Accepted Manuscript

Title: Enzymatic transesterification for production of biodiesel

using yeast lipases: An overview

Author: M. Aarthy P.Saravanan M.K. Gowthaman C. Rose

N.R. Kamini

PII: S0263-8762(14)00184-1
DOI: http://dx.doi.org/doi:10.1016/j.cherd.2014.04.008
Reference: CHERD 1552

To appear in:

Received date: 2-8-2013

Revised date: 4-3-2014
Accepted date: 15-4-2014

Please cite this article as: Aarthy, M., Gowthaman, M.K., Rose, C., Kamini,
N.R.,Enzymatic transesterification for production of biodiesel using yeast
lipases: An overview, Chemical Engineering Research and Design (2014),
http://dx.doi.org/10.1016/j.cherd.2014.04.008

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Research Highlights

Highlights

 Greener production of biodiesel by enzymatic transesterification

 Potential feedstocks that could be used as substrates for fatty acid alkyl esters
 Optimum transesterification conditions for various yeast lipases
 Utilization of whole cell as biocatalyst in the reaction

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*Manuscript

Enzymatic transesterification for production of biodiesel using yeast lipases: An overview

M. Aarthya, P.Saravananb, M.K. Gowthamana, C. Rosea, N.R. Kaminia,*

a
Department of Biotechnology, CSIR–Central Leather Research Institute, Chennai 600 020,India
b
Tannery, CSIR–Central Leather Research Institute, Chennai 600 020, India

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*Corresponding author. Tel.: +91 44 24430273; fax: +91 44 24911589

E-mail address: [email protected], [email protected] (N.R. Kamini).

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ABSTRACT

Biodiesel has provided an eco-friendly solution to fuel crisis as it is renewable,

biodegradable and a non-toxic fuel that can be easily produced through enzymatic

transesterification of vegetable oils and animal fats. Enzymatic production of biodiesel has many

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advantages over the conventional methods as high yields can be obtained at low reaction

temperatures with easy recovery of glycerol. Microbial lipases are powerful biocatalysts for

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industrial applications including biodiesel production at lower costs due to its potential in

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hydrolyzing waste industrial materials. Among them, lipases from yeasts, Candida antarctica,

Candida rugosa, Cryptococcus sp., Trichosporon asahii and Yarrowia lipolytica are known to

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catalyze such reactions. Moreover, stepwise addition of methanol in a three step, two step and

single step reactions have been developed using yeast lipases to minimize the inhibitory effects
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of methanol. The latest trend in biodiesel production is the use of whole-cell as biocatalysts,
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since the process requires no downstream processing of the enzyme. Synthesis of value added
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products from the byproduct glycerol further reduces the production cost of biodiesel. This

review aims at compiling the information on various yeast lipase catalyzed transesterification
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reactions for greener production of biodiesel.

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Keywords: Biodiesel; Enzymatic transesterification; Oils and fats; Yeast lipase; Biocatalyst
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1. Introduction

Increased demand for energy, increasing price of crude oil, global warming due to

emission of green house gases, environmental pollution, and fast diminishing supply of fossil

fuels have led to the development of alternate fuels, especially biofuels like ethanol and biodiesel

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(Abbaszaadeh et al., 2012; Lee et al., 2011). Owing to its environmental advantages and the

increase in petroleum price, a rapid increase in biodiesel production is observed. Until 2010, the

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supply of biodiesel was equivalent to its demand and it is estimated that in future, the demand

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would outweigh its production and increase in all regions of the world from 40.5 billion liters

(2015) to 58.5 billion liters by 2020 (Fig. 1) (Pinto, 2011).

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Biodiesel is defined as the non-petroleum-based diesel fuel consisting of fatty acid alkyl

esters, typically made by transesterification of vegetable oils or animal fats with alcohols, which
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could be used alone, or blended with routine petrodiesel in unchanged diesel-engine vehicles.
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Various alcohols have been used for the transesterification process including methanol, ethanol,
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iso-propanol and butanol, but methanol is considered for industrial production because of its low

cost and availability (Tan et al., 2010). Moreover, the fatty acid esters produced from methanol
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have lower viscosities, slightly higher cloud and pour points than the corresponding fatty acid
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ethyl esters (Robles-Medina et al., 2009). Biodiesel has many benefits such as it is
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biodegradable, non-toxic, has a low emission profile and is a renewable resource. In addition, it

does not contribute to the increase in carbon dioxide levels in the atmosphere and thus minimizes

the intensity of the greenhouse effect (Abbaszaadeh et al., 2012).

Four primary ways to make biodiesel are direct use and blending, micro-emulsions,

thermal cracking (pyrolysis) and transesterification (Bisen et al., 2010). The most popular and

commonly used method is transesterification of oils and fats that can be broadly classified into

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two categories as chemical and enzymatic. Chemically, transesterification reaction could be acid

or alkali catalyzed which has many disadvantages such as high energy consumption and the

difficulty in transesterification of triglycerides with high free fatty acid (FFA) content. Moreover,

the downstream processes such as the recovery of glycerol, the removal of inorganic salts and

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water from the product, catalyst removal and the treatment of alkaline wastewater are complex

and incur additional cost (Bajaj et al., 2010). On the other hand, enzyme catalyzed reaction using

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lipases provides a solution to the aforementioned problems as it is more efficient, highly

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selective, involves less energy consumption, and produces less waste (Akoh et al., 2007).

Glycerol recovery is easier in enzymatic process and high grade glycerol would be produced

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compared to an alkaline process (Robles-Medina et al., 2009). It is reported that the enzymatic

reactions are insensitive to FFA and water content in the raw material (Kulkarni and Dalai,
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2006). The problem of inactivation of lipase in the presence of high concentration of methanol
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could be overcome by stepwise addition of methanol (Shimada et al., 1999b). A comparison of

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the chemical and enzymatic process is given in Table 1.

Lipases (triacylglycerol acylhydrolases, E.C.3.1.1.3) are serine hydrolases that catalyze

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the hydrolysis of triglycerides to glycerol and free fatty acids at an oil-water interface (Feng et
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al., 2013; Gupta et al., 2004; Kamini and Iefuji, 2001). However, under certain conditions, they
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catalyze synthetic reactions like acidolysis, alcoholysis, aminolysis, esterification and

transesterification (Saxena et al., 2003). The biocatalytic properties of lipases in both aqueous

and non aqueous media enable its use in medical and therapeutic fields, food process industries,

organic chemistry and in treatment of wastewater. Among various lipases, yeast lipases have

been widely used in polymer degradation (Masaki et al., 2005), as an additive in detergent

formulation (Thirunavukarasu et al., 2008), flavor and cosmetic industry (Ozyilmaz and Gezer,

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2010; Shimada et al., 1999a), food industry (Ferrer et al., 2005), ester synthesis (Jin et al., 2013),

biocatalytic resolution of pharmaceuticals (Henke et al., 2000), biosensors (Kartal et al., 2007),

wastewater treatment (Tsuji et al., 2013) and production of biodiesel (Fedosov et al., 2013;

Kamini and Iefuji, 2001). Although intensive investigations have been focused on production of

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biodiesel by various researchers using yeast lipases (Huang et al., 2010; Kumari and Gupta,

2012; Talukder et al., 2011), reviews are not available on enzymatic transesterification of

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triglycerides by yeast lipases. Therefore, this review describes the current status of research on

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biodiesel production using yeast lipases, the potential feedstocks utilized in the process and the

latest trends in improving the productivity using whole cells as biocatalyst.

2. Sources of lipase an
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Lipases are ubiquitous enzymes and are produced by animals (pancreas of cattle,
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sheep, hogs and pigs), plants (papaya latex, rapeseed, oat and castor seeds) and microorganisms
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(bacteria, fungi and yeast). Most of the plant lipases are not commercially used, while the lipases

from animal and microbial origin have wider applications because of their broad pH and thermal
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stability (Pahoja and Sethar, 2002). Among them, microbial lipases have gained special
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industrial attention because of its stability, broad substrate specificity, possible high yields, ease
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of genetic manipulation, regular supply due to absence of seasonal fluctuations and rapid growth

of microorganisms on inexpensive media (Hasan et al., 2006) and they share about 5% of the

world enzyme market after proteases and carbohydrases (Christopher et al., 2014).

In general, microbial lipases are 19–60 kDa proteins and belong to the α/β hydrolase

family with its core composed of a central β sheet consisting of up to eight different β strands (β1

– β8) connected by up to six α helices (A– F). The active site is formed by a catalytic triad of the

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amino acids serine, aspartic (or glutamic) acid and histidine. The nucleophilic serine residue is

located at the C-terminal end of strand β5 in a highly conserved pentapeptide G-X-S-X-G (X

may be any amino acid), forming a characteristic β-turn-α motif named the ‘nucleophilic elbow’.

Interfacial activation occurs in presence of a substrate which takes place by the movement of a

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lid and exposure of the hydrophobic pocket and active site structure above the critical micellar

concentration (CMC) of the substrate. This interfacial activation is unique to the class of lipases

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for its use in transesterification of fats and oils (Jaeger and Reetz, 1998) owing to its ability in

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utilizing all mono, di, and triglycerides as well as the FFA, low product inhibition, high activity

and yield in non-aqueous media, low reaction time, temperature and alcohol resistance (Bajaj et

al., 2010).

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Lipases are broadly classified as intracellular and extracellular enzymes. In case of
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extracellular lipases the enzyme would be recovered from the culture broth and then purified,
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while intracellular lipase remains either inside the cell or in the cell producing walls (Gog et al.,
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2012). The methanolysis reactions have been carried out from major lipase producing

microorganisms like Mucor miehei (Al-Zuhair et al., 2006), Rhizopus oryzae (Zarei et al., 2014),
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Candida antarctica (Talukder et al., 2011) and Pseudomonas cepacia (Kuan et al., 2013). In the
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industrialization of these processes, immobilization of lipase has become vital because it could
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be recovered easily and used in continuous reactions (Park et al., 2006). Immobilized enzymes

are more stable towards temperature, chemical as well as shear denaturation and also enable easy

handling, recovery and recycling of the biocatalyst. The most widely used immobilized lipases

are extracellular and commercially available as Novozym 435, C. antarctica lipase, immobilized

on a macroporous acrylic resin, Lipozyme RM IM, Rhizomucor miehei lipase, immobilized on an

anionic resin, and Lipozyme TL IM, Thermomyces lanuginosus lipase, immobilized on a gel of

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granulated silica respectively (Robles-Medina et al., 2009). However, the supports used in the

processes are of high cost and the activity of immobilized enzymes decreases on continuous

recycling due to desorption, substrate inactivation and product inhibition (Bajaj et al., 2010). In

addition, with low cost crude oil as substrate, the immobilized lipases could not be used for many

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cycles as they become rapidly inactivated in feedstock containing phospholipids (Watanabe et

al., 2002). Another strategy which has recently been used to reduce the cost of enzyme is to

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make use of microbial cells producing intracellular lipases as whole cell biocatalyst

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(Ranganathan et al., 2008). Soluble lipases are also used in transesterification reactions that are

carried out in the presence of organic solvents in low water-containing systems because of their

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higher activities and elimination of immobilization (Sharma and Kanwar, 2014). But, there are

only limited reports available on soluble lipase-catalyzed biodiesel production (Zhao et al., 2007;
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Chen et al., 2008) including the transesterification of low cost crude (non-degummed) soybean
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oil (Cesarini et al., 2013).

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3. Potential feedstock for biodiesel production

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The cost of raw material accounts for 60–80% of the total production cost of biodiesel,
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which indicates that selecting the appropriate feedstock is of considerable importance for
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ensuring the economic feasibility of the process (Juan et al., 2011). Edible oils, such as palm oil

(Talukder et al., 2011), rapeseed oil (Jeong and Park, 2008), sunflower oil (Modi et al., 2007),

ricebran oil (Kamini and Iefuji, 2001) and soybean oil (Yu et al., 2010) are the major sources for

biodiesel production. However, there are serious concerns regarding the use of edible oil in

biodiesel production, since they are more expensive and the mass propagation of these plants

would lead to deforestation as reported by Leung et al. (2010). In order to overcome these

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drawbacks, researchers have focused on alternate feedstock such as non-edible oil, waste oil and

microbial oil for biodiesel production. The fatty acid composition of different oil feedstocks used

in transesterification reaction are summarized in Table 2.

Non-edible oils or the second generation feedstocks obtained from jatropha, pongamia,

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crambe and castor are less expensive than edible oils and potentially available for producing

biodiesel. Non-edible oil plants are well adapted to arid, semi-arid conditions and they do not

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require addition of fertilizer and moisture. These plants do not compete with food and the seed

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cake after oil expelling could be used as fertilizer for soil enrichment. The selection of these oils

would depend on the estimated availability of oil seeds in each country. In particular, Jatropha

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curcas oil is considered as one of the promising feedstocks for biodiesel production (Atabani et

al., 2013; Juan et al., 2011). The high oil contents in the jatropha seed, ranging from 30% to 50%
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based on seed weight, makes it a viable source for biodiesel production (Azocar et al., 2010).
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Biodiesel manufacturers are also focusing their attention on using low-cost feedstock
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such as waste oils (waste cooking oil, grease) and animal fats (lard, tallow). The generation of

waste cooking oil is huge and would result in environmental contamination, if proper disposal
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method has not been adopted. Waste cooking oils are generated by repeated use of vegetable oils
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fried at high temperatures. During this process, chemical reactions such as hydrolysis,
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polymerization and oxidation may occur leading to the formation of many detrimental

compounds and their toxicological effects upon human consumption are not known. However,

they could be used as a potential feedstock for biodiesel production. But the transesterification

reaction may greatly be affected due to the high content of FFA in waste cooking oil ranging

between 0.5 and 15 wt.%, while refined oil usually has less than 0.5 wt.% FFA (Lam et al.,

2010).

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Recently, interest has been growing in the development of microbial oils and it is found

that many microorganisms, such as algae, yeast, bacteria and fungi, have the ability to

accumulate oils under some special cultivation conditions. Compared to other plant oils,

microbial oils have many advantages, such as short life cycle, less influence by season, climate

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and land space, requirement of less labor and easier to scale up (Li et al., 2008). Different

cultivation modes, including fed-batch, and continuous fermentations, have been used to increase

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cell density of oleaginous microbes. However, the cost of microbial oil production is currently

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higher than those of vegetable oil which could be reduced by using lignocellulose-based

carbohydrates as substrates for the growth of microbes (Li et al., 2007b). Microalgae such as

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Botryococcus, Chlorella, Cylindrotheca, Nitzschia, and Schizochytrium utilize carbon dioxide

and sunlight for oil accumulation under specific environmental conditions. The oil contents
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obtained from several yeast strains such as Cryptococcus, Lipomyces, and Rhodotorula reach 60–
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70% of their dry weight (Meng et al., 2009). The potential fungal species which produce unique
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lipids such as docosahexaenoic acid, γ-linolenic acid, eicosapentaenoic acid and arachidonic acid

are Aspergillus oryzae, Mortierella isabellina, Humicola lanuginosa, and M. vinacea (Azocar et
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al., 2010). The direct biosynthesis of fatty acid alkyl esters, referred to as microdiesel, was also
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achieved using metabolically engineered Escherichia coli (Kalscheuer et al., 2006).

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4. Enzymatic transesterification using yeast lipases

Transesterification of triglycerides with alcohol gives fatty acid alkyl esters as main

product and glycerol as by product in the presence of a catalyst. The first step is the conversion

of triglycerides to diglycerides, which is followed by the conversion of diglycerides to

monoglycerides and of monoglycerides to glycerol, yielding one methyl ester molecule from

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each glyceride at each step (Abbaszaadeh et al., 2012). The basic transesterification reaction is

illustrated in Fig. 2. Numerous reports are available on transesterification reactions catalyzed by

yeast lipases. The following section reviews some of the potential yeast lipases that have been

studied in transesterification of oils and fats with emphasis given on the substrate and optimum

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conditions used for production of biodiesel.

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4.1 Candida antarctica

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Extensive studies on transesterification reactions have been carried out using C.

antarctica lipase (Novozym 435), using a wide variety of substrates. Alcoholysis of tallow with

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primary and secondary alcohols in the presence of solvents was first investigated using lipase

from C. antarctica by Nelson et al. (1996) and it was reported that the lipase was most suitable
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for transesterifing triglycerides with secondary alcohols for production of branched alkyl esters.
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Addition of short chain alcohols like methanol in the transesterification reaction mixture at
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moderate concentrations leads to inactivation of the enzyme and decrease the yield of ester.

Therefore, stepwise addition of methanol was suggested as a solution. Shimada et al. (1999b)
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developed a three step reaction for methanolysis of vegetable oil (a mixture of soybean and
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rapeseed oil) because the C. antarctica lipase was inactivated in a reaction mixture containing
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more than 1.5 molar equivalent of methanol. According to stoichiometry of the reaction, three

molar equivalents of methanol are needed for the complete methanolysis of oil. Thus, one molar

equivalent of methanol was added three times after 10, 14 and 24 h, respectively to obtain a final

conversion of 98.4%. Later, Watanabe et al. (2000) developed a two step methanolysis reaction,

since the C. antarctica lipase was not inactivated when the reaction contained 2:3 molar

equivalents of methanol, a mixture of acylglycerols and 33% methyl esters. They carried out the

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first step at 30 °C for 12 h with shaking in a reaction mixture containing vegetable oil, 1:3 molar

equivalent of methanol and 4% immobilized lipase; the second step for 24 h by adding 2:3 molar

equivalent of methanol (36 h in total) (Fig. 3).

Samukawa et al. (2000) found that preincubation of the enzyme, Novozym 435 in methyl

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oleate for 0.5 h and subsequently in soybean oil for 12 h enhanced the methanolysis of soybean

oil to 97% in 3.5 h by stepwise addition of 0.33 molar equivalent of methanol at 0.25–0.4 h

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intervals. Later, Watanabe et al. (2002) removed the phospholipids (PLs) present in the soybean

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oil by degumming, since PLs inhibit the methanolysis of triacylgycerols, which could be due to

the interference of the interaction of the lipase molecule by PLs bound on immobilized

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preparation. A three step methanolysis successfully converted 93.8% degummed soybean oil to

its corresponding methyl esters and the C. antarctica lipase could be reused for 25 cycles without
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any loss of activity. Similarly, when degummed ricebran oil was used as substrate, a methyl ester
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yield of 98% was obtained (Lai et al., 2005). When cotton seed oil (Kose et al., 2002) and canola
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oil (Hajar et al., 2008) were used as substrates, the fatty acid methyl ester (FAME) yield was

91.5% and 84.4% respectively.

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Royon et al. (2007) developed an alternative method using t-butanol as low cost non-
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toxic solvent to dissolve methanol, since moderate concentrations of methanol inactivates the C.
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antarctica lipase and the enzyme does not act on tertiary alcohols. They carried out methanolysis

in a reaction mixture containing 32.5% t-butanol, 13.5% methanol, 54% cottonseed oil and 0.017

g enzyme/g oil and obtained a methanolysis yield of 97% after 24 h at 50 °C. Later, Jeong and

Park (2008) also used t-butanol as solvent for the transesterification of rapeseed oil and obtained

a FAME yield of 76.1%. Methyl acetate (MA) has been used as a novel acyl acceptor for

biodiesel production from soybean oil using Novozym 435 to eliminate the inactivation of

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enzyme by methanol. A molar ratio of methyl acetate/soybean oil of 12:1 gave a methyl ester

yield of 92% in a solvent free medium (Xu et al., 2003). A method for enhanced Novozym 435-

catalyzed transesterification of palm oil to biodiesel using a mixture of methanol and methyl

acetate as acyl acceptors was also developed. Biodiesel yield using methanol–MA mixture

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reached 95–96% after 8 h at a methanol to palm oil molar ratio of 1:1, a MA to palm oil molar

ratio of 12:1, 2 g of palm oil, and 0.6 g of Novozym 435 (Talukder et al., 2011). Ethyl acetate

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was explored as an acyl acceptor for preparation of biodiesel from the crude oils of Jatropha

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curcas (jatropha), Pongamia pinnata (karanj) and Helianthus annuus (sunflower). The maximum

yield of ethyl esters was 91.3%, 90% and 92.7% with crude jatropha, karanj and sunflower oils,

respectively (Modi et al., 2007).

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Watanabe et al. (2000) carried out a two step transesterification of a mixture of vegetable
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oil and methanol in a reactor fixed with an impeller. However, the enzyme carrier was destroyed
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and the lipase was not stable in the process. Concurrently, they attempted a continuous flow
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reaction using three columns packed with 3.0 g immobilized C. antarctica lipase. The reaction

mixture containing vegetable oil and 1:3 molar equivalent of methanol was fed into the first
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column at a constant flow rate of 6.0 ml/h. The eluate and 1:3 molar equivalent of methanol were
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mixed and then fed into the second column at the same flow rate. The final step reaction was
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done by feeding a mixture of eluate from the second column and 1:3 molar equivalent of

methanol at the same flow rate. The ME content in the final step reached 93%. When soybean oil

(Shaw et al., 2008) and waste cooking palm oil (Halim et al., 2009) were used as substrates for

continuous biosynthesis of biodiesel in a packed bed reactor using C. antarctica lipase, the

FAME yield reached 75.2% and 79%, respectively.

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Ultrasound assisted lipase-catalyzed reaction method witnesses a fast development. The

enhancement of enzyme activity of Novozym 435 by ultrasound irradiation has been reported

earlier (Lin and Liu, 1995). Yu et al. (2010) achieved a 96% yield of FAME from soybean oil in

4 h with a combination of 50% of ultrasonic power and 50 rpm vibration. The improved reaction

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rate was attributed to the rapid emulsification by ultrasonic irradiation and sufficient contact

between enzyme and substrate by vibration which caused accelerated transportation of reactants.

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The transesterification process could also be improved by using a combination of lipases

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due to the variation in substrate specificity of the lipases. Huang et al. (2010) produced biodiesel

from lard by the combined use of Novozym 435 (non-specific) and Lipozyme TL-IM (1,3-

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specific) lipases in the reaction mixture and obtained a methyl ester yield of 97.2% in 20 h.
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4.2 Candida sp. 99-125
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Deng et al. (2003) isolated Candida sp. 99-125 strain from sewage water of northern
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China. Lipase was produced in a medium containing coconut oil as an inducer and immobilized

on a fiber cloth. Transesterification of rapeseed oil and methanol using the immobilized lipase
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gave a FAME yield of 83%. Tan et al. (2006) immobilized Candida sp. 99-125 lipase on a cheap
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cotton membrane and used the membrane-immobilized lipase for at least six times without a
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decrease in yield of FAMEs. Methyl ester conversion of 91, 91.8, 90.8, 91.2, 88.5% were

obtained with soybean oil, safflower oil, linseed oil, corn oil, palm oil, respectively. Li et al.

(2010) used immobilized Candida sp. 99-125 lipase to catalyze biodiesel production of non-

edible crude rice bran oil extracted from white rice bran which gave a FAME yield of 87.4%.

Continuous reaction in a fixed bed reactor was investigated by Nie et al. (2006) using a

series of columns packed with immobilized Candida sp. 99–125 lipase (Fig. 4). As substrate of

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the first reaction step, plant or waste oil was used with 1:3 molar equivalent of methanol against

total fatty acids in the oil. Mixtures of the first and second step eluates and 1:3 molar equivalent

of methanol were used for the second and third reaction steps. A hydrocyclone was used in order

to separate the by-product glycerol after every 1:3 molar equivalent of methanol addition.

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Petroleum ether was used as solvent and the pump was operated with a flow rate of 15 l/h. The

final conversion ratio of FAME from plant oil and waste oil under the optimal condition was

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93% and 92%, respectively at 40 ºC and the immobilized lipase was stable for more than 10

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days. This method is feasible for an industrial scale production of biodiesel because of its

advantages such as low pollution, environmental friendly and low energy costs.

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Li et al. (2007a) concentrated on biodiesel production from a heterotrophic microalga,

Chlorella protothecoides, instead of using vegetable oils as substrate. Microalgal oil produced by
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green microalga C. protothecoides was converted to 98.15% of monoalkyl esters of fatty acids in
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12 h, with 75% immobilized Candida sp. 99–125 lipase, 10% water and 3:1 ratio of methanol to
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oil, batch-fed at three times at 38 ºC. Lu et al. (2007) used lard as a substrate and the FAME

yield was 87.4%.

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4.3 Candida rugosa (formerly Candida cylindracea)

In vegetable oil refining process, the upgrading of crude oils of vegetable origin requires
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the use of activated bleaching earth (ABE), an adsorbent, for the removal of carotene,

chlorophyll, and other components formed during the refining process (e.g., phosphatides and

soaps). Nearly 35–40% of the weight of waste ABE is vegetable oil, a substrate that could be

utilized for the synthesis of a wide range of products. Hence, lipase from C. cylindracea was

used for transesterification of waste palm oil adsorbed on activated bleaching earth (ABE), and

during the first 4 h of reaction more than 78 % FAME was produced (Lara and Park, 2004).

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Later, Park et al. (2008) investigated large scale production of FAMEs from waste ABE

discarded by the crude oil refining industry using C. cylindracea lipase in a 50-L pilot plant.

Diesel oil or kerosene was used as an organic solvent for the transesterification of triglycerides

embedded in the waste ABE. When 1% (w/w) lipase was added to waste ABE, the FAME

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content reached 97% (w/w) at 12 h and 25 ºC with an agitation rate of 30 rpm and 1:3.5 oil to

methanol molar ratio. These results show a promising reutilization method of industrial waste

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resources containing waste vegetable oils for the production of FAMEs targeted for biodiesel

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application.

Nasratun et al. (2009) immobilized C. rugosa lipase on chitosan beads to catalyze the

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transesterification of cooking oil with methanol which gave a FAME yield of 72.25%. Moreno-

Pirajan and Giraldo (2011) immobilized C. rugosa lipase with activated carbon and used it for
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the enzymatic transesterification of palm oil with methanol and ethanol, which gave 70 and 85
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mol% of methyl and ethyl esters, respectively.

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4.4 Cryptococcus sp. S-2

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A reaction system without an organic solvent in an aqueous medium is desirable for the
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industrial production of diesel fuel. The crude lipase from the yeast Cryptococcus sp. S-2
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efficiently catalyzed the methanolysis of vegetable oils and the methyl ester contents increased

with increasing the water content from 60 to 100 wt.%. The optimal methanolysis conditions for

rice bran oil was an oil/methanol molar ratio of 1:4, a water content of 80 wt.% by weight of the

substrate containing 2000 U of crude lipase with shaking at 160 rpm for 120 h at 30 °C (Kamini

and Iefuji, 2001). Thus, the reaction was conducted in a single step to avoid stepwise addition of

methanol and the methyl ester contents reached 80.2 wt.% at 120 h (Fig. 5).

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Page 16 of 43
4.5 Trichosporon asahii MSR54

The yeast strain was isolated from petroleum sludge and grown in a medium containing

corn oil as an inducer for lipase production. The enzyme was immobilized by physical

adsorption on basic alumina. Transesterification using T. asahii lipase gave a FAME yield of

t
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87.6% with coconut oil/methanol ratio of 1:4, 100 U of immobilized enzyme/g of oil using 1,4-

dioxane as solvent at 45 ºC in 24 h (Kumari and Gupta, 2012) .

cr
us
4.6 Yarrowia lipolytica

Two step catalysis of soybean oil, hydrolysis followed by esterification, with

an
immobilized Y. lipolytica lipase was reported by Meng et al. (2011). First, soybean oil was

hydrolyzed at 40 °C with 100 U of lipase broth, 1 g of oil and 30% to 60% (v/v) of water. The
M
FFA distilled from the hydrolysis mixture was then used for the esterification of FFA to fatty
d

acid ethyl ester by immobilized lipase. A mixture of 2.82 g of FFA and equimolar ethanol
te

(addition in three steps) were shaken at 30 °C with 18 U of lipase per gram of FFA. The degree

of esterification reached 85% after 3 h and the reaction could be repeated for 25 batches without
p

loss in enzyme activity and esterification yield.

ce
Ac

5. Use of whole cell biocatalysts in transesterification reactions

The efficiency of the transesterification process could be increased by using microbial

cells that are producing intracellular lipase as whole-cell biocatalysts, in the place of extracellular

lipase which requires complex purification steps before immobilization, since the cost of lipases

significantly limits their applicability for the bulk production of biodiesel (Ranganathan et al.,

2008). This prompted research into the potential use of microorganisms such as bacteria, yeast

16

Page 17 of 43
and filamentous fungi as promising whole cell biocatalysts for biodiesel production and the

process of immobilization could also be carried out spontaneously during the process of cell

cultivation (Abbaszaadeh et al., 2012). The immobilization of the filamentous fungi, Rhizopus

oryzae (Ban et al., 2002; Tamalampudi et al., 2008), on biomass support particles (BSPs)

t
ip
allowed the separation of the whole cell biocatalyst and facilitated its reuse. However, substrate

conversion yield was limited by toxicity of the solvent and low mass transfer rate of high

cr
molecular weight substrates from the solvent phase to the whole cell biocatalyst in aqueous

us
phase. Later, this limitation was overcome by surface immobilization of enzyme on microbial

cells (Wernerus and Stahl, 2004). In 2006, Jung et al. immobilized a thermostable lipase (TliA)

an
from Pseudomonas fluorescens on the cell surface of a solvent resistant bacterium, P. putida

GM730. Gao et al. (2009) developed a genetically engineered E. coli whole cell biocatalyst
M
expressing lipase (LipK107) from Proteus sp. and the engineered E. coli produced a 100% yield
d

of biodiesel under optimal conditions.

te

Yeast whole-cell biocatalyst was constructed by intracellularly overproducing Rhizopus

oryzae lipase (ROL) in Saccharomyces cerevisiae MT8-1. These whole-cell biocatalysts were
p

permeabilized by air-drying and used for the synthesis of methyl esters, from soybean oil and
ce

methanol in a solvent-free system and the methyl ester content in the reaction mixture was 71%
Ac

at 165 h and 37 ºC (Matsumoto et al., 2001). However, when the ROL was expressed on the cell

surface of S. cerevisiae using a FLO1 gene, encoding a lectin like cell wall protein (Fig. 6), a

FAME yield of 78% was obtained in 72 h (Matsumoto et al., 2002). This difference in yield and

conversion rate of methyl ester might be attributed to the facilitated access of substrate molecules

to the cell-surface displayed ROL, indicating that permeabilization of cells is not required.

17

Page 18 of 43
Recently, Jiang et al. (2008) reported Pichia pastoris to be a more robust expression system for

surface display of lipases with better stability and higher biomass production.

Certain strains of yeasts naturally produce cell-bound lipase and could be cultivated to a

high density in an inexpensive medium. The cell-bound lipase produced by the yeast

t
ip
Rhodotorula glutinis was freeze dried and a conversion of 60–62% was obtained upon

esterification of palmitic or oleic acid with butanol in 96 h. The enzyme preparation was reused

cr
in four consecutive batch reactions with only 10% loss of activity (Hatzinikolaou et al., 1999).

us
Srimhan et al. (2011) reported the transesterification of refined palm oil and methanol using

Rhodotorula mucilagenosa P11I89 isolated from oil contaminated soil. FAME yield of 51.26%

an
was obtained, when the reaction was catalyzed by whole cell of R. mucilagenosa P11I89 in the

presence of 3 molar equivalents of methanol. As the molar equivalent of methanol against palm
M
oil increased, FAME yield was dramatically enhanced. Maximum FAME yield of 83.29% was
d

achieved at 72 h with a methanol to palm oil molar ratio of 6:1, 3 U/g substrate of lipase and10%
te

water in the reaction mixture. Use of the methanol-tolerant lipase producing yeast as a whole cell

biocatalyst could effectively resolve major technical obstacles in terms of enzyme stability and
p

high cost of lipase, leading to the feasibility of green biodiesel industrialization.

ce
Ac

6. Economic assessment of biodiesel production

A comparative economics diagram of plant investment costs and manufacturing costs for

one tonne capacity of biodiesel by alkali, immobilized lipase and soluble lipase catalyzed

transesterification is shown in Fig. 7. Biodiesel production cost using alkali catalyst process was

found to be the lowest ($ 1166.67/tonne) compared to soluble lipase catalyst ($7821.37/tonne)

and immobilized lipase catalyst ($2414.63/tonne) process. The total plant costs (103 tonne) for

18

Page 19 of 43
the alkali, soluble and immobilized catalyst processes were $2.10 million, $3.30 million and

$3.31 million, respectively. The above results showed that the plant cost for biodiesel production

using immobilized enzyme was 57.18% higher than the alkali catalyst process and 0.40% higher

than soluble enzyme catalyst process. The high cost for both the soluble and immobilized

t
ip
catalyst process was due to the process time variation with respect to the alkali catalyst.

Likewise, a marginal increase of plant cost for immobilized catalyst process over soluble enzyme

cr
process was due to the addition of encapsulation unit as reported by Jegannathan et al. (2011).

us
Due to the advantages of enzymatic transesterification over chemical method, there is an

increase in commercialization of the process at pilot and industrial scale. In 2007, Lvming Co.

an
Ltd., Shanghai, China, established an enzymatic biodiesel production plant with a capacity of

10,000 tons using waste cooking oil as feedstock and immobilized lipase of Candida sp. 99–125
M
($0.03/kg biodiesel) as catalyst. In 2012, Piedmont Biofuels, North Carolina, developed a new
d

technology (FAeSTER) for a continuous biodiesel production using C. antarctica Lipase B

te

(CALB) from Novozymes (Christopher et al., 2014).

p

7. Utilization of the byproduct glycerol

ce

As biodiesel production is increasing exponentially, crude glycerol, the byproduct

Ac

generated from the transesterification of oils has also been generated in a large quantity.

Approximately, it constitutes about 10 wt% of the total product during the biodiesel production

(Ayoub and Abdullah, 2012). Therefore, much effort has been made to transform the low-value

glycerol into a value-added byproduct by different approaches. The salt content in crude glycerol

ranges from 5% to 7% (Yang et al., 2012) in biodiesel production using homogeneous alkaline

19

Page 20 of 43
catalysts, which makes the conventional purification technique more costly whereas in a lipase

catalyzed transesterification process, improved quality of crude glycerol could be obtained.

The value added products could be obtained from glycerol through chemical or biological

catalysis. The potential products of chemical conversion include propylene glycol, propionic

t
ip
acid, acrylic acid, propanol, isopropanol, allyl alcohol and acrolein (Bajaj et al., 2010). The

anaerobic fermentative production of 1,3-propanediol (1,3-PD) by bacteria such as Klebsiella

cr
pneumoniae (Mu et al., 2006) and Clostridium butyricum (Gonzalez-Pajuelo et al., 2004) is the

us
most promising option for biological conversion of glycerol. A novel integrated bioprocess

combining biodiesel production by Novozym 435 with microbial production of 1,3-PD by K.

an
pneumoniae using a hollow fiber membrane was investigated by Mu et al. (2008). Glycerol

produced during the transesterification process, could pass through the membrane and was
M
converted to 1,3-PD directly by K. pneumoniae. The highest conversion of biodiesel obtained in

20 h was 84%, and the molar yield of 1,3-PD was 0.47 mol/mol and 1.7 g l−1 h−1, respectively.
d
te

Crude glycerol could be converted to polyhydroxy butyrate by Paracoccus denitrificans,

Cupriavidus necator, Zobellella denitrificans and Pseudomonas oleovorans. Docasahexanoic

p

acid has been produced by fermentation of the alga Schizochytrium limacinum in a medium
ce

containing crude glycerol as carbon source. As a sole carbon source, crude glycerol has been
Ac

used for the production of phytase in industrial scale high cell density fermentations with

recombinant Pichia pastoris possessing a pGAP-based constitutive expression vector (Yang et

al., 2012).

8. Conclusion

20

Page 21 of 43
Biodiesel has received increasing attention in recent years as it is the only alternate fuel to

petroleum derived diesel that can be used directly in existing engines. Yeast lipase catalyzed

transesterification reactions for biodiesel production is a growing area of research and has

immense potential to generate an eco-friendly economic fuel. Yeast lipases allow the use of

t
ip
waste industrial materials of low quality and high content FFA, and non-edible oils as substrates

for transesterification reactions. Methanol as acyl acceptor has been extensively used owing to its

cr
low cost, availability and several methods such as stepwise addition of methanol in a three step,

us
two step and single step reactions have been developed by researchers. Other acyl acceptors like

methyl acetate and ethyl acetate have been reported to minimize the inhibitory effects of

an
methanol. Utilization of yeast as whole-cell biocatalyst provides an inexpensive method of

catalyst preparation for its excellent operational stability. For industrial applications, the reaction
M
rate of whole-cell catalyzed biodiesel production could be increased by designing packed-bed
d

reactors for continuous operations. Moreover, recombinant DNA technology, protein engineering
te

and directed evolution methods could be used to improve lipase stability, substrate specificity

and catalytic efficiency, which will facilitate lowering the cost of the overall process. Additional
p

research and development is required to directly utilize the by-product glycerol during biodiesel
ce

production on a large scale. Further exploration and integration of these technologies would
Ac

promote commercial biodiesel production as a green process.

Acknowledgments

The authors thank Dr. A. B. Mandal, Director, CSIR–CLRI, Chennai, India, for his kind

permission to publish this work. Financial support received for carrying out this work from

ZERIS-CSC0103-WP09 is gratefully acknowledged.

21

Page 22 of 43
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Figure legends

Fig. 1 – Supply and demand of biodiesel in different regions, 2010–2020 (Pinto, 2011).

Fig. 2 – Transesterification of triglycerides with alcohol.

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Fig. 3 – Two and three step methanolyses of vegetable oil triacylglycerols by immobilized

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Candida antarctica lipase (Watanabe et al., 2000). Arrows indicate the addition of methanol.

Fig. 4 – Biodiesel production in a fixed bed reactor using immobilized Candida sp. 99-125 lipase

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(Nie et al., 2006).

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Fig. 5 – Single step methanolysis of ricebran oil using Cryptococcus sp. lipase (Kamini and

Iefuji, 2001).
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Fig. 6 – Yeast as whole cell biocatayst. ROL (Rhizopus oryzae lipase) expressed on the cell
d

surface of Saccharomyces cerevisiae using a FLO1 anchor (Fukuda et al., 2008).

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Fig. 7 – Economic assessment of biodiesel production (Jegannathan et al., 2011).

p
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Fig. 1

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Fig. 2

CH2-OOC-R1 R1-COO-R’ CH2-OH

Catalyst +
CH2-OOC-R2 + 3R’OH R2-COO-R’ + CH2-OH
+
CH2-OOC-R3 R3-COO-R’ CH2-OH

Triglycerides Alcohol Alkyl esters Glycerol

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Fig. 7

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Table 1

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Table 1 – Comparison of the chemical and enzymatic method for production of biodiesel (Gog et al., 2012; Robles-Medina et

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al., 2009).

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Parameter Chemical method Enzymatic method

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Acid process Alkaline process

Biodiesel yield >90% >96% >96%

FFA content in the substrate Converted to biodiesel Soap formation Converted to biodiesel

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Water in the substrate Interference with Interference with No influence
reaction reaction
Purification of methyl esters Repeated washing Repeated washing None
Glycerol recovery Complex, low grade glcerol Complex, low grade glcerol Easy, high grade glycerol
Reaction rate
Reaction temperature
Catalyst recovery
Slow
>100 ºC
ed
Difficult, the catalyst ends up
High
60-80 ºC
Difficult
Low
20-50 ºC
Easy
pt
in the by-products
Catalyst reuse No reusability Partially lost in post-processing Reusable
steps
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Cost of catalyst Low Low High

Waste water generation High High Low
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Table 2

Table 2 – Fatty acid (%) composition for different oil source (Atabani et al., 2013; Li et al.,
2008; Ribeiro et al., 2011).

Feedstock % Fatty acid

Palmitic Stearic Oleic Linoleic Linolenic
(C16:0) (C18:0) (C18:1) (C18:2) (C18:3)

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Edible oil

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Soybean 7–14 1.4–5.5 19–30 44–62 4–11
Rapeseed 2.5–6.5 0.8–3 53–70 15–30 5–13

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Sunflower 3–10 1–10 14–35 55–75 <0.3
Olive 10–11.7 2.1 73.8–78 7.0–9.8 n.d

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Coconut 7–10 1–4 5–8 1–3 n.d
Safflower 5.2 2.2 76.3 16.2 n.d
Peanut 6–11 3–6 39–66 17–38 n.d

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Cotton seed 17–23 1–3 23–41 34–55 n.d
Corn 8–10 1–4 30–50 34–56 n.d
Mustard 3 1.5 15–60 12 5–10
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Non-Edible oil
Pongamia 9.8 6.2 72.2 11.8 n.d
Jatropha curcas 10–17 5–10 36–64 18–45 n.d
d

Babassu 5.2–11 1.8–7.4 9–20 1.4–6.6 n.d

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Crambe 1.8–2 0.7–1 16–17.2 8–8.7 5.2–7

Camelina 5 2.2 17.7 18 37.9
Castor bean 1.1 1 3.3 3.6 0.32
p

Neem 14.9 20.6 43.9 17.9 0.4

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Animal Fat
Lard 23.8–25 12–13.5 41.2–45 10–10.2 1
Tallow 24.9–27 7–18.9 36–48 3.1 0.6
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Oleaginous yeasts
Lipomyces starkeyi 33 4.7 55.1 1.6 n.d
Rhodosporidium toruloides 24.3 7.7 54.6 2.1 n.d
Lipomyces lipofera 37 7 48 3 n.d
Trichosporon pullulan 15 2 57 24 1
Rhodotorula glutinis 18 6 60 12 2
Yarrowia lipolytica 11 1 28 51 1

n.d – not determined

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