Enzymatic transesterification for biodiesel production from used

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Review
Enzymatic transesterification for biodiesel production from used
cooking oil, a review
Faegheh Moazeni a, *, Yen-Chih Chen a, Gaosen Zhang b, c
a Environmental Engineering Department, School of Science, Engineering & Technology, Penn State Harrisburg University, 777 W Harrisburg Pike,
Middletown, PA, 17057, USA
b Key Laboratory of Desert and Desertification, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou, 730000,
China
c Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000, China
a r t i c l e i n f o
Article history:
Received 12 September 2018
Received in revised form
13 January 2019
Accepted 16 January 2019
Available online 21 January 2019
Keywords:
Biodiesel production process
Used cooking oil
Enzymatic transesterification
Lipase
Biocatalysts
Enzyme immobilization
* Corresponding author.
E-mail address: fxm53@psu.edu (F. Moazeni).
https://doi.org/10.1016/j.jclepro.2019.01.181
0959-6526/© 2019 Elsevier Ltd. All rights reserved.
a b s t r a c t
This paper reviews various aspects of the enzymatic transesterification method to convert used cooking
oil to biodiesel. The goal of this paper is to provide a thorough overview from general biodiesel pro-
duction processes, reaction conditions, challenges, and solutions for higher biodiesel production yield
through introducing various microorganisms that are capable of producing the enzymes required to
convert used cooking oil into biodiesel. The characteristics, composition, and advantages of the used
cooking oil, as feedstock for biodiesel, is also discussed. In addition, the existing transesterification
methods including homogeneous alkali-catalyzed, homogeneous acid-catalyzed, non-catalytic reaction
under super-critical conditions, and enzyme-catalyzed reactions are explained. Furthermore, the ad-
vantages of the enzymatic method over other methods, and the enzymes, which are the key elements of
such reactions, are discussed. Lipases are the most promising enzymes currently known for biodiesel
conversion. The physiological and physical properties of microbial lipases, the catalytic mechanisms of
the enzymes, various methods of enzyme immobilization such as adsorption, covalent and affinity
binding, entrapment, and the whole-cell immobilization are also reviewed. At the end, three case studies
demonstrating unique and efficient enzymatic transesterification approaches are presented.
© 2019 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
1.1. Problem statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
1.2. Why used cooking oil? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
1.3. Biodiesel production process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
1.3.1. Feedstock pre-treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
1.3.2. Transesterification/esterification reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
1.3.3. Polishing and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
1.4. Contribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
2. Used cooking oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
2.1. Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
2.1.1. Ignition quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
2.1.2. Viscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
2.1.3. Heating value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
2.1.4. Specific temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3. Transesterification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4. Enzymes as catalysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
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F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128118
4.1. Physiological and physical properties of microbial lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.2. Catalytic mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.3. Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.3.1. Various immobilization techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.3.2. Whole-cell immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 124
5. Case studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.1. Biodiesel production using lipase immobilized on epoxychloropropane-modified Fe3O4 sub-microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.2. Ultrasound assisted intensification of biodiesel production using enzymatic inter-esterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.3. Converting oils high in phospholipids to biodiesel using immobilized Aspergillus oryzae whole-cell biocatalysts expressing Fusarium heterosporum
lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
6. Conclusions and recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Table 1
Comparison of fatty acid composition in weight percentage (wt%) for different oil
sources (Lam et al., 2010; Leung and Guo, 2006; Yun et al., 2013; Akbar et al., 2009).
Fatty acid Soybean Cottonseed Palm Coconut Canola UCO
Myristic 0.1 0.7 1.0 19.2 1.0 0.9
Palmiric 0.2 20.1 42.8 9.8 5.5 20.4
Stearic 3.7 2.6 4.5 3.0 2.2 4.8
Oleic 22.8 19.2 40.5 6.9 55 52.9
Linoleic 53.7 55.2 10.1 2.2 24 13.5
1. Introduction
1.1. Problem statement
It is vastly understood that the era of fossil fuels will soon be
over. The increasing accumulation of carbon dioxide in the atmo-
sphere resulting in a drastic increase of global temperature does not
allow the current rate of these fuels' consumption in the world, and
thereby man must seek out clean and renewable resources of en-
ergy to minimize dependence on fossil fuels (Rehan et al., 2018).
Similar to our electricity demandwhich can potentially bemet with
energy resources such as wind, geothermal, and solar, there is also a
need for alternative fuels for transportation. Given that currently
almost 60% of the total crude oil consumption in the world goes
towards transportation, the demand for which is increasing every
year, the need for an alternative non-fossil-based fuel for trans-
portation seems even more crucial. One of the best candidates is
biodiesel, as diesel engine vehicles could use it with little to no
modifications. However, producing biodiesel is too expensive to
compete with petroleum-based fuels. A major portion of the bio-
diesel production's cost comes from the costs of the raw materials
(Ragauskas et al., 2006; Gołaszewski, 2009). In addition, the feed-
stock of the biodiesel cannot compete with agriculture, which was
the drawback to edible-oil-based fuels. Another candidate to pro-
duce oil could be algae, which requires water and is costly to har-
vest (Chye et al., 2018; J€ams€a et al., 2017). Animal fats are also not
great candidates due to the high saturated fatty acid content which
imposes difficulty during the transesterification. Besides, the glyc-
erin produced as by-product of converting animal fats to fuel is low
grade (also known as tech-grade), which will bring very little rev-
enue to the biodiesel plants compared to high quality glycerin that
can be sold out at a high price. Hence, the best candidate should be
non-fossil-edible, cheap to produce, and rather easy to process.
Studies have shown that various types of used cooking oils (UCOs)
are promising candidates for biodiesel production (Gui et al., 2008),
as they do not compete with food (Akbar et al., 2009), they are
inexpensive to obtain (Kumar and Sharma, 2011), and rather easy to
process (Knothe et al., 2015). However, used cooking oil contains a
high amount of free fatty acids which will be converted to soap
during the conventional transesterification process, a reaction
called saponification. Saponification drastically lowers the biodiesel
production yield and the purity of the biodiesel, while it increases
the cost of production due to the need for additional feedstock pre-
treatment processes, catalysts, and polishing and purification pro-
cesses. Despite the disadvantages of utilizing used cooking oil as
feedstock, if biodiesel is converted under the processes that avoid
saponification, UCO is still a better candidate than other types of
feedstock to supply oil for biodiesel.
1.2. Why used cooking oil?
Biodiesel is a composition of mono alkyl esters derived fromoils,
and thus the higher the oil content of the feedstock, the higher the
biodiesel production yield, which also corresponds to a lower cost
of production. Among various crops that are considered for bio-
diesel, such as jatropha, rubber seed, castor, Pongamia pinnata, sea
mango, soybean, palm, and rapeseed, palm oil produces the highest
amount of oil per area with 5000 kg oil per hectare (Gui et al.,
2008), equivalent to 5.3 weight percent (wt%) of fatty acids. On
the other hand, palm oil is edible which would put biodiesel in
competition with agriculture. The palm oil after it is disposed as
waste cooking oil contains more than 20wt% fatty acid (Kusdiana
and Saka, 2004). Table 1 compares the fatty acid content of the
most common virgin oils and a sample of used cooking oil obtained
from a Chinese restaurant, where most of the food preparation is
through frying.
A comparison between the main fatty acids' composition, in
both virgin oils and UCO (Table 1), determines UCO a more prom-
ising feedstock for biofuel. Not only does UCO offer higher fatty
acids content but also it is not food.
1.3. Biodiesel production process
In the following subsection, the key steps of producing biodiesel
are discussed: (Gurunathan and Ravi, 2015; Sebastian et al., 2016;
Sotoft et al., 2010):
1.3.1. Feedstock pre-treatment
Depending on the type of the oil used as feedstock (virgin
vegetable oil, used cooking oil, algal oil, or tallow), a range of
different processes, often in sequence, are applied on the crude oil
to refine it before mixing with other material to be sent to the
reactor. Some of these processes include removing impurities and
solids coming from cooking and handling (in the case of UCO)
(Yaakob et al., 2013). Different forms of sieving are used to separate
solids and particles. Also, degumming to remove phospholipids,
Linolenic 8.6 0.6 0.2 0.0 8.8 0.8
F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128 119
and de-watering in the case of a basic-catalyzed transesterification
method should be done. Water hydrolyzes triglycerides (TGs)
which can result in soap formation during the transesterification
process. Soap formation is one of the most important reasons of
having a low biodiesel production yield. Phospholipids can be
either washed away with hot water or removed via centrifugation
and paper filtration.Water is often removed by heating the solution
up to 100 �C (Demirbas, 2009; Banerjee and Chakraborty, 2009).
Some of other common methods of pre-treatment include steam
injection (Lertsathapornsuk et al., 2005), column chromatography
(Lee et al., 2002), neutralization, film vacuum evaporation
(Cvengro�s and Cvengro�sov�a, 2004), and vacuum filtration (Dias
et al., 2008). However, some studies replaced pre-treatment with
regular esterification of free fatty acids (FFAs) with sulfuric acid
prior to the alkali transesterification, through which FFA content of
the UCO was reduced (Leung et al., 2010). In addition, the appli-
cation of acidic ion-exchange resins as a pre-treatment process has
demonstrated a reductionin the FFAs content of the UCO. Though,
the early catalyst activity deteriorationwas the disadvantage of this
method (Jeromin et al., 1987; Shibasaki-Kitakawa et al., 2007; Li
et al., 2008). One of the most recent pre-treatment methods of
UCO is to utilize glycerin for the acidic rawmaterial, inwhich a high
temperature (about 200 �C) and a catalyst such as zinc chloride are
required. FFAs present in the UCO react with glycerin to produce
mono- and diglycerides which will result in a reduction of FFAs in
the UCO (Van Gerpen et al., 2004). The high associated cost of this
method makes its application economically infeasible in most
cases.
1.3.2. Transesterification/esterification reactions
Transesterification reaction is the way of converting oils and
fatty acids into alkyl esters, also known as biodiesel. In the trans-
esterification process, triglycerides react with an alcohol (methane
or ethane) to generate methyl or ethyl esters of fatty acids and
glycerol. In most countries, alkyl esters are prepared in the form of
methyl esters, rather than ethyl esters, mainly because methanol is
less costly than ethane (Knothe et al., 2015). Transesterification
methods have been the subject to several studies since the idea of
biodiesel was proposed. In 1986, a comparative analysis was con-
ducted on using vegetable oil as an alternative fuel for diesel en-
gines (Ziejewski et al., 1986). Then in 1988, lipases were used as
catalysts in transesterification reactions (Zaks and Klibanov, 1988).
Traditionally, transesterification can be performed heterogeneously
and homogeneously by acid and base catalysts. Alkali catalysts in
the form of sodium or potassium hydroxide are more common in
the homogeneous transesterification reactions, as they offer a
higher speed compared to the acidic catalysts. In the case of a high
acid content, acid-catalyzed esterification can also be used to react
fatty acids with alcohol to produce biodiesel. The biodiesel con-
version can be carried out in the presence or the absence of a
catalyst (Knothe et al., 2015). In addition to the type of the catalysts,
the effect of other parameters such as alcohol to oil molar ratio,
temperature, reaction time, and water content on trans-
esterification results have also been studied (Watkins et al., 2004).
1.3.3. Polishing and purification
The products of the carried out reactions include biodiesel, as
well as un-reacted free fatty acids, soap, glycerol, excess alcohol,
excess water, and catalyst residue. Various procedures are in place
to separate all the impurities from biodiesel in the polishing and
purification step. Glycerol can be separated via gravity and sold out
as by-product. Water can be flashed out and methanol can be
recovered to re-use through distillation. Soaps are often converted
into acids to remove. Process flowcharts related to the conventional
and enzymatic reactions are shown in Figs. 1 and 2 (Gurunathan
and Ravi, 2015; Sebastian et al., 2016; Sotoft et al., 2010).
1.4. Contribution
This paper reviews various methods and their challenges that
are available to produce biodiesel from UCOs that are inexpensive
and abundant, yet rich in fatty acids. Lipase enzymes extracted from
microorganisms serve as bio-catalysts in the enzymatic trans-
esterification reactions (Demirbas, 2008). Based on the types of the
alcohol used in the trasesterification process, various microorgan-
isms can be used as the source for the enzymatic catalysts. For
instance, it was shown that the biodiesel conversion with primary
alcohols wasmost effective by using enzymes extracted fromMucor
miehei, whereas in the presence of the secondary alcohols, the
lipase extracted from Candida antarcticawas most effective (Nelson
et al., 1996). Studies show that the enzymatic catalysts are most
effective and less costly if they are immobilized on a specific carrier
(Amini et al., 2017a) or, intracellularly, as a whole-cell (Kuratani
et al., 2018). Several researchers have studied this topic in recent
years, but finding the best microorganisms that offer the most
effective enzymes for the biodiesel applications and how to
immobilize and re-use them are still the subjects of ongoing
studies. The rest of this review paper is categorized as the
following:
Section 2- Used Cooking Oil: Section 2 will cover characteristics,
composition, advantages, and disadvantages of used cooking oil.
Section 3- Transesterification Methods: Section 3 will explain
the four existing transesterification methods used for biodiesel
conversion, including the process conditions, reactants, pros, and
cons.
Section 4- Enzymes as Catalysts: Section 4 will cover all aspects
of enzymes used for biodiesel conversion, including properties,
mechanisms, and immobilization techniques of the enzymes.
Section 5- Case Studies: The three case studies chosen for re-
view by this paper will propose unique methods in various steps of
enzymatic transesterification. In the first study, the enzymes were
immobilized on a super magnetic surface which facilitated the
enzyme recovery by using an external magnetic field (Zhang et al.,
2016). In the second study, an ultrasound system was used to
intensify the solution which improved enzymatic inter-
esterification reactions (Subhedar and Gogate, 2016). Finally, in
the third case, a particular enzyme was used that demonstrated
high capability of converting oil containing high phospholipids into
biodiesel. The results of this study allow biodiesel production
plants to eliminate the degumming step in the feedstock pre-
treatment process which will cut down the cost of production, as
well as increasing the production yield (Amoah et al., 2016).
2. Used cooking oil
As the name suggests, used cooking oil refers to the vegetable oil
or lard in food industries including the food preparation processes
in restaurants, hotels, and fast food outlets. Such oils and fats
cannot be used any further due to oxidation and pollution with
small food parts. As waste, UCOs cannot be discharged directly into
the environment or wastewater because it can clog the collection
pipes which could potentially lead to the sanitary sewer overflow.
In addition, at the wastewater facilities, the high content of oil of
the UCO can coat the activated sludge which prevents oxygen
transfer for waste degradation. As useless and even wasteful as it
sounds, UCO can be a great feedstock for biodiesel (Gui et al., 2008;
Chhetri et al., 2008). The used cooking oil can come from palm,
sunflower, corn, canola, or any other oil that was originally used for
the food preparation. Similar to many oleaginous vegetable species,
UCO is composed of the esters of glycerol and fatty acids, also called
Fig. 1. Conventional biodiesel production process.
Fig. 2. Enzymatic biodiesel production process (Gurunathan and Ravi, 2015; Sebastian et al., 2016; Sotoft et al., 2010).
F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128120
triglycerides (TGs) or glycerides (Knothe et al., 2015). Fatty acids, in
general, contain the very polar carboxyl group at one end of a hy-
drocarbon molecule. There are two different types of fatty acids;
saturated and unsaturated fatty acids. Saturated fatty acids contain
a single carbon bond, while the unsaturated fatty acids include at
least one C:C doubled bonds (Leung and Guo, 2006). In vegetable
oils, the concentrations of unsaturated fatty acids are higher than in
animal fats. These oils, when undergone the food preparation
processes, change in terms of the physical and chemical charac-
teristics (Van Gerpen, 2005). Among different processes of food
preparation (such as boiling, frying, and steaming), frying is the
most common cooking approach in restaurants as it gives a better
F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128 121
taste to the food (Canakci, 2007). Also, due to economical factors,
frying is conducted in the same oil for several times under high
temperature and in the presence of oxygen and light, which can
cause major changesin the oil. Some of these physical changes are
as the following: higher viscosity, higher specific heat, and higher
tendency to generate foam (Demirbas, 2009). The main types of
reactions causing the properties of the oils to change in the UCO
are: thermolytic reactions under high temperature (180 �C) in the
absence of oxygen; oxidative reactions under high temperature in
air; and hydrolytic reaction under high temperature in the presence
of steam (Talebian-Kiakalaieh et al., 2013). As a result of a combi-
nation of these reactions and the original oils that were used for
cooking, the UCOs demonstrate different characteristics than of
vegetable crude or refined oils (Ullah et al., 2015). The physical and
chemical properties of UCO can vary based on its original fresh
vegetable oil. Some of the UCO properties are shown in Table 2
(Guti�errez-Zapata et al., 2017; Çetinkaya et al., 2005; Yaakob
et al., 2013).
Compared to the fresh oil, the triglycerides of the vegetable oil
break down during the cooking process into FFAs, leading to a
higher free fatty acids content in UCO. Also, the oxidation and
polymerization process occurring during the cooking and frying
will cause an increase in the viscosity and the saponification
number in UCO (da Silva C�esar et al., 2017). High viscose biodiesel is
not favored as it will ultimately damage the fuel injector in the
engine of the cars, since it will result in an incomplete combustion
and thus accumulating unburned deposits in the engine (Wei et al.,
2017). Moreover, because of the heat and mass transfer between
the food and the oil during frying and cooking, UCO contains higher
amount of water than fresh vegetable oil. Such high water content
along with the high FFA content and high saponification number of
UCO cause the saponification reactions to occur during the trans-
esterification process. The saponification reduces the biodiesel
production yield and increases the amount of utilized catalyst.
Hence, an oil pre-treatment process, as well as a suitable trans-
esterification method should be employed to achieve a high bio-
diesel production yield at a feasible cost (Buffi et al., 2017). As
mentioned above, the distribution of various fatty acids in UCO
varies with the original oil and the process done on the oil before it
reaches the biodiesel plants (for instance, how many times the oil
was used for cooking or frying). However, among the various
vegetable oils viable for biodiesel production, olive, sunflower, and
palm oil are the most common ones used in cooking. Thus, the fatty
acidsmainly present in these oils (i.e. palmitic acid, stearic acid, and
oleic acids) are considered to be the major fatty acids in UCOs
(Banerjee et al., 2014). For instance, the FFAs present in UCO was
identified as palmitic acid 30%, stearic acid 10%, and oleic acid 60%
Table 2
UCO physical and chemical properties.
Property Value
Palmiric acid (wt%) 8.5
Stearic acid (wt%) 3.1
Oleic acid (wt%) 21.2
Linoleic acid (wt%) 55.2
Linolenic acid (wt%) 5.9
Others (wt%) 4.2
Density (g/cm3) 0:91� 0:924
Kinetic viscosity (mm2/s) at 40+C 36:4� 42
Saponification value (mgKOH/g) 188:2� 207
Acid value (mgKOH/g) 1:32� 3:6
Iodine number (gI2/100 g) 83� 141:5
Water content (wt%) 0:8� 1:9
Average sodium content (mg/kg) 6.9
Average peroxide value (mg/kg) 23.1
(Yun et al., 2013). Another classification of the UCOs can be done
based on the sources from which the used cooking oil was
collected. Table 3 demonstrates density of and the content of sulfur,
nitrogen, hydrogen, and carbon present in various sources of UCOs
(Bezergianni and Kalogianni, 2009).
2.1. Characterization
The ultimate goal of producing biodiesel made from any source
of oil (fresh or waste) would be to use it in the car engines.
Therefore, the UCO, as one the sources of the biodiesel, must
demonstrate the engine performances that are similar to those
with diesel-based fuels. The crucial characteristics are listed in the
following:
2.1.1. Ignition quality
The cetane number of the biofuel should be sufficiently high
enough to avoid knocking the engine, which is the result of a long
ignition. The satisfactory cetane number varies between 40 and 60
(Ramadhas et al., 2004).
2.1.2. Viscosity
As stated before, the proper value of fuel viscosity affects the
combustion and thermal efficiency of the engine. This is particu-
larly important in conditions under which the speed is low and the
load is light. According to the ASTM D6751-12 standard, the kine-
matic viscosity at 40+C of the biodiesel can vary within the range of
1.9e6.0mm2/s (Charter, 2008).
2.1.3. Heating value
Similar to the other properties, the calorific value of the bio-
diesel should be close to that in diesel-based fuel 43.350 kJ/kg. The
combustion chamber of the engine can generally take in a broad
range of heating values, however, it only operates efficiently with
high heating value fuels. The highest calorific values among various
UCO types belong to the UCO originated from sunflower oil which
demonstrates 40.579 kJ/kg of calorific value (Altın et al., 2001).
2.1.4. Specific temperatures
Pour point, cloud point, and flash point of fuel are important
temperatures in the engine operation. If used cooking oil is utilized
to make the fuel, then both pour point and cloud point of that
should fall below its freezing point, in order for the engine to
operate in cold weather environments. However, the flash point
should be as high as feasible to maintain the safety of ignition.
According to ASTM D6751-12 standard, the minimum flash point is
93 �C. Even in the case of blending biodiesel with diesel (such as in
BD10, BD25), the final flash point should not decrease (Bezergianni
and Kalogianni, 2009; Charter, 2008).
3. Transesterification methods
The feedstock oil (UCO) will be converted to biodiesel through
chemical reactions of transesterification and esterification. Trans-
esterification is the catalyzed process of trading the alkoxy group of
Table 3
Physical and chemical properties of various sources of UCO.
Domestic Restaurant, taverns Fast food outlets
Density (kg=m3) 0.8929 0.8929 0.8929
S (wppm) 0.00 187.70 26.30
N (wppm) 0.40 49.10 61.90
H (wt%) 11.56 11.52 11.58
C (wt%) 77.24 76.53 76.32
Fig. 3. Step-by-step reactions 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)
(Yun et al., 2013).
F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128122
an ester by an alcohol such as methanol and ethanol (acyl acceptor)
to convert the triglycerides (TGs) of the UCO to fatty acid methyl
esters (FAME) and glycerol (Demirbas, 2005, 2008; Cavonius et al.,
2014). A direct esterification could also generate biodiesel, inwhich
FFAs with alcohols produce FAME and water as the by-product.
Temperature is one of the key elements affecting the production
yield of biodiesel through transesterification. Table 4 exhibits the
yield of biodiesel production made from various UCOs. The yield of
biodiesel conversion increased in all types of UCO feedstocks as the
reaction temperature was increased (Bezergianni and Kalogianni,
2009).
The type of catalysts used in the transesterification reactions is
another critical element affecting the biodiesel production. They
could be chemical compounds, such as acids and/or bases, and
enzymes, depending on the method used for biodiesel production.
The overall process involves three consecutive, reversible reactions
which produce intermediate molecules of di- and monoglycerides
(Kayode and Hart, 2017; Vicente et al., 2004). (Fig. 3)
FFAþMeOH/FAME þ H2O (1)
Such reactions currently are carried out under the following
major forms of process (Banerjee et al., 2014):
� The homogeneous alkali-catalyzed reaction, using sodium
hydroxide (NaOH) or potassium hydroxide (KOH) - This
method is vastly used in industry due to its simplicity, the rather
mild reaction conditions it requires (e.g. low temperature and
low pressure), and the highconversion it offers in a short re-
action time. Despite the advantages, this method is not suitable
for processing UCO, as it is highly limited by the FFAs content of
the feedstock. In this method, free fatty acids react with alkali
forming soap. The soap can convert to a gel under ambient
temperature, which will substantially decrease the production
yield (Meher et al., 2006; Leung et al., 2010). To avoid such, the
content of FFA cannot go beyond 2wt% which is not the case in
the lipid-rich UCOs. To avoid saponification, an acidic pre-
treatment process is added in which FFAs are esterified to
FAME (biodiesel) in the presence of an alcohol such asmethanol.
The acid step is usually performed under 0.5e1.0% sulfuric acid
at a high temperature (60� 100+C) and the ratio of meth-
anol:substrate about 30:1 (Fig. 1). Therefore, a two-step process
involving acid and base is designed for the biodiesel feedstock
with high FFA content such as UCO, in which the water resulted
from the acid-catalyzed esterification is removed before
entering the alkali-catalyzed step, to prevent saponification.
Despite the advantage that this two-step acid-base process of-
fers, the high cost of production, which is the result of adding an
extra step to the entire process, is still a great burden when it
comes to a large scale biodiesel production (Vicente et al., 2004;
Dorado et al., 2004).
� The homogeneous acid-catalyzed reaction, using mostly
sulfuric acid (H2SO4) and hydrochloric acid (HCl) - The
advantage of this method is that it can simultaneously catalyze
esterification and transesterification (Wallis et al., 2017). How-
ever, compared to the previous method, it offers a slower re-
action rate, consumes high energy and high amount of methanol
Table 4
Effect of temperature on the yield of biodiesel production.
Temperature (K) Sunflower oil (%) Corn oil (%) Cottonseed oil (%)
620 79.6 80.5 82.3
630 93.6 95.8 96.5
640 96.8 97.2 97.6
due to its high molar ratio of methanol to oil demand, requires
catalyst separation, and causes corrosion (Fig. 1) (Aranda et al.,
2008; Demirbas, 2005). The disadvantages of the chemical-
catalyzed reactions (acid- or alkali-based) can be summarized
as the following: (1) a low purity and low biodiesel production
yield as the results of side reactions of saponification and hy-
drolysis; (2) the high capital cost and energy required for the
process; (3) the high cost of separation and purification of cat-
alysts and glycerol; (4) the need for neutralization and waste-
water treatment (Yun et al., 2013; Kayode and Hart, 2017). The
issues of catalysts recovery and saponification have led re-
searchers towards heterogeneous non-enzymatic catalysts such
as amorphous zirconia, titanium-, aluminum-, and potassium-
doped zirconias, metal oxides, hetero-polyacids, and sulfated
zeolites (Guldhe et al., 2017; Qadri et al., 2017). Though the
heterogeneous catalysts appear to be easier to separate and
more stable on the feedstock oil with high FFA content (such as
UCO), they are often costly and energy intensive because of their
demands for high temperature and high ratio of alcohol:-
substrate in the reactions (Martinez-Guerra et al., 2018).
� The non-catalytic reaction under supercritical conditions -
Though it offers more simplified products separation, polishing,
and purification procedures due to the absence of a catalyst, the
catalyst-free supercritical method is very expensive because of
the critical operating conditions, high consumption of meth-
anol, and energy that the entire process demands (Demirbas,
2005; Patil et al., 2011).
� The enzyme-catalyzed reactions - This is a fairly new method
which still is a subject of ongoing research. In this type of re-
actions, an enzyme such as lipase serves as a bio-catalyst for the
transesterification reactions (Amini et al., 2017b). Similar to the
non-catalyzed reaction method, the enzyme-catalyzed re-
actions offer simple biodiesel purification procedures but with a
substantial reduced energy requirement due to the mild oper-
ating conditions (Amini et al., 2017a). The enzymatic method
demonstrates the following advantages over other methods of
transesterification (Kuratani et al., 2018): requiring mild reac-
tion conditions, providing high selectivity of transesterification
with regards to the feedstock, offering a vast range of substrate
because of the capability to esterify both glyceride-linked and
non-esterified fatty acids in one step; not resulting in side-
reactions such as saponification; producing high-grade glyc-
erol as by-product; and being environmentally accepted. How-
ever, the price and stability of enzymes are the main challenges
yet to be studied (Yun et al., 2013; Nelson et al., 1996; Zhang
et al., 2003; Demirbas, 2008).
Table 5
Lipase-producing microorganisms.
Type Microbial Source Ref. (Shah and Gupta, 2007)
Fungi Alternaria brassicicola (Dutra et al., 2008)
Aspergillus sp. (Dutra et al., 2008)
Candida antarctica (Dutra et al., 2008)
Mucor miehei (Dutra et al., 2008)
Penicillium cyclopium (Dutra et al., 2008)
Rhizomucor miehei (Dutra et al., 2008)
Rhizopus sp. (Dutra et al., 2008)
Streptomyces sp. (Dutra et al., 2008)
Aspergillus niger (Dutra et al., 2008)
Thermomyces lanuginous (Dutra et al., 2008)
Fusarium heterosporum (Dutra et al., 2008)
Humicola lanuginose (Dutra et al., 2008)
Oospora lactis (Dutra et al., 2008)
Rhizopus oryzae (Dutra et al., 2008)
Yeasts Candida sp. (Dutra et al., 2008)
Pichia sp. (Dutra et al., 2008)
Saccharomyces lipolytica (Dutra et al., 2008)
Geotrichum candidum (Dutra et al., 2008)
Yarrowia lipolytica (Dutra et al., 2008)
Bacteria Microbial Source Ref. (Shah and Gupta, 2007)
Achromobacter sp. (Hsu et al., 2002)
Bacillus sp. (Hsu et al., 2002)
Burkholderia glumae Jaeger and Reetz (1998)
Chromobacterium viscosum (Ziejewski et al., 1986)
Micrococcus freudenreichii (Ziejewski et al., 1986)
Moraxella sp. (Ziejewski et al., 1986)
Mycobacterium chelonae (Ziejewski et al., 1986)
Pasteurella multocida (Ziejewski et al., 1986)
Propionibacterium sp. (Ziejewski et al., 1986)
Proteus vulgaris (Ziejewski et al., 1986)
Pseudomonas sp. (Ziejewski et al., 1986)
Proteus vulgaris (Ziejewski et al., 1986)
Psychrobacter immobilis (Ziejewski et al., 1986)
Serratia marcescens (Ziejewski et al., 1986)
Staphylococcus sp. (Ziejewski et al., 1986)
Sulfolobus acidocaldarius (Ziejewski et al., 1986)
Vibrio chloreae (Ziejewski et al., 1986)
Pseudomonas alcaligens (Ziejewski et al., 1986)
Chromobacterium visosum (Ziejewski et al., 1986)
Pseudomonas putida (Ziejewski et al., 1986)
Statphylococcus stolonifer (Ziejewski et al., 1986)
Enterococcus faecalis (Dutra et al., 2008)
F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128 123
4. Enzymes as catalysts
The most common enzymes used for biodiesel production are
lipases. Lipases convert TGs to glycerol and fatty acids via hydrolysis
and thereby are classified as hydrolases (Wu et al., 2017; Hwang
et al., 2014). In general, a carboxylesterase or carboxylic-ester hy-
drolase (EC 3.1.1.1) refers to an enzyme that catalyzes chemical
reactions of the following reaction:
carboxylic esterþ H2O/alcoholþ carboxylate (2)
Comparing the above template with the application of lipases in
the biodiesel production process, we can conclude that lipases are
categorized under carboxylesterases because they catalyze both the
hydrolysis and synthesis of long-chain acylglycerols (Pourzolfaghar
et al., 2016; Chourasia et al., 2015). Lipases can be extracted from
various sources of plant, fungal, animal, and bacterial. Lipases
contributed from plants are located in their energy reserve tissues
such as oilseeds (Pizarro and Park, 2003). While plant lipases offer a
high substrate specificity and low production cost, they are not very
common in industrial applications due to their low expression and
uneconomical fold purity (Shah and Gupta, 2007). Sources of ani-
mal lipases are pancreatic glands of cattle and pigs, as well as the
pre-gastric juices of calves, lambs or baby goats(Jolly and
Kosikowski, 1975). Animal lipases are used extensively in dairy
and food industry, but are less applied in other commercial appli-
cations (Goswami et al., 2013). Microbial lipases, however, are the
most common sources of enzymes used in biodiesel processes (Fan
et al., 2017). Compared to other forms of enzymes, microbial en-
zymes are cheap and fast to produce because they are abundant,
not affected by seasons, grow fast, and can be grown and immo-
bilized on inexpensive media. They offer high yields and can be
genetically modified. In addition, microbial-based lipases are more
stable than the plant- and animal-based enzymes and are safe to
the environment (Christopher et al., 2014). Table 5 shows the most
common lipase-producing microorganisms including bacteria,
fungi, and yeasts (Yun et al., 2013; Seth et al., 2014). Those strains
that are close taxonomically can generate different types of lipases.
For instance, two strains of Mucor miehei (IM 20) and Candida
antarctica (SP 382) lipases were used for free fatty acids esterifi-
cation or fatty acid methyl esters transesterification (Hasan et al.,
2006). Another example of microbial lipases for biodiesel produc-
tion is the immobilized Pseudomonas cepacia. This lipase was used
for the transesterification of soybean oil withmethanol and ethanol
(Akoh, 1993). Also, two commercial lipases of Novozym 435 and
Lipozyme IM were used to produce fatty acid ethyl esters from
castor oil (Noureddini et al., 2005). Immobilized enzymes origi-
nated from Thermomyces lanuginosa and C. antarctica served as
biocatalysts in alkyl ester production made from UCO (Lanza et al.,
2004).
4.1. Physiological and physical properties of microbial lipase
Most microbial-origin lipases are extracellular, responding best
under the pH range of 7.5e9. Their molecular weights range from
30 to 50 kDa and can be categorized into the mesophilic- and
thermophilic-origin source, demonstrating an optimum activity
under 35e50 �C for the former and 60e80 �C for the latter (Zaks
and Klibanov, 1988; Wang et al., 2007; Kazanina et al., 1981;
Kakugawa et al., 2002). The thermophilic enzymes are often used in
the processes requiring high temperature, as they remain active
and stable up to 100 �C evenwhen an organic solvent is present. For
example, lipases from Pyrobaculum calidifonti, Pyrococcus furiosus,
Thermoanaerobacter thermohydrosulfuricus, and Caldanaerobacter
subterraneus, can tolerate temperatures as high as 90 �C (Rehman
et al., 2017; Hotta et al., 2002; Ikeda and Clark, 1998; Royter et al.,
2009; Lusk et al., 2018; Mishra et al., 2017).
Lipases, in general, can become active under mild conditions
such as ambient temperature and pressure which will result in a
low energy consumption and thus a low operational cost in bio-
diesel production. Lipases are specific, meaning that they react
towards specific substrate(s). Such property will avoid producing
unwanted products which will lead to fewer side reactions and
post-reaction separation problems. Once immobilized lipases are
used, organic solvents are often introduced in the process. Lipases
stay active in the presence of the solvent, which is crucial to the
production yield and the economy of the biodiesel.
4.2. Catalytic mechanism
Lipase serves as a biocatalyst for three types of reactions: hy-
drolysis, esterification, and transesterification, among which the
last one includes four categories of alcoholysis, inter-esterification,
acidolysis, and aminolysis. During hydrolysis, molecules of acyl are
transferred betweenmolecules of glyceride in the presence of extra
water. Hydrolysis of oils or fats results in free fatty acids and glyc-
erol formation. This is a reversible reaction, in which the reverse
direction happens when the rate of hydrolysis and the final prod-
ucts vary with the concentration of fatty acid dissolved in the oil
F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128124
phase, while glycerol concentrates in the water phase (Zaharudin
et al., 2018; Alin et al., 2011; Murty et al., 2002; Adlercreutz, 2017).
4.3. Immobilization
Immobilization can be conducted as enzyme immobilization or
whole-cell immobilization (Kawaguti et al., 2006). All the reactants,
substrates, and products in the process of biodiesel production are
in liquid form, thus, enzyme immobilization occurs only when li-
pases are attached to a solid support. Substrates pass over the
support, to which the enzyme is attached, and are converted to
products (Datta et al., 2013). Immobilized enzymes exhibit several
advantages over free enzymes such as the ability of frequent use of
the enzyme, convenient separation of the enzyme from the reac-
tion solution, high enzyme stability, and more pure products.
However, selecting the proper solid support for the enzyme and
efficient immobilization techniques which could provide optimum
enzyme activity and stability in the given substrate medium still
remains challenging (Fai et al., 2017; Hellmers et al., 2018). Various
immobilization methods are discussed in the following, and sum-
marized in Fig. 4.
4.3.1. Various immobilization techniques
1- Adsorption: In this method, the solid support is soaked in
enzyme or the enzyme is dried on surfaces of electrode. In both
cases, hydrophobic interactions and salt linkages allow physical
adsorptionwhichwill lead to the enzyme immobilization. Themost
common lipase enzymes immobilized via adsorption are the
C. antartica lipase, which is immobilized on acrylic resin (Nov-
ozymO435), Mucormiehei lipase, which is immobilized on a mac-
roporous ion-exchange resin (Lipozyme IM), T. lanuginosus acrylic
lipase (Lipozyme TLIM), immobilized on resin, Rhizomucor miehei
lipase which is immobilized on macroporous anion exchange resin
(Lipozyme RM IM), and Candida sp. 99125 lipase, immobilized on
textile membranes (Tan et al., 2010; Bahulekar, 2017). Another
point that must be taken into consideration is that lipase becomes
deactivated bymethanol adsorption onto the immobilized enzyme.
Therefore, some sort of enzyme regeneration method such as
regeneration with higher alcohols like butanol is necessary.
2- Covalent Binding: This method, also known as carrier
binding, occurs due to the presence of side chain amino-acids such
as arginine, aspartic acid, histidine and some degree of reactivity
based on various functional groups such as imidazole, indolyl, and
phenolic hydroxyl. Some of the carriers used for lipase immobili-
zation are polyurethane foam, silica, sepabeads, and cellulosic
nanofibers (Awang et al., 2007; Tran et al., 2012; Krauss et al., 2017;
Zhang et al., 2017).
Fig. 4. Enzyme immobilization methods.
3- Affinity Immobilization: This method harnesses the selec-
tivity of an enzyme towards its support, which can vary under
various physiological circumstances. There are twoways to achieve
such immobilization: the support can be pre-attached to an affinity
ligand specific to a certain enzyme, or the enzyme itself can be
coupled to a component developing affinity towards the support
(Bandikari et al., 2018).
4- Entrapment: As the name suggests, in this approach en-
zymes are entrapped by bonds (either covalent or non-covalent)
inside a gel or a fiber (Rehman et al., 2017). Carriers should be
specific to the enzyme. For instance, to immobilize lipase, poly-
urethane foam, silica, sepabeads, and cellulosic nanofibers can be
used as carriers (Cazaban et al., 2017; Singh et al., 2009; Reetz et al.,
1996; Noureddini et al., 2005).
4.3.2. Whole-cell immobilization
All of the above methods are used to immobilize free (extra-
cellular) enzymes. While efficient, they all demand extremely ac-
curate enzyme purification. However, if the entire cell is
immobilized, the intracellular lipase is immobilized along with the
cell, but at a lower cost and fewer steps. Such allows biodiesel in-
dustry to use readily available industrial cultures which eventually
decreases the cost of enzyme transesterification process. This type
of biocatalyst is called whole-cellimmobilized lipase. For instance,
surface-displayed strategy was used to intracellularly immobilize
the whole-cell yeast Rhizopus oryzae lipase (ROL) in Saccharomyces
cerevisiae MT81 (Matsumoto et al., 2002). With the help of genetic
engineering, rProROL from R. oryzae IFO4697 was constructed un-
der the control of the isocitrate lyase gene of Candida tropicalis
(UPR-ICL) 5'-upstream region at 30 �C for 98 h through a two-stage
cultivation using semidefined medium (SDC medium, which is SD
medium with 2% casamino acids) including 2.0% and 0.5% glucose.
Once this rProROL was constructed, the intracellular lipase activity
increased significantly. This whole-cell immobilized lipase was
then used to form biodiesel from plant-based oil with methanol,
without the presence of solvents and water at the production rate
of 71wt% methyl esters. In another study biomass support particles
(BSPs) strategy was used to intracellularly immobilize the whole-
cell Rhizopus oryzae lipases in order to use them for biodiesel
production from soybean oil (Ban et al., 2002). The R. oryzae cells
were inoculated into a basal medium and incubated for 80e90 h at
35 �C on a reciprocal shaker (150 oscillations/min; amplitude
70mm) with BSPs. As a result, while growing during the shake-
flask incubation, R. oryzae cells became immobilized within the
BSPs.
The BSPs were in the form of 6-mm cubes of reticulated poly-
urethane foam containing a particle porosity beyond 97% with a
size of 50 pores per linear inch. The immobilized cells were then
separated from the BSPs by filtration, washed with tap water to
produce biocatalysts containing about 5% water content. The re-
sults showed that the biodiesel production rate reached up to 80%,
in the presence of the BSP-immobilized lipase. Methanol was added
twice after 25 h and 50 h to ensure the biodiesel conversionwas not
limited by the methanol concentration (Ban et al., 2002). The entire
reaction lasted 70 h. Another research demonstrated that the bio-
diesel production yield was increased up to 95.45% using immo-
bilized MAS1 lipase compared to the biodiesel production yield of
89.50% when there were no biocatalysts (Wang et al., 2017). MAS1
lipase was immobilized in 75mg lipase solution/g XAD1180 resin,
containing an equal volume of sodium phosphate buffer (0.02M,
pH 8.0) under 30 �C and a speed of 200 rpm for 8 h. After rinsed
with sodium phosphate buffer (0.02M, pH 8.0), the resin-free
immobilized MAS1 lipase was dried in a vacuum desiccator at
temperature of 40 �C for 8 h and stored in closed vials under 4 �C
until needed. The Novozymes propyl laurate unit (PLU)methodwas
F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128 125
applied to determine the esterification activity of immobilized
MAS1 lipase, which was 1605 ± 30.7 U/g (Wang et al., 2017).
5. Case studies
5.1. Biodiesel production using lipase immobilized on
epoxychloropropane-modified Fe3O4 sub-microspheres
Because enzymes are mixed with reactants during the process
and separating them from the resultant products is challenging,
they are often used in the immobilized form (Gupta et al., 2013).
This would not only improve the stability and recycling of the en-
zymes but also reduce the production costs (Xie and Wang, 2012).
This case study was chosen because it demonstrates a unique
method of enzyme immobilization to produce biodiesel from UCO.
In this study, super-para-magnetic particles were used to immo-
bilize the enzymes, which later were separated from the system
through an external magnetic field (Zhang et al., 2016). The parti-
cles also provided high specific surface areas, as well as great
diffusion values. Fe3O4 sub-microspheres, which were modified
with epoxychloropropane (shown in Fig. 5), were used for the
immobilization of the lipase enzymes extracted from Candida
sp.99� 125. The studied UCO sample was an acidified waste
cooking oil that mainly contained FFAs (96.23%), diglycerides
(1.23%), and triglycerides (2.53%).
The esterification reaction process involved dodecanoic acid
(1.0 g) as a measurement for transesterification reactions activity,
immobilized lipase (0.02 g) as the enzyme of the reactions, and n-
octanol (158 L), n-hexane (5mL), and methanol (150 L) as the
organic solvents. Fe3O4 sub-microspheres were synthesized by
dissolving FeCl3.6H2O (1.35 g) in ethylene glycol (40mL), producing
a clear solution, then adding sodium glutamate (2 g), sodium ace-
tate (1.5 g), and polyethylene glycol (1.5 g). The solution was mixed
thoroughly, and then autoclaved at 200 �C for 8 h. Once they were
cold, the products were separated by magnetic methods, washed
five times each with de-ionized water and ethanol, and then vac-
uum dried at 60 �C for 6 h, generating Fe3O4 sub-microspheres. It
was also shown that the immobilization of the enzyme was suc-
cessful which resulted in an enhanced esterification yield of the
lipase. The activities of immobilized lipases on epoxy-modified
Fe3O4 sub-microspheres were reported as protein bound of
41mg/g, esterification yield of 57.35%, and hydrolysis yield of 2.43%.
Due to the significant magnetic responses of the materials, the
enzyme easily was separated from the reaction medium using a
magnetic field. The results of this study exhibited 97.11% biodiesel
production yield under the optimum reaction conditions of
Fig. 5. (a) Scanning electron microscope (SEM), (b) Enlarged SEM, and (c) Transmission
electron microscope (TEM) images, and (d) X-ray diffraction (XRD) pattern of Fe3 O4
sub-microspheres (Zhang et al., 2016).
methanol:FFA molar ratio of 1:10, amount of hexane of 1.33mL/g
acidified UCO, and 40 �C. The results also showed an initial increase
in the biodiesel production yield as methanol:FFA molar ratio
increased, but then decreased once the methanol concentration
increased above a certain level due to the harmful effects of
methanol on the enzyme active center. Also, the biodiesel pro-
duction yield increased with the solvent n-hexane concentration,
but then it decreased once the solvent concentration reached a
level at which the substrate became too dilute.
5.2. Ultrasound assisted intensification of biodiesel production
using enzymatic inter-esterification
This case was carried out on enzymatic transesterification at a
faster speed, lower content of enzymes, and higher efficiency
compared to those in the conventional methods. An enzymatic
inter-esterification was applied to produce biodiesel from used
cooking oil via ultrasound intensification, rather than the conven-
tional technique of simple stirring. The results showed that
although the amount of enzyme used in the ultrasound assisted
method was less and the reaction time was shorter than those in
the conventional method, the production yield of biodiesel
increased by 6% (from 90.1% to 96.1%), offering a more efficient,
quicker, and less costly method of biodiesel production (Subhedar
and Gogate, 2016). The immobilized enzyme was extracted from
Thermomyces lanuginosus (Lipozyme TLIM). The following optimum
experimental conditions were established for the stirring (con-
ventional) and the ultrasound assisted method, respectively: Molar
ratio of 1:12 (oil:methyl acetate), enzyme loading of 6% (w/v),
temperature of 40 �C and reaction time of 24 h compared to molar
ratio of 1:9 (oil:methyl acetate), enzyme loading of 3% (w/v),
temperature of 50 �C and reaction time of 3 h. The UCO for this
research was collected from various restaurants in India and had
the following composition (Table 6):
Tributyrin was used as a substrate for enzyme, while 0.2mL
tributyrin was incubated with the 200mg lipase enzyme for 5min
in phosphate buffer (pH 7). Then, 20mLmethanol was added to the
reaction and the solution was titrated against alcoholic NaOH
(0.1M) using phenolphthalein as an indicator (Subhedar and
Gogate, 2016). The activity of the immobilized enzyme was calcu-
lated through the following formula (Eq. (1)):
Enzyme activity ðTBU=gÞ ¼ V*M*1000
E*T
(3)
where V¼ volume in mL of NaOH (a measure oftributyrin
consumed during reaction), M¼molarity of NaOH, E¼ amount of
enzyme employed in mg, T¼ time of reaction in min. For the con-
ventional method (stirring), a glass reactor of 100mL was used in
which amechanical stirrer and baffles, to prevent vortex formation,
were installed. The reactor was also supplemented with a
condenser to ensure complete reflux conditions which helped with
recycling the methyl acetate vapors back to the reaction solution. A
Table 6
UCO composition.
Property Value
Oleic acid (%) 18.3
Palmitic acid (%) 6.7
Stearic acid (%) 1.6
Saponification value (mg KOH/g of oil) 198
Density (kg/m3) 930
Acid value (mg KOH/g oil) 4.3
Viscosity (mm2/s) 54.3
F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128126
water bath maintained a constant temperature 50 �C for the reac-
tion. First, a mixture of UCO and methyl acetate was introduced to
the reactor to heat up to the proper temperature and then lipase
was added to the mixture. Samples were drawn to measure methyl
ester content with HPLC. For the ultrasound assisted method, they
used ultrasonic irradiation at a frequency of 20 kHz to pass ultra-
sound through titanium cylindrical horn. The ultrasonic hornwith a
diameter of 1.1 cm andmaximum rated power output of 120Wwas
lowered 2.0 cm into the reaction mixture. Similar to the conven-
tional method, a water bath maintained the reaction temperature.
In this method, oil and methyl acetate were fed to the reactor first
to be heated and then lipase enzymes were added to the solution.
Samples were taken for methyl ester analysis with high-
performance liquid chromatography (HPLC). This research
demonstrated a significant impact of the ultrasound power in the
degree of intensification and cavitational activity of the biodiesel
production process. The results showed that when the ultrasound
power was increased from 40 to 80W, the biodiesel production
yield increased from 57.23% to 96.1%. This is probably because of the
boosted interaction between enzyme and substrate due to a higher
cavitational activity in the solution which resulted in a higher re-
action rate. A better cavitation would improve the process of mass
transfer and diffusion of the substrate towards the enzyme, which
will eventually lead to a higher yield. However, a further increase
from 80 to 100W of the ultrasound power did not result in an in-
crease nor decrease in the biodiesel yield. (Subhedar and Gogate,
2016). In order to maintain an economic status for the enzymatic
process, lipases were recycled and reused. At the end of each cycle,
the immobilized lipase enzyme was filtered, washed with acetone,
dried in oven for 30min at 40 �C, and kept in the desiccator at room
temperature. The enzyme recycled through this process was used
in the following cycle. However, the results indicated a decrease in
biodiesel yield as the number of the cycles increased. After seven
cycle, only 25% of the original enzyme activity was recovered.
5.3. Converting oils high in phospholipids to biodiesel using
immobilized Aspergillus oryzae whole-cell biocatalysts expressing
Fusarium heterosporum lipase
This case study was selected for review because it offers an
effective technique for biodiesel production made out of any form
of feedstock that contains high amount of phospholipids (10%e
30%), without the need for oil pre-treatment (Amoah et al., 2016).
The studied phospholipids sample was extracted from soybean and
the result was also compared to a sample of soybean refined oil.
Nevertheless, the employed methodology was not designed to
focus solely on biodiesel made of soybean oil, rather to remove
phospholipids from oil regardless of the source and origin of it.
Generally, such high content of phospholipids causes the formation
of water-in-oil phospholipid-based reverse micelles. The reverse
micelles trap the water required for activating the enzymes,
resulting in deactivation of the immobilized enzymes and thus
reducing the biodiesel production yield. To reduce the reverse
micelles formation, a simple yet effective technique was to apply a
gentle agitation in the presence of a higher content of water
(Amoah et al., 2016). The whole-cell immobilized Aspergillus oryzae
enzymes were used as biocatalysts. The following protocol was
conducted to obtain the enzyme from A. Oryzae strains. After
cultivating the strains on Czapek-Dox (CD) agar plates at 30 �C for 6
days, the axenic spores were extracted in 5ml of distilled water
which then were transferred into 500ml Sakaguchi flask contain-
ing approximately 850mg of reticulated polyurethane foam BSPs in
100mL of DP medium. The size of the BSPs used for cell immobi-
lization was 6 � 3 � 3mmwith the pore size of 50 pores per linear
inch. The whole-cell immobilized enzyme was filtered out, washed
with distilled water, and then lyophilized for 48 h before used for
the biodiesel conversion. The reaction was carried out in a block
rotator (Nissin Thermo Block Rotator SN-06BN) at 30 �C with a
rotation speed of 7.5 (an arbitrary scale on the instrument) with or
without pre-agitation. Themixture consisted of oil (with or without
phospholipids), distilled water, and immobilized A. oryzae whole-
cell enzymes, while the reaction was initiated through the addi-
tion of an initial amount of methanol (1:1M ratio of the oil). The
remaining methanol was added to the solution step-by-step after
24, 48, and 72 h (1:1M ratios of the oil at each time leading to a
total of 1:4), inhibiting the lipase enzymes to deactivate by meth-
anol. The biodiesel content was measured in samples taken regu-
larly at certain times during the course of the reaction. The results
showed an approximate of a 3-fold increase in the conversion ef-
ficiency which led to a biodiesel production yield increase from
29.7% (before applying the technique) to 91.0% (after the proposed
technique was carried out). This method can eliminate the cost of
oil pre-treatment processes such as degumming, dewaxing, and
synchronizing lipase with phospholipase in which phospholipids
are removed before the enzymes are added to the oil (Amoah et al.,
2016).
6. Conclusions and recommendations
The enzymatic transesterification method offers more advan-
tages than other methods: the catalysts (i.e. the enzymes) are
environmentally friendly; little to no pre-treatment is required
prior to transesterification; and the operational conditions are
moderate and in most cases ambient. In addition, the ratio of
alcohol to oil is lower than previous methods (which can lower the
cost of production); and the quality of the by-product glycerin is
decent (>90% purity), whichwill add an extra source of revenue for
the plant. Furthermore, the purity of the final product is high and
thus little to no polishing process is necessary at the end. Finally,
since there is no saponification process involved, the enzymatic
method allows for higher free fatty acids content in feedstock
which will permit the usage of a broad variety of oils as feedstock.
In particular, the enzymatic method enables utilizing the used
cooking oil, containing high free fatty acids content, which other-
wise would have been difficult or infeasible to process due to the
economical and operational burdens it imposes on the process of
biodiesel. Such is greatly beneficial to biodiesel production because
of UCO's abundant availability, low cost, and not competing with
food and agriculture.
Despite all the advantages of the enzymatic method, the cost of
the enzymes, the ability to recycle and re-use them, the sensitivity
of enzymatic activities against the reaction conditions and the re-
actants involved in the reaction (such as methanol and ethanol),
and the slow kinetic rate of reaction are yet to be studied. For
instance, genetic engineering could play a great role here by
creating a strain of lipase-producing microorganisms with higher
resistance towards extreme reaction conditions and the presence of
alcohol in the reaction system. Also, developing more efficient
lipase immobilizationmethods can improve the enzyme recovery.
At the end, another approach to improve the economics of enzy-
matic transesterification is to find efficient methods to regenerate
the activities of lipase, after being used in the process.
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