Biotechnology Advances 28 (2010) 500–518

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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Homogeneous, heterogeneous and enzymatic catalysis for transesterification of high

free fatty acid oil (waste cooking oil) to biodiesel: A review
Man Kee Lam, Keat Teong Lee ⁎, Abdul Rahman Mohamed
School of Chemical Engineering, Universiti Sains Malaysia, Engineering Campus, Seri Ampangan, 14300 Nibong Tebal, Pulau Pinang, Malaysia

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

Article history: In the last few years, biodiesel has emerged as one of the most potential renewable energy to replace current
Received 10 December 2009 petrol-derived diesel. It is a renewable, biodegradable and non-toxic fuel which can be easily produced
Received in revised form 16 March 2010 through transesterification reaction. However, current commercial usage of refined vegetable oils for
Accepted 20 March 2010
biodiesel production is impractical and uneconomical due to high feedstock cost and priority as food
Available online 31 March 2010
resources. Low-grade oil, typically waste cooking oil can be a better alternative; however, the high free fatty
Keywords:
acids (FFA) content in waste cooking oil has become the main drawback for this potential feedstock.
Biodiesel Therefore, this review paper is aimed to give an overview on the current status of biodiesel production and
Waste cooking oil the potential of waste cooking oil as an alternative feedstock. Advantages and limitations of using
Homogeneous catalyst homogeneous, heterogeneous and enzymatic transesterification on oil with high FFA (mostly waste cooking
Heterogeneous catalyst oil) are discussed in detail. It was found that using heterogeneous acid catalyst and enzyme are the best
Enzymatic option to produce biodiesel from oil with high FFA as compared to the current commercial homogeneous
Transesterification base-catalyzed process. However, these heterogeneous acid and enzyme catalyze system still suffers from
serious mass transfer limitation problems and therefore are not favorable for industrial application.
Nevertheless, towards the end of this review paper, a few latest technological developments that have the
potential to overcome the mass transfer limitation problem such as oscillatory flow reactor (OFR),
ultrasonication, microwave reactor and co-solvent are reviewed. With proper research focus and
development, waste cooking oil can indeed become the next ideal feedstock for biodiesel.
© 2010 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
2. Biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
2.1. Composition of vegetable oils and fats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
2.2. Composition of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
3. Current status of biodiesel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
4. Biodiesel costing and potential of waste cooking oil as feedstock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
5. Waste cooking oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
5.1. Thermolytic reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
5.2. Oxidative reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
5.3. Hydrolytic reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
6. Catalysis in transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
6.1. Homogeneous base-catalyzed transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
6.2. Homogeneous acid-catalyzed transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507
6.3. Homogeneous acid and base-catalyzed transesterification: two steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507
6.4. Heterogeneous base-catalyzed transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
6.5. Heterogeneous acid-catalyzed transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
6.5.1. Zirconium oxide (ZrO2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
6.5.2. Titanium oxide (TiO2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
6.5.3. Tin oxide (SnO2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510

⁎ Corresponding author. Tel.: +60 4 5996467; fax: +60 4 5941013.

E-mail address: chktlee@eng.usm.my (K.T. Lee).

0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2010.03.002
 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518 501

6.5.4. Zeolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

6.5.5. Sulfonic ion-exchange resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
6.5.6. Sulfonic modified mesostructure silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
6.5.7. Sulfonated carbon-based catalyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
6.5.8. Heteropolyacids (HPAs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
6.6. Enzyme (biocatalyst) catalyzed transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
6.6.1. Mucor miehei (Lipozym IM 60) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
6.6.2. Pseudomonas cepacia (PS 30) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
6.6.3. C. antarctica (Novozym 435). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
6.6.4. Bacillus subtilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
6.6.5. Rhizopus oryzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
6.6.6. Penicillium expansum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
7. Other methods or technologies for biodiesel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
7.1. Oscillatory flow reactor (OFR) for transesterification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
7.2. Microwave technology in transesterification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
7.3. Ultrasonic technology in transesterification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
7.4. Co-solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516

1. Introduction among other renewable resources as shown in Fig. 2 (International Energy

To date, fossil fuels account over 80.3% of the primary energy
consumed in the world, and 57.7% of that amount is used in
transportation sector (International Energy Agency I, 2006). On the
other hand, the global consumption of diesel fuel is estimated to be
934 million tonnes per year (Kulkarni and Dalai, 2006). Thus, The
World Energy Forum predicted that fossil oil will be exhausted in less
than 10 decades, if new oil wells are not found (Sharma and Singh,
2009). The main reason that caused the fast diminishing of energy
resources is due to rapid population and industrialization growth
globally (Pimentel and Pimentel, 2006). Due to this phenomenon, the
era of cheap crude oil no longer exists leading to high sky rocketing
price of petroleum, bellicose conflicts and increasing the number of
undernourished people especially from undeveloped countries. Fig. 1
presents the projection of world energy demand in the near future
indicating that there is an urgent need to find more new renewable
energies to assure energy security worldwide (Exxon Mobile, 2004).
Renewable energy has been highlighted in the last ten years due to its
potential to replace fossil fuel especially for transportation. Renewable
energy sources such as solar energy, wind energy, hydro energy, and
energy from biomass and waste have been successfully developed and
used by different nations to limit the use of fossil fuels. Nevertheless, based
on recent study from International Energy Agency (IEA), only energy
produced from renewable sources and waste has the highest potential
Agency I, 2008). Combustible renewable and waste accounted for 10.1%,
compared to hydro energy 2.2% and other 0.6% (included geothermal,
solar wind and heat). Hence, it is predicted that renewable energy from
combustible energies such as biodiesel will enter the energy market
intensively in the near future to diversify the global energy sources.
2. Biodiesel
Biodiesel is an alternative diesel fuel derived from vegetable oils or
animal fats (Vasudevan and Briggs, 2008). The main components of
vegetable oils and animal fats are triglycerides or also known as esters
of fatty acids attached to a glycerol. Normally, triglycerides of
vegetable oils and animals fats consist of several different fatty
acids. Different fatty acids have different physical and chemical
properties and the composition of these fatty acids will be the most
important parameters influencing the corresponding properties of a
vegetable oils and animal fats (Gerhard Knothe and Krahl, 2004).
Direct use of vegetable oils and animal fats as combustible fuel is not
suitable due to their high kinematic viscosity and low volatility.
Furthermore, its long term use posed serious problems such as
deposition, ring sticking and injector chocking in engine (Muniyappa
et al., 1996). Therefore, vegetable oils and animal fats must be subjected

Fig. 2. World total energy supply by fuel (Mtoe) in year 2006 (excluding electricity and
Fig. 1. Projection of energy demand for the near future. heat tread). Total: 11,741 million tonnes of oil equivalent (Mtoe).
502 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518

to chemical reaction such as transesterification to reduce the viscosity of Table 2

oils. In that reaction, triglycerides are converted into fatty acid methyl Chemical structures of common FAME.

ester (FAME), in the presence of short chain alcohol, such as methanol or Methyl ester Formula Common acronym Molecular weight
ethanol, and a catalyst, such as alkali or acid, with glycerol as a by-
Methy palmitic C17H34O2 C16:0 270.46
product (Vasudevan and Briggs, 2008). Eq. (1) depicts the transester- Methy stearate C19H38O2 C18:0 298.51
ification reaction. Another alternative way to produce biodiesel is Methy oleate C19H36O2 C18:1 296.50
through thermal cracking or pyrolysis. However, this process is rather Methy linoleate C19H34O2 C18:2 294.48
Methy linolenate C19H24O2 C18:3 292.46
complicated to operate and produce side products that have no
commercial value (Sharma and Singh, 2009).

3. Current status of biodiesel production

ð1Þ
2.1. Composition of vegetable oils and fats
Vegetable oils and animal fats usually have hydrophobic properties,
which mean they are insoluble in water. As mention earlier, triglycerides
are made up of 1 mol glycerol and 3 mol fatty acids. Fatty acids vary in
terms of carbon chain length and number of unsaturated bonds (double
bonds). Typical fatty acids compositions found in several vegetable oils
are summarized in Table 1 (Ma and Hanna, 1999). Fatty acids that have
no double bonds are termed “saturated” such as stearic acid. These
chains contain maximum number of possible hydrogen atoms per atom
carbon. Fatty acids that have double bonds are termed “unsaturated”
such as Linoleic acid. These chains do not contain maximum number of
hydrogen atoms due to the presence of double bond(s) on some carbon
atoms. Normally, natural vegetable oils and animal fats are obtained in
the crude form through solvent extracting or mechanically pressing,
containing a lot of impurities such as free fatty acids, sterols and water. In
fact, these free fatty acids and water content will have significant effect
on the transesterification reaction, especially if a base catalyst is used.
They could also interfere with the separation of FAME and glycerol
during water washing (purification step) because of soap formation.
2.2. Composition of biodiesel
With the crude fossil fuel prices near all-time high, biodiesel has
emerged as the fastest growing industries worldwide. Several
countries especially United State and members of European Union
are actively supporting the production of biodiesel from the
agriculture sector. In year 2006, nearly 6.5 billion liters of biodiesel
was produced globally as shown in Fig. 3 (TBW, 2008). It is interesting
to note that 75% of the total biodiesel production comes from
European countries. This is mainly due to substantial support from the
European government such as consumption incentives (fuel tax
reduction) and production incentive (tax incentives and loan
guarantees) which will further catalyzed the growth of the biodiesel
market in the next ten years. Besides that, the United States spent
around US$ 5.5 billion to 7.3 billion a year (TBW, 2008) to accelerate
biofuels production. As a result, around 450 million gallons of
biodiesel was produced in the United States in the year 2007
compared to only 25 million gallons in year 2004 (Thurmond,
2008). This 1700% increment was indeed a shocking increase in the
entire history of biodiesel production.
However, by the year 2020, it is predicted that biodiesel
production from Brazil, China, India and some South East Asia
countries such as Malaysia and Indonesia could contribute as much
as 20% (Thurmond, 2008). The driving forces for development of
biodiesel in these countries are economic, energy and environmental
security, improving trade balances and expansion of agriculture sector
(Zhou and Thomson, 2009). In addition, Brazil, China and India each
have set targets to replace 5% to 20% of total diesel with biodiesel by
the year 2010 with emphasis on second generation non-edible
feedstock (Thurmond, 2008). If governments from these countries
continue to aggressively promote biodiesel generation and continue
to invest in research and development for non-edible feedstocks such
as jatropha, castor, algae and grease, the prospects to achieve
biodiesel targets will be realized faster than anticipated. Fig. 4 depicts

Biodiesel is a mixture of fatty acid alkyl esters. In the case when

methanol is used as reactant, it will be a mixture of fatty acid methyl
esters (FAME) whereas if ethanol is used as reactant, the mixture will
be fatty acid ethyl esters (FAEE). However, methanol is commonly and
widely used in biodiesel production due to their low cost and
availability. Based on different feedstock, the biodiesel produced will
have different composition of FAME. Table 2 shows the common
composition of FAME in biodiesel (Ma and Hanna, 1999).

Table 1
Typical fatty acid composition (%) for different common oil source.

Fatty acid Soybean Cottonseed Palm Lard Tallow Coconut

Lauric (C12:0) 0.1 0.1 0.1 0.1 0.1 46.5

Myristic (C14:0) 0.1 0.7 1.0 1.4 0.8 19.2
Palmitic (C16:0) 0.2 20.1 42.8 23.6 23.3 9.8
Stearic (C18:0) 3.7 2.6 4.5 14.2 19.4 3.0
Oleic (C18:1) 22.8 19.2 40.5 44.2 42.4 6.9
Linoleic (C18:2) 53.7 55.2 10.1 10.7 10.7 2.2
Linolenic (C18:3) 8.6 0.6 0.2 0.4 0.4 0.0
Fig. 3. Biodiesel production in year 2006. Total production: 6.5 billion liters.
 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
Fig. 4. World biodiesel production and capacity.
a more recent world biodiesel production and capacity in the recent
years (Thurmond, 2008).
4. Biodiesel costing and potential of waste cooking oil as
feedstock
Currently, the major concern for biodiesel production is economic
feasibility. In a reality scenario, biodiesel production will not be
favored without tax exemption and subsidy from government; as the
production cost is higher than fossil derived diesel (Demirbas and
Balat, 2006). The overall biodiesel cost consists of raw material
(production and processing), catalyst, biodiesel processing (energy,
consumables and labour), transportation (raw materials and final
products) and local and national taxes (Haas et al., 2006). To date,
most biodiesel plants are using refined vegetable oils as their main
feedstock. Therefore, the cost of refined vegetable oils contributed
nearly 80% of the overall biodiesel production cost (Lam et al., 2009b).
Thus, it is undeniable that feedstock will be the most crucial variable
affecting the price of biodiesel in the global market.
A generic process model to estimate biodiesel capital and
operating cost had been developed by Haas et al. and the result is
shown in Fig. 5 (Haas et al., 2006). The model was designed on the
basis of continuous transesterification process using crude,
degummed soybean oil as the main feedstock and is dependent on
the price of feedstock. In addition, the model was based on a process
plant with an annual production capacity of 378,541,181 L
(10 × 106 gal). However, some economic factors were excluded, such
as internal rate of return, economic life span, corporate tax rate,
salvage value, debt fracture, construction interest rate and long term
interest rate, working capital, environment control equipment,
marketing and distribution expenses, the cost of capital, and the
Fig. 5. Impact of feedstock prices on the predicted unit cost of biodiesel production from
crude degummed soybean oil with crude glycerol co-product assigned a value of $0.15/
lb ($0.33/kg).
503
existence of governmental credits or subsidies. Based on Fig. 5, when
the feedstock cost is at US$ 0.52/kg (US$ 0.236/lb), the model
estimated a final biodiesel production cost of US$ 0.53/L ($ 2.00/gal).
It is clear that the cost of feedstock contributed the most, which
accounted 88% of the total production cost. In addition, the model also
estimated the process economic from the recovery of co-product,
glycerol as illustrated in Fig. 6. It was assumed that glycerol was sold
as low commercial grade glycerol with purity 80% w/w aqueous
solution. It can be noted that returns from selling glycerol was not
significant, only accounted to 6% reduction in the overall biodiesel
production cost.
In order to overcome this limitation, biodiesel manufacturer are
focusing their attention on using low-cost feedstock such as waste
cooking oil in order to ensure economic viability in biodiesel production.
Waste cooking oil is far less expensive than refined vegetable oils and
therefore has become a promising alternative feedstock to produce
biodiesel. In fact, generation of waste cooking oil in any country in the
world is huge, and may result to environmental contamination if no
proper disposal method is implemented. Table 3 shows the estimated
waste cooking oil produced in selected countries (Gui et al., 2008). Based
on the table, waste cooking oil generated is more than 15 million tonnes.
However, it should be noted that the actual amount of waste oil
produced is much higher based on global production. If such amount of
waste oil is successfully collected and converted to biodiesel, it will be
sufficient to meet the European biodiesel production target at 10 million
tonnes in year 2010 (Lam et al., 2009b).
Apart from that, a recent study on the production cost of biodiesel
using waste cooking oil as feedstock shows that the overall production
costs of biodiesel can be reduced by more than half compared to virgin
vegetable oil (Escobar et al., 2009). Furthermore, the production costs
are even lower than fossil derived diesel as illustrated in Fig. 7
(Escobar et al., 2009). Hence, the high cost of feedstock can be
overcome if waste cooking oil is used for biodiesel production.
5. Waste cooking oil
Currently, world oil and fats production is about 154 million
tonnes (MPOC, 2008). This figure refer to the production of 17 major
oils and fats, comprising from vegetable oils (i.e. soybean, cottonseed,
groundnut, sunflower, rapeseed, sesame, corn, olive, palm, palm
kernel, coconut, linseed, and castor) and animal fats/oils (i.e. butter,
lard, tallow, grease and fish oil). Most of this oil is used for deep-frying
processes, after which could cause disposal problem. Serious water
contamination may occur if no proper disposal method is implemen-
ted. Such scenario does not only contribute to pollution problems but
is also harmful to human beings. In fact, EU has enforced a ban on the
utilization of all waste oils as domestic animal feed because during
frying process, many harmful compounds are formed. Eventually,
Fig. 6. Impact of market value of 80% (w/w) glycerol on the unit cost of biodiesel
production with the soy oil feedstock assigned a value of $0.236/lb ($0.520/kg).
504 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518

Table 3 5.2. Oxidative reaction

Quantity of waste cooking oil produced in selected countries.

Country Quantity (million tonnes/year) Oxidative reaction occurs when oxygen in air dissolved in the oil or
fat and reacts mainly with unsaturated acyglycerols (AG) resulting in
China 4.5
European 0.7–1.0 the formation of various oxidation products. The main reactions
United States 10.0 involved in the oxidation reactions are summarized in Fig. 8 (Velasco
Japan 0.45–0.57 and Dobarganes, 2002). RH represents triacylglycerol undergoing
Malaysia 0.5
oxidation in one of its unsaturated fatty acyl groups. Initially, radicals —
Canada 0.12
Taiwan 0.07 alkyl radicals (R˙) are formed. By the addition of oxygen, eventually
alkylperoxyl radicals (ROO˙) are produced. Finally, alkoxyl radicals
(RO˙) are formed due to the decomposition of hydroperoxides
these harmful compounds will enter the human food chain during (ROOH) which produce various saturated and unsaturated alde-
meat consumption (Kulkarni and Dalai, 2006). hydes, ketones, hydrocarbons, lactones, alcohols, acids and esters.
Since frying improves the taste of food, it has become a common Most of these compounds will remain within the oil or fat, e.g.
method in food preparation. During frying, oil is heated under dimeric and polymeric acid, dimeric AG and polyglycerols as prod-
atmospheric condition at temperature of 160–190 °C (Gazmuri and ucts of the radical reactions and increase the viscosity of the cooking
Bouchon, 2009) for relative long period of time. In addition, the same oil. Others might be further decomposed through alkoxyradicals to
oil or fat is used several times, mainly because of economical reasons. volatile polar compounds, e.g. hydroxyl- and epoxyacids that escape
However, continuously using the same oil or fat for frying will causes from the oil (Cvengros and Cvengrosova, 2004).
various physical and chemical changes in the oil, depending on the
type of oil and oil composition. Some physical changes observed in
5.3. Hydrolytic reaction
vegetable oil after frying are (i) an increase in viscosity, (ii) an
increase in specific heat, (iii) a change in surface tension, and (iv) a
The hydrolysis of triglycerides occurs when steam produced
change in color (Cvengros and Cvengrosova, 2004).
during the preparation of food. Part of the water quickly evaporates,
Apart from that, oils are also subjected to three types of reactions
but a certain part dissolved in the oil or fat and induces its cleavage to
during frying, mainly thermolytic, oxidative and hydrolytic (Mittel-
give higher fatty acids, glecerol, monoglycerides and diglycerides
bach and Enzelsberger, 1999; Nawar, 1984). These three reactions will
concentration (Kulkarni and Dalai, 2006).
continuously cause the formation of many undesired and harmful
compounds if the oil is used repeatedly. The toxicological effects of
these compounds upon human consumption are still not completely 6. Catalysis in transesterification
known. However, if waste cooking oil is to be made feedstock for
biodiesel production, the amount of polar compound in the waste The following section describes various catalysis methods for
cooking oil, especially free fatty acid (FFA) must be taken into transesterification reaction of waste cooking oil and the potential of
consideration as it will greatly affect the transesterification reaction. heterogeneous acid catalysts and enzymes towards a more sustainable
Refined oil usually contains less than 0.5 wt.% FFA whereas for waste biodiesel industry. However, due to limited availability of information
cooking oil, FFA contents range between 0.5 and 15 wt.% (Gerhard regarding the transesterification of waste cooking oil for certain type of
Knothe and Krahl, 2004). catalysts; other oils with high FFA oils are taken to re-present waste
cooking oil so that the potential of these catalysts for transesterification
can be easier understood. Nonetheless, study that uses waste cooking oil
5.1. Thermolytic reaction as feedstock will be given priority when discussing various technologies.
Table 4 summarizes the reaction conditions for various types of catalysts
A thermolytic reaction occurs in the absence of oxygen at high
temperatures. A series of alkanes, alkenes, lower fatty acids,
symmetric ketones, oxopropyl esters, CO, and CO2 are produced
from the saturated fatty acids in the oil. For unsaturated fatty acids,
basically diametric compounds including dehydrodimers, saturated
dimers and polycyclic compounds are formed. In addition, dimers and
trimers may be formed when unsaturated fatty acids react with other
unsaturated fatty acids through Diels–Alder reaction (Kulkarni and
Dalai, 2006).

Fig. 7. Range of production costs for biodiesel and diesel in year of 2006. Fig. 8. Simplified mechanism for oil oxidation reaction during frying.
 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518 505

Table 4
Comparison of reaction conditions and performance for various types of catalysts used in transesterification of waste cooking oil.

Catalyst Reaction conditions Performance Comments Reference

Temperature, °C Type of alcohol Catalyst Reaction

(alcohol to oil loading, time, h
molar ratio) wt.%

Homogeneous base catalyst

NaOH 60 Methanol (7:1) 1.1 0.33 Yield = 88.8% Excess catalyst used Leung and Guo
led to soap formation (2006)
KOH 87 Methanol (9:1)a 6 2 Yield = 87%a – Demirbas (2009)

Homogeneous acid catalyst

H2SO4 95 Methanol (20:1) 4 20 Conversion = N 90% – Wang et al. (2006)
H2SO4 70 Methanol (245:1) 41.8a 4 Yield = 99% – Zheng et al. (2006)
H2SO4 65 Methanol (30:1) 1 69 Conversion = 99% – Freedman et al.
(1984)

Two-step: Acid catalyst follow by base catalyst

Ferric sulfate follow Acid: 95 Acid: Methanol (10:1) Acid: 2 Acid: 2 Conversion = 97% – Wang et al. (2006)
by KOH Base: 65 Base: Methanol (6:1) Base: 1 Base: 1
Ferric sulfate follow Acid: 100 Acid: Methanol (9:1) Acid: 2 Acid: 2 Yield = 96% – Patil et al. (2010)
by KOH Base: 100 Base: Methanol (7.5:1)a Base: 0.5a Base: 1
Ferric sulfate follow Acid: 60 Acid: Methanol (7:1) Acid: 0.4 Acid: 3 Yield = 81.3% – Wan Omar et al.
by CaO (2009)
Base: 60 Base: Methanol (7:1) Base: Not Base: 3
clearly
specified

Heterogeneous base catalyst

CaO Reflux methanol Methanol (12:1) 0.85 1 Yield = 66% Calcium soap was Kouzu et al. (2008a)
temperature: 60–65 detected
K3PO4 60 Methanol (6:1) 4 2 Yield = 97.3% – Guan et al., 2009
Oil palm ash 60 Methanol (18:1) 5.35 0.5 Yield = 71.7% – Chin et al. (2009)

Heterogeneous acid catalyst

WO3/ZrO2 75 Methanol to Oleic acid Not clearly 20 FFA Packed-bed reactor Park et al. (2008)
(19.4:1) specified Conversion = 85% was used
Zeolite Y (Y756) 460 Methanol (6:1) Not clearly 0.37 Yield = 26.6% – Brito et al. (2007)
specified
Carbon-based catalyst 80 Methanol (30:1) 10 8 Yield = 92% – Lou et al. (2008)
derived from starch
H3PW12O40·6H2O 65 Methanol (70:1) 3.7a 14 Yield = 87% 4 Å zeolite was used Cao et al. (2008)
(PW12) simultaneously
to adsorb water
Zr0.7H0.2PW12O40 65 Methanol (20:1) 2.1 8 Yield = 98.9% Nanostructured Zhang et al. (2009)
(ZrHPW) catalyst contain
double acid sites
ZS/Si 200 Methanol (18:1) 3 5 Yield = 98% – Jacobson et al.
(2008)
SO2−
4 /TiO2–SiO2 200 Methanol (9:1) 3 4 a
Yield = 90% a
– Peng et al. (2008)
SO2−
4 /SnO2–SiO2 150 Methanol (15:1) 3 3 Yield = 92.3% – Lam et al. (2009a)

Enzymatic catalyst
Pseudomonas cepacia 38.4 Ethanol (6.6:1) 13.7 2.47 Yield = 96% Second portion of lipase Wu et al. (1999)
(PS 30) SP 435 (5 wt %) was
added into the reaction
media after 1 h
Candida antarctica 30 Methanol (3:1) 4 50 Conversion = 90.4% Three-step batch Watanabe et al.
(Novozym 435) methanolysis (2001)
Novozym 435 30 Methanol (3:1) 4 50 Conversion = 90.9% Three-step continuous Watanabe et al.
methanolysis at different (2001)
flow rate
Novozym 435 40 Methanol (4:1) 4 12 Yield = 88 Tert-butanol was used as Halim and Harun
co-solvent Kamaruddin (2008)
Bacillus subtilis 40 Methanol (1:1) 3 72 Yield = 90 Methanol was added in Ying and Chen
encapsulated in two stepwise (2007)
magnetic particles
(Magnetic cell
biocatalyst)
Rhizopus oryzae 40 Methanol (4:1) 30 30 Yield = 88–90% Methanol was added in Chen et al. (2006)
three stepwise.
Immobilized Penicillium 35 Methanol (1:1) Not clearly 7 Yield = 92.8% Methanol was added in Li et al. (2009)
expansum on resin specified three stepwise.
D4020 Blue silica gel was used
to adsorb excess water.
a
Self-estimation.
506 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518

Table 5
Advantages and disadvantages of different types of catalysts used in transesterification of waste cooking oil.

Type of catalyst Advantages Disadvantages

Homogeneous base catalyst
Heterogeneous base catalyst
Homogeneous acid catalyst
Heterogeneous acid catalyst
Enzyme
•Very fast reaction rate — 4000 times faster than acid-catalyzed
transesterification
•Reaction can occur at mild reaction condition
and less energy intensive
•Catalysts such as NaOH and KOH are relatively
cheap and widely available
•Relatively faster reaction rate than acid-catalyzed
transesterification
•Reaction can occur at mild reaction condition and
less energy intensive
•Easy separation of catalyst from product
•High possibility to reuse and regenerate the catalyst
•Insensitive to FFA and water content in the oil
•Preferred-method if low-grade oil is used
•Esterification and transesterification occur simultaneously
Reaction can occur at mild reaction condition and less
energy intensive
•Insensitive to FFA and water content in the oil
•Preferred-method if low-grade oil is used
•Esterification and transesterification occur simultaneously
•Easy separation of catalyst from product
•High possibility to reuse and regenerate the catalyst
•Insensitive to FFA and water content in the oil
•Preferred-method if low-grade oil is used
•Transesterification can be carried out at low reaction
temperature, even lower than homogeneous base catalyst
•Only simple purification step is required
•Sensitive to FFA content in the oil
•Soap will formed if the FFA content in the oil is
more than 2 wt.%
•Too much soap formation will decrease the biodiesel yield and
cause problem during product purification especially generating
huge amount of wastewater
•Poisoning of the catalyst when exposed to ambient air
•Sensitive to FFA content in the oil due to its basicity property
•Soap will be formed if the FFA content in the oil is more
than 2 wt.%
•Too much soap formation will decrease the
biodiesel yield and cause problem during product
purification
•Leaching of catalyst active sites may result to product
contamination
•Very slow reaction rate
•Corrosive catalyst such as H2SO4 used can lead to corrosion on
reactor and pipelines
•Separation of catalyst from product is problematic
•Complicated catalyst synthesis procedures lead to higher cost
•Normally, high reaction temperature, high alcohol to oil molar
ratio and long reaction time are required.
•Energy intensive
•Leaching of catalyst active sites may result to product
contamination
•Very slow reaction rate, even slower than acid-catalyzed
transesterification
•High cost
•Sensitive to alcohol, typically methanol that can deactivate
the enzyme

in transesterification of waste cooking oil whereas Table 5 lists out their and base catalyst (KOH). This reaction is highly undesirable because it
advantages and disadvantages. will deactivate the catalyst from accelerating the transesterification
reaction. Furthermore, excessive soap in the products can drastically
6.1. Homogeneous base-catalyzed transesterification reduce the fatty acid methyl ester (FAME) yield and inhibit the
subsequent purification process of biodiesel, including glycerol
Currently, biodiesel is commonly produced using homogeneous separation and water washing (Nag, 2008, Kulkarni and Dalai, 2006).
base catalyst, such as sodium hydroxide (NaOH) or potassium
hydroxide (KOH) (Felizardo et al., 2006, Kulkarni and Dalai, 2006).
These catalysts are commonly used in the industries due to several
reasons: (i) able to catalyze reaction at low reaction temperature and
atmospheric pressure; (ii) high conversion can be achieved in a
minimal time, (iii) widely available and economical (Lotero et al., ð2Þ
2005). In fact, it was reported that the rate for base-catalyzed reaction
would be 4000 times faster compared to acidic catalyst (Fukuda et al., Apart from that, high water content in waste cooking oil also
2001, Kulkarni and Dalai, 2006). However, the use of this catalyst is affects the methyl ester yield. When water is present, particularly at
limited only for refined vegetable oil with less than 0.5 wt.% FFA high temperatures, it can hydrolyze triglycerides to diglycerides and
(Wang et al., 2006) or acid value less than 1 mg KOH/g (Felizardo et form free fatty acid. Eq. (3) shows the hydrolysis reaction. With the
al., 2006). Some researchers reported that base catalyst can tolerate presence of base catalyst, the FFA will subsequently react to form soap
higher content of FFA as shown in Table 6. Nevertheless, it is clear that as shown in Eq. (2). Thus, when water is present in the reactant, it
the FFA content in oil feedstock should be as low as possible (ranging
from less than 0.5 wt.% to less than 2 wt.%) for base-catalyzed
transesterification reaction. Thus, if waste cooking oil with an average Table 6
Level of FFA recommended for homogeneous base catalyst transesterification.
FFA content more than 6 wt.%, base catalyst is definitely not suitable
to be used (Lotero et al., 2005). Author/reference Recommended FFA (wt.%)
FFA consists of long carbon chain that is disconnected from Ma and Hanna (1999) b1
glycerol backbone. They are sometimes called carboxylic acids. If an Ramadhas et al. (2005) ≤2
oil or fat containing high FFA such as oleic acid is used to produce Zhang et al. (2003a) b 0.5
biodiesel, alkali catalyst will typically react with FFA to form soap, Freedman et al. (1984) b1
Kumar Tiwari et al. (2007) b1
which is highly undesirable (Nag, 2008, Yan et al., 2009, Kulkarni and
Sahoo et al. (2007) ≤2
Dalai, 2006). Eq. (2) shows a typical reaction between FFA (oleic acid)
 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518 507

generally manifests itself through excessive soap production. Apart

from that, the soaps of saturated fatty acids tend to solidify at ambient
temperatures and thus a reaction mixture with excessive soap may
gel-up and form a semi-solid mass which is very difficult to recover
(Felizardo et al., 2006).

ð3Þ

6.2. Homogeneous acid-catalyzed transesterification

Since liquid base-catalyzed transesterification process poses a lot

of problems especially for oil or fat with high FFAs concentration,
liquid acid catalysts are proposed in order to overcome the
limitations. To date, the most investigated catalysts for acid-catalyzed
system are sulfuric acid (H2SO4) and hydrochloric acid (HCl). Acid-
catalyzed transesterification holds an important advantage with
respect to base-catalyzed process: acid catalyst is insensitive to the
presence of FFAs in the feedstock (Kulkarni and Dalai, 2006) and can
catalyzes esterification and transesterification simultaneously (Jacob-
son et al., 2008). Esterification is a chemical reaction in which two
reactants, typically an alcohol (e.g. methanol) and an acid (e.g. FFA)
react to form an ester as the reaction product. It was reported that acid
catalysis is more efficient when the amount of FFA in the oil exceeds
1 wt.% (Zhang et al., 2003a, Canakci and Van Gerpen, 1999, Freedman
et al., 1984). In addition, economic analysis has proven that acid-
catalyzed procedure, being a one-step process, is more economical
than the base-catalyzed process which requires an extra step to
convert FFA to methyl esters (Zhang et al., 2003a, b).
However, acid-catalyzed system is not a popular choice for
commercial applications due to slower reaction rate, requirement of
high reaction temperature, high molar ratio of alcohol to oil,
separation of the catalyst, serious environmental and corrosion
related problem (Jacobson et al., 2008, Wang et al., 2006). In a study
of acid-catalyzed transesterification of waste cooking oil using H2SO4,
Wang et al. reported that the yield of FAME increased with longer
reaction time, higher methanol to oil ratio and higher catalyst loading.
The conversion of waste cooking oil was more than 90% at a reaction
time of 10 h with ratio of methanol to oil at 20:1 and 4 wt.% H2SO4
(with reference to weight of oil) (Wang et al., 2006). In another study,
Freedman et al. reported 99% oil conversion by using 1 mol% of H2SO4
and methanol to oil ratio 30:1 for 69 h reaction time (Freedman et al.,
1984). These data indicates that acid-catalyzed transesterification
process requires more severe reaction conditions (such as longer
reaction time) than base-catalyzed reaction.
Lotero et al. investigated the reasons for the low activity of acid-
catalyzed compared to base-catalyzed transesterification reaction. Fig. 9
shows the mechanism of acid-catalyzed reaction (Lotero et al., 2005).
From the figure, it can be seen that the protonation of carbonyl group is
the key step in the catalyst–reactant interaction. However, this initial
chemical pathway has in turn increases the electrophilicity of the
adjoining carbon atom, resulting in the intermediate molecules
susceptible to nucleophilic attack. In contrast, the base catalysis takes
on a more direct route, at which alkoxide ion is created initially and
directly acts as a strong nucleophile. Fig. 10 shows the mechanism of
base-catalyzed reaction (Lotero et al., 2005). This crucial different
Fig. 9. Homogeneous acid-catalyzed reaction mechanism for the transesterification of
triglycerides: (1) protonation of the carbonyl group by the acid catalyst; (2)
nucleophilic attraction of the alcohol, forming a tetrahedral intermediate; (3) proton
migration and breakdown of the intermediate. The sequence is repeated twice for R2
and R3.
pathway (formation of electrophilic species by acid catalysis versus
formation of stronger nucleophile by base catalysis) is ultimately
responsible to the difference in catalytic activity in the transesterifica-
tion reaction.
6.3. Homogeneous acid and base-catalyzed transesterification: two steps
Since homogeneous acid and base catalysts have their own
advantages and limitations, some studies have attempted to use a
combination of both catalysts to synthesis biodiesel from oil contain-
ing high FFAs. Initially, acid catalyst was employed to convert FFAs to
ester through esterification. When the FFAs content in the oil drops to
lower than 0.5–1 wt.%, transesterification of the oil can then be
performed by using a base catalyst.
Canakci and Van Cerpen have developed a pilot plant to produce
biodiesel from feedstock with high FFAs content via two steps
method; esterification and transesterification (Canakci and Van
Gerpen, 2003). The feedstock was first treated with H2SO4 to reduce
the level of FFAs to below 1 wt.%, followed by transesterification
process catalyzed by homogeneous base KOH. Although high FAME
yield can be obtained, but the rate of FFA esterification reaction was
relatively very slow. Thus, higher amount of acid catalyst are required
to accelerate the rate of reaction. The drawback of this two-step
process is even more pronounced due to the requirement of extra
separation steps to remove the catalyst in both stages. Although
problem of catalyst removal from the first stage can be avoided by
using base catalyst from the second stage through neutralization
508 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
Fig. 10. Homogeneous base-catalyzed mechanism for the transesterification of triglycer-
ides: (1) production of the active species, RO−; (2) nucleophilic attack of RO− to carbonyl
group on triglycerides, forming a tetrahedral intermediate; (3) intermediate breakdown;
(4) regeneration of the RO− active species. The sequence is repeated twice for R2 and R3.
process, the use of extra base catalyst will add to the cost of biodiesel
production (Kulkarni and Dalai, 2006).
6.4. Heterogeneous base-catalyzed transesterification
To date, many solid base catalysts have been developed for
biodiesel production, such as basic zeolites, alkaline earth metal
oxides and hydrotalcites. On top of that, alkaline earth metal oxides
especially calcium oxide, CaO have attracted much attention due to
their relatively high basic strength, low solubility in methanol and can
be synthesized from cheap sources like limestone and calcium
hydroxide (Zabeti et al., 2009). Kouzu et al. (2008a) reported that
CaO obtained from calcinations of pulverized limestone, CaCO3 at
900 °C for 1.5 h in the flow of helium gas exhibited substantially good
result in transesterification of refined soybean oil. The yield of FAME
was 93% after 1 h reaction time at methanol reflux temperature and
methanol to oil ratio 12:1. Fig. 11 shows the mechanism of CaO as
heterogeneous base catalyst in transesterification. However, the yield
of FAME dropped to 66% when waste cooking oil with FFA content
2.6 wt.% was used under the same reaction condition. It is obvious that
the basic sites of CaO were poisoned by strong adsorption of FFAs on
the surface of the catalyst (Kouzu et al., 2008a). Consequently, a
portion of the catalyst changed into calcium soap by reacting with the
FFAs adsorbed, resulting in low recovery of catalyst. Kouzu et al.
further reported that the concentration of Ca in reaction product was
Fig. 11. CaO as heterogeneous base catalyst mechanism in transesterification of
triglycerides: (1) abstraction of proton from methanol by the basic sites to form
methoxide anion; (2) methoxide anion attacks carbonyl carbon in a molecule of the
triglyceride leading to the formation of alkoxycarbonyl intermediate; (3) alkoxycarbo-
nyl intermediate further transformed into a more stable form: FAME and anion of
diglyceride; (4) methoxide cation attracts the anion of diglyceride leading to the
formation of diglyceride. The sequence is repeated twice for R2 and R3.
3065 ppm which exceeded the basic standard of biodiesel, the
concentration of mineral matter should be below 200 ppm (Kouzu
et al., 2008a).
In addition, some researchers also pointed out that soluble
substance from CaO can leached out during transesterification.
Gryglewicz (1999) stated in his paper that calcium oxide slightly
dissolves in methanol. Lopez et al. conducted transesterification of
sunflower oil with methanol in which a slight amount of calcium
oxide was found to dissolved in the reaction product (Granados et al.,
2007). Kouzu et al. (2008b) further identified the soluble substance as
calcium diglyceroxide in which CaO reacted with glycerol during
transesterification of soybean oil with methanol. Thus, an extra
purification step is needed such as ion-exchange resin to remove the
soluble content in the biodiesel (Kouzu et al., 2009).
Apart from that, Granados et al. (2007) used activated CaO as a
solid base catalyst in the transesterification of sunflower oil to
investigate the role of water and carbon dioxide on the deterioration
of the catalytic performance upon contact with air for different period
of time. The study showed that CaO was rapidly hydrated and
carbonated in the air. No calcium oxide peak was detected in the
samples after exposed to air for more than 20 days. It was further
reported that the active sites of CaO were poisoned due to
chemisorption of carbon dioxide and water on the surface sites to
form carbonates and hydroxyl groups, respectively. However, the
catalytic activity of CaO can be regenerated if CaO is subjected to an
 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
activation treatment at 700 °C in order to remove the main poisoning
species (the carbonate groups) from the surface. However, leaching of
the catalyst was still observed in the transesterification reaction
although prior thermal treatment was employed.
Besides, magnesium oxide (MgO) which is produced by direct
heating of magnesium carbonate or magnesium hydroxide was also
investigated on its catalytic activity in transesterification. Di Serio et al.
(2006) reported that MgO was efficient in transesterification of soybean
oil, however high reaction temperature (180 °C) is required. At low
reaction temperature (100 °C), MgO catalyst exhibited very low
catalytic performance since the FAME yield observed was less than
20%. This result was in agreement with Cantrell et al. (2005) and
Gryglewicz (1999), who reported low or even no activity when using
MgO in transesterification reactions performed at 60 °C. In fact, MgO has
the weakest basic strength among group II oxides, e.g. CaO and
strontium oxide (SrO) (Kouzu et al., 2008a). Nevertheless, mixed
magnesium–alumina (Mg–Al) oxide prepared by using hydrotalcites
(Mg6Al2(OH)16CO34H2O) as precursor and calcined at high temperature
has manifested the basic sites of the catalyst (Xie et al., 2006). More than
90% of FAME yield was observed when using Mg–Al oxide as catalyst but
high reaction temperature is still required (Di Serio et al., 2006). Besides
that, high concentration of Mg and Al ions were found to leach out from
the catalyst, resulting in the requirement of extra purification step (Oku
et al., 2005). However, a more in-depth study on using Mg–Al in
transesterification of high FFAs oil is required since the effect of FFAs
content towards the catalyst performance is still unknown.
6.5. Heterogeneous acid-catalyzed transesterification
Currently, biodiesel research is focused on exploring new and
sustainable solid acid catalysts for transesterification reaction. In
addition, it is believed that solid acid catalysts have the strong
potential to replace liquid acid catalyst (Jacobson et al., 2008). The
advantages of using solid acid catalyst are (1) they are insensitive to
FFA content, (2) esterification and transesterification occurs simulta-
neously (Kulkarni and Dalai, 2006), (3) eliminate the washing step of
biodiesel (Jitputti et al., 2006), (4) easy separation of the catalyst from
the reaction medium, resulting in lower product contamination level,
(5) easy regeneration and recycling of catalyst and (6) reduce
corrosion problem, even with the presence of acid species (Suarez
et al., 2007). In fact, the development of heterogeneous catalyst
system holds an important factor to be incorporated into a continuous
flow reactor (Lotero et al., 2005). Such continuous process can
minimize product separation and purification costs, making it
economically viable and able to compete with commercial petro-
leum-based diesel fuel (de Almeida et al., 2008).
The ideal solid acid catalyst for transesterification reaction should
have characteristics such as an interconnected system of large pores, a
moderate to high concentration of strong acid sites, and a hydropho-
bic surface (Kulkarni and Dalai, 2006). However, research on direct
use of solid acid catalyst for biodiesel production has not been widely
explored due to its limitation of slow reaction rate and possible
undesirable side reactions. Furthermore, there is a knowledge gap on
the fundamental studies dealing with reaction pathway of triglycer-
ides on solid acids. The following section will give an overview of
various solid acid catalysts reported so far for biodiesel production.
6.5.1. Zirconium oxide (ZrO2)
There have been several studies on the usage of zirconium oxide
(ZrO2) as a solid acid catalyst for transesterification of different
feedstock due to its strong surface acidity. The acidity property can
even be enhanced by coating the surface of this metal oxide with
anions like sulfate and tungstate. This can be done by impregnating
ZrO2 with acidic solution such as sulfuric acid (H2SO4) to become
sulfated zirconia, SO2−
4 /ZrO2 (Miao and Gao, 1997). Jitputti et al.
(2006) reported that SO2− 4 /ZrO2 can gives promising results in
509
transesterification of palm kernel oil and crude coconut oil with
methyl ester yield reaching as high as 90.3% and 86.3%, respectively.
However, when unsulfated ZrO2 was used as catalyst instead of SO2− 4 /
ZrO2, only 64.5% (palm kernel oil) and 49.3% (crude coconut oil) of
methyl ester yield were attained, respectively. This eventually
indicates that modification of metal oxide surface acidity is the key
factor in obtaining high conversion of triglycerides.
Apart from that, combination of alumina, Al2O3 with ZrO2 and
modification of ZrO2–Al2O3 with tungsten oxide (WO3) not only
provides high mechanical strength but also enhances the acidity of the
catalyst (Jacobson et al., 2008). The addition of Al2O3 further stabilizes
the tetragonal phase of ZrO2 support and also prevents the growth of
WO3 particles. Furuta et al. (2004a) evaluated the performance of
tungstated zirconia–alumina (WZA) and sulfated zirconia–alumina
(SZA) in the transesterification of soybean oil with methanol at 200–
300 °C using a fixed bed reactor under atmospheric pressure. WZA
was found to have a higher activity in transesterification as compared
to SZA. However, the authors did not elaborate much on the causes of
the improved activity of WZA catalyst. Nevertheless, high reaction
temperature (250 °C) and long reaction time (20 h) were needed in
order to achieve 90% conversion. Similar results was also reported by
Jacobson et al. where by catalyst was prepared by impregnating 10 wt.
% of WO3 on to ZrO2–Al2O3. Relatively low ester yield was obtained
(65%) when the transesterification reaction was carried out at lower
reaction temperature of 200 °C and shorter reaction time of 10 h
(Jacobson et al., 2008). On the other hand, Faria et al. (2009) proposed
a reaction mechanism for ZrO2 supported on SiO2 in transesterifica-
tion of soybean oil, as shown in Fig. 12. Thus, a more detail
understanding on reaction pathway by ZrO2 catalyst in transester-
ification can be obtained.
Application of ZrO2 in esterification of waste cooking oil was also
reported by Park et al. (2008). In their study, WO3 was incorporated
into ZrO2 rather than impregnating with H2SO4. It was found that
WO3/ZrO2 has higher stability than SO2− 4 /ZrO2, and therefore avoiding
the leaching of acid sites into the reaction media. Even if WO3 leached
into the reaction media, it does not contaminate the product (Park et
al., 2008, Park et al., 2010). From the study, it was found that 85% of
FFA conversion was attained in a packed-bed reactor after 20 h of
reaction time at 75 °C, but decrease to 65% and remained stable
thereafter. The reason given was due to the oxidation of WO3 after
long term exposure to FFA (a reducing agent) that resulted to a
decrease in catalytic activity. Therefore, leaching of WO3 was rule out
as the main reason for catalyst deactivation. In addition, WO3/ZrO2
could be simply regenerated by air re-calcination. However, further
study on catalyst and process optimization and also the oxidation
state of WO3 are still required.
Despite the high acidity of SO2− 4 /ZrO2, however it is known to
suffer significant deactivation during liquid-phase transesterification,
possibly due to sulfate leaching (Omota et al., 2003). Catalyst leaching
was tested by dissolving fresh SO2− 4 /ZrO2 catalyst with water. The pH
of the suspension was found to decrease quickly as a result of sulfate
groups hydrolyzed to become H2SO4 and HSO− 4 . This will then cause
the transesterification to occur via homogeneous acid catalysis and
therefore interfere with the measurements of heterogeneous catalytic
activity. Recently, a new preparation method for SO2− 4 /ZrO2 was
proposed by using chlorosulfonic acid, HSO3Cl instead of the
conventional impregnation of H2SO4 (Yadav and Murkute, 2004).
The prepared SO2− 4 /ZrO2 exhibited higher catalytic activity in
transesterification and no sulfate leaching was observed. However,
HSO3Cl is a very hazardous chemicals, even an exposure for a very
short time can cause death or major fatal injury (Kapias and Griffiths,
2001).
6.5.2. Titanium oxide (TiO2)
Titanium dioxide (TiO2) is among transition metal oxides that
have attracted attention for biodiesel production due to their acidic
510 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
Fig. 12. Proposed catalytic cycle for SiO2/ZrO2 in transesterification.
properties. In addition, introduction of sulfuric group on the surface of
TiO2 will enhance the acid strength of the catalyst. However, there are
still very limited study reported in the literature describing the
application of such catalyst for transesterification of vegetable oils (de
Almeida et al., 2008). Recently, Chen et al. (2007) had evaluated the
catalytic activity of SO2− 2−
4 /TiO2 and SO4 /ZrO2 for transesterification of
cotton seed oil high in FFAs content to FAME. It was interesting to find
that the activity of this catalyst is proportional to its specific surface
area. SO2− 2
4 /TiO2 with a specific surface area of 99.5 m /g can achieved
a higher yield of 90% as compared to SO2− 4 /ZrO2 with a specific surface
area of 91.5 m2/g, which can only achieved a yield of 85%. However,
this catalyst requires high reaction temperature (230 °C), a negative
factor for industrial application. When lower reaction temperature
(120 °C) and reaction time (1 h) were used, the FAME yield obtained
was only 40% (de Almeida et al., 2008).
It was proposed that the reactivity of SO2−4 /TiO2 can be increased by
introducing a secondary metal, SiO2 to produce SO2− 4 /TiO2–SiO2 (Peng
et al., 2008). By adding SiO2 to SO2−
4 /TiO2, the specific surface area of the
catalyst increased to 258 m2/g with an average pore diameter of
10.8 nm. The synthesized SO2− 4 /TiO2–SiO2 was then subjected to
transesterification of refined cotton seed oil blended with 50% oleic
acid. The optimum FAME yield obtained was more than 90% at reaction
temperature 200 °C, methanol to oil ratio 9:1, catalyst loading 3 wt.%
and a shorter reaction time of 3 h. However, the reaction temperature is
still considered very high if compared to homogeneous catalyst which
lies between 60 and 100 °C. Apart from that, more in-depth research
especially on the leaching characteristic of this catalyst must be carried
out to justify its potential for industrial biodiesel production.
6.5.3. Tin oxide (SnO2)
Tin oxide, SnO2 is n-type semiconductor material with a wide band
gap (∼ 3.6 eV); it is transparent to visible light and reflects infrared
light (Gutierrez-Baez et al., 2004). This optical and electrical
properties make SnO2 an excellent material for several applications
in catalysis, conductivity, gas sensing, ceramics, plastics and biomed-
icine (Toledo-Antonio et al., 2003). Normally, SnO2 with mesostruc-
ture form can be obtained by hydrolyzing inorganic precursor such as
meta-stannic acid (SnO·H2O) (Furuta et al., 2004a) in the presence of
cationic, anionic and neutral surfactants as structure director
(Gutierrez-Baez et al., 2004). However, most of SnO2 mesostructure
is not stable and collapsed during calcination. Thus, in order to
increase the mesostructure stability, anionic surfactant with phos-
phate or sulfate has been proposed such as the synthesis of
mesoporous sulfated tin oxide, SO2− 4 /SnO2 (Gutierrez-Baez et al.,
2004). The phosphate or sulfate groups will then remain bounded to
the surface of SnO2, which will further stabilized its mesostructure
walls and increasing its thermal stability.
Apart from SO2− 2−
4 /ZrO2, SO4 /SnO2 is another solid super acid
catalyst that has potential in transesterification reaction. Results from
temperature programmed desorption (TPD) of ammonia and adsorp-
tion heat of argon (Ar) indicates that the acid strength of SO2− 4 /SnO2
was higher than that of SO2− 2−
4 /ZrO2. Consequently, SO4 /SnO2 have
shown superior catalytic activity than SO2− 4 /ZrO2 in esterification of n-
octanoic acid with methanol at temperature below 150 °C (Furuta et
al., 2004b). However, report concerning the application of SO2− 4 /SnO2
in transesterification reaction is still very limited, typically for oil or fat
containing high FFA. In addition, among the few studies reported in
the literature, the findings reported on its catalytic performance in
transesterification reaction have not been conclusive. The limited
study carried out on SO2− 4 /SnO2 may be due to its complicated
preparation procedure particularly owing to the difficulty in obtaining
oxide gels from its salt as compared to SO2− 4 /ZrO2 that can be easily
prepared (Khder et al., 2008, Matsuhashi et al., 2001). Therefore, a
more widespread testing of SO2− 4 /SnO2 should be carried out to obtain
more data and information for this promising material as potential
solid catalyst for transesterification reaction.
 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
6.5.4. Zeolites
Zeolites are microporous crystalline solids which contain silicon
(Si), aluminium (Al) and oxygen in their framework. One of the
common applications of zeolite (inorganic solid catalyst) is for the
production of organic compounds such as ester (Balaji and Chanda,
1998). This is because the characteristic of zeolite can be tailored
made to suit its function. For instance, zeolite can be synthesized with
different crystal structures, pore sizes, Si/Al ratios and proton-
exchange levels (Lotero et al., 2005). Therefore, the acid strength of
the catalyst can be controlled by changing the aluminosilicate
framework such that it fits specific reaction requirements (Corma
and Garcia, 1997). However, it should be noted that low surface
acidity may caused slow reaction rate whereas extremely high acidity
may cause deactivation due to coking or possible formation of
undesirable by-products. Apart from that, zeolite also has another key
feature. Since zeolite with specific pore structure and surface
hydrophobicity can be tailored made according to substrate's size
and polarity (Lotero et al., 2005), therefore only molecules with
appropriate dimension are allowed to enter the zeolite cavity and
diffusing through the pores (Xavier et al., 2009).
Although zeolite has numerous advantages over other heteroge-
neous catalysts, however its catalytic activity in transesterification
reactions is relatively low. This is mainly due to diffusion limitation of
bulky reactants (triglycerides) into the microporous structure of
zeolite (Kiss et al., 2006). Triglycerides with an average molecular size
of 2 nm suffered mass transfer resistance in the zeolite's micropore
(1–2 nm). Therefore, it is believed that transesterification reaction
only occurs on the external surface of zeolite crystal. In order to
overcome this limitation, the pore size and structure of zeolite must
be adjusted by varying the Si/Al ratio. Generally, higher Si/Al ratio
resulted in zeolite with larger-pore size (Okuhara, 2002) but with
weaker acidic strength (Chung et al., 2008). Thus, although zeolite
with larger-pore size may overcome the diffusion limitation problem
but the reaction rate is still rather slow due to low acidity strength.
Brito et al. (2007) reported the application of different zeolite Y
catalysts in transesterification of waste cooking oil. Several types of
zeolite Y with different concentration of Al2O3 and Na2O were utilized
in the reaction, however, the catalysts were found to give poor
performance. The highest biodiesel yield attained was only 26.6%
although the reaction was carried out at high reaction temperature of
460 °C, methanol to oil molar ratio of 6 and reaction time of 22 min.
Similar reports on the low activity of zeolite catalyst when used in
transesterification were also reported in recent publications (Shu et
al., 2007, Kiss et al., 2006, Okuhara, 2002).
6.5.5. Sulfonic ion-exchange resin
Ion-exchange resins are insoluble macroporous polymer that is
capable to exchange specific ions within the polymer itself with other
ions in a solution or reaction media. Normally, sulfonic ion-exchange
resins are co-polymers of divinylbenzene (DVB), styrene and sulfonic
acid groups (as the active sites-Brønsted acidity) (Özbay et al., 2008).
The polymer structure of the resin is mainly characterized by the
composition of the cross-linking component (normally DVB), which
will then determine its surface area and pore size distribution
(Pääkkönen and Krause, 2003). Besides that, their catalytic activity
is also strongly dependent on their swelling properties because the
swelling capacity limits reactant accessibility to the acid sites and thus
affects their overall activity (Feng et al., 2010). Common types of
acidic ion-exchange resin are such as Amberlyst-15, Amberlyst-35
and Nafion SAC-13. These catalysts were reported to give good
performance in FFA esterification, but, weak in transesterification
(Chen et al., 1999; Vicente et al., 1998).
Application of Amberlyst-15 with acidic functional groups has
exhibited excellent catalytic activity in esterification reaction. Kiss et
al. tested the activity of several solid acid catalysts in the esterification
of dodecanoic acid with 2-ethylhexanol at 150 °C. Amberlyst-15 was
511
found to require the least reaction time to achieve 90% conversion
compared to sulfated zirconia and Nafion-NR50. Similar good results
in esterification by using acidic Amberlyst-15 were also reported by
Heidekum et al. (1999) and Chen et al. (1999). However, Amberlyst-
15 was found to give low performance in transesterification reaction.
At a relative low reaction temperature (60 °C), the conversion of
sunflower oil to FAME was reported to be only 0.7%, using the
following reaction conditions: atmospheric pressure for 8 h reaction
time and 6:1 methanol to oil molar ratio (Vicente et al., 1998). In
another study, Dos Reis et al. reported the transesterification of
Babassu coconut oil using Amberlyst-15 (Dos Reis et al., 2005). It was
reported that a rather good triglycerides conversion of 80% can be
achieved only if the methanol to oil ratio used is increased to 100:1.
The reaction temperature and time are at 60 °C and 8 h respectively.
This finding was rather expected as the activity of solid acid catalysts
in transesterification is normally low at low reaction temperature.
Consequently, if Amberlyst-15 is to be used, it is necessary to increase
the reaction temperature to 150–200 °C to obtain sufficiently fast
reaction rate. However, most ion-exchange resins such as Amberlyst-
15 have low thermal stability and become unstable at temperature
above 140 °C (Lotero et al., 2005). Thus, this problem certainly limits
their application to reactions that require high temperatures. Study
regarding the deactivation of polystyrene sulfonic acid resins in
esterification of high FFAs oils at a higher reaction temperature was
reported and discussed extensively by Tesser et al. (2005).
6.5.6. Sulfonic modified mesostructure silica
Mesostructure materials such as silica have an exceptional
potential to be utilized as heterogeneous acid catalyst in biodiesel
production. This mesoporous materials (silica) consist of large
mesopores in which can significantly minimize the diffusion problem
for reactants to access into the active sites of the catalyst (Mbaraka
and Shanks, 2006). Apart from that, the physical and chemical
properties of these mesoporous materials can be manipulated by
incorporating suitable organic or inorganic functional groups into the
mesoporous silica matrix (Mbaraka et al., 2006). For instance, in order
to obtain a solid acid catalyst, organosulfonic groups can be
incorporated onto the mesoporous silica material as shown in
Fig. 13 (Melero et al.). The organosulfonic acid anchored on
mesoporous silica acts as Brønsted acid (active sites) that is suitable
to catalyze esterification and transesterification reactions. Mesos-
tructure silica materials functionalized with propylsulfonic acid
(SO3H) groups have been reported to have good catalytic activity in
esterification of refined and unrefined oil (Melero et al.; Melero et al.,
2009).
To date, application of sulfonated silica in transesterification of
waste cooking oil is still limited. Most of the recent studies reported
focused on transesterification of refined oil rather than oil with high
FFA and waste cooking oil. Mbaraka et al. (2006) reported the
utilization of acidic mesoporous silica in esterification of beef tallow.
From the study, it was found that propylsulfonic acid-functionalized
mesoporous silica (SBA-15-SO3H) has an excellent catalytic activity in
esterification. Nearly 95% conversion of FFA from beef tallow was
attained at relatively short reaction time of 30 min, at reaction
temperature of 120 °C and methanol to FFA ratio of 20. Unfortunately,
the catalytic activity of SBA-15-SO3H reduced drastically for the
subsequent reaction cycles due to accumulation of organic or
carbonaceous matter on the catalyst surface that blocks the acidic
sites. However, leaching of the active sites was rule out as the reason
for catalyst deactivation since the authors overcome this problem by
incorporating hydrophobic agent (propyltrimethoxysilane) onto SBA-
15-SO3H in order to increase its hydrophobicity. This bi-functionized
(acidic and hydrophobic) mesostructure silica would have lower
pores polarity, thereby reducing the interaction between polar
impurities (contained in the beef tallow) with the sulfonic acid
groups. Results showed that high FFA conversion (84%) was
512 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
Fig. 13. Organosulfonic acid-functionalized mesoporous silica.
maintained for the second cycle of reaction. However, in-depth
research and analysis work using sulfonic modified silica in
transesterification reaction (typically from high free fatty acid oil)
should be carried out to justify the potential of this solid acid catalyst.
6.5.7. Sulfonated carbon-based catalyst
Carbon-based catalyst is classified as a type of sugar catalyst, in which
sugar, starch or cellulose is carbonized incompletely at temperature
below 500 °C (Hara, 2009). The incomplete carbonized carbon is then
immersed in concentrated sulfuric acid, H2SO4 (purity N96%) and heated
to 150 °C for 15 h under the flow of N2 (Lou et al., 2008, Takagaki et al.,
2006, Nakajima et al., 2007). The resulting catalyst was claimed to be the
first of its kind and is called as sulfonated carbon-based catalyst which
comprise high density functional group such as sulfonic group (SO3H)
and carboxyl group (COOH) (Hara, 2009). The structure of a typical
carbon-based catalyst derived from D-glucose is schematically shown in
Fig. 14 (Takagaki et al., 2006). The minimum unit in this material is a
nanographene sheet (ca. 1 nm) comprised of 10–20 carbon six-
membered rings (Hara, 2009). In addition, the sulfonated carbon
material has a high Hammett acid strength (H0) of −8 to −11, which
is almost comparable to concentrated H2SO4 (Okamura et al., 2006).
Furthermore, leaching of SO3H group was not observed in the
esterification of high free fatty acid oil (Takagaki et al., 2006). Such
finding not only open up a new route to minimize extensively the usage
of H2SO4 in the industries, but also lead to a greener approach for
Fig. 14. Proposed schematic structure of the carbon materials prepared from D-glucose.
The materials are amorphous carbons consisting of polycyclic aromatic carbon sheets
with phenolic OH and COOH groups in addition to SO3H groups.
producing acid-based catalyst. However, it should be noted that the
carbon-based catalyst could not be prepared by sulfonation of an
incomplete carbonized resin, amorphous glassy carbon, activated carbon
or natural graphite (Hara, 2009). Heating these carbon materials with
H2SO4 will only result to low density SO3H groups and do not function as
a solid acid catalyst.
Up to date, there are only minimum study that reported on the use
of carbon-based material for biodiesel production, typically from
waste cooking oil. In a recent study reported by Lou et al., various
sulfonated carbon-based catalysts were derived, mainly from starch,
cellulose, sucrose and D-glucose (Lou et al., 2008). It was found that
carbon-based catalyst derived from starch has a relatively larger-pore
volume (0.81 cm3/g) and pore size (8.2 nm) allowing better reactants
access to the SO3H sites. Apart from that, starch derived carbon-based
catalyst also have a relatively higher total acid sites (1.97 mmol/g)
and higher sulfur content (5.9 wt.%). In addition, high FAME yield
(92%) was attained from transesterification of waste cooking oil at
reaction temperature of 80 °C, methanol to oil molar ratio of 30,
catalyst loading of 10 wt.% (referred to weight of oil) and reaction
time of 8 h. Furthermore, the catalyst was found to be stable even after
fifty cycles of repeated reaction. Nevertheless, this catalyst must still
be further improved such as proper optimization work on the catalyst
preparation as well as transesterification reaction conditions.
6.5.8. Heteropolyacids (HPAs)
Heteropolyacids (HPAs) catalyst has attracted researcher's atten-
tion recently due to their excellent water tolerant ability, poses strong
Brønsted acidity (stronger than conventional homogeneous acid,
H2SO4) and high catalytic activity and stability (Sivasamy et al., 2009,
Narasimharao et al., 2007). Typical HPAs which are easily available are
H3PW12O40, H4SiW12O40, H3PMo12O40 and H4SiMo12O40 (Zhang et al.,
2010). In addition, adding appropriate ratio of salt (Cs+, NH+ 4 and
Ag+) to HPAs will dramatically increase its surface area and allow
easier accessibility of reactant to its active sites (Narasimharao et
al., 2007). However, it should be noted that HPAs can be slightly
soluble in the reaction media and resulting to homogeneous
reaction, in which contributed to the overall reaction rate (Sivasamy
et al., 2009). Therefore, leaching of active sites may also easily occur
and cause serious catalyst deactivation.
Application of HPAs in biodiesel synthesis from waste cooking oil
was reported by Cao et al. (2008). H3PW12O40·6H2O (PW12) was used
as the HPAs catalyst and the waste cooking oil contained high amount
of FFA (15.65%) and water content (0.1%). Optimum yield of 87%%
biodiesel was attained at reaction temperature of 65 °C, methanol to
oil molar ratio of 70:1 and 14 h of reaction time. At the same time, 4 Å
zeolite was introduced into the reaction media as an adsorbent to
adsorb water. Although PW12 has a high tolerance towards FFA
content and is stable even after 5 reaction cycles, however, relatively
high methanol to oil molar ratio and long reaction time may restrict
the application of this catalyst in industrial scale. Apart from that, the
catalyst was found not stable when reaction temperature was
 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
increased more than 60 °C. The authors claimed that this observation
is due to the nature of waste cooking oil that contains many
undesirable compounds that will cause side reactions when carrying
out transesterification reaction at higher temperature.
On the other hand, attempt was made to synthesize HPA with
higher acidity by introducing Lewis acid into HPA, making it contains
both Brønsted and Lewis acid sites. This was done by loading Brønsted
acidic HPAs on Lewis acidic supporters, such as ZrO2, TiO2 or Ta2O5
(Zhang et al., 2009). Proper coordination of a Lewis acid to a Brønsted
acid could enhance its original acidity and therefore forming a bi-
functional acid site catalysts. Zhang et al. (2009) studied the potential
of this double acidic sites of HPAs in transesterification of waste
cooking oil. Zr0.7H0.2PW12O40 (ZrHPW) with nanotube structure was
successfully synthesized using natural cellulose fiber as a template.
ZrHPW exhibited high acidity capacity with 1350 μmol/g acid sites
was detected, much higher than its original HPW (892 μmol/g). This
high density acid sites is comparable to recent studies reported in the
literature, such as carbonized glucose (1550 μmol/g) and mesoporous
sulfated silica zirconia (1260 μmol/g). Apart from that, high biodiesel
yield of 98.9% was attained at reaction temperature of 65 °C, methanol
to oil molar ratio of 20, catalyst loading of 2.1% and reaction time of
8 h. Moreover, after 5 reaction cycles, biodiesel yield still remain at
95%.
6.6. Enzyme (biocatalyst) catalyzed transesterification
Enzymatic transesterification especially those using lipase has
drawn researcher's attention in last ten years due to the downstream
processing problem posed by chemical transesterification. Huge
amount of wastewater generation and difficulty in glycerol recovery
are among problems that eventually increase the overall biodiesel
production cost and being not environmental benign. In contrast,
enzyme catalysis proceeds without the generation of by-products,
easy recovery of product, mild reaction condition, insensitive to high
FFA oil and catalyst can be reuse (Kulkarni and Dalai, 2006). These
advantages prove that enzyme catalyzed biodiesel production has
high potential to be an eco-friendly process and a promising
alternative to the chemical process. However, it still has its fair
share of constraints especially when implemented in industrial scale
such as high cost of enzyme, slow reaction rate and enzyme
deactivation (Bajaj et al., 2010). The following section reviews some
of the potential enzymes which have been studied in transesterifica-
tion of waste cooking oil to biodiesel with emphasis given on the
reaction and optimum condition used.
6.6.1. Mucor miehei (Lipozym IM 60)
In a study by Nelson et al., Lipozym IM 60 was found to show a
promising result in transesterification of tallow with high FFA
content. Based on the study, two different types of alcohols were
investigated: (1) primary alcohols, such as methanol, ethanol,
propanol, butanol and isobutanol and (2) secondary alcohol, such
as isopropanol and 2-butanol. The effect of adding solvent (hexane)
into the reaction mixture was also studied. The purpose of
introducing solvent into enzymatic transesterification process is to
increase the solubility between methanol and glycerol and therefore
minimizes the possibility of enzyme deactivation caused by
methanol and glycerol (Halim and Harun Kamaruddin, 2008). It
was found that Lipozym IM 60 is a promising enzyme in
transesterification of tallow with primary alcohol resulting in
biodiesel yield as high as 93–99%. The optimum reaction conditions
were: reaction temperature at 45 °C, stirring speed of 200 rpm, 5 h
reaction time, 0.34 molar of triglyceride in hexane, methanol to oil
molar ratio of 3 and 12.5–25% enzyme (by weight of tallow).
However, for secondary alcohol, the yield of biodiesel obtained was
relatively low. Only 19–24% of biodiesel yield was obtained using the
same reaction condition as primary alcohols. No explanation was
513
given for this observation. On the other hand, in a solvent-free
system (without the addition of hexane), biodiesel yield dropped
significantly to merely 20–65% when methanol or ethanol were used
as primary alcohol. Nevertheless, if branched primary alcohol
(isopropanol) was used in a solvent-free system, high yield of
biodiesel (97%) could be maintained (Nelson et al., 1996). The main
advantage of using secondary alcohol in biodiesel production is to
reduce the solidification point and subsequently improve its cloud
point and pour point characteristic (Salis et al., 2005).
6.6.2. Pseudomonas cepacia (PS 30)
Other than Lipozym IM 60, Nelson et al. (1996) also screened the
potential of PS 30 in transesterification of tallow to biodiesel.
However, the efficiency of the enzyme in catalyzing the transester-
ification reaction was low, even when solvent was introduced into the
reaction mixture. Biodiesel yield obtained was merely 13.9–28.8% for
primary alcohols (methanol, ethanol and isobutanol) and 44.1% for
secondary alcohol (isopropanol). The reaction conditions reported
are; reaction temperature at 45 °C, stirring speed of 200 rpm, 5 h
reaction time, 0.34 molar of triglyceride in hexane, methanol to oil
molar ratio of 3 and 12.5–25% enzyme by weight of tallow.
Although Nelson et al. reported that PS 30 was not a good enzyme,
nevertheless, Wu et al. still attempted to study the same enzyme but
by optimizing the transesterification reaction using response surface
methodology (RSM) (Wu et al., 1999). In his study, restaurant grease
was used as feedstock and ethanol with 95% purity was used as the
source of primary alcohol. Using the regression equation developed, it
was predicted that an optimum biodiesel yield of 85.4% can be
obtained at the following optimum conditions: 38.4 °C, 2.47 h, 13.7%
lipase (PS-30), and grease to ethanol molar ratio of 1:6.6. However,
the apparent yield of biodiesel obtained was way below 85% when
using the predicted optimum conditions. Consequently, the authors
improved the biodiesel yield by introducing a second portion of lipase
PS-30 into the reaction mixture after 1 h of reaction. Unfortunately,
this method did not improve the biodiesel yield significantly. On the
other hand, if the second portion was changed to lipase SP 435
(Candida antarctica) with 5% (wt) of loading, 96% of ethyl ester yield
can be obtained. Nevertheless, neither lipase PS-30 nor SP435 when
used individually could give high yield as predicted using RSM.
Recently, enzymatic transesterification reached another important
milestone with the use of immobilization technology. The purpose of
immobilization is to provide a more rigid external backbone for lipase
molecule which will result in a faster reaction rate (Knezevic et al.,
1998). Lipase can be immobilized into ion-exchange resin, photo-
cross linkable resin, silica beads and alumina through adsorption,
cross-linking, entrapment and covalent bonding method. The advan-
tages of immobilized enzymes over free enzymes are easier lipase
recovery, high stability, insensitive to solvent, and reusable. Hsu et al.
immobilized PS-30 within a phyllosilicate sol–gel matrix (IM PS-30)
and was subsequently subjected to transesterification using waste
grease oil (Hsu et al., 2002). It was found that IM PS-30 was able to
convert 84–94% of grease oil to biodiesel for both primary and
secondary alcohols. The reaction was conducted at 50 °C, alcohol to
grease molar ratio of 1:4, 100 mg IM PS-30, and reaction time of 18 h.
In addition, the enzymatic activity of IM PS-30 did not deactivate even
after 48 h of reaction as the lipase was strongly constrained within the
matrix. Furthermore, by adding molecular sieve (to remove water) in
the reaction mixture, the biodiesel yield increased by as much as 20%
with shorter reaction time.
6.6.3. C. antarctica (Novozym 435)
C. antarctica was first used by Nelson et al. (1996) in transester-
ification of high FFA tallow to produce biodiesel. It was observed that
Novozym 435 has high enzymatic activity when secondary alcohol
(2-butanol) was used in a solvent-free system. Biodiesel yield of
96.4% was obtained at the following reaction conditions: 0.34 molar
514 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
of tallow, reaction temperature at 45 °C, alcohol to tallow molar ratio
of 3:1, stirring speed of 200 rpm and reaction time of 16 h. Besides
that, adding suitable amount of water (6 mol% based on tallow) into
the system was found to promote ester formation when secondary
alcohol was used with Novozym 435 (Nelson et al., 1996). This is
because water is an essential element to maintain the catalytic
activity of enzymes. However, the presence of too much water may
cause hydrolysis of oil in which is undesirable and eventually will
lead to lower conversion of oil to biodiesel.
In another study reported by Watanabe et al. (2001), stepwise
methanol addition was introduced during transesterification in order
to avoid deactivation of enzyme by methanol and to prolong its
durability. Waste cooking oil was used as the main feedstock whereas
immobilized Novozym 435 was used as the enzyme. The reaction was
conducted with three different ways: (1) three-step batch (fixed flow
rate) methanolysis, (2) three-step flow methanolysis and (3) one-
step flow methanolysis. For the three-step batch reaction, the amount
of methanol used was divided equally in each step. 90.4% conversion
of waste oil to biodiesel was obtained after the third step. In addition,
for the three-step continuous feed, methanol at a flow rate of 6.1, 6.3
and 4.2 mL/h was introduced into the first, second and third reactor,
respectively and 90.9% of biodiesel conversion was achieved. On the
other hand, for one-step methanolysis, waste oil was diluted initially
with 90% methyl ester (biodiesel) with weight ratio of 3:1 and
equimolar amount of methanol, relative to total fatty acids in the
waste oil. The mixture was then fed into a bioreactor containing 3 g of
Novozym 135 at a flow rate of 4 mL/h and 90% of biodiesel conversion
was achieved.
Apart from that, in order to reduce the overall production cost of
enzyme, Novozym sp. 99–135 was immobilized on low-cost textile
cloth (Chen et al., 2009). The immobilization carrier of textile cloth
was initially activated by mixing with co-fixing agents consisted of
polyethylene glycol, tween and span (surfactants), gelatin and
lecithin. The lipase solution was then blended with activated
immobilization carrier and dried in air prior to use. Three reactors
are connected in series to form a three-step reaction system in
which glycerol was separated at each step. Waste cooking oil was
used as the main feedstock with high acid value of 143 mg KOH/g.
The optimum reaction condition of lipase/hexane/water/waste
cooking oil weight ratio of 25:15:10:100, reaction temperature of
45 °C and reactant flow of 1.2 ml/min gave 91% yield of biodiesel.
However, for this continuous process, biodiesel yield was found to
decrease to 76% after running the reaction at optimum condition
continuously for 100 h. The expected reasons are: (1) the glycerol
absorbed on the surface of immobilized lipase restricts mass
transfer between substrate and enzyme, and (2) enzyme was
poisoned by methanol.
Most researchers prefer to use hexane as co-solvent in enzymatic
transesterification as it can enhance higher activity and posed good
stability. However, hexane is a hydrophobic solvent in which
hydrophilic compounds used as subtract (alcohol) or obtained by-
product (glycerol) are easily immiscible in hydrophobic reaction
medium. This has resulted to low solubility of both mediums and high
possibility to deactivate the enzyme (Halim and Harun Kamaruddin,
2008). In a study done by Halim and Harun Kamaruddin, tert-butanol
was used as an ideal solvent in a reaction mixture containing waste
cooking palm oil, methanol and Novozym 435. High solubility of
methanol and glycerol in tert-butanol can eventually eliminate the
negative effect of methanol and glycerol on enzyme activity. From the
study, it was found that 88% of biodiesel yield can be obtained at
reaction temperature 40 °C, methanol to oil molar ratio of 4:1, 4%
Novozym and 12 h reaction time.
6.6.4. Bacillus subtilis
Ying et al. was the first group of researchers that used B. subtilis for
transesterification of waste cooking oil to biodiesel (Ying and Chen,
2007). B. subtilis was initially encapsulated within the net of
hydrophobic carrier with magnetic particles (Fe3O4), and then the
secreted lipase can be conjugated with carboxyl at the magnetic poly-
microsphere surface. This magnetic cell biocatalyst (MCB) was
claimed to have better dispersion during transesterification and can
easily be separated from the reaction mixture by subjecting to an
external magnetic field. From the study, it was found that biodiesel
yield can reached up to 90% at reaction temperature 40 °C, pH 6.5,
loading of 3.0% MCB, adding methanol in two stepwise and 72 h
reaction time. Furthermore, MCB can be easily regenerated without
losing its enzymatic activity.
6.6.5. Rhizopus oryzae
Chen et al. investigated the enzymatic conversion of waste cooking
oil using immobilized R. oryzae lipase (Chen et al., 2006). A three-step
batch transesterification reactor was used and stepwise process was
introduced in the reactor to reduce the poisoning of enzyme by
methanol. The optimum reaction condition was reported at 40 °C,
methanol to oil molar ratio of 4, immobilized lipase to oils weight ratio
of 30%, pressure of 1 atm and reaction time of 30 h. Biodiesel yield in
the range of 88–90% can be obtained under these conditions.
6.6.6. Penicillium expansum
Immobilized P. expansum on resin D4020 was reported by Li et al.
(2009) to have high enzymatic activity in transesterification of waste
oil to biodiesel. The process was further enhanced with the addition of
adsorbents such as molecular sieve and blue silica gel into the reaction
mixture with the purpose to absorb excess water produced during
esterification of FFA with methanol. Excess water will not only result
to aggregation of the enzyme in hydrophobic media, hence reducing
its enzymatic activity, but also have a negative effect on the enzyme's
stability. Optimum biodiesel yield of 92.8%% was obtained when the
reaction was carried out at 35 °C, stirring speed of 200 rpm, 2 g of
waste oil, 0.4 g t-amyl alcohol, 0.96 g blue silica gel, 168 U immobi-
lized penicillium expansum and 7 h of reaction time. One molar
equivalent of methanol was added at reaction time of 0, 1 and 3 h.
Apart from that, the synthesized enzyme displayed higher stability in
waste oil with 68.4% of the original enzymatic activity retained even
after recycle and reuse for 10 batches.
7. Other methods or technologies for biodiesel production
Currently, most commercial scale biodiesel plants use batch or
continuous-type reactor to produce biodiesel from refined vegetable
oils. In addition, homogeneous base catalysts such as potassium
hydroxide and sodium hydroxide are the most widely used catalysts
due to their fast reaction rate and mild reaction conditions. However,
if waste cooking oil is to be used as the main feedstock to produce
biodiesel, a two- step process may be required in which the first step
is esterification process to reduce FFA content in the oil and the
second step is transesterification process. However, this two-step
process generates a lot of waste water and is not environmental
benign. Alternatively, as presented earlier, heterogeneous and
enzymatic catalysts have the potential to overcome the problems
posed by homogeneous catalysts. However, most of the studies
reported are carried out at laboratory scale. Therefore, if heteroge-
neous or enzymatic based transesterification process need to be
scaled up to industrial scale, mass and heat transfer limitation must be
carefully addressed. The following section reviews some of the
potential technologies that can facilitate heterogeneous and enzy-
matic transesterification system to overcome the mass and heat
transfer limitation and thus obtaining higher yield of biodiesel in a
shorter reaction time.
 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
7.1. Oscillatory flow reactor (OFR) for transesterification reaction
Oscillatory flow reactor (OFR) was first introduced by Harvey et al.
(2003) to produce biodiesel through some improvement in mixing
intensity between reactants. OFR is a novel type of continues flow
reactor, consisting of tubes containing equally spaced orifice plate
baffles. Therefore, an oscillatory motion is superimposed upon the net
flow of the process fluid, creating flow patterns conducive for efficient
heat and mass transfer, whilst maintaining plug flow regime (Harvey
et al., 2003). In addition, each baffle essentially behaves as a stirred
tank that lead to excellent mixing and suspension by creating vortices
between orifice baffles and oscillating fluid (Zheng et al., 2007). This is
an essential element in designing a biodiesel reactor especially when
heterogeneous catalysts are used due to the presence of three
immiscible phases (oil–alcohol–catalyst) at the initial stage of
reaction. Thus, improvement in mixing and suspension of catalysts
tend to produce higher yield of biodiesel in a shorter reaction time
compared to conventional batch-type stirred tank reactor. Apart from
that, OFR allows longer residence time as the mixing is independent of
the net flow and hence the reactor length-to-diameter ratio can be
reduced. This is an important plus point if the process is scaled up for
commercial application in order to reduce the overall capital and
pumping cost.
Harvey et al. applied OFR in the production of biodiesel from waste
cooking oil and pure rapeseed oil (Harvey et al., 2003). The reaction
was performed at temperature of 20–70 °C, residence time of 10–
30 min and molar ratio of methanol to oil was maintained at 1.5. Pure
sodium hydroxide (32.4 g) was dissolved in pure methanol initially at
40 °C for 1 h. It was found that at 50 °C and 30 min of reaction time,
nearly 99% of biodiesel was produced. Moreover, the product contains
negligible amount of triglyceride and diglyceride. However, some
traces of monoglyceride were detected (Noureddini and Zhu, 1997).
Nevertheless, Harvey et al. (2003) concluded that in-depth study on
OFR in transesterification with heterogeneous catalyst is promptly
required as OFR is ideal for suspending solid catalysts or polymer
supported catalysts.
7.2. Microwave technology in transesterification reaction
In recent years, electromagnetic energy utilization and develop-
ment has gained much interest by many research groups (Corsaro et
al., 2004). Microwave irradiation is one of the examples as this process
posed several advantages such as higher yields of cleaner product,
minimum energy consumption and environmentally benign com-
pared to conventional heating in various chemical reactions (Grois-
man and Gedanken, 2008). In fact, conventional heating process
suffers significant drawback due to its limitation such as it is
dependent on the thermal conductivity of materials, specific heat
and density (Groisman and Gedanken, 2008). Apart from that,
conventional heating is rather slow and heat is not distributed
uniformly in a reaction vessel, resulting to more energy (than the
theoretical value) is required for a particular chemical reaction
(Mutyala et al., 2010). Moreover, direct contact between the hot
reaction vessel surface with reaction media (reactants) may results to
product decomposition especially when heated for long period of
time. In contrast, microwaves transfer energy in a form of electro-
magnetic and not thermal heat reflux. The oscillating microwave field
tends to oscillate polar ends of molecules or ions continuously (Marra
et al., 2010; Azcan and Danisman, 2007). Consequently, collisions and
friction between the moving molecules is created and generate heat
(Marra et al., 2010). Heat is therefore directly deposited into the
reaction media and resulted to rapid temperature increase through-
out the sample (Liu and Cheng, 2009, Azcan and Danisman, 2007).
Thus, higher yield of products can be obtained in a short reaction time.
However, the major drawbacks of using microwave irradiation for
biodiesel synthesis are the scaling-up of the process from laboratory
515
scale to industrial production scale and process safety (Vyas et al.,
2010).
A few reports concerning the usage of microwave irradiation in
transesterification have been reported recently (Lertsathapornsuk et
al., 2008, Barnard et al., 2007, Leadbeater and Stencel, 2006). One of
the interesting studies is the development of a continuous flow
microwave reactor to produce biodiesel from waste cooking oil as
reported by Barnard et al. (2007). In that study, transesterification
reaction was performed using a commercially available multimode
microwave apparatus (CEM MARS). Initially, a 10 L mixture of waste
cooking oil, methanol and catalyst was prepared (1:6 ratio of oil to
methanol and 1 wt.% KOH as catalyst) and placed in a holding tank.
The mixture was then pumped into the microwave reactor vessel at a
flow rate of 2 L/min and heated to 50 °C using microwave power of
1600 W. After 10 min, the products were pumped out from the
reactor. It was found that the overall biodiesel conversion achieved
97.9%. Furthermore, when the flow rate of the reaction mixture was
increased to 7.2 L/min, 98.9% of biodiesel conversion can still be
obtained. Apart from that, the study also reported the overall energy
consumption by microwave irradiation and conventional heating as
shown in Table 7. The results clearly show that microwave irradiation
process would be significantly more energy-efficient than conven-
tional heating in a continuous biodiesel production process.
7.3. Ultrasonic technology in transesterification reaction
Ultrasonic technology has been recognized as an effective method
to enhance mass transfer rate between immiscible liquid–liquid
phases within a heterogeneous system (Ji et al., 2006). Therefore, it
has been widely used in various biological and chemical reactions to
improve the yield within a shorter reaction time. Ultrasound is
defined as sound with frequency beyond human ear can respond. The
normal sound frequency that can be detected by human lies between
16 and 18 kHz, but frequency for ultrasound generally lies between
20 kHz and 100 MHz (Vyas et al., 2010). This high frequency sound
wave will compresses and stretches the molecular spacing of a
medium in which it passes through. Thus, molecules will be
continuously vibrated and cavities will be created. As a result, micro
fine bubbles are formed through sudden expansion and collapse
violently, generating energy for chemical and mechanical effects
(Colucci et al., 2005). Furthermore, the collapsed bubbles will disrupt
the phase boundary and impinging of the liquids to create micro jets,
leading to intensively emulsification of the system (Ji et al., 2006).
Ultrasonic technology in transesterification has proven to be an
efficient mixing tool and provides sufficient activation energy to
initiate the reaction (Singh et al., 2007). Ultrasonic-assisted transes-
terification does not only shorten reaction time, but also minimize the
molar ratio of alcohol to oil and reduce energy consumption
Table 7
Energy estimation for the preparation of biodiesel using conventional and microwave
heating.
Entry Reaction conditions Energy consumption (kJ/L)a
1 Conventional heatingb 94.3
2 Microwave continuous flow 26.0
(7.2 L/min feedstock flow)
3 Microwave continuous flow 60.3 (92.3)d
(2 L/min feedstock flow)c
4e Microwave heating (4.6 L batch reaction) 90.1
a
Normalized for energy consumed per liter of biodiesel prepared.
b
On the basis of values from the joint U.S. Department of Agriculture and U.S.
Department of Energy 1998 study into the life cycle inventory of biodiesel and
petroleum diesel for use in an urban bus.
c
Assuming a power consumption of 1700 W and a microwave input of 1045 W.
d
Assuming a power consumption of 2600 W and a microwave input of 1600 W.
e
Assuming a power consumption of 1300 W, a microwave input of 800 W, a time to
reach 50 °C of 3.5 min, and a hold time at 50 °C of 1 min.
516 M.K. Lam et al. / Biotechnology Advances 28 (2010) 500–518
compared to conventional mechanical stirring method (Vyas et al.,
2010). However, up to now, there are only few studies applying
ultrasonic technology in transesterification of waste cooking oil
(Refaat and El Sheltawy, 2008, Wang et al., 2007). Wang et al.
explored the potential of ultrasonic technology in enzymatic
transesterification of high acid value waste oil (Wang et al., 2007).
Commercial immobilized Novozym 435 from C. antarctica was utilized
as a biocatalyst in the system. It was found that the enzymatic activity
was enhanced with the assistance of low frequency and mild energy
ultrasonic sound wave. Under the optimal ultrasonic assistant
reaction conditions, such as 8% oil quantity of Novozym 435, molar
ratio of propanol to oil 3:1, frequency of ultrasonic assistant 28 kHz
occupied at power 100 W, reaction temperature at 40–45 °C, an
overall biodiesel (propyl oleate) conversion of 94.86% was achieved in
only 50 min. Furthermore, ultrasonic sound wave tends to reduce the
adsorption of biodiesel and glycerol on the surface of immobilized
Novozym 435. As a result, Novozym 435 can be recycle to use with
clean appearances, well decentralizations, no agglomeration, easy
washing and well operational stability.
7.4. Co-solvent
Transesterification reaction is a slow reaction process which
typical requires 30 min to few hours to drive the reaction towards
completion, depending on the type of catalyst used. This is because
the reactants used in transesterification (triglyceride and alcohol) are
not miscible and therefore causing mass transfer limitation especially
at the initial time of the reaction. In order to accelerate the rate of
transesterification reaction, researchers have recently introduced co-
solvent such as tetrahydrofuran (THF), hexane and diethyl ether
(DME) into the reaction mixture with the aim to increase the
solubility and subsequently improve the mass transfer rate between
oil and methanol phase (Guan et al., 2009). Experimental results have
shown that THF is indeed a good co-solvent that can accelerate
biodiesel production within a shorter reaction time, either for
homogeneous or heterogeneous system (Chai et al., 2007, Yang and
Xie, 2007, Peña et al., 2009). However, one of the disadvantages of
using THF is the co-solvent must be separated out from the final
product upon completion of reaction. Although it can be easily
distilled out together with methanol, however separation between
methanol and THF becomes rather problematic since both chemicals
have close boiling point. Note that distilled methanol should be
further purified before it is subjected to the next cycle of reaction. In
another study, biodiesel itself was used as co-solvent since it is
miscible in oil and methanol phase (Park et al., 2009). Furthermore,
biodiesel does not need to be separated out since it itself is the final
product in the transesterification reaction. In the study by Park et al., it
was reported that when biodiesel was added as co-solvent, transes-
terification reaction can be completed within a relatively shorter
reaction time (20 min) as compared to system without co-solvent
(60 min).
Nevertheless, when co-solvent was used in heterogeneous
transesterification of waste cooking oil, a contra observation was
reported (Guan et al., 2009). Result showed that the addition of co-
solvent THF or DME into the reaction media, caused a drop in biodiesel
yield. Based on this finding, the authors suggested that mass transfer
between the methanol and oil phase in a heterogeneous catalytic
system was not affected significantly even with the addition of co-
solvent. Furthermore, the authors observed that when THF was added
into the reaction mixture, the solid catalyst easily stick with glycerol
and agglomerate on the reactor wall. Thus, catalyst was deactivated as
a result of agglomeration. A similar observation was also reported in a
homogeneous system when waste cooking oil was utilized as the
feedstock (Sabudak and Yildiz, 2010). The authors concluded that the
positive effect of THF addition was found to be insignificant because
the reactor used for the transesterification reaction was equipped
with a mechanical agitator and circulation pump that is capable to
homogenize the mixture sufficiently even without the use of co-
solvent. Furthermore, it is more economical to improve the mass
transfer limitation through mechanical stirring rather than using co-
solvent as additional energy is required to distillate the co-solvent.
Nevertheless, more studies are required to verify the effect of co-
solvent in heterogeneous transesterification system particularly for
enzyme catalyst.
8. Conclusion
Biodiesel is a renewable and alternative fuel to petroleum-based
diesel that is non-toxic, biodegradable and does not contribute net
carbon emission to the atmosphere. Currently, biodiesel is produced
through transesterification reaction from vegetable oils such as
rapeseed, soybean and palm oil. However, the high prices of these
oils in the global market have sharply increased the overall biodiesel
production cost and making it not economically viable as compared to
petrol based diesel. Furthermore, the oils are important commodities
in the human food supply chain and therefore its conversion to
biodiesel in a long run may not be sustainable. As an alternative, this
paper has addressed the potential of using waste cooking oil, as a
cheap and economical feedstock for biodiesel production.
Nevertheless, current commercial technology (homogeneous base
catalyst) was found not suitable for the transesterification of waste
cooking oil due to its high FFA content. On the other hand, using
homogeneous acid catalyst requires longer reaction time and could
potentially cause corrosion on equipment. Therefore, it is clear that
research on heterogeneous catalyst, either base or acid type, should be
carried out extensively to develop a suitable catalyst to convert waste
cooking oil to biodiesel with special emphasis on catalyst deactivation
and regeneration. Apart from that, enzymatic transesterification is
another possible way to produce biodiesel from waste cooking oil due
to its high stability towards FFA content in oils. However, the enzyme
should be synthesized in a cheaper way and available for commercial
use.
Apart from the development of suitable heterogeneous and
enzymatic catalysts for biodiesel production from waste cooking oil,
one major drawback still exist. Up-scaling of laboratory scale process
involving immiscible phases to commercial scale is not an easy task
due to mass and heat transfer limitation. Nevertheless, recent
advances in technologies such as oscillatory flow reactor (OFR),
microwave irradiation, ultrasonic technology and co-solvent have
shown high potential in overcoming the limitation. These technolo-
gies not only facilitate transesterification reaction in terms of
increasing mixing intensity, heat and mass transfer rate, but also
proved to be more energy-efficient as compared to conventional
heating process. Thus, in order to materialize the technology for
converting waste cooking oil to biodiesel using heterogeneous and
enzyme catalysts, extensive research work on process scaling-up and
more advance research on developing sustainable catalysts should be
conducted simultaneously.
Acknowledgements
The authors would like to acknowledge the funding given by
Universiti Sains Malaysia (Research University Grant No. 1001/
PJKIMIA/814062, Short-term Grant No. 304/PJKIMIA/6039015, Re-
search University Postgraduate Research Grant Scheme No. 1001/
PJKIMIA/8031018 and USM Fellowship) for this project.
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