluding remark and recommendation for future work

economic evaluations are performed based on process simulation

Lipase-catalyzed production of biodiesel has attracted great

X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182197

monoglyceride and diglyceride should be determined. For