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Lipase-catalyzed process for biodiesel production: Enzyme immobilization,

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 Renewable and Sustainable Energy Reviews 44 (2015) 182–197

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
b
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

art ic l e i nf o a b s t r a c t

Article history: Transesterification of oil feedstocks using immobilized lipase (IL) is a promising process for biodiesel
Received 1 May 2014 production. However, the running cost of this process is still higher than that of conversional chemical-
Received in revised form catalyzed approaches. To address this challenge, both upstream and downstream processes have to be
12 November 2014
optimized. This review provides an overview of recent progresses in improving IL-catalyzed biodiesel
Accepted 12 December 2014
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
Keywords: lipase is a costly process. Most of the commercial ILs are prepared by adsorption of free lipase on
Biodiesel polymeric materials. However, to further reduce cost, works should be focused on developing cheap
Immobilized lipase
carriers and strengthening the interaction between enzyme and carrier but without significant loss of
Transesterification
lipase activity. Running cost of lipase also can be reduced by improving its lifetime during transester-
Process optimization
Techno-economic evaluation ification. 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 effluents. A global process integration and optimization with economic evaluation also should be
figured out to improve the economic feasibility of Il-catalyzed production of biodiesel.
& 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2. Immobilization of lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
2.1. Techniques for immobilization of lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
2.1.1. Overview of lipase immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
2.1.2. Immobilization of lipase by physical adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
2.1.3. Immobilization of lipase by ionic bonding or covalent bonding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
2.1.4. Immobilization of lipase by entrapment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
2.1.5. Immobilization of lipase by cross-linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
2.1.6. Commercialization of immobilized lipase for biodiesel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
3. Process optimization and reactors for IL-catalyzed transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
3.1. Parameter optimization for transesterification process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
3.2. Reactors for IL-catalyzed transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3.2.1. Stirred tank reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3.2.2. Packed-bed reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3.2.3. Airlift loop reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3.2.4. Other heterogeneous reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
4. Process simulation and optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

n
Corresponding author. Tel./fax: þ 86 10 62772130.
nn
Corresponding author. Tel./fax: þ 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.

http://dx.doi.org/10.1016/j.rser.2014.12.021
1364-0321/& 2014 Elsevier Ltd. All rights reserved.
 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197 183

4.1. Kinetic modeling of IL-catalyzed transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

4.2. Process simulation and product purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
4.2.1. Process simulation and unit optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
4.2.2. Biodiesel purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
5. Techno-economic evaluation of IL-catalyzed production of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
6. Concluding remark and recommendation for future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

1. Introduction
Lipase-catalyzed transesterification 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 scientific publications
including articles, review papers, book chapters, patents and
conference abstracts have been published (Fig. 1). However, con-
ventional biodiesel plants adopt transesterification processes
using chemical catalysts such as alkalis and acids. Only a few
plants have employed enzymatic process for industrial produc-
tions 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 2000–2013 found in Web of Science
the term “biodiesel and lipase” separated by document types.
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 appli-
cation 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 transesterification,
strategies can be made in up-, mid- and down-stream processings
(Fig. 2). Specifically, 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 intensification 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
transesterification. At this point, reaction media, operation para-
meters 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 introdu-
cing 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-efficiency reactor for IL-catalyzed production
of biodiesel goes slowly. Commonly used reactors are stirred tank
TM
database by 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 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197
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 transesterification
reaction, and lack of recently reported data. In this article, we
have comprehensively reviewed the mid-and down-stream pro-
cessing 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 first immobilized enzyme was reported by Nilson and
Grifflin more than a century ago [4], while Chibata and co-workers
developed the first industrial immobilized enzyme, i.e., Aspergillus
oryzae aminoacylase for synthetizing racemic D–L amino acids [5].
In recent years, production of biodiesel using immobilized lipases
has attracted great interest. Significant progresses have been made
on both of the immobilization techniques and process develop-
ment for IL-mediated biodiesel production.
An immobilized enzyme is defined as the enzyme physically
confined to a certain defined 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 effluent problems,
greater pH and thermal stability, and higher tolerance to reactants
and products. However, the current ILs still show several draw-
backs for industrial applications, including: (1) loss of enzymatic
activity during immobilization; (2) high cost of the carriers;
(3) low stability in oil–water 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 transesterification 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 immobiliza-
tion (Fig. 3) as intensively reviewed in some recently published
papers [2,12]. Generally, these techniques can be classified into
three types: carrier bonding, cross-linking and entrapment.
Depending on the type of interactions between enzymes and
carriers, these techniques can be further classified into irreversible
and reversible immobilization techniques [5]. In irreversible
immobilization, once enzymes are attached to supporting materi-
als, 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 immobi-
lization of lipases. Physical adsorption and various non-covalent
bondings, such as affinity bonding and chelation bonding, are
well-known reversible immobilization procedures. Each immobi-
lization technique has its own merits and inevitably some dis-
advantages 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 immobiliza-
tion, including non-specific physical adsorption, bio-specific
adsorption, affinity adsorption, electrostatic interaction (also
 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197 185

Table 1
Some reported immobilizations of lipase by different techniques for biodiesel preparation in recent years.

Carrier Lipase source Immobilization techniques References

– Penicillium expansum Cross-linking [38]

Accurel EP-100 Pseudomonas sp. Physical adsorption [90]
Activated carbon Candida antarctica Physical adsorption [108]
Aldehyderesin Thermomyces lanuginosus Covalent bonding [109]
Carbon cloth Pseudomonas cepacia Physical adsorption [108]
Celite Yarrowia lipolytica Physical adsorption [111]
Celite supported sol-gel Candida antarctica Entrapment [36]
Ceramics Pseudomonas cepacia Physical adsorption [110]
Chitosan Candida rugosa Covalent bonding [112]
Epoxy–SiO2–PVA Penicillium camembertii Covalent bonding [31]
Hydrophilic resins Rhizomucor miehei Physical adsorption [113]
Hydrotalcite Thermomyces lanuginosus Physical adsorption [114]
Lewatit Thermomyces lanuginosus Covalent bonding [115]
Magnetic particles Candida rugosa Covalent bonding [82]
Thermomyces lanuginosus Covalent bonding [116]
MANAE-agarose Penicillium camembertii Ionic adsorption [31]
MCM-41 materials Candida rugosa Physical adsorption [117]
Mg-Al hydrotalcites Saccharomyces cerevisiae Physical adsorption [118]
Nb2O5 and SiO2-PVA Burkholderia cepacia Covalent bonding [119]
Olive pomace Thermomyces lanuginosus Covalent bonding [120]
Organosilicate Pseudomonas fluorescens Physical adsorption [121]
Poly (methacrylate-co-divinyl benzene) Staphylococcus haemolyticus Physical adsorption /entrapment [40]
Polyacrylic bead Steapsin lipase Covalent bonding [53]
Polyacrylonitrile Pseudomonas cepacia Physical adsorption [122]
Polymethacrylate Pseudomonas fluorescens Physical adsorption [123]
Polypropylene Candida rugosa Physical adsorption [121]
Pseudomonas fluorescens Physical adsorption [121,123]
Polystyrene Pseudomonas cepacia Physical adsorption [124]
Polyurethane foam Thermomyces lanuginosus Covalent bonding [125]
Pretreated textile Candida sp. Physical adsorption [126]
PVA/chitosan film Lipolases (Aspergillus oryzae) Entrapment [127]
Resin D4020 Penicillium expansum Physical adsorption [128]
Silica Pseudomonas fluorescens Physical adsorption [123]
Burkholderia cepacia Entrapment [129]
Silica aerogel Candida antarctica Entrapment [35]
Silica gel Rhizopus orizae and Candida rugosa Covalent bonding [130]
Enterobacter aerogenes Covalent bonding [131]
Styrene–divinylbenzene copolymer Thermomyces lanuginosus Covalent bonding [132]
Zeolites Thermomyces lanuginosus Physical adsorption [114]
κ-carrageenan Candida antarctica Entrapment [33]

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 addi-
tives 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-specific 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, polyacry-
late etc. These polymers are cheap to obtain. One of the most
common commercial ILs, Novozyms 435, is prepared by immo-
bilizing lipases on acrylic resin by adsorption [14]. This IL has been
widely tested to catalyze the transesterification of various vege-
table oil feedstocks, animal fats, yeast lipids and algae oils [15–20].
Both carrier properties and immobilization conditions have sig-
nificant influences on the immobilization efficiency (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 significant influence 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 immobiliza-
tion [21]. However, the hydrophobicity of carrier is not an indepen-
dent parameter dominating lipase immobilization efficiency.
Polarity also showed important impact on the microenvironment
of lipase after immobilization. It is reported that native and hydro-
phobic lipase derivatives showed higher specific activities when
immobilized on polar polymers compared with non-polar polymers
[22]. More specifically, the carriers with well established hydro-
phobicity 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 lyophiliza-
tion, 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 immobi-
lization efficiency. The recent work by Zhao et al. [15] has shown that
the kinetics of lipase adsorption on cross-linked polystyrene resin
beads can be significantly affected by resin-to-lipase ratios. Increase
of resin-to-lipase ratio can enhance the adsorption rate constant but
186 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197
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 transesterification process [6]. Increasing the interaction
between lipase and carrier without decreasing enzyme activity is
important for further improvement of this immobilization techni-
que. Generally, the interaction can be intensified 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 monomethoxypolyethy-
lene glycol, acetyl-dehyde, dodecyl-dehyde, methyl acetonimidate,
methyl 4-phenylbutyrimidate on lipase molecule, the hydrophobi-
city of lipase can be increased, and thus the modified 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 proce-
dure. 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 transesterification 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 strate-
gies, 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 pre-
paration 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 κ-carrageenan with an encapsulation
efficiency 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 transesterification 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 multi-
functional 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 fermenta-
tion broth and recovered as cross-linked enzyme aggregates
(CLEAs). The formed CLEAs demonstrate significantly 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
 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197
lipolyticum lipase M37 increased significantly 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 cross-
linking reagents that can change the conformation of lipases and
potentially lead to significant loss of activity. Other disadvantages
associated with cross-linking immobilization are low immobiliza-
tion 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 immobiliza-
tion 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 $1000/kg [8]. To reduce cost, the carrier must be
easy to synthesize or commercially available at low prices. The
immobilization process should be efficient for recovering proteins
and retaining their enzymatic activities. The ILs should have high
stability to avoid enzyme leaching or activity loss. Some commercial
ILs are listed in Table 2. Among these commercial ILs, Novozyms
435 is the most widely-reported in literatures for biodiesel produc-
tion. In 2006, the first industrial plant for enzymatic production of
biodiesel was built in China, with a capacity of 20,000 t/yr. Lipo-
zymes TL IM and Novozyms 435 were both used in this plant. tert-
butanol was selected as the reaction medium. The highest biodiesel
yield of the plant can reach  95% using rapeseed oil as feedstock [1].
Another biodiesel production line was established in 2007 with a
capacity of 10,000 t/yr. In this process, lipase of Candida sp. 99–125
immobilized on textile membranes was used as catalyst. The cost of
lipase was estimated to only  32$/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
transesterification
3.1. Parameter optimization for transesterification process
Parameter optimizations for IL-catalyzed transesterification of oil
feedstocks have been extensively studied in recent years. Hundreds of
papers have been published on optimization of parameters for
transesterification process with various lipases. Although the optimal
operation conditions vary depending the type of lipases and immo-
bilization techniques, the type of parameters that needs to be
Table 2
Some commercial immobilized lipases used for biodiesel production [2].
Commercial name Enzyme origin Support
Novozyms 435 Candida antarctica form B Lewatit VP OC 1600
Lipozymes RM IM Rhizomucor miehei Duolite A568
Lipozymes TL IM Thermomyces lanuginosa Silica granules
Lipase PS Amano IM Burkholderia cepacia Diatomaceous earth
– Candida sp. 99–125 Textile membrane
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 final 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 transester-
ification is a reversible reaction. However, high concentrations of
alcohol (especially methanol) significantly reduce the activity of
lipases, and stepwise addition of methanol is, therefore, performed
to minimize the deactivation of lipase [2].
The transesterification reaction can be performed in either
organic solvent or solvent-free systems. Because oil and metha-
nol is not complete miscible in a solvent-free system, the water
content of the system can significantly 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 oil–water 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 trans-
esterification 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 solvent-
free 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 sol-
vents periodically [48]. However, these strategies cannot be
easily adapted to large scale operations. Alternatively, introdu-
cing organic solvents to the reaction can covert the liquid–liquid
bi-phase into a homogenous phase. Hydrophobic organic sol-
vents such as n-hexane and petroleum ether [54], and tert-
butanol, have been used as reaction media for IL-catalyzed
transesterification. For example, the stability of Novozyms 435
in tert-butanol was greatly enhanced [55–57]. With the intro-
duction 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-
Hydrophobicity/philicity Producer or inventor
Medium hydrophobic Novozymes (Denmark)
Hydrophilic Novozymes (Denmark)
Hydrophilic Novozymes (Denmark)
Hydrophilic Amano (Japan)
Hydrophobic Beijing University of Chemical Technology (China)
188 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197
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 tert-
butanol 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 transesterification
Most of the IL-catalyzed biodiesel productions in lab are
performed in shaking flasks, 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], fluidized 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 sunflower 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
transesterification 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 five 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 continu-
ously for over 70 days with regeneration of the lipase by tert-
butanol 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 transesterification using two CSTRs in series, with a
bottom sintered glass filter 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 flow 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 produc-
tivity 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 transesterification 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 immobi-
lized 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 con-
straints for these variables and their correlation. As firstly pro-
posed 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 reac-
tants [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 para-
meters that should be considered for optimizing biodiesel produc-
tion with PBR mainly include the flow rate, reaction temperature,
 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197
molar ratio of methanol to oil [73] and solvent dosage if used.
Flow rate is the most important operational variable. A low flow
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 3–7 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
efficiency [2]. To solve this issue, process with stepwise methano-
lysis 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 Séverac et al. [76],
when Novozyms 435 was used for conversion of crude high-oleic
sunflower 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 transesterification
were in the range of 70–96% 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 flow
velocity or obstruction of the catalyst bed by accumulation of
insoluble components from the reaction mixture [7]. In solvent-
free system, pressure drop becomes more significant 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 flow 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 flow pattern inside PBR because of the limited
flow velocity relative to the pressure drop allowance [7]. According
to the study of Halim et al. [62], flow velocity significantly 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
influence on the apparent kinetics of biodiesel formation in PBR.
As analyzed by Fjerbaek et al. [78], the effectiveness factor, η,
defined 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 intensification 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).
Superficial 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 sacrifice 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
transesterification of oils in solvent system. ALR has a channel for
189
gas–liquid upflow—the riser, and a separate channel for the
downflow [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 liquid–solid (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 intensified. 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 transesterification of oil feedstocks. A biodiesel yield
as high as 98.95% can be obtained by transesterification of babassu
oil with ethanol under the catalysis of Novozyms 435 in a
continuous fluidized bed reactor (FBR) combined with a packed
column to remove glycerol [81]. Ricca et al. [63] employed FBR for
continuous transesterification 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) defined as
the ratio of fresh feed flow rate (F) and the recycle rate (R) had an
important influence on the productivity and oil conversion.
Increasing R may cause more dilution of the stream flowing 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 considera-
tion of productivity, biodiesel yield and catalyst lifetime. Com-
pared 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 fluidization may cause
damage of carrier as well as leaching of lipase from the carriers.
Productivity and lifetime of ILs can be improved through
modification 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 modifications also increase the complexity of
equipments as well as operation.
4. Process simulation and optimization
4.1. Kinetic modeling of IL-catalyzed transesterification
Understanding the kinetics of IL-catalyzed transesterification 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 etherification processes [85], pseudo-homogenous
transesterification is usually modeled by ping-pong bi–bi mechan-
ism, by which a product is released before the addition of the other
substrate [86–89]. For the simplest mechanisms in which the oil
transesterification is considered to consist of three consecutive
reversible reactions, the initial reaction rate can be described as the
190 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197
CH3OH CH3OOCR
CH2OOCR CH2OH
k1
CHOOCR CHOOCR
k2
CH2OOCR CH2OOCR
+ k5
H2O
CH2OOCR
k13 CHOH
k14
CH2OOCR
Glycerol
k15
RCOOH + CH3OH
k16
Fig. 5. A comprehensive kinetic model for lipase-mediated methanolysis of glyceride for formation of biodiesel and glycerol with consideration of esterification,
transesterification and acyl migration (adapted from with permission [93]).
following equation [86,87]:
V max ½TG½A
vi ¼
K m;TG ½Að1 þ ½A=K i;A Þ þ K m;A ½TG þ ½TG½A
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 fitted 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 transesterification of high oleic
sunflower 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 μmole
min  1 g  1, 5.3 mM, 13 mM and 55 mM, respectively. According to the
study of Xu et al. [87], when refined soybean oil was inter-esterified by
Novozyms 435 with methyl acetate as the acyl acceptor in solvent-
free system, the kinetic constants were determined as Vmax ¼ 1.9
mol min  1 L  1; Km,TG ¼ 1 mol/L; Km,A ¼16 mol/L, and Ki,A ¼ 0.0455
mol/L, and the first step reaction, triacylglyceride to diacylglyceride
was the rate-limiting step for the overall reaction. Nevertheless, Eq. (1)
is just a simplified 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 esterification
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 transesterification mechanisms on the kinetic modeling of
biodiesel production by immobilized lipase [90]. They found that
hydrolysis and transesterfication occur simultaneously, and the reac-
tion mechanism regarding to stepwise hydrolysis followed by ether-
ification 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
transesterification, hydrolysis, esterification, as well as acyl migration
[91]. Acyl migration has been often observed during enzymatic
conversion of triglycerides to biodiesel using 1,3-positional specific
lipase. The acyl migration can happen between 1,2-diglyceride (1,2-
DG) and 1,3-diglyceride (1,3-DG), or 2-monoglyceride (2-MG) and 1-
monoglyceride (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
CH3OH CH3OOCR
CH2OH
k3
CHOOCR
k4
CH2OH
k6 k7 k8
CH3OH CH3OOCR CH3OH CH3OOCR
k9 CH2OH k11 CH2OH
k10 CHOH k12 CHOH
CH2OOCR CH2OH
CH3OOCR + H2O
catalysis. The acyl migration can be described as first-order
reversible reactions, and temperature and water activity were
ð1Þ two crucial factors influencing acyl migration kinetics [92].
However, the mechanism of acyl migration still needs further
investigation. Particularly, the thermodynamic and kinetic driv-
ing forces of this phenomenon should be figured out. Moreover,
it seems that the “apparent” kinetics of lipase-catalyzed produc-
tion 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 transesterification is the negli-
gence 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 film 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 purification
4.2.1. Process simulation and unit optimization
Process simulation can obtain general mass and energy bal-
ances, 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 transesterification [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 40–80% 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 transesterification reactions
occur in a highly non-ideal chemical system. However, sometimes
 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197
P-101
101-A
Crude Oil 101 M-101
P-102
102-A
Tert-Butanol 102
P-103
103-A
103
Methanol
303-A
P-302
GAS 501
T-501
FAME 502
P-401
403
401
Waste Oil 503
Glycerol 402
Fig. 6. A process simulation for IL-catalyzed production of biodiesel with ASPEN PLUS [98].
the binary NRTL coefficients are not included in the database,
which should be estimated by corresponding thermody-
namic methods. For example, Zheng [98] estimated the binary
NRTL coefficients 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 opti-
mized, 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 recom-
mended for the purification 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 cata-
lysts, production capacity is very important. Too small scale usually is
not economically feasible.
4.2.2. Biodiesel purification
Biodiesel products need purification 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 first removed out after
transesterification. 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
191
P-201 M-102
R-101 P-203
201-A
P-104
105
203
104
201
202-A
202
Sumpmentary Methanol
P-202
M-103
303 CDS2 204
CDS3
CDS4 CDS1 STEAM
H-201 QB3 QB2 QB1
S-401
P-301 EA3 EA2 EA1
Q1
302
301
OUT3 OUT2 OUT1 R-201
triple effect evaporator
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
rectification. Compared with those of alkali or acid-catalyzed
processes, the purification of biodiesel produced by IL-catalysis is
less complex. As reviewed by Atadashi et al. the catalyst used in
transesterification, oil to alcohol ratio, water and free fatty acids
contents are found to have significant effects on the complexity of
downstream processing and purification [100]. Conventionally,
biodiesel is purified by washing with distilled water. For the
alkali or acid-catalyzed transesterification, a large amount of
water is usually consumed in order to remove soap and other
contaminants, and reduce the alkaline metal (Na, K) concentra-
tions less than 5 ppm or acid value less than 0.5 mg KOH/g.
However, for IL-catalyzed transesterification these requirements
are easy to achieve since no alkaline metals and mineral acid are
introduced into the reaction system. Nevertheless, another pro-
blem 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 significant emulsion
of the system, resulting to the difficult of phase separation.
Therefore, FFA should be removed by any chemical or enzymatic
treatment. Preferably, FFA can be converted to biodiesel by
esterification with alcohols. This treatment not only increases
the conversion of oil feedstock to biodiesel, but also reduces the
acid value of the product.
Another purification 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 purified form can be obtained
by separation of mixture with a drum filter or related filtration
techniques [102]. When ion-exchange resins are used, the process
is usually conducted by a packing bed column filled with the
resins, where the crude biodiesel passes through resin bed in a
continuous flow until the impurities are removed. According to the
results of Berrios et al. adsorption and resin treatment can remove
192 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197

Table 3
Biodiesel standard in different countries or area.

Europe Germany USA China

Specification EN 14214:2003 DIN V 51606 ASTM D 6751-12 GB/T20828-2007

Density 15 1C g/cm³ 0.86–0.90 0.875–0.90 0.82–0.90
Viscosity 40 1C mm²/s 3.5–5.0 3.5–5.0 1.9–6.0 1.9–6.0
Distillation %@1C 90%, r 360 1C 90%, r 360 1C
Flashpoint (Fp) 1C Z 120 Z 110 Z 130 Z 130
n
CFPP 1C country specific Summer 0, spr/aut  10, winter  20 Report
n
Cloud point 1C Report
Sulfur mg/kg r 10 r 10 r 15 r 50
CCR 100% %mass r 0.05 r 0.05
Carbon residue (10%dist.residue) %mass r 0.3 r 0.3 r 0.05 r 0.3
Sulphated ash %mass r 0.02 r 0.03 r 0.02 r 0.02
Water mg/kg r 500 r 300 r 500 r 500
Total contamination mg/kg r 24 r 20 Trace
Cu corrosion max 3 h/50 1C No. 1 No. 1 No. 3 No. 1
Oxidation stability hrs;110 1C Z6 Z3 Z6
Cetane number r 51 Z 49 Z 47 Z 49
Acid value mgKOH/g r 0.5 r 0.5 r 0.5 r 0.5
Methanol %mass r 0.20 r 0.3 r 0.2 or FpZ 130 1C
Ester content %mass Z 96.5
Monoglyceride %mass r 0.8 r 0.8 r 0.4
Diglyceride %mass r 0.2 r 0.4
Triglyceride %mass r 0.2 r 0.4
Free glycerol %mass r 0.02 r 0.02 r 0.02 r 0.02
Total glycerol %mass r 0.25 r 0.25 r 0.24 r 0.24
Iodine value r 120 r 115
Linolenic acid ME %mass r 12
C(x:4) & greater unsaturated esters %mass r1
Phosphorus mg/kg r 10 r 10 r 10
Alkalinity mg/kg r5
Gp I metals (Na, K) mg/kg r5 r5
GpII metals (Ca, Mg) mg/kg 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 Solvent-free Rapeseed oil Yes 1000 1.95 [97]

200,000 Solvent-free Rapeseed oil Yes 1000 0.96 [97]
8000 tert-butanol Rapeseed oil Yes 1000 3.12 [97]
200,000 tert-butanol Rapeseed oil Yes 1000 2.23 [97]
8000 Solvent-free Rapeseed oil Yes 10 0.98 [97]
200,000 Solvent-free Rapeseed oil Yes 10 0.065 [97]
8000 tert-butanol Rapeseed oil Yes 10 2.87 [97]
200,000 tert-butanol Rapeseed oil Yes 10 1.97 [97]
1000 Solvent-free Palm oil Yes 1200 2.414 [105]
 8500a tert-butanol Waste oil Yes 2000 $/klU  0.9b [133]
8000 Supercritical CO2 Waste cooking sunflower oil Yes 800€/kg 1.64€/L [134]
8000 Supercritical CO2 Waste cooking sunflower oil Yes 8€/kg 0.75€/L [134]

a
Calculated according to authors’ data.
b
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 purification. This
process exhibits several advantages over the conventional ones,
such as minimization of capital cost and other related production
costs, and high specific area of mass transfer [100]. Membrane
separation can be conducted simultaneously with the transester-
ification using membrane reactors [84,103], or after the transes-
terification process [104]. It has been found that membrane
separation was able to reduce the amount of soap in crude
biodiesel, and ultrafiltration membrane of 10 kDa could reduce
glycerol effectively to be less than 0.02 wt% [104]. Although these
purification processes are considered promising on industrial
scale, the process still needs optimization with systematic con-
sideration 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 pro-
duction 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 flow sheets, time
charts, equipment lists followed by estimations of equipment cost,
plant cost and manufacturing cost [104]. Most of the techno-
 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197
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 flowsheet, 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 expres-
sion:
 
New size n
New cost ¼ ðBase costÞ ð2Þ
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 0.6–0.7. However
in cases of process plants where capacity is increased by duplicat-
ing 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 techno-
economic 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 conver-
sion ratio, retention time for transesterification, 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 significant influence 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
193
100 $/kg, respectively. Another promising solution is to increase
the specific activity of IL to decrease enzyme loading used for
transesterification, which can be done by lipase modification 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
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 immobiliza-
tion is usually performed by a combination of two or more of these
approaches. Most of the commercial ILs are prepared by adsorp-
tion 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 bio-
diesel 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 conver-
sion 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 IL-
catalyzed transesterification. 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 bi–bi mechanism.
194 X. Zhao et al. / Renewable and Sustainable Energy Reviews 44 (2015) 182–197
Two possible reaction mechanisms have been proposed for oil-to-
biodiesel conversion. One refers to the direct transesterification 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 esterification with the alcohols. Both hydrolysis and trans-
esterfication take place simultaneously during IL-catalyzed bio-
diesel 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 triglycer-
ide, 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 pro-
ducts should be well established. By process simulation, unit
operation can be optimized to minimize consumptions of water
and energy.
Biodiesel needs to be purified in order to meet corresponding
standard. Compared with those of alkali or acid-catalyzed pro-
cesses, the purification 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. Adsorp-
tion and membrane separation also may be used for biodiesel
purification.
Techno-economic evaluation is important for IL-catalyzed pro-
duction 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 specific 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 trasesterficiation: 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 efficient.
To improve the tolerance of IL towards alcohol and glycerol,
biological or chemical modification of the lipase by protein
engineering may be a promising pathway.
(2) Develop novel reactors and figure 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 modified 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 figured out to guide the reactor improvement.
(3) Comprehensively and intensively study the kinetics of IL-
catalyzed 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, transesterification, esterification,
hydrolysis acyl migration and lipase activity loss should be
considered in the models. However, for micro-kinetic study-
ing, 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 14–18 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 purification.
(5) Process integration and optimization should be further inves-
tigated. 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 final 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 financial support of National Natural
Science Foundation of China (No. 21406130) and Tsinghua Scien-
tific Research Funding (Nos. 2012Z02295 and 2012Z98148) to
this work.
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