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Chapter 21

Lipases as Biocatalyst for Biodiesel Production

Marcos Vargas, Xochitl Niehus, Leticia Casas-Godoy,
and Georgina Sandoval

Global shortages of fossil fuels, significant rise in the price of crude oil, and increased environmental
concerns have stimulated the rapid growth of biodiesel production. Biodiesel is generally produced through
transesterification reaction catalyzed either chemically or enzymatically. Enzymatic transesterification is of
interest since it shows advantages over the chemical process and, in addition, is considered a “green”
process. This chapter reviews the current status of biodiesel production with a lipase biocatalysis approach,
including sources of lipases, kinetics, lipase immobilization techniques, and lipase reaction mechanism for
biodiesel production. Factors affecting biodiesel production and the economic feasibility of lipase biodiesel
production are also covered.

Key words Biodiesel, Oils, Transesterification, Esterification, Biocatalysis, Lipase

1 Introduction

Depletion of resources and rise in world population, as well as

environmental pollution, have led to a worldwide environmental
and energetic crisis. The increased demand for crude oil and limited
resources of fossil oil have stimulated the search of alternative fuels
from renewable sources that are environmentally more acceptable.
In the last few years, biodiesel has gained a lot of attention because
it is one of the most promising renewable energies to replace fossil
diesel. Its use reduces CO and polycyclic aromatic hydrocarbon
emissions and has also been favored by the legislation and mandates
in several countries in the last years [1].
As a renewable, biodegradable, and nontoxic fuel, biodiesel can
be derived from vegetable oils, animal fats, or microbial oils [2]
through transesterification or esterification. Both reactions are with
an alcohol (methanol is the most common) in the presence of a
catalyst which could be basic (generally, NaOH), acid (usually, HCl
or H2SO4), or enzymatic [1, 3].

Georgina Sandoval (ed.), Lipases and Phospholipases: Methods and Protocols, Methods in Molecular Biology, vol. 1835,, © Springer Science+Business Media, LLC, part of Springer Nature 2018

378 Marcos Vargas et al.

Fig. 1 Transesterification general reaction

In the transesterification reaction, the ester group from the

triglycerides is detached to form three alkyl ester molecules
(Fig. 1) [1]. This reaction is normally modeled as a three-step
process. In the first step, triglycerides are converted to diglycerides
and alkyl esters, which in turn are converted to monoglycerides and
alkyl esters in the second step. In the third and final step, mono-
glycerides are converted to glycerol and alkyl ester. In each step,
one molecule of alkyl ester is formed for every molecule of glyceride
reacted [1, 4].
Degraded or reused oils contain great quantities of free fatty
acids (FFA), which can also be converted into biodiesel by lipases
via esterification reaction (Fig. 2) [1, 4].
Virgin oils commonly used for biodiesel production include
soybean [5], canola [6], sunflower [7, 8], corn [9], cottonseed
[10], rapeseed [11], and palm oil [12]. High biodiesel yield can
be easily achieved using vegetable oils as feedstock due to their low
free fatty acid content and high quality. However, the high cost of
these oils makes biodiesel production from these sources less com-
petitive than petro-diesel, since these feedstocks account for about
60–88% of the overall biodiesel production costs [1, 13]. Moreover,
the rapid expansion of biofuel production from edible oils has
raised serious concerns on preserving the food security of the planet
and has caused a food vs. fuel debate. Therefore, many researchers
have focused on the use of low-cost feedstock for biodiesel produc-
tion, such as yellow grease, brown grease [14], and nonedible oils,
including jatropha (Jatropha curcas) [15], karanja (Pongamia pin-
nata) [16], mahua (Madhuca indica) [17], linseed (Linum usita-
tissimum) [18], rubber seed [19], castor [20, 21], and rice bran
oil [22].
Microorganisms, such as algae, yeast, bacteria, and fungi, have
the ability to accumulate oils under specific cultivation conditions
[23]. Those that accumulate more than 20% of their dry weight
mass as lipids are considered to be oleaginous microorganisms
[23]. Compared to plant oils, microbial oils have many advantages,
such as short life cycle, less process steps required, not affected by
season or climate, and easier to scale up. Although currently there is
no large-scale production process, microbial oils might become one
of the potential oil feedstocks for biodiesel production.
Lipases as Biocatalyst for Biodiesel Production 379

Fig. 2 Esterification general reaction

It should be pointed out that most used vegetable oils contain

high levels of FFA. Therefore, base-catalyzed transesterification is
not feasible, since homogeneous alkali can react with FFA to form
unwanted soap and water, which would affect final biodiesel quality
and require extra investment for downstream separation and purifi-
cation. Acid-catalyzed esterification is preferred over base-catalyzed
transesterification to process high FFA feedstock due to its ability to
convert FFA into biodiesel. However, excess of alcohol is needed to
reach high biodiesel yield, and if sulfuric acid is used, it is difficult to
recover after reaction; therefore, it produces toxic wastewater and
high residual sulfur content in biodiesel.
Contrary to alkaline catalysts, lipase-catalyzed transesterifica-
tion process does not form soaps and can esterify both FFA and
triglycerides in one step without the need of a subsequent wash-
ing step. This is an added advantage when considering the use of
low-cost feedstocks, such as tallow and recycled restaurant grease,
with high FFA to improve the sustainability of biodiesel produc-
tion. Currently, the application of lipases as biocatalyst for bio-
diesel production is a worldwide growing area of interest.
Compared to alkaline or acid catalysts, lipases show the following
1. Able to produce biodiesel in fewer process steps with a drasti-
cally reduced amount of wastewater
2. Able to improve product separation and generate a higher-
quality glycerol
3. Able to process low-quality (with high FFA) feedstock without
yield loss
4. Are highly selective and can act at lower temperatures and
normal pressure conditions
This chapter presents a detailed review of the use of lipases for
biodiesel production, including sources of lipases, kinetics, lipase
immobilization techniques, and lipase reaction mechanism for bio-
diesel production. Factors affecting lipase-catalyzed biodiesel pro-
duction and the economic feasibility of lipase biodiesel production
are also discussed.
380 Marcos Vargas et al.

2 Sources of Lipases

Lipases (EC, triacylglycerol acylhydrolases, are widely

employed to catalyze hydrolysis, esterification, and transesterifica-
tion of carboxylic esters. Lipases can be found in all living organisms
and can be produced intracellularly and extracellularly. Lipases can
be obtained from plants (papaya latex, oat seed lipase, and castor
seed lipase), microorganisms (bacteria and fungi), and animals
(pancreatic lipases). The ideal lipases for biodiesel production
should demonstrate the following characteristics:
non-stereospecific so that all tri-, di-, and monoglycerides can be
converted to biodiesel, capable of catalyzing the esterification of
FFA, easily produced, temperature and alcohol tolerant, reusable,
low reaction time, low product inhibition, and high biodiesel yield.
Many researchers have studied various lipases from different
sources for their transesterification activity on different feedstocks.
Table 1 provides an overview of these results for enzymatic biodie-
sel production. Data in Table 1 shows that the reaction time varies
greatly from 8 to 90 h and reaction temperature range is between
25 and 50  C. In general, most lipases can achieve >90% biodiesel
yield. For industrial biodiesel production, some extensively studied
lipases are Candida antarctica lipase B immobilized on acrylic resin
and Candida sp. 99–125 lipase-immobilized on inexpensive textile
membranes [24]. These lipases are used due to their relatively high
stability and activity.

3 Kinetics and Reaction Mechanism of Biodiesel Production Using Lipases

A two-step mechanism is widely accepted to explain lipase transes-

terification of triglycerides (oils) with an alcohol. The first step is
the hydrolysis of the ester bond and release of the alcohol moiety
followed by an esterification with the second substrate [1, 7, 25].
This mechanism conforms with a Ping-Pong Bi-Bi mechanism
(Fig. 3, where E, enzyme; S and T, substrates; P and Q, products;
Esc and EscT, intermediate complex of enzyme and substrate; ET,
inactive complex enzyme-substrate T) since each product is
released between additions of the substrates, especially considering
lipase-catalyzed esterifications of long-chain fatty acids [1, 4, 7, 25,
26]. The mechanism (Fig. 3) consists of the following steps:
(a) nucleophilic addition to form enzyme-substrate complex,
where the nucleophile is the oxygen in the O–H group on the
enzyme. (b) A proton is transferred from the conjugate acid of
the amine to the alkyl oxygen atom of the substrate, and a glycerol
moiety is formed. If a triglyceride is the initial substrate, then a
diglyceride would be formed, whereas if diglyceride is the substrate,
then monoglyceride would be formed and so on. (c) The oxygen
Table 1
Various lipases for biodiesel production

Immobilization Yield
Source of lipases Source of oil Alcohol medium (%) Other conditions References

Burkholderia cepacia Jatropha curcas Methanol Glutaraldehyde, 100 35 C, 24 h, 1:10 mol ratio of oil to ethanol, [32]
L. oil sodium alginate, 1 g water, 5.25 g immobilized lipase
and κ-carrageenan
Rhizopus oryzae Jatropha curcas Methanol Lewatit VP OC 1600 96.7a 30  C, 4 h, 4% (w/w) water content in the [15]
L. oil reaction medium, substrate molar ratio
Rhizopus oryzae Jatropha curcas Methanol Lifetech™ 97.1a (methanol/jatropha oil) of 3:1, and
L. oil ECR1030M 7 methanol additions. A load of 5% (w/w)
Rhizopus oryzae Jatropha curcas Methanol Lifetech™ 90.6a of biocatalyst in relation to the amount of
L. oil ECR1090M jatropha oil (10 g)
Rhizopus oryzae Jatropha curcas Methanol Lifetech™ 91.3a
L. oil ECR8285M
Rhizopus oryzae Jatropha curcas Methanol Amberlite IRA-96 95.7a
L. oil
Carica papaya Jatropha curcas Methanol Lewatit VP OC 1600 77.5a
L. oil
Thermomyces lanuginosus Rubber seed Methanol – 92.83 37  C, 24 h, 170 rpm, 6:1 methanol-to-oil [56]
oil molar ratio with three-step addition
Rhizopus oryzae Rubber seed Methanol – 33.3 24 h, 1:11 Mol ratio of ethyl acetate. And [19]
oil Ethyl catalyst concentration 10 (w/w) % of oil.
acetate In the case of methanol, molar ratio of 1:4
and catalyst concentration of 15(w/w) % of
oil after 48 hours
Pseudomonas fluorescens Algal oils Methanol Immobead 150 >95 35  C, methanol to oil ratio of 3:1 with 10% [52]
enzyme amount, and 2.5% water content
based on oil weight
Lipases as Biocatalyst for Biodiesel Production

Candida antarctica Algal oils Methanol Acrylic resin 99.1 25  C, 4 h, t-butanol weight ratio of 1:1, an [57]
t-Butanol oil to methanol molar ratio of 1:12

Table 1


Immobilization Yield
Source of lipases Source of oil Alcohol medium (%) Other conditions References

Candida cylindracea Sludge palm oil Ethanol – 57.5 41 C, 250 rpm, 4:1 alcohol-to-SPO molar [54]
Candida sp. 99–125 Jatropha curcas Ethanol Hydrophobic silicate 79 40  C, 24 h, 200 rpm, molar ratio of oleic [58]
L. oil acid to ethanol is 1:2
Marcos Vargas et al.

Rhizomucor miehei mixed Soybean oil Ethanol – 90 30  C, 10 h, 200 rpm, 25 wt% enzyme [55]
with Thermomyces content based on weight of oil, 80% TLL in
lanuginosus (TLL) the mixture, 7.5:1 ethanol-to-oil molar
Rhizopus oryzae mixed with Soybean oil Methanol Silica gel 99 45  C, 21 h, 300 rpm, 4.5:1 methanol-to-oil [53]
Candida rugosa molar ratio, added in 10 steps
The theoretical maximum FAME production is 66.7 (%, w/w) for sn-1,3 regioselective lipases
Lipases as Biocatalyst for Biodiesel Production 383

Fig. 3 Ping-Pong Bi-Bi mechanism with competitive inhibition from the

nucleophile T

atom from a methanol molecule is added to the carbon atom of the

C¼O of the acyl-enzyme intermediate to form acylated enzyme-
alcohol complex. (d) Finally, the enzyme oxygen atom of the
complex is eliminated, and a proton is transferred from the conju-
gate acid of the amine, resulting in fatty acid methyl ester (biodie-
sel). As Al-Zuhair et al. [27] pointed out, this mechanism is based
on the assumption that the first product coming out of the reaction
is biodiesel, followed by the glycerol moiety. In addition, alcohol
and substrate inhibition effects were not taken into consideration.
Therefore, Al-Zuhair et al. [27] modified this mechanism and
developed a sturdy reaction kinetic model which considered alco-
hol, substrate, and product inhibition effects and thus avoided any
unexpected responses. This revised model can successfully deter-
mine the optimal conditions for biodiesel production in their study
using lipase (Rhizomucor miehei)-catalyzed transesterification of
palm oil with methanol in n-hexane organic medium. Calabrò
et al. [28] carried out a theoretical kinetic analysis to identify the
reaction path using the same lipase for ethanolysis of triolein. The
proposed model was successfully validated, which cannot only help
optimize operating conditions but also provide designers with a
reliable tool for reactor sizing. More importantly, the kinetic equa-
tion was proved to be intrinsic, which is crucial in reactors’ perfor-
mance modeling, where kinetics and mass transport within the mass
balance can be taken into account separately. Cheirsilp et al. [29]
proposed three kinetic models for biodiesel production from palm
oil and ethanol with immobilized Pseudomonas sp. lipase. These
models considered the effect of substrates and products during the
entire reaction. The kinetic study showed that the hydrolysis of the
TAG ester bond and esterification of the free fatty acid occurred
simultaneously instead of hydrolysis followed by esterification. Li
et al. [30] used free lipases as catalysts for the reaction between
soybean oil and methanol. As for lipase-catalyzed esterification
[31], the system of Li et al. [30] followed a Ping-Pong Bi-Bi
mechanism with methanol inhibition, showing that the methano-
lysis reaction and the hydrolysis followed by esterification occurred
simultaneously. Furthermore, the enzymatic rate constants showed
that direct transesterification is the preferred pathway for this
384 Marcos Vargas et al.

4 Lipases and their Immobilization Techniques Used for Biodiesel Production

Both free and immobilized lipases show excellent catalytic activity

in nonaqueous media, which enhances the esterification and trans-
esterification process during biodiesel production [32, 33]. How-
ever, for practical and economic reasons, it is advantageous to use
lipase in its immobilized form. Immobilized enzymes are defined as
“enzymes physically confined or localized in a certain defined
region of space with retention of their catalytic activities, and
which can be used repeatedly and continuously” [34]. Enzyme
immobilization has generally been used to obtain reusable enzyme
Immobilized lipases are more stable toward temperature, che-
micals, as well as shear denaturation [35]. Immobilized enzymes
also offer several other advantages such as ease of product separa-
tion and greater control over catalysis and process. However,
immobilization strategies may influence the catalytic and enantio-
selective properties of the enzyme [34]. Therefore, various immo-
bilization strategies may provide immobilized enzymes with
different activity and selectivity characteristics [35, 36]. The selec-
tion of an immobilization strategy should be based upon process
specifications for the catalyst, inactivation and regeneration char-
acteristics, procedure cost, toxicity of immobilization reactants, and
the desired final properties of the immobilized derivative
[32, 37]. Based on chemical retention and physical retention, the
methods for lipase immobilization can be classified into two basic
categories as shown in Fig. 4 [38]. Various methods for lipase
immobilization include adsorption, covalent bonding, entrapment,
encapsulation, and cross-linking. All these methods have been used
to improve lipase stability for biodiesel production.

Fig. 4 Classification of immobilization methods

Lipases as Biocatalyst for Biodiesel Production 385

5 Factors Affecting Biodiesel Production Using Lipases

There are several factors to be considered when producing lipase-

catalyzed biodiesel. These factors depend on each specific lipase and
the feedstock used, but in general the following aspects should be
(a) Specificity for certain triglycerides or fatty acid chain length.
Depending on the feedstock for biodiesel production, differ-
ent lipases are suitable for the process (see Table 1).
(b) Temperature. In general, the optimal temperature can be
expected to increase when using an immobilized lipase
because binding to the carrier material gives stability to the
enzyme and, therefore, decreases the effect of thermal deacti-
vation compared to the free enzyme [39].
(c) Presence of water. Water molecules join to the protein structure
creating hydrogen bonds inside the molecule participating in
the structural integrity, active site polarity, and protein stability
[40]. An excess of water causes inhibition of the lipase in the
transesterification reaction. At high water content, diffusive
limitations of the substrate can occur, and water can promote
the hydrolysis of the substrate, thus decreasing product
yield [41].
(d) Presence or absence of solvent. In free solvent medium, higher-
purity products can be achieved; this also facilitates down-
stream processing since fewer components would be present
in the reaction mixture at the end of the reaction. However,
this medium causes high viscosity which may result in poor
mixing, causing low mass transfer and, hence, slow reaction
rates. In the presence of organic solvents, the water activity is
kept low as same as viscosity, and it decreases the thermody-
namic or kinetic barrier, increasing the reaction ratio [31, 39,
42, 43]. On the other hand, this type of media increases the
production cost due to the solvent and its final recovery. The
most frequently used organic solvents are hexane, cyclohex-
ane, and isooctane [31, 39, 44].
(e) Alcohol concentration. A major part of the studies done on
enzymatic production of biodiesel from vegetable oils involve
the effect of alcohol. At constant substrate concentration (oils
in this case), high alcohol concentration inhibits the lipases
[41]. Therefore, it is of great relevance to determine the
optimal oil-alcohol molar ratio. Particular enzyme kinetics
and thermodynamic effects of the alcohol concentration
must be also considered [43, 45, 46].
(f) Type of alcohol. The length of the alcohol chain influences
directly on the quality of the final biodiesel obtained. The
386 Marcos Vargas et al.

larger the alcohol is, the greater the quality of biodiesel

obtained, and, in addition, it causes less inhibition of the lipase
but increases the cost of the final product [41, 43, 47].
(g) Lipase quantity. In general, the final biodiesel yield increases
with an increasing amount of lipase, until a constant yield is
reached even if more enzyme is added. Therefore, there is an
optimal lipase concentration [41]. It has been reported that
the addition of large amounts of lipase was unpractical, since
the matrix with the lipase made the solution extremely viscous,
without further increasing the biodiesel yield [7].
(h) Agitation speed. With immobilized catalysts, the reactants
need to diffuse from the bulk liquid to the external surface
of the particle and from there into the interior pores of the
catalyst. External mass transfer limitations can be minimized
by carrying out the reaction at an optimum agitation speed
[43, 48].

6 Economic Feasibility of Biodiesel Production Using Lipases

Recent changes in world energetics legislation and tax incentives in

many countries have stimulated reduction in biodiesel prices
[1, 49] and became key factors in biodiesel industry development.
But feedstocks for biodiesel production still account for the major-
ity cost of the process [1, 13]. In general lipases are more expensive
than commonly used chemical catalysts, but considering the fact
that lipases allow to use cheap feedstocks such as brown grease and
oils rich in FFA or with considerable amounts of impurities, they
may become a profitable option. As well as the facts that it are
possible to reuse lipases, less purification steps are required (e.g.,
removal of alcohol excess and water), and less side products are
Moreover, sales of coproduced glycerol can also improve bio-
diesel profits, and since the quality of this by-product is greatly
improved in the lipase-catalyzed process, purification steps are
It has been reported that capital investment is paid back in
about 2 years for alkali-catalyzed process [1], and for feedstocks
with high FFA content, such as waste cooking oil, a global process
including acid neutralization is reported to be affordable
[50, 51]. The use of the commercial lipase Novozym 435 was
also evaluated and was found to be also affordable but still more
expensive than the basic or acid-catalyzed process [51], but other
aspects can also be taken into account in order to further reduce
costs, such as reaction condition optimization.
Research efforts continue to focus in the development of
cheaper lipases and their optimized reactions [15, 19, 32, 52–58].
Lipases as Biocatalyst for Biodiesel Production 387

In conclusion, enzymatic biodiesel, as a green alternative to

chemical biodiesel, has a potential economic growth in the near

7 Concluding Remarks

Lipases have been proved as suitable catalysts for biodiesel produc-

tion process. Lipases can esterify/transesterify many different types
of oils and greases, no matter the quantity of FFA or the presence of
some amounts of water without forming soaps. Using lipases to
produce biodiesel is also less energy intensive, allows easy recovery
of glycerol, and offers more safety, cleaner, and environmentally
friendlier processes.
There are many types of lipases produced by different biological
sources, such as microorganisms, mammalian cells, and plants.
They have different characteristics which make them suitable for
different specific purposes. For biodiesel production, lipases should
be non-stereospecific so that all tri-, di-, and monoglycerides can be
converted to biodiesel, capable of catalyzing the esterification of
FFA as well, easy to produce, temperature and alcohol resistant, and
reusable and should have low reaction time, low product inhibition,
and high biodiesel yield.
The main factors affecting the biodiesel production using
lipases as catalysts are specificity for certain triglycerides or fatty
acid chain length, temperature, presence of water in excess, pres-
ence or absence of solvent in the reaction medium, type of alcohol,
alcohol concentration and oil-alcohol molar ratio, lipase quantity,
and agitation speed.
More research still needs to be explored in order to accomplish
the lipase-catalyzed biodiesel production at suitable costs.


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