Applied Energy 119 (2014) 497520

Contents lists available at ScienceDirect

Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Enzymatic biodiesel: Challenges and opportunities

Lew P. Christopher a,b,, Hemanathan Kumar c, Vasudeo P. Zambare d
a
Center for Bioprocessing Research and Development, South Dakota School of Mines and Technology, Rapid City, SD, USA
b
Department of Civil and Environmental Engineering, South Dakota School of Mines and Technology, Rapid City, SD, USA
c
Department of Chemistry, Laboratory of Applied Chemistry, University of Jyvskyl, Finland
d
Biotechnology Division, Rossari Biotech Ltd., Naroli, Silvassa, India

h i g h l i g h t s

 A comprehensive overview of recent progress in enzymatic biodiesel production.

 Critical assessment of merits and demerits of enzymatic biodiesel process.
 Economic considerations and large scale developments in enzymatic biodiesel.
 Current trends and future directions for enzymatic biodiesel R&D.

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

Article history: The chemical-catalyzed transesterication of vegetable oils to biodiesel has been industrially adopted
Received 31 August 2013 due to its high conversion rates and low production time. However, this process suffers from several
Received in revised form 21 November 2013 inherent drawbacks related to energy-intensive and environmentally unfriendly processing steps such
Accepted 6 January 2014
as catalyst and product recovery, and waste water treatment. This has led to the development of the
Available online 5 February 2014
immobilized enzyme catalyzed process for biodiesel production which is characterized by certain envi-
ronmental and economical advantages over the conventional chemical method. These include room-tem-
Keywords:
perature reaction conditions, elimination of treatment costs associated with recovery of chemical
Biodiesel
Lipase
catalysts, enzyme re-use, high substrate specicity, the ability to convert both free fatty acids and triglyc-
Enzyme-catalyzed transesterication erides to biodiesel in one step, lower alcohol to oil ratio, avoidance of side reactions and minimized impu-
Feedstock rities, easier product separation and recovery; biodegradability and environmental acceptance. This
Glycerol paper provides a comprehensive review of the current state of advancements in the enzymatic transeste-
rication of oils. A thorough analysis of recent biotechnological progress is presented in the context of
present technological challenges and future developmental opportunities aimed at bringing the enzyme
costs down and improving the overall process economics towards large scale production of enzymatic
biodiesel. As the major obstacles that impede industrial production of enzymatic biodiesel is the enzyme
cost and conversion efciency, this topic is addressed in greater detail in the review. A better understand-
ing and control of the underpinning mechanisms of the enzymatic biodiesel process would lead to
improved process efciency and economics. The yield and conversion efciency of enzymatic catalysis
is inuenced by a number of factors such as the nature and properties of the enzyme catalyst, enzyme
and whole cell immobilization techniques, enzyme pretreatment, biodiesel substrates, acyl acceptors
and their step-wise addition, use of solvents, operating conditions of enzymatic catalysis, bioreactor
design. The ability of lipase to catalyze the synthesis of alkyl esters from low-cost feedstock with high
free fatty acid content such as waste cooking oil, grease and tallow would lower the cost of enzymatic
biodiesel. Discovery and engineering of new and robust lipases with high activity, thermostability and
resistance to inhibition are needed for the establishment of a cost-effective enzymatic process. Opportu-
nities to create a sustainable and eco-friendly pathway for production of enzymatic biodiesel from
renewable resources are discussed.
2014 Elsevier Ltd. All rights reserved.

Corresponding author. Address: 501 E. St. Joseph Street, South Dakota School of
Mines and Technology, Rapid City 57701, SD, USA. Tel.: +1 605 394 3385.
E-mail address: lew.christopher@sdsmt.edu (L.P. Christopher).

0306-2619/$ - see front matter 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.apenergy.2014.01.017
498 L.P. Christopher et al. / Applied Energy 119 (2014) 497520

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
2. Biodiesel production methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
2.1. Chemical production of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
2.2. Enzymatic production of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
3. Factors affecting enzymatic biodiesel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
3.1. Enzyme source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
3.2. Enzyme properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
3.3. Enzyme and whole cell immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
3.3.1. Enzyme immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
3.3.2. Whole cell immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
3.4. Enzyme pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
3.5. Biodiesel feedstock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
3.6. Acyl acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
3.7. Enzyme operating variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
3.8. Bioreactor design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
3.9. Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
4. Current trends and future directions for enzymatic biodiesel R&D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
5. Economic considerations and large scale developments in enzymatic biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514

1. Introduction global biodiesel market is estimated to reach 37 billion gallons

Alternative fuels for internal combustion engines have recently
attracted considerable attention due to the ever diminishing petro-
leum reserves and environmental consequences of increasing
greenhouse gas emissions. The growing environmental concerns,
tougher Clean Air Act Standards [1,2] and Renewable Fuel Standard
(RFS) Mandates [3] are among the major drivers for development
of alternative fuels from renewable resources that are sustainable
and environmentally acceptable. The U.S. RFS sets a goal of 36 bil-
lion gallons of biofuels production by year 2022, of which 21 bil-
lion gallons should come from the so-called advanced biofuels
and a minimum of 1 billion gallons of biodiesel.
Over the last decade, biodiesel has attracted considerable atten-
tion as a renewable, biodegradable, non-toxic and clean-burning
substitute for petroleum based diesel fuel which reduces emissions
of carcinogenic compounds by as much as 85% compared to petro-
diesel, essentially free of sulfur, polycyclic aromatic hydrocarbons
and metals. The biodiesel properties such as cetane number, en-
ergy content, viscosity and phase changes are similar to those of
petrodiesel fuel [46], however, the engines fueled with biodiesel
emit signicantly fewer particulates, hydrocarbons, and less car-
bon monoxide than those engines operating on conventional diesel
fuel. The greenhouse gas (GHG) emission of biodiesel (B100) are
4.5-times lower than gasoline, and 3-fold lower than petrodiesel
and 85% ethanolgasoline fuel (E85). Although the NOx levels of
biodiesel are slightly higher than those of conventional diesel fuel,
biodiesel is believed to be eco-friendly alternatives to fossil fuels
that can help reduce the risk of global warming by reducing the
net carbon emissions to the atmosphere [79].
One of the great advantages of biodiesel is that it can be used in
existing engines, vehicles and infrastructure with practically no
changes [10]. B100 can be blended at any level with petroleum die-
sel, and its higher ash point makes it a safer fuel to use, handle,
and store using existing diesel tanks and equipment. Biodiesel
has excellent energy balance as compared to fossil fuels; biodiesel
contains 3.2 times the amount of energy it takes to produce it [11].
The biodiesel production in the US increased dramatically in the
past few years [12]. According to the National Diesel Board [13],
there were 105 plants in operation as of early 2007 with a total
production capacity of 864 million gallons and additional 1.7 bil-
lion gallons coming online from plants under construction. The
by 2016 with an average annual growth of 42%. In 2012, the US
produced 1.143 billion gallons of biodiesel which was 14.3% more
than what the RFS called for. In 2013, the biodiesel production in
the US is projected to reach 1.28 billion gallons, a 10.7% increase
over the 2012 blended gallons. Since 2005, the biomass-based die-
sel production increased more than 18-fold in the US only. The bio-
diesel industry in the US in on the rise, and federal agencies such as
the U.S. Department of Transportation and Department of Defense
invest in research and development to dramatically reduce depen-
dence on foreign oil, and spur the creation of a domestic bio-indus-
try for sustainable production of biofuels including biodiesel [14].
The cost of biodiesel, however, is currently approximately 30%
higher than that of petroleum-based diesel [1517]. This is mainly
due to the use of expensive, high quality and mostly non-rened
virgin oils such as soybean, sunower, olive, palm, sh, jatropha,
canola, cottonseed, peanut and linseed oil, known as rst genera-
tion biodiesel feedstock. In the enzyme-catalyzed biodiesel pro-
duction, the high enzyme cost signicantly impacts the process
protability. The cost of commercial enzyme products for indus-
trial use is approximately $1,000/kg which is signicantly higher
than that of the alkali catalyst ($0.62/kg) [18]. Biodiesel fuel is
expensive in comparison with petroleum-based fuel as 6080% of
the cost is associated with the feedstock oil [19]. Previous studies
have estimated that biodiesel production costs range between
$1.50 and $2.50 per gallon depending on the feedstock used in
the production process. These costs exceeded the wholesale price
of petroleum-based diesel by anywhere from $0.20 to $0.82 per
gallon depending on the time period when these studies were con-
ducted [20]. Currently, biodiesel production from soybean oil at
$0.50/lb with a 7.35 lbs/gallon efciency is estimated to cost about
$3.675/gallon [14].
Since the biodiesel production costs are proportional to the
costs of the raw materials, use of alternate, low-cost feedstock such
as waste frying oil, non-edible oil and oil extracted from other
feedstocks such as waste restaurant oil, yellow grease, lard, animal
fats and others, known as second generation biodiesel feedstock,
instead of virgin oil is estimated to reduce the production cost
and make it competitive in price with petroleum diesel [16,17].
Oils extracted from algae are particularly viewed as the most sus-
tainable feedstock for the future due to the signicantly higher
yields, 15,000 gal/acre/year, as compared to only 48 gal/acre/year
 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
for soybean oil, 113 gal/acre/year, for peanut oil, and 127 gal/acre/
year, for canola oil (also known as rapeseed oil).
Rudolph Diesel, the inventor of the internal compression igni-
tion engine now known as the Diesel engine, rst used vegetable
oils (such as peanut oil) to fuel his engine [21,22]. The vegetable
oils performed well in short term engine tests [23,24], but failed
in long term operations [21,25,26]. Problems such as cooking of
injectors, carbon deposits, oil ring sticking, thickening and gelling
of the lubricating oils were encountered. High viscosity (almost
10 times that of No. 2 diesel fuel) and a tendency for polymeriza-
tion within the cylinder appeared to be the root cause for many
problems (such as high cloud point temperature), associated with
the direct use of vegetable oils as fuel [23,2634].
In order to reduce these difculties, the idea of converting the
vegetable oils into their ester derivatives was introduced. Reacting
triglycerides (TGs) in oil with an alcohol in presence of a catalyst
can produce esters of free fatty acids (FFA) such as fatty acid methyl
esters (FAME). As a result, the viscosity of these alkyl esters is re-
duced, however, their cetane numbers and heating values remain
unchanged. FAME are termed biodiesel and the process is known
as trans-esterication [35,36]. The catalysts used for transesteri-
cation of biodiesel are alkali, acid (chemical) and enzyme (bio-
based). The alkali-catalyzed process gives higher conversion of TG
at short reaction times. The foremost drawback of the alkali process
is its sensitivity towards FFA in oils that leads to soap formation
during the transesterication process. The acid-catalyzed processes
are insensitive to FFA, however, with a drawback of slower reaction
rates. Furthermore, the use of chemical catalysts can cause techni-
cal problems related to biodiesel purication and separation from
catalysts and the glycerol by-product [37,38].
To minimize the problems associated with the use of acid and/
or alkali for biodiesel production, a lipase-catalyzed process has
been proposed and extensively researched in the last decade
[18]. Advantages of the enzyme-based biodiesel process include:
(1) simplied production process; (2) lower energy consumption;
(3) higher purity of glycerol by-product; (4) no soap formation in
the system; (5) easy separation and reuse of immobilized enzymes.
Although the enzyme catalyst has some drawbacks, mainly associ-
ated with lower reaction rates, higher costs, and loss of activity or
enzyme inhibition, the enzymatic route for biodiesel production is
nowadays considered as an environmentally-friendly alternative
that is becoming more realistic as new, more efcient and less
expensive enzymes are being developed [39]. To minimize enzyme
costs, the enzymatic catalyst can be reused by immobilization,
which leads to improved efciency of biodiesel recovery. In addi-
tion, the enzyme catalytic activities can be enhanced by screening
and selection of high alcohol-tolerant microorganisms and by
genetic engineering [40].
OCOR 2
1. R1 OCO OCOR 3 + ROH
Triglyceride Alcohol
OCOR 2
2.
HO OCOR3 + ROH
Diglyceride Alcohol
OH
3. HO OCOR3 + ROH
Monoglyceride Alcohol
Fig. 1. Step-wise reaction pathways for biodiesel production (R is a small alkyl group, R1, R2 and R3 are fatty acid chains; k1, k2, k3, k4, k5, k6 are chemical or enzymatic
catalysts).
499
The following sections provide a state of the art review of the
current biodiesel process technologies with emphasis on the enzy-
matic transesterication method. A comprehensive analysis of
recent biotechnological advancements in the enzyme process
biodiesel is presented in the context of current technical challenges
and future developmental opportunities aimed at bringing the
enzyme costs down and improving the overall process economics
towards large scale production of enzymatic biodiesel.
2. Biodiesel production methods
Biodiesel can be made via three production routes: microemul-
sions, thermal cracking (pyrolysis), and trans/inter/esterication
[26]. The most cost-efcient method for production of biodiesel
with higher quality is transesterication of vegetable oils and ani-
mal fats. In lipid chemistry, transesterication is the catalytic pro-
cess of exchanging the alkoxy group of an ester by an alcohol (acyl
acceptor) that converts the TG in oils to fatty acid alkyl esters
(FAAE) and glycerol (Fig. 1). This process is also known as alcohol-
ysis and makes use of short chain alcohols such as methanol and
ethanol as acyl acceptors. Interesterication is the transformation
of TG to biodiesel in presence of an ester (such as methyl acetate)
as the acyl acceptor. In this process, instead of glycerol, another tri-
acylglycerol is formed as the by-product. Lastly, biodiesel can be
also produced by a direct esterication of FFA with alcohols to pro-
duce FAAE and water as the by-product. Chemical (acids and/or
bases) and biological (enzymes) agents serve as catalysts. The over-
all process of alcoholysis includes three reversible reactions in
which di- and mono-glycerides are formed as intermediates
[26,41]. From each TG molecule, three molecules of biodiesel and
one molecule of glycerol are produced (Fig. 1). The process equilib-
rium can be shifted toward product formation in excess of alcohol
or by continuous product removal.
2.1. Chemical production of biodiesel
The base-catalyzed transesterication of oils and fats to biodiesel
proceeds at faster rates and greater yields than the acid-catalyzed
process [4143]. Hence, biodiesel at yields of 9499% is convention-
ally manufactured via chemical catalysis that uses sodium or potas-
sium hydroxide at concentrations in the range of 0.51 wt%, a
methanol to oil ratio of 6:1, and temperatures of 4580 C to com-
pletely transesterify the lipids in several hours. The 6:1 ratio of acyl
acceptor to vegetable oil is higher than the stoichiometrically re-
quired 3:1; this, however ensures excess of methanol in the reaction
mixture and increased methyl ester yields. However, molar ratios
higher than 6:1 increase glycerol solubility and complicate glycerol
OCOR 2
k1
R 1OOCR + HO OCOR 3
k4
Ester Diglyceride
OH
k2
R 2OOCR + HO OCOR 3
k5
Ester Monoglyceride
OH
k3
R 3OOCR + HO OH
k6
Ester Glycerol
500 L.P. Christopher et al. / Applied Energy 119 (2014) 497520

separation. Sodium hydroxide is the preferred alkaline catalyst due
to its lower price and higher biodiesel yields [44].
Although biodiesel is currently exclusively produced at com-
mercial scale utilizing alkali, mainly sodium hydroxide, there are
process limitations that are considered drawbacks of chemical bio-
diesel. These are normally associated with the presence of FFA in
quantities higher than 0.5% that can lead to soap formation, and
with the presence of water exceeding 0.3% that can results in con-
sumption of the catalyst thereby reducing the reaction yield.
Saponication not only consumes the alkali catalyst, but the result-
ing soaps can cause the formation of emulsions that create difcul-
ties in downstream recovery of biodiesel, and can lower the ester
yields [45]. Therefore, to prevent saponication, an acid pretreat-
ment step is introduced that esteries the FFA to FAME in presence
of methanol. The acid step is normally carried out with 0.51.0%
sulfuric acid at a higher temperature (60100 C) and alcohol/sub-
strate molar ratio of approximately 30 to 1. Esterication of oils
containing high FFA using acid catalyst results in reduction of
FFA to less than 1 wt%. Other mineral acids such as hydrochloric
acid, p-toluenesulfonic acid, methanesulfonic acid and phosphoric
acid are also commonly used. For instance, Guan et al. [46] used p-
toluenesulfonic acid as catalyst for a complete transesterication
of corn oil to biodiesel in dimethyl ether. Following the initial con-
version of FFA to FAME, the remaining TG are then transesteried

(a)
Alcohol

Preesterification
Transesterification

Evaporation

Oil
Separation

Fatty Acid Alkyl Esters

Alcohol
Water
Evaporation

Water
Washing
Catalyst Catalyst
(Acid) (Base)
Waste Water
Alcohol
Crude Glycerol

Drying

Acid
Acidification Glycerol
Biodiesel

Saponified product

(b)
Alcohol

Transesterification

Evaporation
Oil

Separation

Fatty Acid Alkyl Esters

Alcohol

Enzyme Biodiesel
Glycerol

Fig. 2. Biodiesel production from feedstocks containing high free fatty acids: (a) two-step chemical process; (b) enzymatic process.
 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
to biodiesel in presence of a base [4754]. A two-step acidbase
process has been developed to utilize oils such as used cooking
oil and restaurant grease with a high FFA content (2050%) in bio-
diesel production (Fig. 2a). The water formed in the acid-catalyzed
esterication is separated from the organic layer of FAME prior to
the alkali-catalyzed step. The presence of hydroxide ions in water
increases the saponication which produces soap by reaction of
the hydroxide ions with esters, FFA and with other glycerides, thus
rendering the downstream separation expensive and affecting the
ester yields [41,5558]. Liu et al. [59] studied the affect of water on
sulfuric acid-catalyzed esterication of carboxylic acid and re-
ported a signicant decrease in catalytic activity as water was pro-
duced from the condensation of carboxylic acids and alcohols. The
acid-catalyzed process is corrosive in nature causing damage to
equipment, proceeds at low rates, and requires long reaction times
to achieve conversion in excess of 90% [55]. For a full completion of
esterication (up to 99%), more than 48 h is normally needed
[41,46,55]. However, the major demerit of the acidbase process
is the increased number of process steps and required equipment,
which in turn results in increased production cost.
There are several drawbacks associated with the chemical pro-
duction of biodiesel [19,37]: (1) side reactions of saponication
and hydrolysis affect biodiesel yield and purity; (2) the process,
especially the acid pretreatment step, is energy and capital inten-
sive; (3) recovery and purication of catalysts and glycerol is
expensive; (4) neutralization and waste water treatment is re-
quired. Problems with separation and soap formation have
prompted research with heterogeneous non-enzymatic catalysts
such as amorphous zirconia, titanium-, aluminum-, and potas-
sium-doped zirconias, metal oxides, hetero-polyacids, sulfated
zeolites and others [6062]. A mesoporous silica catalyst function-
alized with hydrophobic allyl and phenyl was shown to hydropho-
bically exclude water from the catalyst active sites while effectively
esterifying FFA [63]. Compared to homogenous catalysts, heteroge-
neous catalysts have the advantages of easier recovery and usability
on substrates containing higher concentrations of FFA. However,
some heterogeneous catalysts could be cost and energy intensive
due to requirements for high reaction temperatures and high
alcohol to substrate molar ratios [64].
2.2. Enzymatic production of biodiesel
As elaborated in the previous section, the main disadvantages of
the traditional acid/base transesterication methods are the high
reaction temperatures, high energy consumption, corrosive nature
of acids, glycerol purication and recovery, separation of catalysts
and unreacted alcohol accompanied with additional washing steps
to remove impurities from biodiesel. These problems can be mini-
mized and even eliminated by using environmentally-friendly bio-
catalysts (enzymes) for biodiesel production (Fig. 2b). Enzymes
such as lipases offer a biological route of biodiesel production with
a number of environmental and economic advantages over the
chemical route [6568]:
 Use of mild reaction temperatures.
 High selectivity and specicity of trans/esterication towards
substrates.
 Broader substrate range due to ability to esterify both glyceride-
linked and non-esteried fatty acids in one step; use of lower
alcohol to oil ratios.
 Avoidance of side reactions, easier separation and product
recovery due to the production of a glycerol side stream with
minimal impurities and water content [12].
 Elimination of treatment costs associated with recovery of
chemical catalysts.
 Enzyme biodegradability and environmental acceptability.
501
 Opportunity for enzyme reuse and improved stability through
enzyme immobilization.
The presence of FFA in the starting material poses a major
problem for transesterication via traditional methods, while this
problem can be easily overcome by using enzymes that can catalyze
both esterication of FFA and transesterication of TGs. The FFA
contained in waste oils and fats can be completely converted to bio-
diesel. In fact, the enzyme-catalyzed transesterication is more
suitable for use on FFA-rich feedstocks such as waste oils, greases,
beef tallow and lard since the FFA are directly esteried enzymati-
cally into FAME [12]. Therefore, lipase can be used on oils with var-
iable chemical composition which broadens the feedstock base and
is of great advantage when waste oils and fats are considered for the
establishment of a cost-effective biodiesel production process
[65,69]. However, there are several technical challenges that need
to be overcome to improve the economic feasibility of the lipase bio-
process: high cost of enzymes, loss of activity during the process, en-
zyme inhibition by reactants and products, and slow reaction rates.
3. Factors affecting enzymatic biodiesel production
The yield and conversion efciency of enzymatic biodiesel is
inuenced by a number of factors: the nature and properties of
the enzyme catalyst; enzyme and whole cell immobilization tech-
niques; enzyme pretreatment; biodiesel substrates; acyl acceptors,
their step-wise addition and use of solvents; operating conditions
of enzymatic catalysis; and bioreactor design.
3.1. Enzyme source
Lipases are enzymes (biocatalysts) which carry hydrolysis of TG
to glycerol and fatty acid, hence, they are categorized in the class of
hydrolases (acylglycerolacylhydrolases, EC 3.1.1.3) [70,71]. They
are best dened as carboxylesterases that catalyze both the hydro-
lysis and synthesis of long-chain acylglycerols [72]. These enzymes
are ubiquitously present and based on their origin are classied as
plant, animal or microbial lipases. Plant lipases have been reported
from papaya latex, rapeseed, oat and castor seeds [73]. Plant
lipases are not commercially used whereas the animal and microbial
lipases are used extensively. Sources of the animal lipases are pan-
creas of cattle, sheep, hogs and pigs [73]. Microbial lipases have
gained wide industrial importance and they now share about 5% of
the world enzyme market after proteases and carbohydrases [74
78]. Lipases of microbial origin are more stable than plant and animal
lipases and are available in bulk at lower cost compared to lipases of
other origin. Yeasts lipases are easy to handle and grow compared to
bacterial lipases [79]. Among the yeast lipases, Candida rugosa has
gained good commercial importance. The most commonly used bio-
catalyst for biodiesel production are the microbial lipases that are
produced by a number of fungal, bacterial and yeast species
(Table 1).
As it can be seen from Table 1, a large number of microbial strains
have been used for lipase production, however, the most frequently
reported enzyme sources are Candida sp., Pseudomonas sp. and Rhi-
zopus sp.[132]. Lipase producing microorganisms have been
screened from various sources including soil, marine water, waste
water and industrial wastes [76]. Traditionally, screening is carried
out on batch cultures using agar substrates which is time-consum-
ing, however, continuous enrichment cultures overcome this prob-
lem by using fermentors for growth and selection of desired
microbial isolates [133]. Soil isolates of Aspergillus, Mucor, Candida
and Sclerotina species were reported to produce lipase [129].
Lipase-producing strains of P. uorescens, P. alcaligenes, Enterobacter
intermedium, Geotrichum asteroids and Bacillus acidophilus were iso-
lated from vegetable oil processing plants [129,134,135].
502 L.P. Christopher et al. / Applied Energy 119 (2014) 497520

Table 1 Table 1 (continued)

Lipase-producing microorganisms.
Microbial type Microbial source Refs.
Microbial type Microbial source Refs.
Candida quercitrusa [109]
Bacteria Achromobacter lipolyticum [77] Pichia burtonii [128]
Acinetobacter radioresistens [80] Pichia sivicola [128]
Acinetobacter calcoaceticus [81] Pichia xylosa [129]
Acinetobacter pseudoalcaligenes [82] Saccharomyces lipolytica [100]
Aeromonas hydrophila [83] Geotrichum candidum [130]
Archaeglobus fulgidus [84] Yarrowia lipolytica NRRL YB-423 [131]
Bacillus acidocaldarius [72]
Bacillus megaterium [76]
Bacillus pumilus [72]
3.2. Enzyme properties
Bacillus sp. [77]
Bacillus stearothermophilus [85]
Bacillus subtilis [86] Microbial lipases are mostly extracellular with a molecular
Bacillus thermocatenulatus [87] weight of 3050 kDa and a pH optimum in the slightly alkaline pH
Bacillus thermoleovorans [88] range of 7.59 [136]. Lipases originate mainly from mesophilic
Burkholderia glumae [77]
and thermophilic microorganisms and have an optimum activity
Chromobacterium viscosum [77]
Enterococcus faecalis [89] at 3550 C and 6080 C, respectively [129,135]. Most mesophilic
Micrococcus freudenreichii [90] lipases are unstable at temperatures above 70 C, whereas thermo-
Moraxella sp. [73] stable lipases show activity at up to 100 C in presence of organic
Mycobacterium chelonae [91]
solvents and detergents [137]. For instance, thermostable lipases
Pasteurella multocida [92]
Propionibacterium acnes [73]
from the hyperthermophilic archaea Pyrobaculum calidifonti [138]
Propionibacterium avidium [93] and Pyrococcus furiosus [139] and from the extreme thermophilic
Propionibacterium granulosum [93] bacteria Thermoanaerobacter thermohydrosulfuricus and Caldanae-
Proteus vulgaris [73] robacter subterraneus [140] exhibited lipase activity at 90 C. In
Pseudomonas aeruginosa [90,94]
addition, the Thermoanaerobacter and Caldanaerobacter lipases were
Pseudomonas cepacia [95]
Pseudomonas fragi [77,96] shown to be resistant to organic solvents, which makes them strong
Pseudomonas mendocina [77] candidates for biodiesel production in water-free environments.
Pseudomonas nitroreducens var. thermotolerans [96] The optimum expression of lipases depends on many factors
Pseudomonas sp. [97]
including carbon and nitrogen sources, growth conditions like
Psychrobacter immobilis [98]
Serratia marcescens [99]
pH, temperature, dissolved oxygen. Numerous literature sources
Staphylococcus aureus [77] are available on lipase producing microorganisms, methods for li-
Staphylococcus canosus [100] pase production and application [74,78,91,141]. As lipases are lar-
Staphylococcus epidermidis [101] gely inducible enzymes, the main factor for the expression of lipase
Staphylococcus haemolyticus [102]
activity is the carbon source which can be a lipid source or a carbon
Staphylococcus hyicus [87,103]
Staphylococcus warneri [103] source [142]. The lipid sources include triacylglycerols, short or
Staphylococcus xylosus [91] long chain fatty acids, hydrolyzable esters, tween, bile salts and
Sulfolobus acidocaldarius [73] glycerol [141,143] and the carbon sources are sugars, sugar alco-
Vibrio chloreae [73] hol, polysaccharides, whey, amino acids and other complex sources
Pseudomonas alcaligens [104]
Chromobacterium visosum [105]
[141]. For maximum lipase production, incubation period usually
Pseudomonas putida [106] range from few hours to few/several days based on the organism
Statphylococcus stolonifer [107] [141] and production method. Fungal species are preferably culti-
Fungi Alternaria brassicicola [108] vated in solid-state fermentation, while bacteria and yeast are best
Aspergillus fumigates [109] produced in submerged fermentation [144]. The lipase reaction
Aspergillus japonicas [110] systems for biodiesel production are complex, consisting of two
Aspergillus nidulans [111]
immiscible phases an aqueous phase containing enzyme, and
Candida antarctica [87]
Mucor miehei [112] an organic phase containing oil/fat. Lipases have the unique feature
Penicilliumcyclopium [113] of acting on the interface between aqueous and non-aqueous
Rhizomucor miehei [114] phases and so they can catalyze many reactions including hydroly-
Rhizopus arrhizus [82] sis, inter-esterication and alcoholysis [78].
Rhizopus chinensis [76]
Rhizopus microsporous [76]
The mode of action of lipases in substrate transesterications to
Rhizopus niveus [115] biodiesel depends on their origin and specic properties. Lipases
Rhizopus nodosus [116] catalyze the trans/esterication reaction between TG and acyl
Rhizopus oryzae [117,118] acceptors (alcohols) through the formation of acyl-enzyme inter-
Streptomyces cinnamomeus [119]
mediates that subsequently donate the acyl moiety to produce
Streptomyces exfoliates [98]
Streptomyces fradiae [82] FAAE [145]. The overall structure of the lipases can be described
Streptomyces sp. [82] as a structure with a central L-sheet with the active serine placed
Aspergillus niger [120] in a loop called catalytic elbow [146]. The activation which is often
Thermomyces lanuginous [121] necessary for the lipase enzyme is the movement of a lid. Some en-
Fusarium heterosporum [122]
Humicola lanuginose [123]
zymes such as Thermomyces lanuginosus lipase have an active site
Oospora lactis [124] and a lid on the surface of the enzyme. Others like C. rugosa lipase
Rhizopus oryzae [125] have an active site at the end of a tunnel containing the lid in its
Yeasts Candida deformans [87] external parts [147]. The structural properties of lipase from differ-
Candida parapsilosis [112] ent sources might be the reason for showing different activity on
Candida rugosa [126,127] different oil substrates, hence, the need to optimize the process
 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
based on the selected enzymes and substrates for biodiesel pro-
duction. Furthermore, the selection of a particular lipase for lipid
modication is based on the type of the desired modication and
may be position-specic modication of triacylglycerol, fatty
acids-specic modication, modication by hydrolysis, and modi-
cation by direct synthesis and transesterication [148]. Based
on their substrate specicity, lipases can be divided into three
groups: 1,3-specic, fatty acid-specic, and non-specic lipases.
The 1,3-specic lipases release and hydrolyze ester bonds in the
position 1 and 3 of TGs [149]. Du et al. [150] reported the use of
T. lanuginosus 1,3-specic lipases to produce a transesterication
yield of 90%. The fatty acid-specic lipases are known to hydrolyze
esters of long chain fatty acids with double bonds in cis-position at
C9, whereas the non-specic lipases randomly cleave the acylgly-
cerols in FFA [149]. For optimal biodiesel production, lipases
should be able to convert all three forms of glycerides (mono-,
di-, and tri-glycerides) to biodiesel, hence, they need to be non-ste-
reospecic and to efciently catalyze the esterication of FFA [18].
3.3. Enzyme and whole cell immobilization
Immobilization is the process of attaching enzymes physically
to a solid support so that the substrate is passed over the enzyme
support and can be converted to the product. Immobilization has
been increasingly used in industrial applications to facilitate sepa-
ration of biocatalysts from the process stream, and hence, the
recovery and purication of products [151]. Immobilized enzymes
are preferred over free enzymes due to their prolonged enzyme-
substrate contact that curtails redundant downstream and puri-
cation processes [152]. There are many advantages of immobilized
enzyme over free enzyme: the repetitive use of a single batch of
enzymes is economical in the development of continuous biopro-
cesses; rapid termination of the enzyme-substrate reaction by
removing the enzyme from the reaction solution; enzyme stabil-
ization due to binding to support; no contamination of the product
and enzyme [153]. Immobilization can dramatically affect enzyme
properties such as pH-dependence, temperature prole, resistance
to proteolytic digestion and denaturants, thermostability and
kinetics. The chief issues for enzyme immobilization are selection
of support matrices and immobilization techniques that permit
both rapid enzyme activity and enzyme stability under the con-
straints imposed by the substrate medium [154,155].
A number of techniques and supports are available for the immo-
bilization of enzymes on a variety of natural and synthetic supports
[156]. The choice of the support and the technique depends on the
enzyme nature, nature of the substrate and the type of reaction it
is used [152,157]. For industrial application, support materials are
selected based on the ow properties, low cost, non-toxicity and
maximum biocatalysts loading while retaining the desirable ow
characteristics, operational durability, availability and ease of
immobilization [158]. The activity of the immobilized enzyme
may be reduced during the immobilization procedure and as a result
of mass-transfer limitations. Enzyme immobilization techniques
have been classied into three categories: entrapment, carrier bind-
ing and cross-linking [159]. The economics of the immobilization
process depends on both the activity and the operational stability
(activity integrated over the operational time) of the biocatalysts.
3.3.1. Enzyme immobilization
Immobilization of lipases was carried out using entrapment
[159], physical adsorption [160], ion exchange [161], and cross-
linking [162]. Carriers for lipase immobilization include polyure-
thane foam [163], silica [164], sepabeads [165], cellulosic nano-
bers [166]. Criteria for selecting the immobilization technique
and carrier depends on the source of lipase, the type of reaction
system (aqueous, organic solvent or two-phase system) and the
503
process conditions (pH, temperature and pressure). Based on the
immobilization technique and carrier, the bioreactor type (batch,
stirred tank, membrane reactor, column and plug-ow) can be de-
signed. The literature is replete with various lipase producing
microorganisms, enzyme immobilization methods and physical
carriers. The challenge will be to select a carrier and immobiliza-
tion technique that will allow maximum lipase activity, retention
and stability on the oil substrate [167]. Adsorption is the most
widely employed method for lipase immobilization. The most fre-
quently immobilized enzymes used for commercial applications
are the C. antartica lipase, immobilized on acrylic resin (Nov-
ozym435), Mucormiehei lipase, immobilized on a macroporous
ion-exchange resin (Lipozyme IM), T. lanuginosus acrylic resin-
immobilized lipase (Lipozyme TLIM), Rhizomucor miehei lipase
immobilized on macroporous anion exchange resin (Lipozyme
RM IM), and Candida sp. 99125 lipase, immobilized on textile
membranes [168]. As lipase is deactivated by methanol adsorption
onto the immobilized enzyme, enzyme regeneration with higher
alcohols such as butanol is required. Due to immiscibility of the
large TG molecules with lower alcohols, diffusion problems to ac-
cess the immobilized biocatalyst may arise due to the small pores
of the carrier. This may cause internal transport problems thereby
reducing the enzyme efciency [18].
Recently, interest has focused on carrier-free immobilized li-
pases by cross-linking of enzyme aggregates for use in a solvent-
free biodiesel production [169]. This technique presents several
interesting advantages over carrier-bound immobilized enzymes
that includes highly concentrated enzymatic activity, high stability
of the produced superstructure, costs savings from omitting the
support material, and no enzyme purication requirement [170].
Lipase from Rhizopus oryzae was immobilized as crosslinked en-
zyme aggregate via precipitation with ammonium sulfate directly
from the fermentation broth and simultaneous crosslinking with
glutaraldehyde [171]. The cross-linked enzyme retained 91%
activity after 10 cycles in aqueous medium. Another recent
development in enzyme immobilization with potential use in bio-
diesel production is the protein-coated microcrystals that consist
of water-soluble micron-sized crystalline particles coated with a
biocatalyst such as lipase [172]. Microcrystals coated with a C. ant-
arctica lipase B retained nearly 90% of the enzyme initial activity
over a period of one year at room temperature. The enzyme
activity of P. aeruginosa lipase protein-coated microcrystals was
enhanced tenfold over that of the free lipase enzyme [173]. A com-
bination of lipase cross-linking and lipase coating was reported to
improve the operational, pH and thermostability as well as the
organic solvent tolerance of a Geotrichum sp. lipase in biodiesel
production [174].
3.3.2. Whole cell immobilization
In an attempt to avoid the complex enzyme recovery and puri-
cation requirements for immobilization of free (extracellular) lipase,
immobilization of intracellular lipase, known as whole cell immobi-
lization, has been extensively researched [175]. Immobilization of
the intracellular, cell- or membrane-bound lipase is believed to offer
an alternative and less expensive source of immobilized biocatalyst
for biodiesel production. Considerably fewer steps are required to
produce whole-cell lipase using inexpensive and readily available
industrial cultures. Consequently, this can signicantly reduce the
cost of the transesterication process [175] as shown in Fig. 3 which
compares the two immobilization methods.
Lipase-producing bacteria, fungi and yeasts have been immobi-
lized to serve as a lipase biocatalyst (Table 2). Matsumoto et al.
[183] developed whole cell biocatalysts by immobilizing R. oryzae
cells, permeabilized by air drying. This biocatalyst was used in a
three-step addition of methanol (known as methanolysis) to plant
oil in a solvent- and water-free system at 37 C. The FAME yield
504 L.P. Christopher et al. / Applied Energy 119 (2014) 497520

(a)
Purification of Lipase Immobilization of Lipase
Extraction Cross linking Immobilized
Cultivation
Adsorption Covalent bonding Lipase Biocatalyst
Chromatography Entrapment
Crystallization

(b)

Cultivation
Whole Cell Lipase
&
Biocatalyst
Immobilization

Fig. 3. Immobilization methods used for production of enzymatic biodiesel: (a) lipase immobilization; (b) whole cell immobilization.

Table 2
Whole cell lipase biocatalysts used for biodiesel production.

Microbial source Support material Substrate Conditions Yield (%) Refs.

P. uorescens Na-alginatea Jatropha oil 40 C; 3:1 methanol: oil; hexane solvent; 48 h 72 [69]
Pseudomonas sp. Na-alginate Used cottonseed oil 37 C; 6:1 methanol: oil; step-wise; 48 h 70 [176]
R. mucilagenosa None Palm oil 30 C; 3:1 methanol: oil; 72 h 51 [177]
A. niger BSPsb Palm oil 25 C; 3:1 methanol: oil; step-wise; 72 h 87 [178]
A. niger BSPs Palm oil 40 C; 3:1 methanol: oil; 3 step-wise; 72 h 69 [179]
A. niger BSPs + GAc Palm oil 40 C; 3:1 methanol: oil; 3 step-wise; 72 h 69 [179]
R. oryzae BSPs Used cooking oil 35 C; 3:1 methanol: oil; 3 step-wise; 72 h 98 [180]
R. oryzae BSPs + GA Jatropha oil 30 C; 3:1 methanol: oil; 72 h 89 [181]
R. orzyae BSPs Soybean oil 35 C; 3:1 methanol: oil; 3 step-wise; 72 h 8085 [182]
R. orzyae BSPs + GA Soybean oil 35 C; 3:1 methanol: oil; 3 step-wise; 72 h 8992 [182]
a
Na-alginate, sodium alginate.
b
BSPs, biomass support particles.
c
GA, glutaraldehyde.

was 71 wt% after 165 h of reaction. Some of the researcher reported
that olive oil and oleic acid enhanced the methanolysis activity of
immobilized R. oryzae cells with a step-wise addition of methanol
(in presence of 15% water) yielding 90% biodiesel [175,184,185].
Cross-linking of R. oryzae cells with 0.1% glutaraldehyde (GA) stabi-
lized and maintained lipase activity at the same level even after six
batches of methanolysis, with ester yields of 7083% that repre-
sented a 20 to 33% increase over the control without cross-linking.
Also, Tamalampudi et al. [181] compared the performance of whole
cell R. oryzae immobilized on polyurethane biomass support parti-
cles (BSPs) to Novozym435 in methanolysis of jatropha oil. The
presence of water in jatropha oil had a positive effect on the meth-
anolysis with immobilized cells reaching a maximum yield of 89%
(Table 3) at 5% (v/v) water, whereas the activity of Novozym435
was inhibited.
Methanolysis of plant oil using a R. oryzae whole cell biocatalyst
was also investigated in shake asks and in a packed-bed reactor
[66,185]. The latter provided better results by protecting cells from
physical damage and excess methanol. The cells were immobilized
within 6  6  3 mm BSPs during batch cultivation in a 20 L air-lift
bioreactor. Emulsication of the reaction mixture at a ow rate of
25 L/h resulted in maximum FAME yields of 90%. R. oryzaecells
immobilized with polyurethane BSPs and treated with GA have
been extensively researched widely used as a whole cell biocatalyst
for production of biodiesel from various sources of non-edible and
low-cost feedstock [180,182,183]. Oda et al. [229] cultured R. oryzae
cells immobilized in BSPs in a 20 L air-lift bioreactor, and performed
repeated batches of methanolysis of soybean oil. They found that
the hydrolytic activity of the whole-cell biocatalyst was intact after
20 batches of methanolysis that produced 6580% ester yields. Li
et al. [230] studied biodiesel production from different feedstocks
(rened, crude and acidied rapeseed oil) in tertiary-butanol using
whole cell R. oryzae cells immobilized in BSPs. They found that the
reaction rate and nal ester yield were much greater when acidied
rapeseed oil was used. Furthermore, the increase in the FFA in oil,
the presence of phospholipids, and the use of adsorbents for water
removal improved the rate of reaction and biodiesel yields. From
this perspective, based on numerous literature reports, the lipase-
producing R. oryzaeseems to show great promise for further
research and development that could aid in reducing the cost of
biodiesel production [181,230,231].
In addition to R. oryzae, other lipase-producing fungal and yeast
whole cell catalysts have been also investigated in biodiesel pro-
duction: a newly isolated strain of A. niger JN [178]; a methanol-
tolerant yeast R. mucilagenosa [177]; Pseudomonas sp. including
P. zuorescens MTCC 103 [69,232]. A 72% biodiesel yield was ob-
tained in transesterication of Jatropha oil with P. uorescens MTCC
103 immobilized in sodium alginate gel (Table 3). The optimum
conditions were determined as follows: neutral pH, 40 C, oil/
methanol mole ratio of 1:4, 48 h [69,67]. Pseudomonas sp. was used
as a whole cell biocatalyst for biodiesel production from used cot-
tonseed oil: the FAME yield of 70% was attained by a step-wise
addition of methanol in excess (oil/methanol mole ratio of 1:6)
for 48 h [176]. Pseudomonas sp. immobilized in sodium alginate
gel was recommended for industrial use [69,232]. Lu et al. [233]
studied the transesterication of lard using immobilized Candida
sp. 99125 whole cells via a three-step methanolysis. They re-
ported that for processing 1 g of lard the optimum, loading condi-
tions are 0.2 g immobilized whole cell, 8 ml n-hexane as solvent,
20 wt% water, and 40 C. A biodiesel yield of 87.4% was obtained
and the whole cell lipase was stable for 180 h of repeated use.
3.4. Enzyme pretreatment
Pretreatment of lipase is believed to have a positive impact on
enzyme performance by: (1) providing a protective enzyme shield
 L.P. Christopher et al. / Applied Energy 119 (2014) 497520 505

Table 3
Lipase-catalyzed transesterication of oils to biodiesel.

Substrate Enzyme Operating conditions Solvent/ Acyl acceptor Yield Refs.

water (%)
Jatropha oil E. aerogenes lipase 55 C; 48 h NSa Methanol 94 [186]
Palm oil PS 30 Lipase 40 C; 8 h NS Ethanol 72 [187]
NS t-Butanol 62
NS 1-Butanol 42
NS n-Propanol 42
NS 1-Propanol 24
Coconut oil PS 30 Lipase 40 C, 8 h NS Ethanol 35
NS iso-Butanol 40
NS 1-Butanol 40
NS 1-Propanol 16
Soybean oil Novozym435 40 C;14 h NS Methyl acetate 92 [188]
Vegetable oils Lipozyme TL IM 25 C; 7 h NS Ethanol 84 [189]
Novozym435 Methanol >99
Plant oils R. oryzae lipase 35 C; 5 h Water (4 Methanol 8090 [190]
30%)
Rapeseed oil Lipozyme TL IM + Novozym435 35 C; 12 h t-Butanol Methanol 95 [191]
Microalgae Candida sp. lipase IM 38 C; 12 h Hexane Methanol 98 [192]
Sunower oil Pseudomonas lipase 45 C; 5 h Petroleum Methanol 79 [193]
ether
65 C; 5 h Petroleum Methanol 49
ether
45 C; 5 h Petroleum Ethanol 99
ether
Jatropha oil Novozym435 50 C; 8 h NS 2-Propanol 92.8 [194]
Karanj oil 91.7
Sunower oil 93.4
Jatropha oil Novozym435 50 C; 12 h NS Ethyl acetate 91.3 [195]
Karanj oil 90
Sunower oil 92.7
Plant oil Novozym435 Continuous reaction Petroleum Methanol 90 [196]
ether
Tallow oil Lipozyme IM60 45 C; 5 h Hexane Primary 94.8 [197]
alcohols 98.5
Novozym435 NS Secondary 61.2
alcohols 83.8
Lipozyme IM60 NS Methanol 19.4
Lipozyme IM60 NS Ethanol 65.5
Soybean oil IM P. cepacia lipase 35 C; 1 h Water Methanol 67 [154]
Ethanol 65
Cottonseed oil IM C. antarctica lipase 50 C; 24 h t-Butanol Methanol 97 [198]
Triolein Novozym435 6h NS Butanol 100 [199]
Lipozyme RM IM 100
Soybean oil Novozym435 30 C; 3.5 h NS Methanol 97 [200]
Soybean oil deodorizer distillate Novozym435 Molecular sieve adsorbent t-Butanol Methanol 95 [201]
reaction
Acid oil Novozym435 24 h NS Methanol 90 [202]
Waste edible oil Novozym435 Fixed bed reactor NS Methanol 90 [203]
Degummed soybean oil Novozym435 30 C; 6 h NS Methanol 93.8 [204]
Sunower oil Novozym435 NS NS Methanol 97 [205]
Sunower oil Novozym435 45 C; 50 h NS Methanol >99 [206]
Methyl acetate 95.65
Plant oil R. oryzae lipase NS NS Methanol 90 [175]
Soybean oil IM R. oryzae lipase RTb; PBRc NS Methanol 90 [185]
Triolein IM P. ourescens lipase 50 C; 25 h NS Butanol 90 [207]
Soybean oil Lipozyme TL IM 40 C; 12 h NS Methanol 98 [208]
Waste oil adsorbed on activated C. cylindracea lipase 25 C; 12 h Diesel oil Methanol 97 [209]
bleaching earth
Palm oil from waste bleaching earths R. oryzae lipase 35 C; 96 h NS Methanol 55 [210]
Grease PS 30 38.4 C; 2.47 h NS Ethanol 85.4 [211]
Soybean oil Recombinant LipB68 P. uorescens 20 C; 12 h NS Methanol 92 [212]
lipase

(continued on next page)

506 L.P. Christopher et al. / Applied Energy 119 (2014) 497520

Table 3 (continued)

Substrate Enzyme Operating conditions Solvent/ Acyl acceptor Yield Refs.

water (%)

Rice bran oil Cryptococcus spp. S-2 30 C; 96 h Water (40%) Methanol 80 [213]
Waste activated bleaching earth C. cylindracea lipase 37 C; 3 h Diesel oil Methanol 100 [214]
Rened soybean oil Novozym435 40 C; 10 h NS Methyl acetate 92 [215]
Rapeseed oil C. rugosa lipase 37 C; 24 h 2-Ethyl-1- 97 [216]
hexanol
Soybean oil Lipozyme IM-77 NS n-Hexane Methanol 92.2 [217]
Mowrah oil Lipozyme IM-20 60 C; 6 h Water (10%) Alcohols (C4 86.8 [218]
C18) 99.2
Mango oil
Kernel oil
Sal oil
Sunower oil Lipozyme 50 C; 5 h NS Ethanol 83 [219]
Fish oils C. antarctica lipase RT; 22 h NS Ethanol 100 [220]
Soybean oil P. uorescens lipase 35 C; 90 h NS Methanol 80 [190]
Soybean oil C. rugosa lipase 35 C; 90 h NS Methanol 80
Soybean oil P. cepacia lipase 35 C; 90 h NS Methanol 100
Sunower oil R. miehei lipase 40 C; 48 h NS Methanol 95.5 [221]
Sunower oil T. lanuginosus lipase 40 C; 48 h NS Methanol 92.3
Sunower oil IM C. antarctica lipase 50 C; 12 h NS Ethyl acetate 63.3 [222]
Soybean oil IM M. miehei lipase 35 C; 8 h n-Hexane Ethanol 95.6 [223]
Sunower oil IM T. lanuginosus lipase 30 C; 6 h n-Hexane Ethanol 1635 [223]
Crude palm oil Lipozyme RM IM 50 C; 4 h NS Methanol 12 [224]
Lipozyme TL IM NS Methanol 15
Lipozyme RM IM NS Ethanol 16
Lipozyme TL IM NS Ethanol 25
Soybean oil deodorizer distillate Novozym435 50 C; 1.5 h NS Ethanol 83.5 [225]
Jatropha oil IM P. uorescens lipase 40 C; 48 h n-Hexane Methanol 71 [69]
Corn oil P. expansum lipase 40 C; 24 h Ionic liquids Methanol 86 [226]
Palm oil IM B. cepacia lipase 30 C; 72 h NS Methanol 100 [227]
Fats and oils IM T. lanuginosus lipase 50 C; 48 h NS Ethanol/ 70100 [228]
methanol
Restaurant grease IM T. lanuginosus lipase 50 C; 48 h NS Ethanol/ 8090
methanol
a
NS, not specied.
b
RT, room temperature.
c
PBR, packed bed reactor.

to minimize enzyme deactivation caused by lower alcohols
(methanol and ethanol) and glycerol [4,200,234], and (2) enhanc-
ing enzyme activity by maintaining the lipase molecule conforma-
tion in its active form, or reversing the conformation of the active
enzyme sites from close to open [167,235]. The overall effect of li-
pase pretreatment is regeneration of enzyme activity and im-
proved mass transfer and transesterication rates, resulting in
enhanced productivity [236,237], however, the exact mechanism
of enzyme pretreatment is not clearly understood [238].
The most commonly used pretreatment agents include alcohols,
solvents, ethers and salts. Washing with t-butanol and 2-butanol
affected a ten-fold increase in Novozym435 activity [234]. In
comparison, the enzyme was completely deactivated by methanol
without washing. Pretreatment of the completely deactivated en-
zyme with tert-butanol and 2-butanol restored the lipase activities
of their original levels of 56% and 75%, respectively. Incubation of
immobilized C. antarctica in methyl oleate for 30 min, followed
by a step-wise addition of methanol to soybean oil increased the
FAME yields to 97% [200]. Shah et al. [239] reported a 45% increase
in ester conversion (from 34% to 79%) of jatropha oil when an
immobilized lipase from P. uorescens was pretreated via irradia-
tion in presence of an aqueous buffer and organic solvents.
Pretreatment of immobilized Novozym435 using ultrasonic irra-
diation with vibration resulted in 96% methyl ester yields after ve
repeated cycles [240]. Pretreatment of R. oryzae lipase with
soybean oil before immobilization increased the lipase activity
20-times over that of the non-treated lipase thereby maintaining
it at levels exceeding 90% of its original activity after 10 reuses
[241]. Salts as pretreatment agents including calcium or magne-
sium chloride are thought to stabilize the protein molecule and en-
hance its resistance toward methanol inhibition [235].
3.5. Biodiesel feedstock
The choice of feedstocks depends on the process chemistry,
physical and chemical characteristics of virgin or used oils and
economy of the process. First-generation biodiesel has been pro-
duced from vegetable oils extracted from oilgeneous plants like
sunower, soya, canola and palm (Table 3) [242]. At present, cano-
la, soybean and palm oil constitute about 75% of the world vegeta-
ble oil supply. In the rst half of 2013, they traded for $1150, $1050
and $755 on average per metric ton, respectively. Canola oil is the
primary source for biodiesel production globally, whereas soybean
oil is the largest biodiesel feedstock in the US which is also ex-
change-traded. Soybeans are widely grown in the US for their high
protein and lipid content. Because of the value of the products and
 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
the ability to be used in crop rotations with nitrogen-intensive
crops such as corn, soybean oil now accounts for more than 50%
of all bio-based oils in the US. Although soybean oil yields only
48 gallons/acre/year, 2 billion gallons were harvested from more
than 30 million ha in 2002 [35]. Nowadays soybean oil makes up
25% of the total soybean oil demand in the US [14]. Biodiesel qual-
ity is directly inuenced by the fatty acid composition of vegetable
oils [243]. Low cetane numbers have been associated with the
presence of unsaturated fatty acids (C18:2 and C18:3) that are typ-
ically found in soybean oils, whereas saturated fatty acids such as
palmitic (C16:0) and stearic (C18:0) acid increase the cetane num-
ber of biodiesel produced from palm oil [244].
The main constraint for biodiesel production is the cost of the
feedstock. It has been estimated that up to 80% of the total biodie-
sel production cost arises from the cost of raw material [245247].
The high value of edible vegetable oils as a food product makes
production of a cost-effective biodiesel fuel very challenging. In
addition, the recent increase in the use of vegetable (edible) oils
for biodiesel production has caused some major controversy owing
to their inuence on the global imbalance to food market and food
security [248]. Furthermore, there are large amounts of low-cost
feedstock such as waste cooking oils and animal fats that could
be converted to biodiesel [167] thereby also helping solve the
problem of waste oil disposal [249]. This has historically led to
the production of the second-generation biodiesel that is derived
from non-edible oils. In comparison to edible oils, the major short-
comings in the use of non-edible oils are the relatively low oil
yields (with a few exceptions) and feedstock quality. For example,
the yield of palm oil peaks at 5000 kg oil/hectare, whereas yields
from non-edible oils such as jatropha and pongamia pinnata are
250-fold lower, and range from 100 to 2300 kg oil/hectare. There-
fore, in some instances, the use of non-edible oils for biodiesel pro-
duction would require large plantation areas. However, in
comparison, the oil yield of jatropha oil is nearly 5-times higher
than that that of soybean oil (475 kg/hectare), whereas macauba
can produce 10-times more oil than soybean. Furthermore, due
to the presence of high FFA in non-edible oils, biodiesel production
from this feedstock, the use of enzyme catalysts has been favored
[248]. Nevertheless, inedible oils are a viable alternative to edible
vegetable oils due to their lower price and wide adaptability with
minimal production requirements. For instance, jatropha can grow
on waste, sandy and saline soils under a wide variety of climatic
conditions (severe heat, low rainfall, high rainfall and frost), and
produce up to 60 wt% oil in its seeds and kernels [250]. Because
of that, Jatropha oil, despite its toxicity to humans, is regarded as
a promising potential feedstock for biodiesel production in Asia,
Europe and Africa [244]. The advantages of non-edible oils over
their edible counterparts are their lower price, their availability
and portability, higher caloric value and lower sulfur and aro-
matic content. Drawbacks of inedible oils include higher reactivity
due to higher content of unsaturated fatty acids, higher viscosity
and carbon residue content, and lower volatility [251,252].
The waste cooking oils are directly accessible for biodiesel pro-
duction (Table 3) and their quantity is mainly relying on the
amount of edible oil consumed. Cooking oils contain many types
of vegetable-based oils as well as rendered animal oils. There are
enough used cooking oils and fats generated in the US annually,
including 18 billion pounds of soybean oil and 11 billion pounds
of animal fat, to produce an estimated 5 billion gallons of biodiesel
[253]. The cost of waste cooking oil is calculated from the collec-
tion, transportation and pretreatment costs, which are minimal
compared to those for edible oils. The physical properties and
chemical composition of waste cooking oil vary depending on the
oil source and the content of water (0.75%) and FFA (56%) that
is comparatively higher than virgin oil (<0.8% FFA) [248]. Waste
cooking oils having less than 15% free fatty acids (FFA) as a by-
507
product of oxidation during cooking are considered yellow grease.
If the FFA content is higher than 15%, as might occur particularly in
the summer months during storage of waste grease, they are con-
sidered lower value brown grease. It has been reported that the
FAME of oleic acid show greater stability and improved biodiesel
properties [249], hence, waste cooking oils with high content of
oleic acid would be a preferred biodiesel substrate. As discussed
earlier, waste cooking oils with high FFA content can be easily con-
verted to biodiesel in a single step as lipase is capable of catalyzing
both transesterication of TG and interesterication of FFA [250].
Lipases exhibit greater stability and performance in FFA-rich sub-
strates such as waste cooking oils than in TG-rich vegetable oils
[251]. Moreover, crude vegetable oils contain appreciable amounts
of phospholipids that were found to diminish the lipase perfor-
mance during biodiesel production [204]. These phospholipids
can be removed in a degumming pretreatment step which how-
ever adds to the cost of biodiesel production [252].
Animal feedstocks for biodiesel production include tallow, poul-
try fat, pork fat, yellow grease, lard and sh oil [253]. Animal feed-
stocks are priced favorably for a cost-efcient conversion to
biodiesel, however, their availability is limited [252]. The U.S.
Department of Agriculture predicts a continued growth of 1% per
year with a guaranteed and steady supply of tallow and fats. Bio-
diesel produced from animal fats and vegetable oil has a similar
chemical content with some variation in the component propor-
tions [254] which however results in differences in properties of
vegetable oil-derived and animal fat-derived biodiese. The cetane
number of animal fat-derived biodiesel is up to 15% higher than
that of soybean-based and conventional biodiesel. In addition to
improving fuel combustion, increasing cetane levels often reduce
emissions of NOx and particulate matter. Animal fat-based biodie-
sel also provides added lubricity, a measure of protective com-
pounds in the fuel that reduce engine wear and tear. The higher
percentage of saturated fats in animal fat-based biodiesel: (1) pro-
vides greater oxidative stability than its plant oil-based counter-
parts, reducing the risk of sedimentation [254]; (2) reduces the
low-temperature uidity thereby leading to an increase in the cold
lter plugging point of biodiesel [254] because the high levels of
saturates crystallize more readily. Hence, the use of additives and
blends with vegetable oil biodiesel could reduce temperatures be-
low the cloud point. Currently, about 11 billion pounds of rendered
fats are produced annually of which only a small fraction of 38% is
used to produce about 116 million gallons of biodiesel. The tax
incentive for biodiesel made from inedible animal fats and used
vegetable oils is 50/gallon, and it takes about 7.5 lb of waste oils
and fats for each gallon of biodiesel produced [255]. Microalgae
are regarded as the biodiesel feedstock of the future and the only
renewable source of renewable biodiesel that can meet the global
demand for transportation fuels [256]. It has been estimated that
only 2% of the existing US cropping area would be required to pro-
duce microalgae with 3050 wt% oil content that could potentially
replace about a third of the US annual demand for transportation
fuels. To completely replace all transportation fuels of about 180
billion gallons of gasoline and diesel, less than 5% of the cropping
area would be needed for algae production [256]. Microalgae have
lower water requirements compared to soybeans and canola [257].
They can accumulate more than 80% water-free oil of the algal dry
biomass and produce over 250 times greater oil yields per acre
than soybean water-free oil [256,258]. The exploitation of microal-
gae for biodiesel has some apparent advantages over other feed-
stocks: high oil yields; production from carbon dioxide and
sunlight only; use of non-arable land. They can grow in salty and
waste water, and have high growth rates with a generation time
of 24 h [259,260] which allows multiple harvesting during the
year. However, major obstacles to commercialization are the light
intensity requirements, nitrogen deciency, and technological
508 L.P. Christopher et al. / Applied Energy 119 (2014) 497520

challenges related to harvesting and oil extraction from algal reported by several researchers for soybean oil [198], jatropha oil
biomass [247,261]. Harvesting contributes 2040% of the total cost [261] and canola oil [273]. Immobilized C. antarctica lipase medi-
of biomass production [262]. In addition, the microalgae contain ated transesterications of vegetable oil with a maximum conver-
high levels of polyunsaturated fatty acids with four or more double sion of 95% achieved at a methanol/oil molar ratio of 3:1 [274].
bands [263] as eicosapentanoic acid (C20) and docosahexaenoic Methanolysis of Simarouba oil with a fungal immobilized lipase
acid (C22) which are unstable and prone to oxidation during stor- produced a maximum yield of 91.5% FAME using methanol as acyl
age that diminishes biodiesel performance [256]. acceptor [275]. A 95% yield of methyl esters of rape seed oil was
As biodiesel runs in normal compression engines or diesel en- achieved with methanol/oil molar ratio of 4:1 [191]. Biodiesel pro-
gines, its physicochemical properties should meet the quality duction from transgenic corn oil using Lipozyme TL IM peaked at a
requirements that are applicable to the conventional diesel fuel methanol to oil ratio of 6 to 1 [268].
(petrodiesel) [256]. The US and European standards for biodiesel For most of the transesterication reactions, methanol is com-
are stipulated in the American Society for Testing and Materials monly used because of its reactivity, volatile nature, and lower cost
(ASTM) D6751 and EN 14214, respectively [258]. Feedstock than other alcohols. However, methanol is mostly produced from
properties like FFA composition and content, moisture content, non-renewable sources such as natural gas or coal, and is toxic.
unsaponied compounds, impurities, etc. inuence the engine Most lipases can be at least partially inactivated by methanol that
performance of biodiesel [244,249]. A considerable amount of liter- affects their catalytic performance [234]. The degree of lipase deac-
ature has been published on testing the physiochemical properties tivation is believed to be inversely proportional to the number of
of biodiesel using chemical catalysts [259]. Recent studies com- carbon atoms in the linear lower alcohols [4]. On the other hand,
pared the properties of biodiesel obtained from enzymatic transe- the rate of transesterication was reported to increase proportion-
sterication with those of chemically-catalyzed biodiesel and ally to the number of carbon atoms in the alcohol [250]. Therefore,
concluded that enzymatic biodiesel closely meets the stringent ethanol as a longer carbon chain alcohol, presents an eco-friendly
quality requirements of EU and US standards [260,262]. Properties and renewable alternative that is less inhibitory than methanol.
of enzymatic biodiesel such as density, ash point, pour point, However, with ethanol as the acyl acceptor, the lipase activity of
heating value, viscosity and ash content have met the ASTM bio- Novozym435 was completely lost after only six cycles of enzyme
diesel standard 6751-03 (Table 4). reuse [195]. The enzyme activity of Novozym435 with different
substrates and acyl acceptors is shown in Table 5.
Enzyme inhibition by methanol can be overcome by overdosing
3.6. Acyl acceptor
the lipase, which however is not a cost-effective solution to the
problem [18]. It was also reported that some lipases (from Pseudo-
The commercial production of biodiesel requires the use of inex-
monas) were more tolerant to alcohol than others (e.g. from T.
pensive and readily available acyl acceptors such as methanol and
lanuginosus and R. miehei) [18,37]. Three strategies have been
ethanol. Stoichiometrically, the alcoholysis of any oil requires three
developed to minimize alcohol inhibition: (1) stepwise or sequen-
moles of alcohol for each mole of oil. Since transesterication is a
tial addition of alcohol or alcohol aliquots [224,277]; (2) use of sol-
reversible reaction, an excess of alcohol is required to shift the equi-
vents [195]; (3) use of alternative acyl acceptors such as longer
librium to FAAE formation [265]. The yield of biodiesel increases
chain alcohols and alkyl esters [215].
with the increase in the alcohol concentration up to a certain con-
The step-wise addition of alcohol maintains the alcohol concen-
centration, depending on the particular operating conditions such
tration below the critical level to avoid loss of solubility in the oil
as the type and amount of alcohol, biocatalyst, temperature, mixing
and undesirable deactivation of the enzyme. Research has focused
intensity used, the moisture and FFA content of the substrate, and
on stepwise addition of methanol as the most commonly used acyl
mode of bioreactor operation [266]. Overall, the lipase biocatalysts
acceptor with the greatest inhibitory effect on lipase. Shimada
require lower alcohol to oil ratio than chemical catalysts [181,267].
et al. [274] rst used stepwise addition of methanol and attained
Generally, molar ratios of alcohol to oil of between 3:1 and 6:1 have
a 95% conversion after 50 cycles of operation. Watanabe et al.
been commonly used [268270]. A further increase of the alcohol
[278] reported a 90% yield using a two-step batch-wise addition
content beyond the optimum concentration does not improve the
of methanol. The yield was maintained even after 100 batches of
biodiesel yield, however, it has a negative impact on the costs of
operation. Similarly, a 95% conversion was observed following a
alcohol recovery [269,271]. High alcohol to oil molar ratios may in-
step-wise addition of methanol after 50 batches [274]. A biodiesel
hibit lipase activity and interfere in the separation of glycerol due to
yield of as high as 97% was obtained from plant oil by a three-step
its increased solubility in ethanol [268,272]. The effect of methanol
addition of 0.33 M equivalents of methanol at 0.250.4 h intervals
to oil ratio on the FAAE yield catalyzed by immobilized lipase was
[200]. Kaieda et al. [190] reported that the stepwise addition of
methanol prevented inhibition of non-regiospecic lipases such
Table 4 as P. cepacia, C. rugosa and P. ourescens and regiospecic lipases
Properties of enzymatic biodiesel. such as R. oryzae. Yields of 90% were obtained by step-wise addi-
Parameters Biodiesel Biodiesel Biodiesel Biodiesel tion of methanol to waste cooking oil [277]. Similarly, during batch
from from from standard and continuous transesterication with immobilized
stillingia jatropha waste (ASTM
oil [263] oil [262] cooking D6751-
oil [260] 03) [264]
Table 5
Density (g/cm3) 0.900 0.8924 0.87 Acyl acceptors used with Novozym435 in biodiesel production.
(20 C) 0.90
(15 C) Plant oil Acyl Conditions Enzyme activity Refs.
Pour point (C) 5 15 to acceptor
10 Soybean Ethanol 25 C; 7 h 85% After 9 [189]
Flash point (C) 137 82 195 P130 batches
Heating value (MJ/kg) 36.5 3340 Olive Methanol Methanol step-wise 70% After 8 [276]
Viscosity (mm2/s) 4.81 8.2 9.12 1.96 addition batches
(40 C) (20 C) (40 C) Sunower Ethyl Immobilized enzyme; 85% After 12 [195]
Ash content (wt%) 0.037 0.003 60.02 acetate 25 C batches
 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
Novozym435 using step-wise addition of methanol to waste
cooking oil, 96% and 93% yields were achieved for the batch and
continuous process, respectively, with lipase retaining full activity
after 20 days of continuous operation [196]. Yields of 98% were re-
ported for transesterication of soybean oil with T. lanuginosus li-
pase using a step-wise addition of methanol, while iso-propanol
was used to remove glycerol [208]. A 34% increase in the conver-
sion was achieved through a stepwise addition of methanol com-
pared to a batch methanolysis of olive oil [279].
Solvents exert multiple effects on both reactants and products in
biodiesel production. These effects include: (1) increased solubility
of alcohol that protects lipase from denaturation [262]; (2) increased
solubility of glycerol that prevents lipase inhibition [198]; (3) crea-
tion of a single phase reaction mixture that improves mass transfer
and reaction rates, reduces viscosity, and stabilizes immobilized en-
zymes [4,18]. The enzyme stabilization is due to increased water
activity in the vicinity of the enzyme that is caused by the sol-
ventwater immiscibility. The polar, less hydrophobic solvents are
not suitable for biocatalytic processes since they can distort the
water microlayer around the enzyme, inuencing its native struc-
ture and leading to denaturation [280]. Organic solvents such as
hexane, ionic liquids and t-butanol have been widely used
[70,198]. Most of the literature concentrated on the use of solvents
with commercial enzymes for production of biodiesel from vegeta-
ble oils. Transesterication of sunower oil with methanol, ethanol
and butanol was studied using Novozym435 with and without
petroleum ether as the solvent [193]. The petroleum ether did not
impact the product yields in presence of ethanol and butanol (as acyl
acceptors), however, it was required for biodiesel production with
methanol. Similar ndings pointed out to the need of a solvent (hex-
ane) in a study of methanolysis of soybean oil, rapeseed oil, tallow,
and recycled restaurant grease catalyzed by M. miehei lipase (Lipo-
zyme IM 60) and C. antarctica (SP 435) [197]. This was attributed
to the inhibition of immobilized enzymes by methanol. The use of
a common solvent for both methanol and oil has proved to be effec-
tive [191,198,207]. Iso et al. [207] investigated the effects of 1,4-
dioxane, benzene, chloroform and tetrahydrofuran as common
solvents for methanolysis with lipase enzymes from
P. uorescens (Lipase AK), P. cepacia (Lipase PS), M. javanicus
(Lipase M), C. rugosa (Lipase AY) and R. niveus (Newlase F). Higher
conversions of rapeseed oil of up to 97% were reported using mixture
of 2-ehtyl-1-hexanol [216]. Lipase AK achieved high conversion
rates without loss of activity when 1,4-dioxane was used as solvent.
Use of tert-butanol resulted in higher yields of methyl esters
[191,198]. Studies showed that often excess alcohols are used to
shift the equilibrium towards products [37,41]. Although similar
biodiesel yields have been obtained in presence and absence of sol-
vent [18,262,280], the use of solvents signicantly increases the
reaction rate in comparison to solvent-free systems. However, the
use of solvents is not economically and environmentally favored
as it requires the addition of a solvent recovery unit for separating
the organic solvent from the reaction mixture, which leads to in-
crease in cost of recovery, losses and gas emissions [247].
Acyl acceptor alternatives to methanol include primary, second-
ary, straight chained and branched alcohols such as isopropanol
[281], t-butanol [191,198], and octanol [67]. The choice of alcohol
can inuence the cold ow properties [282] and lubricity [283] of
biodiesel. The longer chain alcohols have shown higher yields than
methanol [64] as lipases are known to have a higher afnity to-
ward long-chain than short-chain alcohols [65].
3.7. Enzyme operating variables
Generally, transesterication is carried out below the boiling
point of the alcohol used order to prevent the alcohol evaporation.
In the case of methanol and ethanol, the boiling temperatures cor-
509
respond to 65 C and 78 C, respectively. Overall, higher reaction
temperatures reduce oil viscosity, increase the reaction rates and
shorten the reaction time [41]. As most lipases have their temper-
ature optimum between 30 and 60 C, the enzymatic transesteri-
cation is mostly conducted in that temperature range. The free
lipase enzymes, especially bacterial lipases, are known for higher
thermal stability [67]. Lipase immobilization to solid supports de-
creases the effect of temperature deactivation which leads to im-
proved thermostability [18] and increased temperature optimum
of lipase [284]. Overall, the optimum temperature is inuenced
by the enzyme stability, alcohol to oil molar ratio and the type of
organic solvent used [250]. The initial rate of reaction increases
with reaction temperature, however, conversion rates decrease at
temperatures above the optimal due to thermal deactivation of li-
pase [285,286]. A broad range of operational temperatures has
been employed depending on the enzyme source: 2555 C for
C. antarctica IM lipase (temperature optimum of 40 C) [287];
2070 C for P. cepacia IM lipase (50 C) [199]; 2060 C for R. chin-
ensis whole-cell IM lipase (30 C) [288]. The optimal temperature
for lipid transesterications with a mixture of R. oryzae and C. rug-
osa IM lipases was 45 C [289]. Most frequently, the optimum oper-
ating temperature for lipases has been reported to be 40 C on
average [290].
Bacterial lipases have neutral or alkaline pH optima [141,291],
whereas lipases from yeasts and fungi are more active at the near
neutral to slightly acidic pH [292294]. However, some lipases are
active and stable over a broad pH range (pH 312) [295]. Overall,
bacterial lipases have been reported to exhibit a higher activity
and thermostability than other microbial lipases [141,284,291].
The protein conformation of lipase is affected by pH, and upon
immobilization, the optimum pH for reactions catalyzed by lipases
is slightly shifted toward more alkaline values due to the partial
opening of the lid at the enzyme active site [284]. In recent years,
research has focused on the use of lipase-producing fungi as a
source of extracellular lipase, or as a whole cell biocatalyst
[19,277,296]. In terms of cost effectiveness of the biodiesel process,
lamentous fungi have appeared as the most prominent whole cell
biocatalyst for industrial applications [66].
Overall, the reaction times needed for enzymatic transesteri-
cation, although dependant on reaction temperature, reactant
and catalyst concentrations, are generally longer (36 h on average)
than those of the chemically-catalyzed process (9 h) [19]. A simpli-
ed model of the reaction behavior can be presented to explain the
accumulation of FAME as a function of the reaction time: (1) a slow
initial rate to allow for mixing and diffusion of alcohol into the oil;
(2) rate of fatty acid esters conversion increases exponentially with
reaction time [41,45]; (3) reaction rate reaches a peak and pro-
ceeds at a steady state [43,216]; (4) reaction rate declines [18].
Immobilized B. cepacia lipase produced a 90% biodiesel from used
Jatropha oil yield after a 12 h reaction time [297]. Methanolysis
of Simarouba oil and vegetable oils with immobilized lipase was
optimum at 36 h [275] and 48 h [274], respectively. Extending
the reaction time beyond that required for optimum biodiesel pro-
duction can reduce the biodiesel yields due to enzyme inhibition
by methanol and glycerol [275,298].
Water maintains the three dimensional structure of the enzyme
[285] and can directly affect the enzyme structure and activity
[299]. The latter is determined by the amount of enzyme-bound
water which is best dened as water activity [250,300]. The opti-
mal protein conformation is disturbed by removal of water that
surrounds the enzyme macromolecule, whereas excess of water
forms water droplets within the enzyme active site. The hydrolysis
reaction, that competes transesterication, is catalyzed by increas-
ing amounts of water in the reaction mixture [301]. Generally,
minimum amount of water is necessary for biocatalytic activity
that is specic for a given lipase [167]. No transesterication is pos-
510 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
sible in absence of water as the formation of enzyme substrate
complexes (lipase-oil) proceeds only in presence of oilwater
interface. A optimum water content, minimum hydrolysis and
maximum transesterication occur [154,167]. The reaction rate
was reported to increase proportionally to the water content in
many studies [18,65,302,303]. The optimal water content for R.
chinensis lipase [288], C. antarcticaand T. lanuginosalipase [191]
was 2% whereas 20% water was required for maximum transeste-
rication by Candida sp. 99125 lipase [285].
3.8. Bioreactor design
The batch stirred tank reactor (STR) [45,304] and packed-bed
reactor (PBR) [196,277,278,305] are the most extensively studied
bioreactors for enzymatic biodiesel production. In STR, the enzyme
(free or immobilized) is dispersed in the reaction mixture by agita-
tion, whereas in the PBR, the immobilized enzyme is packed into a
column. The batch STR is the simplest bioreactor type consisting of
a reactor and a propeller. It is suitable high-viscous solutions and
immobilized enzymes that are less sensitive to shear stress and
deactivation upon physical stirring. A solidliquid separation using
centrifugation or ltration is normally applied at the end of the
process to recover the immobilized enzyme. The STR operated in
a batch mode has a low throughput due to the need to empty, clean
and reload the reactor before a new batch can start. The low pro-
ductivity disadvantage of the batch STR can be eliminated by using
a continuous STR where the enzyme is retained in the reactor with
a lter placed at the reactor outlet. The PBR can operate in batch or
continuous mode by recirculating the reaction mixture. The recy-
cling method is advantageous as it allows the substrate solution
to be passed through the column at a desired velocity. For indus-
trial applications, the upward substrate ow is generally preferred
over downward ow because it does not compress the enzyme bed
that results in blockages with poor oxygen transfer and pressure
drops [18]. In the continuous PBR, reaction mixture is continuously
pumped through the column and the enzyme can be effectively re-
used without a prior separation. Advantages of using continuous
PBR include high efciency, low cost and ease of construction,
operation and maintenance. In addition, it allows for continuous
removal of glycerol and excess alcohol, and protects the enzyme
particles from mechanical shear stress [197,198]. The continuous
PBR is superior to the batch PBR due to automated control and
operation, reduced labor costs, stable operating conditions, and
easy quality control of products [18,306]. Other bioreactor cong-
urations include uid beds, expanding bed, recirculation mem-
brane reactors, and static mixers [18]. In the membrane
bioreactors, the enzyme is immobilized onto at sheet or hollow
ber membranes; however, membrane fouling presents a problem
that is more challenging than the bed plugging in the PBR.
Although most of the existing biodiesel plants are currently
running in batch mode with stirred tank reactors, recent research
efforts have focused on optimization of PBR for use in different
enzymatic biodiesel production processes [30,179,203,307,308].
Chen et al. [308] tested a PBR for continuous biodiesel production
using methanolysis of soybean oil in a t-butanol solvent system
catalyzed by Novozym435. A molar conversion of 83% was at-
tained with no considerable decline in lipase activity in continuous
operation for 30 days at a ow rate of 0.1 ml/min, 52 C and a 4:1
methanol to oil molar ratio. A 88% conversion was reached in a
continuous PBR system catalyzed by a lipase nanoparticles com-
posite (lipase-Fe3O4) using a mixture of soybean oil, distilled water,
methanol and n-hexane with volumetric ratio of 6:3:1:0.2, respec-
tively, at a ow rate of 0.25 ml/min for 192 h [307]. PBR was used
for production of enzymatic biodiesel from waste cooking oil with
a FAME yield of 90% using a solvent-free system [203]. The
stepwise addition of methanol prevented the deactivation of the
lipase, which allowed a constant enzyme activity during 100 days
of operation. Transesterication of soybean oil in a PBR at a ow
rate of 25 l/h using a R. oryzae whole-cell immobilized catalyst
and a step-wise addition of methanol produced a maximum FAME
yield of 90% [184]. A whole-cell biocatalyst of A. niger immobilized
in a PBR produced 90% FAME from palm oil at a ow rate of 15 l/h
by continuous recycling of the reaction mixture for 72 h [179]. The
biodiesel yield dropped to 85% after 4 consecutive cycles. An opti-
mal continuous production of biodiesel by methanolysis of soy-
bean oil in a packed-bed reactor was developed using response
surface methodology (RSM) and Box-Behnken design with immo-
bilized lipase (Novozym435) as a catalyst in a t-butanol solvent
system [308]. The continuous process over 30 days showed no
appreciable decrease in the molar conversion of 83.3%.
Production of biodiesel from vegetable oils with a high FFA con-
tent requires an additional step for separation of the reaction by-
products from the biodiesel product. This can be achieved by
water-washing or water-free washing of biodiesel [309]. The
water-free washing process is performed by a direct mixing of
the transesterication reaction products and adsorbent particles
or of biodiesel washing pellets. The biodiesel uid containing the
adsorbent particles subsequently requires ltration that is both
monotonous and time consuming. Alternatively, a water-free
washing in PBR has been adopted as a method that effects auto-
matic separation of the adsorbents. Several parameters are fac-
tored into the design of a packed bed water-free washing
biodiesel separator such as the ease with which the biodiesel
stream ows through the bed; bed porosity; adsorption dynamics
of the adsorbents; ratio of convective to ow rate, etc. It should be
noted that the washing requirement complicates the conventional
biodiesel production, but is not a challenge in the production of
enzymatic biodiesel with minimal impurities [12].
3.9. Glycerol
Due to its hydrophilicity, glycerol, the co-product in biodiesel
production, is insoluble in the oils and gets easily adsorbed onto
the surface of the immobilized lipase. This creates mass transfer
limitations that negatively impact lipase activity and stability.
Adding silica gel into the reaction system to absorb the glycerol
or periodically washing the lipase with organic solvents to regen-
erate the lipase activity have been proposed. Studies by Bako-Bela
et al. [205] employed dialysis for glycerol removal which resulted
in a 97% conversion by methanolysis. However, these methods are
impractical for large-scale continuous production of biodiesel. As
discussed before, a novel interesterication process, that makes
use of methyl or ethyl acetate as the acyl acceptor instead of pure
methanol, does not produce glycerol [215]. The triacetylglycerol
co-product does not interfere with the enzyme catalyst or the bio-
diesel main product, which also eliminates the need of complicated
downstream processing.
The stoichiometric yield of glycerol is 10% (w/w) with respect to
the biodiesel produced. Following methanol recovery, glycerol
with up to 90% purity can be used as a marketable commodity.
Most commercial biodiesel manufacturing companies are able to
send the glycerol to a glycerol recovery/rening facility. Pure
grades of glycerol (99.7%) can be used as a raw material in other
industrial sectors such as food, cosmetics, paints, pharmaceutics,
paper, textiles, leather, toiletries, toothpaste, drugs, animal feed,
plasticizers, tobacco, and emulsiers, and for the production of
various chemicals [310]. The increase in biodiesel production is
expected to result in excess of glycerol which has a limited market
thereby reducing the price of glycerol. This necessitates the
development of alternative and viable processes for glycerol as a
value-added co-product [311] that would impact positively on
the overall production cost of biodiesel [312]. For instance, there
 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
is a growing interest in glycerol fermentation to methanol and eth-
anol used in biodiesel production, which would improve the eco-
nomics of the biodiesel process [189,313317].
Fig. 4 shows some major economically-important biochemicals
that can be produced from glycerol-based biorenery setup. Glyc-
erol has been used as an alternative and inexpensive carbon source
in the microbial production of various compounds such as citric
acid, succinic acid, 1,3 propanediol, 2,3-butanediol, carotenoids,
hydrogen, biosurfactants, etc. [318,319]. Using the yeast species
Yarrowia lipolytica, Rymowicz et al.[320] studied the production
of citric acid from raw glycerol derived from the biodiesel produc-
tion of rapeseed oil. A maximum yield of 0.62 g citric acid/g of glyc-
erol was reported. A nal concentration of 1,3-propanediol of
51.3 g/l and 53.0 g/l was obtained with Klebsiella pneumoniae from
crude glycerol (derived from methanolysis of soybean oil) using al-
kali- and lipase-catalyzed process, respectively [321]. Another
strain of K. pneumonia, ATCC 15380, was optimized for production
of 1,3 propanediol from glycerol derived from biodiesel production
of non-edible jatropha oil [322]. The bacterial strain E. coli AC-521
produced lactic acid from crude glycerol with a high yield of
0.9 mol/mol glycerol [323]. Anaerobic digestion of crude acidied
glycerol yielded 0.306 m3 methane/kg glycerol [324]. The natural
occurring polymer polyhydroxybutryate (PHB, also known as bac-
terial polyester, can be synthesized from crude glycerol instead of
glucose as carbon source by Paracoccus denitricans and Cupria vid-
usnecator JMP 134 bacterial strains [325].
Using convention catalysts in addition to microbial conversion,
chemical compounds with a diverse range of industrial applica-
tions can be synthesized from biodiesel glycerol. These include
among others acrolein[326], ethylene glycol [327], syngas [328],
(2,2-dimethyl-1,3-dioxolan-4-yl) methyl acetate [329] and oligo-
mers of glycerol [330]. A 74 mol% acrolein with 81% of selectivity
was synthesized from glycerol using an acid catalyst at supercriti-
cal conditions (673 K and 34.5 MPa pressure) [331]. The fuel
additive (2,2-dimethyl-1,3-dioxolan-4-yl) methyl acetate can be
produced from crude glycerol with acetone using p-toluene sul-
fonic monohydrate catalyst. The addition of (2,2-dimethyl-1,3-
dioxolan-4-yl) methyl acetate to biodiesel was reported to recover
the biodiesel viscosity and meet the U.S. and European fuel stan-
dards [329]. Dichlor-2-propanol was prepared directly from glyc-
erol by the aid of heteropolyacid and acetic acid as catalysts [279].
HO
O
Glycerol
Acrolein
O O
Catalytic
O O
conversions
(2,2-dimethyl-1,3-dioxolan-4-yl)
methylacetate
OH
Cl
Cl
Dichloro-2-Propanol CO/H2
Syngas
Fig. 4. Potential products derived from catalytic and microbial conversion of glycerol.
511
4. Current trends and future directions for enzymatic biodiesel
R&D
Recently, a novel method for production of biodiesel at reduced
costs has been reported [332]. This method employs the use of a
solid whole cell biocatalyst obtained by solid state fermentation
(SSF). The advantages associated with the use of a SSF-generated
biocatalyst are twofold: (i) the microorganisms grow on low-cost
substrates (agro-industrial residues) maintaining low moisture
content; (ii) the crude fermented solid material can be used di-
rectly as a biocatalyst thereby omitting three processing steps: li-
pase extraction, purication and immobilization. This leads to a
signicant reduction of the lipase costs that translates into lower
biodiesel production costs [333,334]. A SSF-derived lipase-contain-
ing whole cell material, produced by Burkholderia cepacia LTEB11,
was used for a direct catalytic synthesis of biodiesel in n-heptane
[335] and co-solvent-free [336] systems A recent study by Liu
et al. [333] reported SSF production of a B. cepacia lipase on a
mixed substrate of sugarcane bagasse and sunower seed meal
with a catalytic activity of 72.3 units/g dry solids over a period of
96 h. A novel mixed substrate for enhanced SSF production of li-
pase from A. niger MTCC 2594 was developed for hydrolyzing tal-
low at 85.2% for 24 h [337].
Another innovative approach, hydroesterication, was recently
developed as a means to overcome problems related to biodiesel
production from feedstocks containing high percentage of FFA
and water [338]. The process of biodiesel production through
hydroesterication occurs in two stages. In the rst stage, mono-
and tri-acylglycerols are hydrolyzed to FFA and glycerol. In the
second, FFA are separated and esteried to biodiesel [339]. The
hydroesterication process can be carried out by: (1) a supercriti-
cal hydrolysis and esterication (catalyst-free); (2) enzymatic-
chemical hydroestrication; and (3) enzymatic hydroesterication.
The catalytic-free supercritical hydrolysis and supercritical esteri-
cation process (both conducted at a temperature of 270 C and
pressure of 20 MPa) in presence of methanol resulted in biodiesel
conversion rates in excess of 90% [340]. In a new hybrid
(enzyme/chemical) hydroesterifcation process, biodiesel was pro-
duced through hydrolysis of physic nut oil by a plant enzyme (veg-
etable enzyme extracted from germinated physic nut seeds),
followed by esterication of the generated FFA with methanol cat-
OH
OH O OH
O O
HO OH
OH
Citricacid
O
Microbial OH
conversions OH
Lacticacid
HO OH
CH4 1,3 Propanediol
R O
Methane
O n
Poly(hydroxyalkanoates)
(PHAs)
512 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
alyzed by a heterogeneous acid (niobic acid in pellets) [339]. A nal
product containing 91% FAME, 1% FFA and 7% of lipophilic com-
pounds was obtained by enzymatic hydroesterication of acid
oil, a byproduct from vegetable oil rening [341]. The initial hydro-
lysis step acid oil was carried with C. rugosa lipase and the subse-
quent esterication of FFA to FAME - by C. antarctica IM lipase.
However, the hydroesterifcation was carried as a two-stage and
energy-intensive process which, although ecofriendly, was consid-
ered a major economic drawback [338,341]. An alternative to
hydroesterication comprised of a two-stage esterication and
transesterication process for biodiesel production from brown
grease was studied by Fan et al. [342]. The lipase deactivation by
methanol was prevented by addition of biodiesel to the reaction
mixture prior to initiation of the conversion process. A FAME yield
greater than 95 wt% was attained after 15 cycles of operation.
Methanol inhibition is addressed through the use of solvents,
continuous removal of glycerol by dialysis or solvent extraction,
and stepwise addition of methanol. However, the use of solvents
adds to biodiesel costs, whereas the glycerol removal and the
stepwise feeding of methanol are impractical and challenging
on commercial scale. A permanent solution to the lipase inhibi-
tion by methanol is offered by recombinant DNA technology
[343]. Protein engineering and directed evolution can improve
the lipase specicity, thermostability, activity and methanol tol-
erance [344346]. A recent example for this effort is the high-le-
vel expression of a thermostable and methanol-tolerant lipase
from Proteus sp. in E. coli. The recombinant strain was able to
produce biodiesel in the presence of high water concentrations.
Alternatively, the recombinant E. coli strain was used as a
whole-cell biocatalyst. Directed evolution was subsequently em-
ployed to increase its thermostability and tolerance to methanol
while retaining the wild-type lipase activity at ambient temper-
ature [347].
One of the latest developments of thermostable and methanol-
tolerant lipases reports on a newly-constructed mutant of P. mira-
billis lipase, known as Dieselzyme 4, that in comparison to the wild
type enzyme, has a 30-fold improved thermostability and a 50-fold
increased methanol tolerance of the half-inactivation time at 50 C
in a 50% aqueous methanol [348]. The mutagenized lipase outper-
formed the commercially available lipase from B. cepacia. In an-
other study, a robust recombinant whole cell thermo- and
solvent-tolerant biocatalyst from A. oryzae (r-BTL) was developed
for biodiesel production [349]. The r-BTL had an increased toler-
ance (up to 30%) toward organic solvents such as dimethyl carbon-
ate, ethanol, methanol, 2-propanol or acetone at 50 C.
Another recent breakthrough in enzymatic biodiesel production
was the development of an enzyme-based bio-hybrid catalyst called
Lipase@Organo-Si(HIPE) [39,350]. The bio-hybrid catalyst was
used in continuous-ow, monolithic micro-reactors resulting in ex-
tremely high biodiesel yields at a superior recycling performance
over the batch reactors. The turnover numbers and frequencies at-
tained demonstrated the real potential of the catalyst for industrial
applications The insolubility of methanol in oil was overcome by
development of an innovative, enzyme-immobilized reactor that
integrated a continuous STR with two PBR connected in series for
a continuous biodiesel production omitting the use of organic sol-
vents and intermittent addition of methanol to the system [351].
Recent research efforts for reducing reaction time and minimiz-
ing catalyst inhibition by methanol also focused on the construc-
tion of a semi-pilot, continuous system for enzymatic biodiesel
synthesis based on the use of near-critical carbon dioxide as reac-
tion medium [352]. The highest conversion of 99.9% was attained
in only 4.5 h with Lipozyme TL IM at 30 C and 100 bar in a carbon
dioxide environment through a continuous mixing of a 1:1.3 oil/
methanol mixture and a three-step addition of methanol. A fore-
most technical development was the use of a PBR coupled to a
glycerol separation system that produced a FAME yield of 94.3%
with an efcient glycerol removal of 99.7% [353].
The production of syngas (synthesis gas) from glycerol is the
furthermost possible application of glycerol. In literature, there
are numerous catalytic processes to produce syngas from glycerol,
which include autothermal reforming, aqueous phase reforming
and supercritical water reforming [328,354,355]. Each process
has different working conditions and various types of catalyst in-
volved in the production of syngas [356]. As methanol used in
the production of biodiesel is mostly derived from nonrenewable
resources, syngas is exploited for methanol production as a sus-
tainable alternative to natural gas [357,358] (Fig. 5a). The product
quality depends on the glycerol purity, which is signicantly high-
er in the enzymatic than chemical process of biodiesel production.
The use of a biocatalyst simplies glycerol separation even if low-
quality feedstocks such as waste cooking oils are utilized.
Despite their current high prices, acyl acceptors such as methyl
or ethyl acetate are currently viewed as promising substitutes for
alcohols [195]. The major advantage of the interesterication of
TGs with methyl or ethyl acetate over the alcoholysis reaction is
that no glycerol is formed which eliminates the need for down-
stream separation and glycerol recovery. Instead, a new, higher-va-
lue by-product of the interesterication with ethyl acetate,
triacetylglycerol (triacetin), is formed, although separation and
recovery of triacetin are still required. Triacetin has a higher value
than glycerol and could help reduce the cost of biodiesel produc-
tion [188]. Triacetin is an antifungal agent and can also be used
as a xative in perfumery or as a solvent in basic dyes [302]. Con-
ferring to the reaction stoichiometry, a single-phase interesterica-
tion product typically contains 80 wt% of biodiesel and 20 wt%
triacetin [359]. Triacetin is miscible with biodiesel in all propor-
tions and can be included in biodiesel formulations [360]. Casas
et al. [359] studied the effects of triacetin on biodiesel quality
and reported that the addition of triactin should not to be limited
considering its positive effects on the fuel quality in accordance
with ASTM D6751-09. However, the European norms (EN
14214:2008) limit the triacetin content in biodiesel mixtures to
3.510 wt% due its effect on increasing density and viscosity of
the fuel. Interesterication also leads to the production of other
byproducts like diactin, monoactin and glycerol in small fractions
[361]. Casas et al. [360] reported that liquidliquid extraction with
a three-stage washing can complete recover these byproducts from
biodiesel-triacetin mixtures with a slight reduction in the triacetin
content. In presence of ethyl acetate, interesterication of soybean
oil with Novozym435 as biocatalyst and methyl acetate as acyl
acceptor produced a biodiesel yield of 92% [188]. After 12 batches
of enzyme reuse, Novozym435 retained its full activity on Jatro-
pha, karanj and sunower oils [194].
The introduction of alternative acyl acceptors offers an innova-
tive and eco-friendly approach to eliminating glycerol-associated
problems (Fig 5b). Methanol is currently the less expensive alter-
native to biodiesel production. However, methanol is considered
to be more toxic than ethanol, and to inhibit the enzyme biocata-
lysts. It is also not suitable for efcient biodiesel conversion of
high-FFA feedstocks. On the other hand, ethyl acetate is currently
more expensive than ethanol and methanol, however, as we con-
sider the current and future trends in enzymatic biodiesel, the
overall process and environmental sustainability of producing bio-
diesel from inexpensive raw materials and waste, and recovery/
conversion of triacetin to high-value products, this solvent appears
as the natural, renewable and sustainable source of acyl acceptors
for biodiesel production. Ethyl acetate is the derivative of two
biodegradable and microbially-produced fermentation products,
ethanol and acetic acid. In the present scenario, where environ-
mental concerns and production costs are given high priorities, a
solvent-free enzymatic process for industrial biodiesel production
 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
(a)
(b)
Fig. 5. Novel and eco-friendly biodiesel production processes using renewable acyl acceptors: (a) use of glycerol-derived biomethanol; (b) use of bioethanol-derived ethyl
acetate.
utilizing renewable materials (enzymes, non-edible and waste oils,
ethyl acetate as a green biochemical) would be encouraged as a
sustainable, cost-effective and environmentally-sound alternative.
With the increase in the production capacity of ethanol and focus
on cellulosic, fermentable sugars from energy crops and agr-waste
in the US and globally, the price of ethanol and acetic acid will be
gradually decreasing which makes ethyl acetate the best candidate
for a green biodiesel production. The use of ethyl acetate avoids the
formation of glycerol which is an enzyme inhibitor and a by-prod-
uct with a costly recovery. Biodiesel produced with ethyl acetate as
acyl acceptor has a higher cetane number, lower pour and cloud
points, higher ash and combustion points that improve cold
starts, and lower smoke opacity and exhaust temperatures [195].
The use of ethyl acetate may also stimulate the development of
new biodieseltriacetin blends to suppress knocking, improve per-
formance and reduce tail pipe emissions of diesel engines [362].
5. Economic considerations and large scale developments in
enzymatic biodiesel
The number of pilot and industrial scale plants for commercial-
ization of enzymatic biodiesel signicantly increased in recent
years (see also Introduction, paragraphs 46). Fjerbaek et al. [18]
roughly estimated the cost of catalyst per kilogram of biodiesel
produced as 0.14 US$ for Lipozyme IM, and 0.006 US$, for alkali
catalyst. Based on a of 100 kg of oil to biodiesel, the cost of the al-
kali and enzyme (Novozym435) catalyst was estimated to be
$0.62/kg and also approximately $1000/kg, respectively. In 2007,
Lvming Co. Ltd., in Shanghai, China, established an enzymatic bio-
diesel production plant with a capacity of 10,000 tons using waste
cooking oil as feedstock, and immobilized lipase of Candida sp. 99
125 as catalyst. The enzyme cost was 200 CNY per ton biodiesel,
513
which translates into about $0.03/kg biodiesel. In 2006, another
Chinese company, Hainabaichuan Co. Ltd., launched production
of enzymatic biodiesel at a 20,000 tons/year scale that was subse-
quently doubled to 40,000 tons per year in 2008. Biodiesel produc-
tion is based on waste palm oil utilizing Novozym435 [168]. A
new enzyme manufacturing company in Israel, TransBiodiesel,
was started to provide the biodiesel market with enzyme-based
catalysts at commercial quantities. In 2012, Piedmont Biofuels,
North Carolina, developed a new technology (FAeSTER) for a con-
tinuous biodiesel production using immobilized or liquid enzyme.
Challenges in the commercialization of enzymatic biodiesel relate
to the retrot of existing conventional processes into enzymatic
and the scale-up of technologies based on whole cell enzyme cat-
alysts. Further progress in the development of innovative, inexpen-
sive enzyme-immobilization techniques with low reaction time,
higher enzyme stability and activity should be developed towards
the global economic acceptance of large-scale enzymatic biodiesel
production.
6. Conclusions
Due to the high conversion rates and short reaction times, the
alkali-catalyzed process is currently the dominant large-scale
method for biodiesel production from virgin oils. However, this
process is characterized by several inherent process drawbacks
including the difculties in glycerol separation and purication,
catalyst recovery and recycling, saponication problems, the need
to wash out biodiesel impurities, treat waste water and eliminate
salts, and their energy-intensive nature. In addition, the production
costs are high as virgin oil constitutes almost two-third of the total
biodiesel price. Taking into account the biodiesel yield and quality,
the alkali process does not offer the best economic and environ-
514 L.P. Christopher et al. / Applied Energy 119 (2014) 497520
mental scenario for biodiesel production from low-cost feedstocks
such as waste oils and animal fats that have high FFA and water;
hence, for optimal results, a selective and specic enzymatic bio-
catalyst should be used. The acid catalysis converts both FFA and
TGs to biodiesel, however, it is corrosive and the transesterication
rates are signicantly lower (several thousand times) than those of
the base catalysis with a requirement for higher reaction tempera-
tures and a greater number of process equipment for separation
and purication of biodiesel from glycerol.
The drawbacks associated with the conventional chemical
catalysis can be overcome with the introduction of an alternative,
enzymatic route for biodiesel production. The enzymatic catalysis
offers a number of environmental and economical advantages over
the chemical method: room-temperature reaction conditions,
elimination of treatment costs associated with recovery of chemi-
cal catalysts, enzyme re-use, high substrate specicity, single-step
conversion of both FFA and TG to biodiesel, lower alcohol to oil ra-
tio, avoidance of side reactions and minimized impurities, easier
product separation and recovery; biodegradability and environ-
mental acceptance. The ability of lipases to catalyze FAME synthe-
sis from low-cost feedstock with high FFA (waste cooking oil,
grease, tallow, etc.) would lower the cost of enzymatic biodiesel.
The major demerits associated with the biocatalyst are the
higher enzyme costs and enzyme inhibition by methanol. Enzyme
costs can be reduced by discovery of new, more efcient lipase pro-
ducers; strain improvement and use of highly productive recombi-
nant strains for production of lipase with improved activity and
methanol tolerance; optimization of fermentation media and
parameters for lipase production; biocatalyst reuse through lipase
and whole cell immobilization; lipase pretreatment for activity
regeneration and extended lipase life, etc. Robust enzymes with
high activity, thermostability and resistance to the harsh condi-
tions of biodiesel production are needed. Such thermostable li-
pases produced from extremophiles are already employed as
detergents in the textile industry.
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