Journal of Cleaner Production 216 (2019) 117e128

Contents lists available at ScienceDirect

Journal of Cleaner Production

journal homepage: www.elsevier.com/locate/jclepro

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 a b s t r a c t

Article history: This paper reviews various aspects of the enzymatic transesterification method to convert used cooking
Received 12 September 2018 oil to biodiesel. The goal of this paper is to provide a thorough overview from general biodiesel pro-
Received in revised form duction processes, reaction conditions, challenges, and solutions for higher biodiesel production yield
13 January 2019
through introducing various microorganisms that are capable of producing the enzymes required to
Accepted 16 January 2019
convert used cooking oil into biodiesel. The characteristics, composition, and advantages of the used
Available online 21 January 2019
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
Keywords:
Biodiesel production process
under super-critical conditions, and enzyme-catalyzed reactions are explained. Furthermore, the ad-
Used cooking oil vantages of the enzymatic method over other methods, and the enzymes, which are the key elements of
Enzymatic transesterification such reactions, are discussed. Lipases are the most promising enzymes currently known for biodiesel
Lipase conversion. The physiological and physical properties of microbial lipases, the catalytic mechanisms of
Biocatalysts the enzymes, various methods of enzyme immobilization such as adsorption, covalent and affinity
Enzyme immobilization 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

* Corresponding author.
E-mail address: [email protected] (F. Moazeni).

https://doi.org/10.1016/j.jclepro.2019.01.181
0959-6526/© 2019 Elsevier Ltd. All rights reserved.
118 F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128

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 Fe3 O4 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

1. Introduction 1.2. Why used cooking oil?

1.1. Problem statement Biodiesel is a composition of mono alkyl esters derived from oils,
and thus the higher the oil content of the feedstock, the higher the
It is vastly understood that the era of fossil fuels will soon be biodiesel production yield, which also corresponds to a lower cost
over. The increasing accumulation of carbon dioxide in the atmo- of production. Among various crops that are considered for bio-
sphere resulting in a drastic increase of global temperature does not diesel, such as jatropha, rubber seed, castor, Pongamia pinnata, sea
allow the current rate of these fuels' consumption in the world, and mango, soybean, palm, and rapeseed, palm oil produces the highest
thereby man must seek out clean and renewable resources of en- amount of oil per area with 5000 kg oil per hectare (Gui et al.,
ergy to minimize dependence on fossil fuels (Rehan et al., 2018). 2008), equivalent to 5.3 weight percent (wt%) of fatty acids. On
Similar to our electricity demand which can potentially be met with the other hand, palm oil is edible which would put biodiesel in
energy resources such as wind, geothermal, and solar, there is also a competition with agriculture. The palm oil after it is disposed as
need for alternative fuels for transportation. Given that currently waste cooking oil contains more than 20 wt% fatty acid (Kusdiana
almost 60% of the total crude oil consumption in the world goes and Saka, 2004). Table 1 compares the fatty acid content of the
towards transportation, the demand for which is increasing every most common virgin oils and a sample of used cooking oil obtained
year, the need for an alternative non-fossil-based fuel for trans- from a Chinese restaurant, where most of the food preparation is
portation seems even more crucial. One of the best candidates is through frying.
biodiesel, as diesel engine vehicles could use it with little to no A comparison between the main fatty acids' composition, in
modifications. However, producing biodiesel is too expensive to both virgin oils and UCO (Table 1), determines UCO a more prom-
compete with petroleum-based fuels. A major portion of the bio- ising feedstock for biofuel. Not only does UCO offer higher fatty
diesel production's cost comes from the costs of the raw materials acids content but also it is not food.
(Ragauskas et al., 2006; Gołaszewski, 2009). In addition, the feed-
stock of the biodiesel cannot compete with agriculture, which was
1.3. Biodiesel production process
the drawback to edible-oil-based fuels. Another candidate to pro-
duce oil could be algae, which requires water and is costly to har-
In the following subsection, the key steps of producing biodiesel
€msa
vest (Chye et al., 2018; Ja € et al., 2017). Animal fats are also not
are discussed: (Gurunathan and Ravi, 2015; Sebastian et al., 2016;
great candidates due to the high saturated fatty acid content which
Sotoft et al., 2010):
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- 1.3.1. Feedstock pre-treatment
enue to the biodiesel plants compared to high quality glycerin that Depending on the type of the oil used as feedstock (virgin
can be sold out at a high price. Hence, the best candidate should be vegetable oil, used cooking oil, algal oil, or tallow), a range of
non-fossil-edible, cheap to produce, and rather easy to process. different processes, often in sequence, are applied on the crude oil
Studies have shown that various types of used cooking oils (UCOs) to refine it before mixing with other material to be sent to the
are promising candidates for biodiesel production (Gui et al., 2008), reactor. Some of these processes include removing impurities and
as they do not compete with food (Akbar et al., 2009), they are solids coming from cooking and handling (in the case of UCO)
inexpensive to obtain (Kumar and Sharma, 2011), and rather easy to (Yaakob et al., 2013). Different forms of sieving are used to separate
process (Knothe et al., 2015). However, used cooking oil contains a solids and particles. Also, degumming to remove phospholipids,
high amount of free fatty acids which will be converted to soap
during the conventional transesterification process, a reaction
Table 1
called saponification. Saponification drastically lowers the biodiesel Comparison of fatty acid composition in weight percentage (wt%) for different oil
production yield and the purity of the biodiesel, while it increases sources (Lam et al., 2010; Leung and Guo, 2006; Yun et al., 2013; Akbar et al., 2009).
the cost of production due to the need for additional feedstock pre-
Fatty acid Soybean Cottonseed Palm Coconut Canola UCO
treatment processes, catalysts, and polishing and purification pro-
cesses. Despite the disadvantages of utilizing used cooking oil as 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
feedstock, if biodiesel is converted under the processes that avoid
Stearic 3.7 2.6 4.5 3.0 2.2 4.8
saponification, UCO is still a better candidate than other types of Oleic 22.8 19.2 40.5 6.9 55 52.9
feedstock to supply oil for biodiesel. Linoleic 53.7 55.2 10.1 2.2 24 13.5
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 and Ravi, 2015; Sebastian et al., 2016; Sotoft et al., 2010).
method should be done. Water hydrolyzes triglycerides (TGs)
which can result in soap formation during the transesterification 1.4. Contribution
process. Soap formation is one of the most important reasons of
having a low biodiesel production yield. Phospholipids can be This paper reviews various methods and their challenges that
either washed away with hot water or removed via centrifugation are available to produce biodiesel from UCOs that are inexpensive
and paper filtration. Water is often removed by heating the solution and abundant, yet rich in fatty acids. Lipase enzymes extracted from
up to 100
C (Demirbas, 2009; Banerjee and Chakraborty, 2009). microorganisms serve as bio-catalysts in the enzymatic trans-
Some of other common methods of pre-treatment include steam esterification reactions (Demirbas, 2008). Based on the types of the
injection (Lertsathapornsuk et al., 2005), column chromatography alcohol used in the trasesterification process, various microorgan-
(Lee et al., 2002), neutralization, film vacuum evaporation isms can be used as the source for the enzymatic catalysts. For
(Cvengros and Cvengrosova , 2004), and vacuum filtration (Dias instance, it was shown that the biodiesel conversion with primary
et al., 2008). However, some studies replaced pre-treatment with alcohols was most effective by using enzymes extracted from Mucor
regular esterification of free fatty acids (FFAs) with sulfuric acid miehei, whereas in the presence of the secondary alcohols, the
prior to the alkali transesterification, through which FFA content of lipase extracted from Candida antarctica was most effective (Nelson
the UCO was reduced (Leung et al., 2010). In addition, the appli- et al., 1996). Studies show that the enzymatic catalysts are most
cation of acidic ion-exchange resins as a pre-treatment process has effective and less costly if they are immobilized on a specific carrier
demonstrated a reduction in the FFAs content of the UCO. Though, (Amini et al., 2017a) or, intracellularly, as a whole-cell (Kuratani
the early catalyst activity deterioration was the disadvantage of this et al., 2018). Several researchers have studied this topic in recent
method (Jeromin et al., 1987; Shibasaki-Kitakawa et al., 2007; Li years, but finding the best microorganisms that offer the most
et al., 2008). One of the most recent pre-treatment methods of effective enzymes for the biodiesel applications and how to
UCO is to utilize glycerin for the acidic raw material, in which a high immobilize and re-use them are still the subjects of ongoing
temperature (about 200
C) and a catalyst such as zinc chloride are studies. The rest of this review paper is categorized as the
required. FFAs present in the UCO react with glycerin to produce following:
mono- and diglycerides which will result in a reduction of FFAs in Section 2- Used Cooking Oil: Section 2 will cover characteristics,
the UCO (Van Gerpen et al., 2004). The high associated cost of this composition, advantages, and disadvantages of used cooking oil.
method makes its application economically infeasible in most Section 3- Transesterification Methods: Section 3 will explain
cases. the four existing transesterification methods used for biodiesel
conversion, including the process conditions, reactants, pros, and
1.3.2. Transesterification/esterification reactions cons.
Transesterification reaction is the way of converting oils and Section 4- Enzymes as Catalysts: Section 4 will cover all aspects
fatty acids into alkyl esters, also known as biodiesel. In the trans- of enzymes used for biodiesel conversion, including properties,
esterification process, triglycerides react with an alcohol (methane mechanisms, and immobilization techniques of the enzymes.
or ethane) to generate methyl or ethyl esters of fatty acids and Section 5- Case Studies: The three case studies chosen for re-
glycerol. In most countries, alkyl esters are prepared in the form of view by this paper will propose unique methods in various steps of
methyl esters, rather than ethyl esters, mainly because methanol is enzymatic transesterification. In the first study, the enzymes were
less costly than ethane (Knothe et al., 2015). Transesterification immobilized on a super magnetic surface which facilitated the
methods have been the subject to several studies since the idea of enzyme recovery by using an external magnetic field (Zhang et al.,
biodiesel was proposed. In 1986, a comparative analysis was con- 2016). In the second study, an ultrasound system was used to
ducted on using vegetable oil as an alternative fuel for diesel en- intensify the solution which improved enzymatic inter-
gines (Ziejewski et al., 1986). Then in 1988, lipases were used as esterification reactions (Subhedar and Gogate, 2016). Finally, in
catalysts in transesterification reactions (Zaks and Klibanov, 1988). the third case, a particular enzyme was used that demonstrated
Traditionally, transesterification can be performed heterogeneously high capability of converting oil containing high phospholipids into
and homogeneously by acid and base catalysts. Alkali catalysts in biodiesel. The results of this study allow biodiesel production
the form of sodium or potassium hydroxide are more common in plants to eliminate the degumming step in the feedstock pre-
the homogeneous transesterification reactions, as they offer a treatment process which will cut down the cost of production, as
higher speed compared to the acidic catalysts. In the case of a high well as increasing the production yield (Amoah et al., 2016).
acid content, acid-catalyzed esterification can also be used to react
fatty acids with alcohol to produce biodiesel. The biodiesel con- 2. Used cooking oil
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, As the name suggests, used cooking oil refers to the vegetable oil
the effect of other parameters such as alcohol to oil molar ratio, or lard in food industries including the food preparation processes
temperature, reaction time, and water content on trans- in restaurants, hotels, and fast food outlets. Such oils and fats
esterification results have also been studied (Watkins et al., 2004). cannot be used any further due to oxidation and pollution with
small food parts. As waste, UCOs cannot be discharged directly into
1.3.3. Polishing and purification the environment or wastewater because it can clog the collection
The products of the carried out reactions include biodiesel, as pipes which could potentially lead to the sanitary sewer overflow.
well as un-reacted free fatty acids, soap, glycerol, excess alcohol, In addition, at the wastewater facilities, the high content of oil of
excess water, and catalyst residue. Various procedures are in place the UCO can coat the activated sludge which prevents oxygen
to separate all the impurities from biodiesel in the polishing and transfer for waste degradation. As useless and even wasteful as it
purification step. Glycerol can be separated via gravity and sold out sounds, UCO can be a great feedstock for biodiesel (Gui et al., 2008;
as by-product. Water can be flashed out and methanol can be Chhetri et al., 2008). The used cooking oil can come from palm,
recovered to re-use through distillation. Soaps are often converted sunflower, corn, canola, or any other oil that was originally used for
into acids to remove. Process flowcharts related to the conventional the food preparation. Similar to many oleaginous vegetable species,
and enzymatic reactions are shown in Figs. 1 and 2 (Gurunathan UCO is composed of the esters of glycerol and fatty acids, also called
120 F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128

Fig. 1. Conventional biodiesel production process.

Fig. 2. Enzymatic biodiesel production process (Gurunathan and Ravi, 2015; Sebastian et al., 2016; Sotoft et al., 2010).

triglycerides (TGs) or glycerides (Knothe et al., 2015). Fatty acids, in oils, the concentrations of unsaturated fatty acids are higher than in
general, contain the very polar carboxyl group at one end of a hy- animal fats. These oils, when undergone the food preparation
drocarbon molecule. There are two different types of fatty acids; processes, change in terms of the physical and chemical charac-
saturated and unsaturated fatty acids. Saturated fatty acids contain teristics (Van Gerpen, 2005). Among different processes of food
a single carbon bond, while the unsaturated fatty acids include at preparation (such as boiling, frying, and steaming), frying is the
least one C:C doubled bonds (Leung and Guo, 2006). In vegetable 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, (Yun et al., 2013). Another classification of the UCOs can be done
frying is conducted in the same oil for several times under high based on the sources from which the used cooking oil was
temperature and in the presence of oxygen and light, which can collected. Table 3 demonstrates density of and the content of sulfur,
cause major changes in the oil. Some of these physical changes are nitrogen, hydrogen, and carbon present in various sources of UCOs
as the following: higher viscosity, higher specific heat, and higher (Bezergianni and Kalogianni, 2009).
tendency to generate foam (Demirbas, 2009). The main types of
reactions causing the properties of the oils to change in the UCO 2.1. Characterization
are: thermolytic reactions under high temperature (180
C) in the
absence of oxygen; oxidative reactions under high temperature in The ultimate goal of producing biodiesel made from any source
air; and hydrolytic reaction under high temperature in the presence of oil (fresh or waste) would be to use it in the car engines.
of steam (Talebian-Kiakalaieh et al., 2013). As a result of a combi- Therefore, the UCO, as one the sources of the biodiesel, must
nation of these reactions and the original oils that were used for demonstrate the engine performances that are similar to those
cooking, the UCOs demonstrate different characteristics than of with diesel-based fuels. The crucial characteristics are listed in the
vegetable crude or refined oils (Ullah et al., 2015). The physical and following:
chemical properties of UCO can vary based on its original fresh
vegetable oil. Some of the UCO properties are shown in Table 2 2.1.1. Ignition quality
(Gutierrez-Zapata et al., 2017; Çetinkaya et al., 2005; Yaakob The cetane number of the biofuel should be sufficiently high
et al., 2013). enough to avoid knocking the engine, which is the result of a long
Compared to the fresh oil, the triglycerides of the vegetable oil ignition. The satisfactory cetane number varies between 40 and 60
break down during the cooking process into FFAs, leading to a (Ramadhas et al., 2004).
higher free fatty acids content in UCO. Also, the oxidation and
polymerization process occurring during the cooking and frying 2.1.2. Viscosity
will cause an increase in the viscosity and the saponification As stated before, the proper value of fuel viscosity affects the
number in UCO (da Silva Ce sar et al., 2017). High viscose biodiesel is combustion and thermal efficiency of the engine. This is particu-
not favored as it will ultimately damage the fuel injector in the larly important in conditions under which the speed is low and the
engine of the cars, since it will result in an incomplete combustion load is light. According to the ASTM D6751-12 standard, the kine-
and thus accumulating unburned deposits in the engine (Wei et al., matic viscosity at 40+ C of the biodiesel can vary within the range of
2017). Moreover, because of the heat and mass transfer between 1.9e6.0 mm2/s (Charter, 2008).
the food and the oil during frying and cooking, UCO contains higher
amount of water than fresh vegetable oil. Such high water content 2.1.3. Heating value
along with the high FFA content and high saponification number of Similar to the other properties, the calorific value of the bio-
UCO cause the saponification reactions to occur during the trans- diesel should be close to that in diesel-based fuel 43.350 kJ/kg. The
esterification process. The saponification reduces the biodiesel combustion chamber of the engine can generally take in a broad
production yield and increases the amount of utilized catalyst. range of heating values, however, it only operates efficiently with
Hence, an oil pre-treatment process, as well as a suitable trans- high heating value fuels. The highest calorific values among various
esterification method should be employed to achieve a high bio- UCO types belong to the UCO originated from sunflower oil which
diesel production yield at a feasible cost (Buffi et al., 2017). As demonstrates 40.579 kJ/kg of calorific value (Altın et al., 2001).
mentioned above, the distribution of various fatty acids in UCO
varies with the original oil and the process done on the oil before it 2.1.4. Specific temperatures
reaches the biodiesel plants (for instance, how many times the oil Pour point, cloud point, and flash point of fuel are important
was used for cooking or frying). However, among the various temperatures in the engine operation. If used cooking oil is utilized
vegetable oils viable for biodiesel production, olive, sunflower, and to make the fuel, then both pour point and cloud point of that
palm oil are the most common ones used in cooking. Thus, the fatty should fall below its freezing point, in order for the engine to
acids mainly present in these oils (i.e. palmitic acid, stearic acid, and operate in cold weather environments. However, the flash point
oleic acids) are considered to be the major fatty acids in UCOs should be as high as feasible to maintain the safety of ignition.
(Banerjee et al., 2014). For instance, the FFAs present in UCO was According to ASTM D6751-12 standard, the minimum flash point is
identified as palmitic acid 30%, stearic acid 10%, and oleic acid 60% 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).
Table 2
UCO physical and chemical properties. 3. Transesterification methods
Property Value
The feedstock oil (UCO) will be converted to biodiesel through
Palmiric acid (wt%) 8.5 chemical reactions of transesterification and esterification. Trans-
Stearic acid (wt%) 3.1
esterification is the catalyzed process of trading the alkoxy group of
Oleic acid (wt%) 21.2
Linoleic acid (wt%) 55.2
Linolenic acid (wt%) 5.9
Others (wt%) 4.2 Table 3
Density (g/cm3) 0:91  0:924 Physical and chemical properties of various sources of UCO.
Kinetic viscosity (mm2/s) at 40+ C 36:4  42 Domestic Restaurant, taverns Fast food outlets
Saponification value (mgKOH/g) 188:2  207
Acid value (mgKOH/g) 1:32  3:6 Density (kg=m3 ) 0.8929 0.8929 0.8929
Iodine number (gI2/100 g) 83  141:5 S (wppm) 0.00 187.70 26.30
Water content (wt%) 0:8  1:9 N (wppm) 0.40 49.10 61.90
Average sodium content (mg/kg) 6.9 H (wt%) 11.56 11.52 11.58
Average peroxide value (mg/kg) 23.1 C (wt%) 77.24 76.53 76.32
122 F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128

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, in which
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. Fig. 3. Step-by-step reactions for biodiesel production (R is a small alkyl group, R1, R2
The overall process involves three consecutive, reversible reactions and R3 are fatty acid chains; k1, k2, k3, k4, k5, k6 are chemical or enzymatic catalysts)
which produce intermediate molecules of di- and monoglycerides (Yun et al., 2013).
(Kayode and Hart, 2017; Vicente et al., 2004). (Fig. 3)

due to its high molar ratio of methanol to oil demand, requires

FFA þ MeOH/FAME þ H2 O (1) catalyst separation, and causes corrosion (Fig. 1) (Aranda et al.,
Such reactions currently are carried out under the following 2008; Demirbas, 2005). The disadvantages of the chemical-
major forms of process (Banerjee et al., 2014): catalyzed reactions (acid- or alkali-based) can be summarized
as the following: (1) a low purity and low biodiesel production
 The homogeneous alkali-catalyzed reaction, using sodium yield as the results of side reactions of saponification and hy-
hydroxide (NaOH) or potassium hydroxide (KOH) - This drolysis; (2) the high capital cost and energy required for the
method is vastly used in industry due to its simplicity, the rather process; (3) the high cost of separation and purification of cat-
mild reaction conditions it requires (e.g. low temperature and alysts and glycerol; (4) the need for neutralization and waste-
low pressure), and the high conversion it offers in a short re- water treatment (Yun et al., 2013; Kayode and Hart, 2017). The
action time. Despite the advantages, this method is not suitable issues of catalysts recovery and saponification have led re-
for processing UCO, as it is highly limited by the FFAs content of searchers towards heterogeneous non-enzymatic catalysts such
the feedstock. In this method, free fatty acids react with alkali as amorphous zirconia, titanium-, aluminum-, and potassium-
forming soap. The soap can convert to a gel under ambient doped zirconias, metal oxides, hetero-polyacids, and sulfated
temperature, which will substantially decrease the production zeolites (Guldhe et al., 2017; Qadri et al., 2017). Though the
yield (Meher et al., 2006; Leung et al., 2010). To avoid such, the heterogeneous catalysts appear to be easier to separate and
content of FFA cannot go beyond 2 wt% which is not the case in more stable on the feedstock oil with high FFA content (such as
the lipid-rich UCOs. To avoid saponification, an acidic pre- UCO), they are often costly and energy intensive because of their
treatment process is added in which FFAs are esterified to demands for high temperature and high ratio of alcohol:-
FAME (biodiesel) in the presence of an alcohol such as methanol. substrate in the reactions (Martinez-Guerra et al., 2018).
The acid step is usually performed under 0.5e1.0% sulfuric acid  The non-catalytic reaction under supercritical conditions -
at a high temperature (60  100+ C) and the ratio of meth- Though it offers more simplified products separation, polishing,
anol:substrate about 30:1 (Fig. 1). Therefore, a two-step process and purification procedures due to the absence of a catalyst, the
involving acid and base is designed for the biodiesel feedstock catalyst-free supercritical method is very expensive because of
with high FFA content such as UCO, in which the water resulted the critical operating conditions, high consumption of meth-
from the acid-catalyzed esterification is removed before anol, and energy that the entire process demands (Demirbas,
entering the alkali-catalyzed step, to prevent saponification. 2005; Patil et al., 2011).
Despite the advantage that this two-step acid-base process of-  The enzyme-catalyzed reactions - This is a fairly new method
fers, the high cost of production, which is the result of adding an which still is a subject of ongoing research. In this type of re-
extra step to the entire process, is still a great burden when it actions, an enzyme such as lipase serves as a bio-catalyst for the
comes to a large scale biodiesel production (Vicente et al., 2004; transesterification reactions (Amini et al., 2017b). Similar to the
Dorado et al., 2004). non-catalyzed reaction method, the enzyme-catalyzed re-
 The homogeneous acid-catalyzed reaction, using mostly actions offer simple biodiesel purification procedures but with a
sulfuric acid (H2SO4) and hydrochloric acid (HCl) - The substantial reduced energy requirement due to the mild oper-
advantage of this method is that it can simultaneously catalyze ating conditions (Amini et al., 2017a). The enzymatic method
esterification and transesterification (Wallis et al., 2017). How- demonstrates the following advantages over other methods of
ever, compared to the previous method, it offers a slower re- transesterification (Kuratani et al., 2018): requiring mild reac-
action rate, consumes high energy and high amount of methanol 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
Table 4 non-esterified fatty acids in one step; not resulting in side-
Effect of temperature on the yield of biodiesel production.
reactions such as saponification; producing high-grade glyc-
Temperature (K) Sunflower oil (%) Corn oil (%) Cottonseed oil (%) erol as by-product; and being environmentally accepted. How-
620 79.6 80.5 82.3 ever, the price and stability of enzymes are the main challenges
630 93.6 95.8 96.5 yet to be studied (Yun et al., 2013; Nelson et al., 1996; Zhang
640 96.8 97.2 97.6 et al., 2003; Demirbas, 2008).
 F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128 123

4. Enzymes as catalysts Table 5

Lipase-producing microorganisms.

The most common enzymes used for biodiesel production are Type Microbial Source Ref. (Shah and Gupta, 2007)
lipases. Lipases convert TGs to glycerol and fatty acids via hydrolysis Fungi Alternaria brassicicola (Dutra et al., 2008)
and thereby are classified as hydrolases (Wu et al., 2017; Hwang Aspergillus sp. (Dutra et al., 2008)
et al., 2014). In general, a carboxylesterase or carboxylic-ester hy- Candida antarctica (Dutra et al., 2008)
drolase (EC 3.1.1.1) refers to an enzyme that catalyzes chemical Mucor miehei (Dutra et al., 2008)
Penicillium cyclopium (Dutra et al., 2008)
reactions of the following reaction:
Rhizomucor miehei (Dutra et al., 2008)
Rhizopus sp. (Dutra et al., 2008)
carboxylic ester þ H2 O/alcohol þ carboxylate (2) Streptomyces sp. (Dutra et al., 2008)
Aspergillus niger (Dutra et al., 2008)
Comparing the above template with the application of lipases in Thermomyces lanuginous (Dutra et al., 2008)
the biodiesel production process, we can conclude that lipases are Fusarium heterosporum (Dutra et al., 2008)
categorized under carboxylesterases because they catalyze both the Humicola lanuginose (Dutra et al., 2008)
hydrolysis and synthesis of long-chain acylglycerols (Pourzolfaghar Oospora lactis (Dutra et al., 2008)
Rhizopus oryzae (Dutra et al., 2008)
et al., 2016; Chourasia et al., 2015). Lipases can be extracted from
Yeasts Candida sp. (Dutra et al., 2008)
various sources of plant, fungal, animal, and bacterial. Lipases Pichia sp. (Dutra et al., 2008)
contributed from plants are located in their energy reserve tissues Saccharomyces lipolytica (Dutra et al., 2008)
such as oilseeds (Pizarro and Park, 2003). While plant lipases offer a Geotrichum candidum (Dutra et al., 2008)
high substrate specificity and low production cost, they are not very Yarrowia lipolytica (Dutra et al., 2008)
Bacteria Microbial Source Ref. (Shah and Gupta, 2007)
common in industrial applications due to their low expression and Achromobacter sp. (Hsu et al., 2002)
uneconomical fold purity (Shah and Gupta, 2007). Sources of ani- Bacillus sp. (Hsu et al., 2002)
mal lipases are pancreatic glands of cattle and pigs, as well as the Burkholderia glumae Jaeger and Reetz (1998)
pre-gastric juices of calves, lambs or baby goats (Jolly and Chromobacterium viscosum (Ziejewski et al., 1986)
Micrococcus freudenreichii (Ziejewski et al., 1986)
Kosikowski, 1975). Animal lipases are used extensively in dairy
Moraxella sp. (Ziejewski et al., 1986)
and food industry, but are less applied in other commercial appli- Mycobacterium chelonae (Ziejewski et al., 1986)
cations (Goswami et al., 2013). Microbial lipases, however, are the Pasteurella multocida (Ziejewski et al., 1986)
most common sources of enzymes used in biodiesel processes (Fan Propionibacterium sp. (Ziejewski et al., 1986)
et al., 2017). Compared to other forms of enzymes, microbial en- Proteus vulgaris (Ziejewski et al., 1986)
Pseudomonas sp. (Ziejewski et al., 1986)
zymes are cheap and fast to produce because they are abundant, Proteus vulgaris (Ziejewski et al., 1986)
not affected by seasons, grow fast, and can be grown and immo- Psychrobacter immobilis (Ziejewski et al., 1986)
bilized on inexpensive media. They offer high yields and can be Serratia marcescens (Ziejewski et al., 1986)
genetically modified. In addition, microbial-based lipases are more Staphylococcus sp. (Ziejewski et al., 1986)
Sulfolobus acidocaldarius (Ziejewski et al., 1986)
stable than the plant- and animal-based enzymes and are safe to
Vibrio chloreae (Ziejewski et al., 1986)
the environment (Christopher et al., 2014). Table 5 shows the most Pseudomonas alcaligens (Ziejewski et al., 1986)
common lipase-producing microorganisms including bacteria, Chromobacterium visosum (Ziejewski et al., 1986)
fungi, and yeasts (Yun et al., 2013; Seth et al., 2014). Those strains Pseudomonas putida (Ziejewski et al., 1986)
that are close taxonomically can generate different types of lipases. Statphylococcus stolonifer (Ziejewski et al., 1986)
Enterococcus faecalis (Dutra et al., 2008)
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- et al., 2017; Hotta et al., 2002; Ikeda and Clark, 1998; Royter et al.,
tion is the immobilized Pseudomonas cepacia. This lipase was used 2009; Lusk et al., 2018; Mishra et al., 2017).
for the transesterification of soybean oil with methanol and ethanol Lipases, in general, can become active under mild conditions
(Akoh, 1993). Also, two commercial lipases of Novozym 435 and such as ambient temperature and pressure which will result in a
Lipozyme IM were used to produce fatty acid ethyl esters from low energy consumption and thus a low operational cost in bio-
castor oil (Noureddini et al., 2005). Immobilized enzymes origi- diesel production. Lipases are specific, meaning that they react
nated from Thermomyces lanuginosa and C. antarctica served as towards specific substrate(s). Such property will avoid producing
biocatalysts in alkyl ester production made from UCO (Lanza et al., unwanted products which will lead to fewer side reactions and
2004). 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
4.1. Physiological and physical properties of microbial lipase production yield and the economy of the biodiesel.

Most microbial-origin lipases are extracellular, responding best

under the pH range of 7.5e9. Their molecular weights range from 4.2. Catalytic mechanism
30 to 50 kDa and can be categorized into the mesophilic- and
thermophilic-origin source, demonstrating an optimum activity Lipase serves as a biocatalyst for three types of reactions: hy-
under 35e50
C for the former and 60e80
C for the latter (Zaks drolysis, esterification, and transesterification, among which the
and Klibanov, 1988; Wang et al., 2007; Kazanina et al., 1981; last one includes four categories of alcoholysis, inter-esterification,
Kakugawa et al., 2002). The thermophilic enzymes are often used in acidolysis, and aminolysis. During hydrolysis, molecules of acyl are
the processes requiring high temperature, as they remain active transferred between molecules of glyceride in the presence of extra
and stable up to 100
C even when an organic solvent is present. For water. Hydrolysis of oils or fats results in free fatty acids and glyc-
example, lipases from Pyrobaculum calidifonti, Pyrococcus furiosus, erol formation. This is a reversible reaction, in which the reverse
Thermoanaerobacter thermohydrosulfuricus, and Caldanaerobacter direction happens when the rate of hydrolysis and the final prod-
subterraneus, can tolerate temperatures as high as 90
C (Rehman ucts vary with the concentration of fatty acid dissolved in the oil
124 F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128

phase, while glycerol concentrates in the water phase (Zaharudin 3- Affinity Immobilization: This method harnesses the selec-
et al., 2018; Alin et al., 2011; Murty et al., 2002; Adlercreutz, 2017). tivity of an enzyme towards its support, which can vary under
various physiological circumstances. There are two ways to achieve
4.3. Immobilization such immobilization: the support can be pre-attached to an affinity
ligand specific to a certain enzyme, or the enzyme itself can be
Immobilization can be conducted as enzyme immobilization or coupled to a component developing affinity towards the support
whole-cell immobilization (Kawaguti et al., 2006). All the reactants, (Bandikari et al., 2018).
substrates, and products in the process of biodiesel production are 4- Entrapment: As the name suggests, in this approach en-
in liquid form, thus, enzyme immobilization occurs only when li- zymes are entrapped by bonds (either covalent or non-covalent)
pases are attached to a solid support. Substrates pass over the inside a gel or a fiber (Rehman et al., 2017). Carriers should be
support, to which the enzyme is attached, and are converted to specific to the enzyme. For instance, to immobilize lipase, poly-
products (Datta et al., 2013). Immobilized enzymes exhibit several urethane foam, silica, sepabeads, and cellulosic nanofibers can be
advantages over free enzymes such as the ability of frequent use of used as carriers (Cazaban et al., 2017; Singh et al., 2009; Reetz et al.,
the enzyme, convenient separation of the enzyme from the reac- 1996; Noureddini et al., 2005).
tion solution, high enzyme stability, and more pure products.
However, selecting the proper solid support for the enzyme and 4.3.2. Whole-cell immobilization
efficient immobilization techniques which could provide optimum All of the above methods are used to immobilize free (extra-
enzyme activity and stability in the given substrate medium still cellular) enzymes. While efficient, they all demand extremely ac-
remains challenging (Fai et al., 2017; Hellmers et al., 2018). Various curate enzyme purification. However, if the entire cell is
immobilization methods are discussed in the following, and sum- immobilized, the intracellular lipase is immobilized along with the
marized in Fig. 4. 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
4.3.1. Various immobilization techniques
of biocatalyst is called whole-cell immobilized lipase. For instance,
1- Adsorption: In this method, the solid support is soaked in
surface-displayed strategy was used to intracellularly immobilize
enzyme or the enzyme is dried on surfaces of electrode. In both
the whole-cell yeast Rhizopus oryzae lipase (ROL) in Saccharomyces
cases, hydrophobic interactions and salt linkages allow physical
cerevisiae MT81 (Matsumoto et al., 2002). With the help of genetic
adsorption which will lead to the enzyme immobilization. The most
engineering, rProROL from R. oryzae IFO4697 was constructed un-
common lipase enzymes immobilized via adsorption are the
der the control of the isocitrate lyase gene of Candida tropicalis
C. antartica lipase, which is immobilized on acrylic resin (Nov-
(UPR-ICL) 5'-upstream region at 30
C for 98 h through a two-stage
ozymO435), Mucormiehei lipase, which is immobilized on a mac-
cultivation using semidefined medium (SDC medium, which is SD
roporous ion-exchange resin (Lipozyme IM), T. lanuginosus acrylic
medium with 2% casamino acids) including 2.0% and 0.5% glucose.
lipase (Lipozyme TLIM), immobilized on resin, Rhizomucor miehei
Once this rProROL was constructed, the intracellular lipase activity
lipase which is immobilized on macroporous anion exchange resin
increased significantly. This whole-cell immobilized lipase was
(Lipozyme RM IM), and Candida sp. 99125 lipase, immobilized on
then used to form biodiesel from plant-based oil with methanol,
textile membranes (Tan et al., 2010; Bahulekar, 2017). Another
without the presence of solvents and water at the production rate
point that must be taken into consideration is that lipase becomes
of 71 wt% methyl esters. In another study biomass support particles
deactivated by methanol adsorption onto the immobilized enzyme.
(BSPs) strategy was used to intracellularly immobilize the whole-
Therefore, some sort of enzyme regeneration method such as
cell Rhizopus oryzae lipases in order to use them for biodiesel
regeneration with higher alcohols like butanol is necessary.
production from soybean oil (Ban et al., 2002). The R. oryzae cells
2- Covalent Binding: This method, also known as carrier
were inoculated into a basal medium and incubated for 80e90 h at
binding, occurs due to the presence of side chain amino-acids such
35
C on a reciprocal shaker (150 oscillations/min; amplitude
as arginine, aspartic acid, histidine and some degree of reactivity
70 mm) with BSPs. As a result, while growing during the shake-
based on various functional groups such as imidazole, indolyl, and
flask incubation, R. oryzae cells became immobilized within the
phenolic hydroxyl. Some of the carriers used for lipase immobili-
BSPs.
zation are polyurethane foam, silica, sepabeads, and cellulosic
The BSPs were in the form of 6-mm cubes of reticulated poly-
nanofibers (Awang et al., 2007; Tran et al., 2012; Krauss et al., 2017;
urethane foam containing a particle porosity beyond 97% with a
Zhang et al., 2017).
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 conversion was 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 75 mg lipase solution/g XAD1180 resin,
containing an equal volume of sodium phosphate buffer (0.02 M,
pH 8.0) under 30
C and a speed of 200 rpm for 8 h. After rinsed
with sodium phosphate buffer (0.02 M, 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
Fig. 4. Enzyme immobilization methods. until needed. The Novozymes propyl laurate unit (PLU) method was
 F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128 125

applied to determine the esterification activity of immobilized methanol:FFA molar ratio of 1:10, amount of hexane of 1.33 mL/g
MAS1 lipase, which was 1605 ± 30.7 U/g (Wang et al., 2017). 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
5. Case studies
increased above a certain level due to the harmful effects of
methanol on the enzyme active center. Also, the biodiesel pro-
5.1. Biodiesel production using lipase immobilized on
duction yield increased with the solvent n-hexane concentration,
epoxychloropropane-modified Fe3 O4 sub-microspheres
but then it decreased once the solvent concentration reached a
level at which the substrate became too dilute.
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). 5.2. Ultrasound assisted intensification of biodiesel production
This would not only improve the stability and recycling of the en- using enzymatic inter-esterification
zymes but also reduce the production costs (Xie and Wang, 2012).
This case study was chosen because it demonstrates a unique This case was carried out on enzymatic transesterification at a
method of enzyme immobilization to produce biodiesel from UCO. faster speed, lower content of enzymes, and higher efficiency
In this study, super-para-magnetic particles were used to immo- compared to those in the conventional methods. An enzymatic
bilize the enzymes, which later were separated from the system inter-esterification was applied to produce biodiesel from used
through an external magnetic field (Zhang et al., 2016). The parti- cooking oil via ultrasound intensification, rather than the conven-
cles also provided high specific surface areas, as well as great tional technique of simple stirring. The results showed that
diffusion values. Fe3O4 sub-microspheres, which were modified although the amount of enzyme used in the ultrasound assisted
with epoxychloropropane (shown in Fig. 5), were used for the method was less and the reaction time was shorter than those in
immobilization of the lipase enzymes extracted from Candida the conventional method, the production yield of biodiesel
sp.99  125. The studied UCO sample was an acidified waste increased by 6% (from 90.1% to 96.1%), offering a more efficient,
cooking oil that mainly contained FFAs (96.23%), diglycerides quicker, and less costly method of biodiesel production (Subhedar
(1.23%), and triglycerides (2.53%). and Gogate, 2016). The immobilized enzyme was extracted from
The esterification reaction process involved dodecanoic acid Thermomyces lanuginosus (Lipozyme TLIM). The following optimum
(1.0 g) as a measurement for transesterification reactions activity, experimental conditions were established for the stirring (con-
immobilized lipase (0.02 g) as the enzyme of the reactions, and n- ventional) and the ultrasound assisted method, respectively: Molar
octanol (158 L), n-hexane (5 mL), and methanol (150 L) as the ratio of 1:12 (oil:methyl acetate), enzyme loading of 6% (w/v),
organic solvents. Fe3O4 sub-microspheres were synthesized by temperature of 40
C and reaction time of 24 h compared to molar
dissolving FeCl3.6H2O (1.35 g) in ethylene glycol (40 mL), producing ratio of 1:9 (oil:methyl acetate), enzyme loading of 3% (w/v),
a clear solution, then adding sodium glutamate (2 g), sodium ace- temperature of 50
C and reaction time of 3 h. The UCO for this
tate (1.5 g), and polyethylene glycol (1.5 g). The solution was mixed research was collected from various restaurants in India and had
thoroughly, and then autoclaved at 200
C for 8 h. Once they were the following composition (Table 6):
cold, the products were separated by magnetic methods, washed Tributyrin was used as a substrate for enzyme, while 0.2 mL
five times each with de-ionized water and ethanol, and then vac- tributyrin was incubated with the 200 mg lipase enzyme for 5 min
uum dried at 60
C for 6 h, generating Fe3O4 sub-microspheres. It in phosphate buffer (pH 7). Then, 20 mL methanol was added to the
was also shown that the immobilization of the enzyme was suc- reaction and the solution was titrated against alcoholic NaOH
cessful which resulted in an enhanced esterification yield of the (0.1 M) using phenolphthalein as an indicator (Subhedar and
lipase. The activities of immobilized lipases on epoxy-modified Gogate, 2016). The activity of the immobilized enzyme was calcu-
Fe3O4 sub-microspheres were reported as protein bound of lated through the following formula (Eq. (1)):
41 mg/g, esterification yield of 57.35%, and hydrolysis yield of 2.43%.
Due to the significant magnetic responses of the materials, the V*M*1000
Enzyme activity ðTBU=gÞ ¼ (3)
enzyme easily was separated from the reaction medium using a E*T
magnetic field. The results of this study exhibited 97.11% biodiesel
production yield under the optimum reaction conditions of where V ¼ volume in mL of NaOH (a measure of tributyrin
consumed during reaction), M ¼ molarity of NaOH, E ¼ amount of
enzyme employed in mg, T ¼ time of reaction in min. For the con-
ventional method (stirring), a glass reactor of 100 mL was used in
which a mechanical 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
Fig. 5. (a) Scanning electron microscope (SEM), (b) Enlarged SEM, and (c) Transmission Acid value (mg KOH/g oil) 4.3
electron microscope (TEM) images, and (d) X-ray diffraction (XRD) pattern of Fe3 O4 54.3
Viscosity (mm2 /s)
sub-microspheres (Zhang et al., 2016).
126 F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128

water bath maintained a constant temperature 50
C for the reac- with distilled water, and then lyophilized for 48 h before used for
tion. First, a mixture of UCO and methyl acetate was introduced to the biodiesel conversion. The reaction was carried out in a block
the reactor to heat up to the proper temperature and then lipase rotator (Nissin Thermo Block Rotator SN-06BN) at 30
C with a
was added to the mixture. Samples were drawn to measure methyl rotation speed of 7.5 (an arbitrary scale on the instrument) with or
ester content with HPLC. For the ultrasound assisted method, they without pre-agitation. The mixture consisted of oil (with or without
used ultrasonic irradiation at a frequency of 20 kHz to pass ultra- phospholipids), distilled water, and immobilized A. oryzae whole-
sound through titanium cylindrical horn. The ultrasonic horn with a cell enzymes, while the reaction was initiated through the addi-
diameter of 1.1 cm and maximum rated power output of 120 W was tion of an initial amount of methanol (1:1 M ratio of the oil). The
lowered 2.0 cm into the reaction mixture. Similar to the conven- remaining methanol was added to the solution step-by-step after
tional method, a water bath maintained the reaction temperature. 24, 48, and 72 h (1:1 M ratios of the oil at each time leading to a
In this method, oil and methyl acetate were fed to the reactor first total of 1:4), inhibiting the lipase enzymes to deactivate by meth-
to be heated and then lipase enzymes were added to the solution. anol. The biodiesel content was measured in samples taken regu-
Samples were taken for methyl ester analysis with high- larly at certain times during the course of the reaction. The results
performance liquid chromatography (HPLC). This research showed an approximate of a 3-fold increase in the conversion ef-
demonstrated a significant impact of the ultrasound power in the ficiency which led to a biodiesel production yield increase from
degree of intensification and cavitational activity of the biodiesel 29.7% (before applying the technique) to 91.0% (after the proposed
production process. The results showed that when the ultrasound technique was carried out). This method can eliminate the cost of
power was increased from 40 to 80 W, the biodiesel production oil pre-treatment processes such as degumming, dewaxing, and
yield increased from 57.23% to 96.1%. This is probably because of the synchronizing lipase with phospholipase in which phospholipids
boosted interaction between enzyme and substrate due to a higher are removed before the enzymes are added to the oil (Amoah et al.,
cavitational activity in the solution which resulted in a higher re- 2016).
action rate. A better cavitation would improve the process of mass
transfer and diffusion of the substrate towards the enzyme, which 6. Conclusions and recommendations
will eventually lead to a higher yield. However, a further increase
from 80 to 100 W of the ultrasound power did not result in an in- The enzymatic transesterification method offers more advan-
crease nor decrease in the biodiesel yield. (Subhedar and Gogate, tages than other methods: the catalysts (i.e. the enzymes) are
2016). In order to maintain an economic status for the enzymatic environmentally friendly; little to no pre-treatment is required
process, lipases were recycled and reused. At the end of each cycle, prior to transesterification; and the operational conditions are
the immobilized lipase enzyme was filtered, washed with acetone, moderate and in most cases ambient. In addition, the ratio of
dried in oven for 30 min at 40
C, and kept in the desiccator at room alcohol to oil is lower than previous methods (which can lower the
temperature. The enzyme recycled through this process was used cost of production); and the quality of the by-product glycerin is
in the following cycle. However, the results indicated a decrease in decent ( > 90% purity), which will add an extra source of revenue for
biodiesel yield as the number of the cycles increased. After seven the plant. Furthermore, the purity of the final product is high and
cycle, only 25% of the original enzyme activity was recovered. thus little to no polishing process is necessary at the end. Finally,
since there is no saponification process involved, the enzymatic
5.3. Converting oils high in phospholipids to biodiesel using method allows for higher free fatty acids content in feedstock
immobilized Aspergillus oryzae whole-cell biocatalysts expressing which will permit the usage of a broad variety of oils as feedstock.
Fusarium heterosporum lipase In particular, the enzymatic method enables utilizing the used
cooking oil, containing high free fatty acids content, which other-
This case study was selected for review because it offers an wise would have been difficult or infeasible to process due to the
effective technique for biodiesel production made out of any form economical and operational burdens it imposes on the process of
of feedstock that contains high amount of phospholipids (10%e biodiesel. Such is greatly beneficial to biodiesel production because
30%), without the need for oil pre-treatment (Amoah et al., 2016). of UCO's abundant availability, low cost, and not competing with
The studied phospholipids sample was extracted from soybean and food and agriculture.
the result was also compared to a sample of soybean refined oil. Despite all the advantages of the enzymatic method, the cost of
Nevertheless, the employed methodology was not designed to the enzymes, the ability to recycle and re-use them, the sensitivity
focus solely on biodiesel made of soybean oil, rather to remove of enzymatic activities against the reaction conditions and the re-
phospholipids from oil regardless of the source and origin of it. actants involved in the reaction (such as methanol and ethanol),
Generally, such high content of phospholipids causes the formation and the slow kinetic rate of reaction are yet to be studied. For
of water-in-oil phospholipid-based reverse micelles. The reverse instance, genetic engineering could play a great role here by
micelles trap the water required for activating the enzymes, creating a strain of lipase-producing microorganisms with higher
resulting in deactivation of the immobilized enzymes and thus resistance towards extreme reaction conditions and the presence of
reducing the biodiesel production yield. To reduce the reverse alcohol in the reaction system. Also, developing more efficient
micelles formation, a simple yet effective technique was to apply a lipase immobilization methods can improve the enzyme recovery.
gentle agitation in the presence of a higher content of water At the end, another approach to improve the economics of enzy-
(Amoah et al., 2016). The whole-cell immobilized Aspergillus oryzae matic transesterification is to find efficient methods to regenerate
enzymes were used as biocatalysts. The following protocol was the activities of lipase, after being used in the process.
conducted to obtain the enzyme from A. Oryzae strains. After
cultivating the strains on Czapek-Dox (CD) agar plates at 30
C for 6
References
days, the axenic spores were extracted in 5 ml of distilled water
which then were transferred into 500 ml Sakaguchi flask contain- Adlercreutz, P., 2017. Comparison of lipases and glycoside hydrolases as catalysts in
ing approximately 850 mg of reticulated polyurethane foam BSPs in synthesis reactions. Appl. Microbiol. Biotechnol. 101 (2), 513e519.
100 mL of DP medium. The size of the BSPs used for cell immobi- Akbar, E., Yaakob, Z., Kamarudin, S.K., Ismail, M., Salimon, J., 2009. Characteristic
and composition of jatropha curcas oil seed from Malaysia and its potential as
lization was 6  3  3 mm with the pore size of 50 pores per linear biodiesel feedstock feedstock. Eur. J. Sci. Res. 29 (3), 396e403.
inch. The whole-cell immobilized enzyme was filtered out, washed Akoh, C.C., 1993. Lipase-catalyzed synthesis of partial glyceride. Biotechnol. Lett. 15
 F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128 127

(9), 949e954. Dutra, J.C., Terzi, S. d. C., Bevilaqua, J.V., Damaso, M.C., Couri, S., Langone, M.A.,
Alin, S.R., de Fa tima FL Rasera, M., Salimon, C.I., Richey, J.E., Holtgrieve, G.W., Senna, L.F., 2008. Lipase production in solid-state fermentation monitoring
Krusche, A.V., Snidvongs, A., 2011. Physical controls on carbon dioxide transfer biomass growth of Aspergillus niger using digital image processing. Appl. Bio-
velocity and flux in low-gradient river systems and implications for regional chem. Biotechnol. 147 (1e3), 63e75.
carbon budgets. J. Geophys. Res.: Biogeosciences 116 (G1). Fai, A., Kawaguti, H., Thomazelli, I., Santos, R., Pastore, G., 2017. Immobilization of
Altın, R., Cetinkaya, S., Yücesu, H.S., 2001. The potential of using vegetable oil fuels fungi b-galactosidase on celite to produce galactooligosaccharides during
as fuel for diesel engines. Energy Convers. Manag. 42 (5), 529e538. lactose hydrolysis. Int. Food Res. J. 24 (1).
Amini, Z., Ilham, Z., Ong, H.C., Mazaheri, H., Chen, W.-H., 2017a. State of the art and Fan, Y., Ke, C., Su, F., Li, K., Yan, Y., 2017. Various types of lipases immobilized on
prospective of lipase-catalyzed transesterification reaction for biodiesel pro- dendrimer-functionalized magnetic nanocomposite and application in bio-
duction. Energy Convers. Manag. 141, 339e353. diesel preparation. Energy Fuels 31 (4), 4372e4381.
Amini, Z., Ong, H.C., Harrison, M.D., Kusumo, F., Mazaheri, H., Ilham, Z., 2017b. Gołaszewski, J., 2009. Renewables and environmental implications. Environ. Bio-
Biodiesel production by lipase-catalyzed transesterification of Ocimum basili- technol. 5, 11e24.
cum L. (sweet basil) seed oil. Energy Convers. Manag. 132, 82e90. Goswami, D., Basu, J.K., De, S., 2013. Lipase applications in oil hydrolysis with a case
Amoah, J., Ho, S.-H., Hama, S., Yoshida, A., Nakanishi, A., Hasunuma, T., Ogino, C., study on castor oil: a review. Crit. Rev. Biotechnol. 33 (1), 81e96.
Kondo, A., 2016. Converting oils high in phospholipids to biodiesel using Gui, M.M., Lee, K., Bhatia, S., 2008. Feasibility of edible oil vs. non-edible oil vs.
immobilized Aspergillus oryzae whole-cell biocatalysts expressing Fusarium waste edible oil as biodiesel feedstock. Energy 33 (11), 1646e1653.
heterosporum lipase. Biochem. Eng. J. 105, 10e15. Guldhe, A., Ramluckan, K., Singh, P., Rawat, I., Mahalingam, S.K., Bux, F., 2017. Cat-
Aranda, D.A., Santos, R.T., Tapanes, N.C., Ramos, A.L.D., Antunes, O.A.C., 2008. Acid- alytic conversion of microalgal lipids to biodiesel: overview and recent ad-
catalyzed homogeneous esterification reaction for biodiesel production from vances. In: Algal Biofuels. Springer, pp. 315e329.
palm fatty acids. Catal. Lett. 122 (1e2), 20e25. Gupta, A., Wilkens, S., Adcock, J.L., Puri, M., Barrow, C.J., 2013. Pollen baiting facil-
Awang, R., Ghazuli, M.R., Basri, M., et al., 2007. Immobilization of lipase from itates the isolation of marine thraustochytrids with potential in omega-3 and
Candida rugosa on palm-based polyurethane foam as a support material. Am. J. biodiesel production. J. Ind. Microbiol. Biotechnol. 40 (11), 1231e1240.
Biochem. Biotechnol. 3 (3), 163e166. Gurunathan, B., Ravi, A., 2015. Biodiesel production from waste cooking oil using
Bahulekar, R.V., 2017. Studies in Polyethyleneimine: Matrix Polymerisation and copper doped zinc oxide nanocomposite as heterogeneous catalyst. Bioresour.
Enzyme Immobilization. Ph.D. thesis. Technol. 188, 124e127.
Ban, K., Hama, S., Nishizuka, K., Kaieda, M., Matsumoto, T., Kondo, A., Noda, H., Gutie rrez-Zapata, C., Martínez, D.B., Collazos, C., Acun  a, H.C., Cuervo, J.,
Fukuda, H., 2002. Repeated use of whole-cell biocatalysts immobilized within Fernandez, C., 2017. Productions of sunflower oil biodiesel and used cooking oil
biomass support particles for biodiesel fuel production. J. Mol. Catal. B Enzym. through heterogeneous catalysts compared to conventional homogeneous
17 (3e5), 157e165. catalysts. In: Journal of Physics: Conference Series, vol. 786. IOP Publishing,
Bandikari, R., Qian, J., Baskaran, R., Liu, Z., Wu, G., 2018. Bio-affinity mediated p. 012025.
immobilization of lipase onto magnetic cellulose nanospheres for high yield Hasan, F., Shah, A.A., Hameed, A., 2006. Industrial applications of microbial lipases.
biodiesel in one time addition of methanol. Bioresour. Technol. 249, 354e360. Enzym. Microb. Technol. 39 (2), 235e251.
Banerjee, A., Chakraborty, R., 2009. Parametric sensitivity in transesterification of Hellmers, F., Takors, R., Thum, O., 2018. Robust enzyme immobilizates for industrial
waste cooking oil for biodiesel production a review. Resour. Conserv. Recycl. 53 isomalt production. Mol. Catal. 445, 293e298.
(9), 490e497. Hotta, Y., Ezaki, S., Atomi, H., Imanaka, T., 2002. Extremely stable and versatile
Banerjee, M., Dey, B., Talukdar, J., Kalita, M.C., 2014. Production of biodiesel from carboxylesterase from a hyperthermophilic archaeon. Appl. Environ. Microbiol.
sunflower oil using highly catalytic bimetallic goldesilver coreeshell nano- 68 (8), 3925e3931.
particle. Energy 69, 695e699. Hsu, A.-F., Jones, K., Foglia, T.A., Marmer, W.N., 2002. Immobilized lipase-catalysed
Bezergianni, S., Kalogianni, A., 2009. Hydrocracking of used cooking oil for biofuels production of alkyl esters of restaurant grease as biodiesel. Biotechnol. Appl.
production. Bioresour. Technol. 100 (17), 3927e3932. Biochem. 36 (3), 181e186.
Buffi, M., Valera-Medina, A., Marsh, R., Pugh, D., Giles, A., Runyon, J., Chiaramonti, D., Hwang, H.T., Qi, F., Yuan, C., Zhao, X., Ramkrishna, D., Liu, D., Varma, A., 2014. Lipase-
2017. Emissions characterization tests for hydrotreated renewable jet fuel from catalyzed process for biodiesel production: protein engineering and lipase
used cooking oil and its blends. Appl. Energy 201, 84e93. production. Biotechnol. Bioeng. 111 (4), 639e653.
Canakci, M., 2007. The potential of restaurant waste lipids as biodiesel feedstocks. Ikeda, M., Clark, D.S., 1998. Molecular cloning of extremely thermostable esterase
Bioresour. Technol. 98 (1), 183e190. gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli.
Cavonius, L.R., Carlsson, N.-G., Undeland, I., 2014. Quantification of total fatty acids Biotechnol. Bioeng. 57 (5), 624e629.
in microalgae: comparison of extraction and transesterification methods. Anal. Jaeger, K.-E., Reetz, M.T., 1998. Microbial lipases form versatile tools for biotech-
Bioanal. Chem. 406 (28), 7313e7322. nology. Trends Biotechnol. 16 (9), 396e403.
Cazaban, D., Wilson, L., Betancor, L., 2017. Lipase immobilization on siliceous sup- €msa
Ja €, M., Lynch, F., Santana-Sa nchez, A., Laaksonen, P., Zaitsev, G., Solovchenko, A.,
ports: application to synthetic reactions. Curr. Org. Chem. 21 (2), 96e103. Allahverdiyeva, Y., 2017. Nutrient removal and biodiesel feedstock potential of
Çetinkaya, M., Ulusoy, Y., Tekìn, Y., Karaosmanog lu, F., 2005. Engine and winter road green alga uhcc00027 grown in municipal wastewater under nordic conditions.
test performances of used cooking oil originated biodiesel. Energy Convers. Algal Res. 26, 65e73.
Manag. 46 (7e8), 1279e1291. Jeromin, L., Peukert, E., Wollmann, G., 1987. Process for the Pre-esterification of Free
Charter, W.F., 2008. Biodiesel Guidelines. Draft, first ed. European Automobile Fatty Acids in Fats and Oils.
Manufacturer’s Association. Jolly, R., Kosikowski, F., 1975. Flavor development in pasteurized milk blue cheese by
Chhetri, A.B., Watts, K.C., Islam, M.R., 2008. Waste cooking oil as an alternate animal and microbial lipase preparations. J. Dairy Sci. 58 (6), 846e852.
feedstock for biodiesel production. Energies 1 (1), 3e18. Kakugawa, K., Shobayashi, M., Suzuki, O., Miyakawa, T., 2002. Purification and
Chourasia, V.R., Gawas, A.S., Menon, A.S., Shinde, P.M., 2015. Production of biodiesel characterization of a lipase from the glycolipid-producing yeast Kurtzmano-
by enzymatic transesterification using immobilized lipase. Int. J. Eng. Res. Gen. myces sp. i-11. Biosc. Biotech. Biochem. 66 (5), 978e985.
Sci. 3, 1e9. Kawaguti, H.Y., Manrich, E., Sato, H.H., 2006. Production of isomaltulose using
Christopher, L.P., Kumar, H., Zambare, V.P., 2014. Enzymatic biodiesel: challenges Erwinia sp. d12 cells: culture medium optimization and cell immobilization in
and opportunities. Appl. Energy 119, 497e520. alginate. Biochem. Eng. J. 29 (3), 270e277.
Chye, J.T.T., Jun, L.Y., Yon, L.S., Pan, S., Danquah, M.K., 2018. Biofuel production from Kayode, B., Hart, A., 2017. An overview of transesterification methods for producing
algal biomass. chapter 3 Bioenergy Biofuels. CRC Press. biodiesel from waste vegetable oils. Biofuels 1e19.
Cvengros, J., Cvengrosova , Z., 2004. Used frying oils and fats and their utilization in Kazanina, G., Selezneva, A., Ruban, E., Volkova, I., 1981. Isolation and characteriza-
the production of methyl esters of higher fatty acids. Biomass Bioenergy 27 (2), tion of lipase from Geotrichum asteroides fkm f-144. Priklad. Biokhim. Mikrobiol.
173e181. 17 (4), 516e522.
da Silva Ce sar, A., Werderits, D.E., de Oliveira Saraiva, G.L., da Silva Guabiroba, R.C., Knothe, G., Krahl, J., Van Gerpen, J., 2015. The Biodiesel Handbook. Elsevier.
2017. The potential of waste cooking oil as supply for the brazilian biodiesel Krauss, U., Ja €ger, V.D., Diener, M., Pohl, M., Jaeger, K.-E., 2017. Catalytically-active
chain. Renew. Sustain. Energy Rev. 72, 246e253. inclusion bodies carrier-free protein immobilizates for application in biotech-
Datta, S., Christena, L.R., Rajaram, Y.R.S., 2013. Enzyme immobilization: an overview nology and biomedicine. J. Biotechnol. 258, 136e147.
on techniques and support materials. 3 Biotech 3 (1), 1e9. Kumar, A., Sharma, S., 2011. Potential non-edible oil resources as biodiesel feed-
Demirbas, A., 2005. Biodiesel production from vegetable oils via catalytic and non- stock: an Indian perspective. Renew. Sustain. Energy Rev. 15 (4), 1791e1800.
catalytic supercritical methanol transesterification methods. Prog. Energy Kuratani, N., Hama, S., Noda, H., Fukuda, H., Jan. 30 2018. Continuous Production of
Combust. Sci. 31 (5e6), 466e487. Biodiesel Fuel by Enzymatic Method. US Patent 9,879,291.
Demirbas, A., 2008. Comparison of transesterification methods for production of Kusdiana, D., Saka, S., 2004. Effects of water on biodiesel fuel production by su-
biodiesel from vegetable oils and fats. Energy Convers. Manag. 49 (1), 125e130. percritical methanol treatment. Bioresour. Technol. 91 (3), 289e295.
Demirbas, A., 2009. Biodiesel from waste cooking oil via base-catalytic and super- Lam, M.K., Lee, K.T., Mohamed, A.R., 2010. Homogeneous, heterogeneous and
critical methanol transesterification. Energy Convers. Manag. 50 (4), 923e927. enzymatic catalysis for transesterification of high free fatty acid oil (waste
Dias, J.M., Alvim-Ferraz, M.C., Almeida, M.F., 2008. Comparison of the performance cooking oil) to biodiesel: a review. Biotechnol. Adv. 28 (4), 500e518.
of different homogeneous alkali catalysts during transesterification of waste Lanza, M., Priamo, W.L., Oliveira, J.V., Dariva, C., De Oliveira, D., 2004. The effect of
and virgin oils and evaluation of biodiesel quality. Fuel 87 (17e18), 3572e3578. temperature, pressure, exposure time, and depressurization rate on lipase ac-
Dorado, M.P., Ballesteros, E., Lo pez, F.J., Mittelbach, M., 2004. Optimization of alkali- tivity in scco2. Appl. Biochem. Biotechnol. 113 (1e3), 181e187.
catalyzed transesterification of Brassica C arinata oil for biodiesel production. Lee, K.-T., Foglia, T.A., Chang, K.-S., 2002. Production of alkyl ester as biodiesel from
Energy Fuels 18 (1), 77e83. fractionated lard and restaurant grease. JAOCS (J. Am. Oil Chem. Soc.) 79 (2),
128 F. Moazeni et al. / Journal of Cleaner Production 216 (2019) 117e128

191e195. Shibasaki-Kitakawa, N., Honda, H., Kuribayashi, H., Toda, T., Fukumura, T.,
Lertsathapornsuk, V., Ruangying, P., Pairintra, R., Krisnangkura, K., 2005. Continuous Yonemoto, T., 2007. Biodiesel production using anionic ion-exchange resin as
transethylation of vegetable oils by microwave irradiation. In: Proceedings of heterogeneous catalyst. Bioresour. Technol. 98 (2), 416e421.
the 1st Conference on Energy Network, vol. 2005. Singh, M., Kathuroju, P.K., Jampana, N., 2009. Polypyrrole based amperometric
Leung, D., Guo, Y., 2006. Transesterification of neat and used frying oil: optimization glucose biosensors. Sensor. Actuator. B Chem. 143 (1), 430e443.
for biodiesel production. Fuel Process. Technol. 87 (10), 883e890. Sotoft, L.F., Rong, B.-G., Christensen, K.V., Norddahl, B., 2010. Process simulation and
Leung, D.Y., Wu, X., Leung, M., 2010. A review on biodiesel production using cata- economical evaluation of enzymatic biodiesel production plant. Bioresour.
lyzed transesterification. Appl. Energy 87 (4), 1083e1095. Technol. 101 (14), 5266e5274.
Li, C., Liu, Y., Luo, A., Ruan, R., Liu, C., 2008. New two-step method of producing Subhedar, P.B., Gogate, P.R., 2016. Ultrasound assisted intensification of biodiesel
biodiesel from waster cooking oil. Cereals Oils Process. production using enzymatic interesterification. Ultrason. Sonochem. 29, 67e75.
Lusk, B.G., Colin, A., Parameswaran, P., Rittmann, B.E., Torres, C.I., 2018. Simulta- Talebian-Kiakalaieh, A., Amin, N.A.S., Mazaheri, H., 2013. A review on novel pro-
neous fermentation of cellulose and current production with an enriched mixed cesses of biodiesel production from waste cooking oil. Appl. Energy 104,
culture of thermophilic bacteria in a microbial electrolysis cell. Microb. Bio- 683e710.
technol. 11 (1), 63e73. Tan, T., Lu, J., Nie, K., Deng, L., Wang, F., 2010. Biodiesel production with immobilized
Martinez-Guerra, E., Muppaneni, T., Gude, V.G., Deng, S., 2018. Non-conventional lipase: a review. Biotechnol. Adv. 28 (5), 628e634.
feedstock and technologies for biodiesel production. In: Advanced Solid Cata- Tran, D.-T., Chen, C.-L., Chang, J.-S., 2012. Immobilization of Burkholderia sp. lipase
lysts for Renewable Energy Production. IGI Global, pp. 96e118. on a ferric silica nanocomposite for biodiesel production. J. Biotechnol. 158 (3),
Matsumoto, T., Fukuda, H., Ueda, M., Tanaka, A., Kondo, A., 2002. Construction of 112e119.
yeast strains with high cell surface lipase activity by using novel display sys- Ullah, Z., Bustam, M.A., Man, Z., 2015. Biodiesel production from waste cooking oil
tems based on the flo1p flocculation functional domain. Appl. Environ. Micro- by acidic ionic liquid as a catalyst. Renew. Energy 77, 521e526.
biol. 68 (9), 4517e4522. Van Gerpen, J., 2005. Biodiesel processing and production. Fuel Process. Technol. 86
Meher, L., Dharmagadda, V.S., Naik, S., 2006. Optimization of alkali-catalyzed (10), 1097e1107.
transesterification of pongamia pinnata oil for production of biodiesel. Bio- Van Gerpen, J., Shanks, B., Pruszko, R., Clements, D., Knothe, G., 2004. Biodiesel
resour. Technol. 97 (12), 1392e1397. Analytical Methods. National Renewable Energy Laboratory, Colorado,
Mishra, S.S., Ray, R.C., Rosell, C.M., Panda, D., 2017. Microbial enzymes in food ap- pp. 37e47.
plications. Microb. Enzyme Technol. Food Appl. 1. Vicente, G., Martınez, M., Aracil, J., 2004. Integrated biodiesel production: a com-
Murty, D., Kirschbaum, M.U., Mcmurtrie, R.E., Mcgilvray, H., 2002. Does conversion parison of different homogeneous catalysts systems. Bioresour. Technol. 92 (3),
of forest to agricultural land change soil carbon and nitrogen? A review of the 297e305.
literature. Glob. Chang. Biol. 8 (2), 105e123. Wallis, C., Cerny, M., Lacroux, E., Mouloungui, Z., 2017. Recovery of slaughterhouse
Nelson, L.A., Foglia, T.A., Marmer, W.N., 1996. Lipase-catalyzed production of bio- animal fatty wastewater sludge by conversion into fatty acid butyl esters by
diesel. J. Am. Oil Chem. Soc. 73 (9), 1191e1195. acid-catalyzed esterification. Waste Manag. 60, 184e190.
Noureddini, H., Gao, X., Philkana, R., 2005. Immobilized Pseudomonas cepacia lipase Wang, L., Chi, Z., Wang, X., Liu, Z., Li, J., 2007. Diversity of lipase-producing yeasts
for biodiesel fuel production from soybean oil. Bioresour. Technol. 96 (7), from marine environments and oil hydrolysis by their crude enzymes. Ann.
769e777. Microbiol. 57 (4), 495.
Patil, P.D., Gude, V.G., Mannarswamy, A., Deng, S., Cooke, P., Munson-McGee, S., Wang, X., Qin, X., Li, D., Yang, B., Wang, Y., 2017. One-step synthesis of high-yield
Rhodes, I., Lammers, P., Nirmalakhandan, N., 2011. Optimization of direct con- biodiesel from waste cooking oils by a novel and highly methanol-tolerant
version of wet algae to biodiesel under supercritical methanol conditions. immobilized lipase. Bioresour. Technol. 235, 18e24.
Bioresour. Technol. 102 (1), 118e122. Watkins, R.S., Lee, A.F., Wilson, K., 2004. LieCaO catalyzed triglyceride trans-
Pizarro, A.V.L., Park, E.Y., 2003. Lipase-catalyzed production of biodiesel fuel from esterification for biodiesel applications. Green Chem. 6 (7), 335e340.
vegetable oils contained in waste activated bleaching earth. Process Biochem. Wei, L., Cheung, C., Ning, Z., 2017. Influence of waste cooking oil biodiesel on
38 (7), 1077e1082. combustion, unregulated gaseous emissions and particulate emissions of a
Pourzolfaghar, H., Abnisa, F., Daud, W.M.A.W., Aroua, M.K., 2016. A review of the direct-injection diesel engine. Energy 127, 175e185.
enzymatic hydroesterification process for biodiesel production. Renew. Sustain. Wu, S., Song, L., Sommerfeld, M., Hu, Q., Chen, W., 2017. Optimization of an effective
Energy Rev. 61, 245e257. method for the conversion of crude algal lipids into biodiesel. Fuel 197,
Qadri, S.S.P., Salman, M., Satyanarayana, M., 2017. Effect of Different Catalysts and 467e473.
Their Performance in Extracting BiodieseleAn Experimental Study. Xie, W., Wang, J., 2012. Immobilized lipase on magnetic chitosan microspheres for
Ragauskas, A.J., Williams, C.K., Davison, B.H., Britovsek, G., Cairney, J., Eckert, C.A., transesterification of soybean oil. Biomass Bioenergy 36, 373e380.
Frederick, W.J., Hallett, J.P., Leak, D.J., Liotta, C.L., et al., 2006. The path forward Yaakob, Z., Mohammad, M., Alherbawi, M., Alam, Z., Sopian, K., 2013. Overview of
for biofuels and biomaterials. Science 311 (5760), 484e489. the production of biodiesel from waste cooking oil. Renew. Sustain. Energy Rev.
Ramadhas, A., Jayaraj, S., Muraleedharan, C., 2004. Use of vegetable oils as IC engine 18, 184e193.
fuelse a review. Renew. Energy 29 (5), 727e742. Yun, H., Wang, M., Feng, W., Tan, T., 2013. Process simulation and energy optimi-
Reetz, M.T., Zonta, A., Simpelkamp, J., 1996. Efficient immobilization of lipases by zation of the enzyme-catalyzed biodiesel production. Energy 54, 84e96.
entrapment in hydrophobic sol-gel materials. Biotechnol. Bioeng. 49 (5), Zaharudin, N.A., Rashid, R., Azman, L., Esivan, S.M.M., Idris, A., Othman, N., 2018.
527e534. Enzymatic hydrolysis of used cooking oil using immobilized lipase. In: Sus-
Rehan, M., Gardy, J., Demirbas, A., Rashid, U., Budzianowski, W., Pant, D., Nizami, A., tainable Technologies for the Management of Agricultural Wastes. Springer,
2018. Waste to biodiesel: a preliminary assessment for Saudi Arabia. Bioresour. pp. 119e130.
Technol. 250, 17e25. Zaks, A., Klibanov, A.M., 1988. Enzymatic catalysis in nonaqueous solvents. J. Biol.
Rehman, S., Bhatti, H.N., Bilal, M., Asgher, M., Wang, P., 2017. Catalytic, kinetic and Chem. 263 (7), 3194e3201.
thermodynamic characteristics of an extracellular lipase from Penicillium Zhang, Q., Zheng, Z., Liu, C., Liu, C., Tan, T., 2016. Biodiesel production using lipase
notatum. Catal. Lett. 147 (1), 281e291. immobilized on epoxychloropropane-modified Fe3O4 sub-microspheres. Col-
Royter, M., Schmidt, M., Elend, C., Ho € benreich, H., Sch€ afer, T., Bornscheuer, U., loids Surfaces B Biointerfaces 140, 446e451.
Antranikian, G., 2009. Thermostable lipases from the extreme thermophilic Zhang, S., Shi, J., Deng, Q., Zheng, M., Wan, C., Zheng, C., Li, Y., Huang, F., 2017.
anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Calda- Preparation of carriers based on ZnO nanoparticles decorated on graphene
naerobacter subterraneus subsp. tengcongensis. Extremophiles 13 (5), 769e783. oxide (GO) nanosheets for efficient immobilization of lipase from Candida
Sebastian, J., Muraleedharan, C., Santhiagu, A., 2016. A comparative study between rugosa. Molecules 22 (7), 1205.
chemical and enzymatic transesterification of high free fatty acid contained Zhang, Y., Dube, M., McLean, D., Kates, M., 2003. Biodiesel production from waste
rubber seed oil for biodiesel production. Cogent Eng. 3 (1), 1178370. cooking oil: 1. process design and technological assessment. Bioresour. Technol.
Seth, S., Chakravorty, D., Dubey, V.K., Patra, S., 2014. An insight into plant lipase 89 (1), 1e16.
researchechallenges encountered. Protein Expr. Purif. 95, 13e21. Ziejewski, M., Goettler, H., Pratt, G., 1986. Comparative Analysis of the Long-term
Shah, S., Gupta, M.N., 2007. Lipase catalyzed preparation of biodiesel from jatropha Performance of a Diesel Engine on Vegetable Oil based Alternate Fuels. Tech.
oil in a solvent free system. Process Biochem. 42 (3), 409e414. rep., p. 12. https://doi.org/10.4271/860301. SAE Technical Paper.