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Recent Patents on Biotechnology 2008, 2, 25-34


Biodiesel: An Alternative Fuel

Maximino Manzanera*, Maria L. Molina-Muoz and Jess Gonzlez-Lpez
Group of Environmental Microbiology, Institute of Water Research, University of Granada, C/Ramn y Cajal No. 4, Edificio Fray
Luis de Granada, Granada 18071, Spain
Received: July 16, 2007; Accepted: August 16, 2007; Revised: September 14, 2007
Abstract: Biodiesel is an alternative energy source and could be a substitute for petroleum-based diesel fuel. To be a viable alternative, a
biofuel should provide a net energy gain, have environmental benefits, be economically competitive, and be producible in large quantities
without reducing food supplies.
Most of the sources, methods and apparatus to produce biodiesel are reviewed here. Some of the patents propose the use of oils and fats
of animal or vegetal origin and other kind of sources. Many others focus on the methods for the production or oxidation stability of the
biofuel in order to make its production economically competitive. Several apparatus comprising reactors and refineries are also presented.
This review article summarizes recent and important patents relating to the production of biodiesel to make its production a viable

Keywords: Alternative fuels, biodiesel, transesterification, FAEEs, FAMEs, lipase, SCF, refinery, NOx, microdiesel.
The biggest challenge modern industrial society is facing today
is the decline and exhaustion of the fossil energy resources. The
primary sources of energy that power our civilization are those
fossil fuels. The International Energy Agency has forecasted that
the world's total energy requirements will rise by half in the next 25
years. On the other hand, global oil product demand has been
revised to 84.3 million barrels a day (mb/d) in 2006 and 85.8 mb/d
in 2007 [1]. Therefore continued use of petroleum-sourced fuels is
now widely recognized as unsustainable because of depleting
supplies and increasing demand.
Oil prices have been rising steadily over the past 3 years and
surged to a record high above $60 a barrel in June 2005, sustaining
a rally built on strong demand for gasoline and diesel and on
concerns about refiners' ability to keep up. This trend continued
into early August 2005, as NYMEX crude oil futures contracts
surged past the $65 mark as consumers kept up the demand for
gasoline despite its high price. Crude oil futures peaked at a close of
over $78.40 per barrel on July 13, 2006 [2].
In addition, the combustion of the fossil fuels used is
considered as the major factor responsible for global warming due
to large-scale carbon dioxide emissions. The present atmospheric
concentration of CO2 is about 383 parts per million (ppm) by
volume. Future CO2 levels are expected to rise due to ongoing
burning of these fossil fuels and land-use change. The IPCC Special
Report on Emissions Scenarios gives a wide range of future CO 2
scenarios, ranging from 541 to 970 ppm by the year 2100. Fossil
fuel reserves are sufficient to reach this level and continue
emissions past 2100, if coal, tar sands or methane clathrates are
extensively used [3]. Fossil fuels are also considered as the main
source of local environmental pollution. Thus, alternative energy
sources based on sustainable, renewable and environmentally
friendly processes are urgently needed. One of the most prominent
alternative energy resources, attracting more and more interest in
recent years with the price for crude oil reaching record heights, is
biodiesel, which is a possible substitute for petroleum-based diesel
fuel. Biodiesel fuels are the alkyl monoesters of fatty acids from
vegetable oils and animal fats. Many studies have shown that the
properties of biodiesel are very close to diesel fuel [4-6]. Therefore,
*Address correspondence to this author at the Group of Environmental
Microbiology, Institute of Water Research, University of Granada,
C/Ramn y Cajal No. 4, Edificio Fray Luis de Granada, Granada 18071,
Spain; Tel: +34 958 242981; Fax: +34 958 243094;
E-mail: manzanera@ugr.es
1872-2083/08 $100.00+.00

biodiesel fuel can be used in diesel engines with little or no

modification. Biodiesel has a higher cetane number than diesel fuel,
no aromatics, no sulfur, and contains 10-11% oxygen by weight.
These characteristics of biodiesel are responsible for a reduction in
the emissions of carbon monoxide (CO), hydrocarbon (HC) and
particulate matter (PM) in the exhaust gas compared to diesel fuel
[7,8]. However, to be a viable alternative, a biodiesel should
provide a net energy gain, be economically competitive, and be
producible in large quantities without reducing food supplies.
1.1. Vegetable Oils
Vegetable oil was exclusively used to run the early diesel
engines since diesel engine was invented in 1892. Nevertheless, the
feedstock shifted to petroleum distillates refined from crude oil
during gasoline production in the 1920s. But while so-called
petrodiesel was cheaper and more abundant than vegetable oil, it
was also lighter and less viscous. Vehicle manufacturers had to
modify engine designs accordingly, and vegetable oil as a fuel
source was sidelined for decades. Then in 1973, the Arab oil
embargo produced a swift price increase. With gas and diesel
suddenly four times more expensive than before, interest in biofuels
returned. Since 1973, many different sources have been proposed in
order to obtain biodiesel. Virgin oil feedstock rapeseed and soybean
oils are most commonly used, with soybean oil alone accounting for
about ninety percent of all biofuel stocks in the US. While in 1994
fuels produced from soybean oil amounted to barely a few thousand
gallons a year, ten years later, that volume increased to 25 million
gallons. Last year (2006) 200 million gallons were sold. Assuming
existing and emerging facilities operate at full capacity, USA
biodiesel production capacity could reach 1.5 billion gallons in
2007 [9].
If soybean oil is the preferred biodiesel feedstock in North
America, rapeseed or canola, is the one preferred in Europe for
biodiesel production. Nevertheless, rapeseed oil is a promising
source for manufacturing biodiesel in North America as well,
evolving into a major cash crop. Canada and the United States
produce between 7 and 10 million metric tons (tonnes) of rapeseed
per year. This is partly because rapeseed produces more oil per unit
of land area as compared with soybean in these areas. Sommerville
estimated that the average yield of soybean oil is 450 pounds per
acre and the average canola seed oil was 650 pounds per acre in
2005 [10]. Although this is in contrast with the much higher yields
of cellulosic biomass that could be grown on the same acres to
produce fuel ethanol, alternative feedstocks are needed to obtain
2008 Bentham Science Publishers Ltd.

26 Recent Patents on Biotechnology 2008, Vol. 2, No. 1

biodiesel where ethanol feedstock can not be grown or do not

render such high yields. In this sense, in 1998 Beer and coworkers
developed a variety of transgenic plant considered to be the most
disease- and drought-resistant variety of rapeseed to date [11] that
allegedly could be translated in an increase in oil production as a
result of growing this variety in locations where neither cellulosic
biomass for ethanol production nor rapeseed was viable before.
A different feedstock of vegetable origin is palm oil, obtained
from the fruit of the oil palm tree. Previously the second-most
widely produced edible oil, after soybean it may have now
surpassed soybean oil as the most widely produced vegetable oil in
the world and certainly is the preferred oil in South East Asia. Thus,
some Asian governments are refocusing on the use of palm oil to
the production of biodiesel to cater to the huge demand from
European countries encouraging the building of biodiesel plants.
These plants will start operating in the middle of next year and
produce 100,000 tons of biodiesel annually. Strong demand for
biodiesel from Europe as well as Colombia, India, South Korea and
Turkey has fueled the industry's growth as more countries seek to
reduce their reliance on fossil fuels. Malaysia has already begun
preparations to change from diesel to bio-fuels by 2008, including
drafting legislation that will make the switch mandatory. From
2007, all diesel sold in this country must contain 5% palm oil.
Being the world's largest producer of crude palm oil, Malaysia
intends to take advantage of the rush to find cleaner fuels. Palmbased biodiesel exhibits a relatively high pour point (approximately
15oC) due to its high content of saturated fatty compounds; hence,
its use is only suitable for tropical countries and unsuitable for low
temperate or cold climate countries where operational temperature
is below its pour point. Nevertheless, palm-based biodiesel
formulations with enhanced cold flow properties are provided using
new formulation comprising at least 60% vol/vol petroleum-based
fuel oil mixed with palm-based fuel oil [12]. Similarly, a novel
alkyl-ester composition derived from specific blend of rapeseed and
sunflower, in particular from Brassica napus and Helianthus
annuus to be used in diesel engines to solve the same kind of
problem [13].
In certain parts of the world, governments and some
corporations consider the jatropha plant, common in hot climates,
one of the most promising sources of biodiesel. The plant can grow
in wastelands, and it yields more than four times as much fuel per
unit of area as soybean, and more than ten times that of corn [14].
Another example of crop, recently proposed for biodiesels
feedstock is the oil extracted from the nuts of the desert plant
Balanites aegyptiaca [15]. Other crops such as cottonseed [16],
mustard, flax, sunflower, hemp and even algae are considered
promising biodiesel feedstocks too.
The advantages of deriving biodiesel from algae include rapid
growth rates, a high per-acre yield. Some species of algae are
ideally suited to biodiesel production due to their high oil content
[17-21]; The National Renewable Energy Laboratory (NREL) in
Golden, Colorado had demonstrated oil production from algae rates
200 times greater per acre than achievable with fuel production
from soybean farming [22]. Unlike other oil crops, microalgae grow
extremely rapid and commonly double their biomass within 24 h.
Biomass doubling times during exponential growth are commonly
as short as 3.5 h. Oil content in microalgae can exceed 80% by
weight of dry biomass [23,24]. Depending on species, microalgae
produce many different kinds of lipids, hydrocarbons and other
complex oils [20,25,26]. And although not all algal oils are
satisfactory for making biodiesel, suitable oils occur commonly.
The strains of race A of microalgae Botryococcus braunii have
been proposed as a renewable source of liquid fuel via CO2 fixation
[27] with hydrocarbon content as high as 30% of biomass. Recent
patents comprise a novel variety of Botryococcus (Ninsei) distinct
from the previously cultured variety in color, biochemistry, size,
shape, and habit in order to facilitate its cultivation [28], or a race

Manzanera et al.

capable of producing different types of hydrocarbons [29] more

suitable for biodiesel production. Genetic and metabolic engineering are likely to have the greatest impact on improving the
economical production of microalgal diesel [17,19,30].
1.2. Non-Vegetable Oils as Biodiesel Feedstock
Although, it has been shown that biodiesel from vegetable
feedstocks have environmental benefits there are as well some
concerns about its use. In this sense, the locations where oilproducing plants are grown is one of the prime worries being that
countries, such as the Philippines and Indonesia are clearing cut
large areas of tropical forest in order to grow such lucrative crops,
in particular, oil palm [31]. Even more, it has been suggested that
biodiesel might increase pressure on world food supplies,
therefore urging to look for alternative feedstocks for biodiesel
such as waste vegetable oils and waste animal fats. The main
sources of waste animal fats are primarily meat animal processin g
facilities. Recycled grease products are referred to waste grease
being generally classified in two categories, yellow grease and
brown grease. Yellow grease is produced from vegetable oil or
animal fat that has been heated and used for cooking. Renderers
filter out the solids and heat the spent cooking oil to drive out
moisture until it meets industry specifications for yellow grease.
Yellow grease is required to have a free fatty acid level of less than
15%. If the free fatty acid level exceeds 15%, it is called brown
grease, sometimes referred to as trap grease. Waste vegetable oil
from restaurants and rendered animal fats are inexpensive
compared with food-grade vegetable oil hence biodiesel production
from waste grease can be expected to benefit from a raw material
cost advantage and it will help to reduce overall biodiesel cost [32].
There are patents that provide methods for producing biodiesel fuel
from waste vegetable or animal oils and fats, and methanol that
does not use a catalyst and does not generate glycerol as a byproduct [33], or smaller processing plant that enables site selection
closer to the raw material supplies, thereby minimizing or
eliminating transportation costs for yellow grease [34]. More
important is the method by Hammond Earl G. and Wang Tong of
converting free fatty acids in acid oil, and the acid fat into FAMEs
(biodiesel) using a small amount of methanol and an acid catalyst.
A method especially useful for the generation of biodiesel using
animal oils and fats that contain a relatively high level of free fatty
acids as starting material [35]. A different application developed in
order to use waste oils and fats as biodiesel feedstock focus on
reducing the viscosity of these wastes in order to facilitate a
filtering process particularly needed for biodiesel synthesis from
this kind of oils and fats [36]. Similarly a mobile refinery has been
developed in order to separate contaminants of waste fryer oil used
for cooking including food particles, and water and emulsified or
congealed grease by vacuuming and heating these wastes into a
container and passing the heated fluid via a strainer [37]. The use of
whole microorganisms producing lipases or the purified lipases
themselves has been proposed as an invention in order to use waste
lipids and fat to produce fatty acid lower alcohol esters as well [38].
Finally, different waste fats are proposed for lower production
cost such as those present in sewage sludge that contains high levels
of lipids, in the form of triglycerides, phospholipids, phosphoglycerides, sphinolipids, glycolipids, and fat-soluble vitamins. In
this method, sludge produced under wastewater treatment is
collected then partially dehydrated by sludge thickeners, filter
presses, centrifuges, and driers to a final solid concentration ranging
from 60% to 100% solids by weight. After drying, lipids are
extracted from the processed material using any of the methods of
chemical extraction such as chemical solvents including aliphatics,
supercritical gases and liquids, hexane, acetone, or primary alcohols
[39]. Alternatively, a different waste material proposed as biodiesel
feedstock is the one produced by the diary industry resulting from
their wastewater processing plants. In these plants, dissolved air
flotation (DAF) systems are commonly used to separate the


insoluble solids from the liquid by injecting gas bubbles into the
wastewater solution. These bubbles adhere to the insoluble particles
and form a floating layer of sludge, which is mechanically scraped
off producing an emulsion of fat and protein in water in significant
quantities whose disposal is both problematic and expensive. The
method by Hamilton RB involves adjustment of the DAF sludge pH
between 2.5 and 4.5, mixing it with an emulsifying agent and
heating the mixture to boiling point. By allowing the mixture to
cool down the fat and protein component parts separate out, and fat
rich component is then used as biodiesel feedstock [40].
Biodiesel production is mainly performed by the transesterification of a triglyceride with an alcohol in a chemical reaction
known as alcoholysis outlined in Fig. (1). In the transesterification
of different types of oils, triglycerides react with an alcohol,
generally methanol or ethanol, to produce esters and glycerol. The
overall process is normally a sequence of three consecutive
reversible reactions [41,42]. During the first reaction, triglycerides
are transformed into diglycerides. Then from diglycerides,
monoglycerides are produced in the second reaction and in the last
reaction glycerol is obtained from monoglycerides. In all these
reactions esters are produced which are the products used as
biodiesel, that finally as the resulting product has to be purified.
The stechiometric relation between alcohol and oil is 3:1. However,
an excess of alcohol is usually added to improve the reaction
towards the desired product. To make the reaction possible, a
catalyst is added as well. This transesterification reaction is affected
by alcohol type, molar ratio of alcohol to oil, temperature, purity of
the reactants and type and amount of catalyst see [43] for a review.
Different types of catalysts are used, from basic ones such as
sodium or potassium hydroxides, acids such as sulfuric acid, ion
exchange resins, lipases and supercritical fluids. For a basic
catalyst, either sodium hydroxide (NaOH) or potassium hydroxide
(KOH) is normally used with methanol or ethanol as well as any
kind of oils, although sodium and potassium alkoxides such as
sodium methoxide, sodium ethoxide, sodium propoxide, sodium
butoxideto have been proposed as alkali catalysts as well. In this
process it is better to produce the alcoxide before the reaction to
obtain a better global efficiency [44]. The alcohol-oil molar ratio
that should be used varies from 1:1-6:1. However, 6:1 is the most
used ratio giving an important conversion for the alkali catalyst
without using a great amount of alcohol. The types of alcohol are
usually methanol and ethanol. The last one has fewer safety
problems because it is less toxic, but methanol is most frequently
used because of its lower cost and its physical and chemical
advantages (polar and shortest chain alcohol), although in some
countries such as Brazil, there is a special interest in developing an
appropriate technology for the use of ethanol instead of methanol
[45] since ethanol is easily available due to the extensive
fermentation industry in the country.

Recent Patents on Biotechnology 2008, Vol. 2, No. 1


2.1. The Alkali-Catalyzed Transesterification

For the alkali-catalyzed transesterification, both the glycerides
and alcohol must be substantially anhydrous, and oils should be
neutral or weak acids since a too high proportion of fatty acids
(higher than 0.5%) would inhibit the reaction by consuming catalyst
to form soaps, lowering the reaction yield and making more
difficult the separation of the products, in a process known as
saponification [46,47]. On the other hand, if absolute alcohol is
used, the formed glycerol does not settle spontaneously and the
alcohol excess must be evaporated or water added, nevertheless not
obtaining a complete conversion. In order to use hydrated alcohol
with success Robert and coworkers proposed a process for
preparing a composition of fatty acid esters, by acid
transesterification where the alcohol could contain up to 60% by
weight of water. This method consists of reducing the free acidity
of the ester phase, followed by a basic esterification in the presence
of a monoalcohol, finished with the recovery of the ester phase
[48]. The process is simplified since, in the presence of hydrated
alcohol, glycerol is easily settled and the ester phase is purer and
the glycerol content lower despite of the high water content of the
alcohol phase. Sometimes, there might be special interest in
obtaining concentrated glycerol in the process of making biodiesel
[49] as added value byproduct for the synthesis of chemicals such
as dichloropropanol [50] polyglycerols [51] or glyceryl ethers or
syngas [52-56] currently extracted from petrochemical resources
that can be alternatively extracted from biological feedstock.
One of the advantages of the alkali transesterification is that
basically any kind of vegetable oil can be used. The amount of
catalyst that should be added to the reactor varies from 0.5% to 1%
w/w [57], but some authors prefer advice values between 0.005%
and 0.35% w/w [43,58]. Other important reason for the great
interest in the alkali process is due to its higher reaction efficiency
and for showing a lower corrosive effect than that for the acid
process, making alkali the preferred catalyst to be used in
industries. However, as mentioned before, in the alkali catalyzed
transesterification, the presence of water causes a partial reaction
change to saponification, which produces soap. The soap consumes
the catalyst and reduces catalyst activity, causing an increase in
viscosity, formation of gels and difficulty in separation of glycerol.
Freedam et al. reported that sodium hydroxide or sodium
methoxide reacted with moisture and carbon dioxide in the air
leading to lowering of their effectiveness to catalyze the
transesterification of oil [47].
Although transesterification processes using alkali catalysts
give high conversion of fatty oil glycerides to esters in relatively
shorter residence time, besides interference of water and free fatty
acids with the reaction, the processes based on alkali catalysts have
several drawbacks. These drawbacks are the intensive energy
required for the reaction, the difficulty in recovering the glycerol,
the fact that the alkaline catalyst has to be removed from the
product, and the high production of alkaline waste water that is

Fig. (1). Transesterification of triglyceride with alcohol.

Triglycerides from oils and fats are reacted with an alcohol in a reaction known as transesterification or alcoholisis in presence of a catalyst. The products of
the reaction are esters of fatty acids (biodiesel) and glycerol.

28 Recent Patents on Biotechnology 2008, Vol. 2, No. 1

required for the treatment. Additionally, further several steps such

as evaporation of methanol, removal of saponified products,
neutrali-zation and concentration are needed to recover glycerol.
2.2. The Acid Transesterification
The acid process is the second method most commonly used for
producing biodiesel. It consists of using an acid as catalyst, either a
homogeneous acidic catalyst or a heterogeneous acidic catalyst. As
homogeneous acidic catalysts, we find compounds such as sulfuric
acid, hydrochloric acid, para-toluene sulfonic acid, methane
sulfonic acid, etc. Although, sulfuric acid is the preferred catalyst
for this kind of reaction [47,42,59,60]. Among the heterogeneous
acidic catalysts, we can use strong acidic sulfonated ion exchange
resins and acidic zeolites.
Similarly to the alkali reaction, if an excess of alcohol is used in
the experiment a better conversion of triglycerides is obtained, but
recovering glycerol becomes more difficult and that is why optimal
relation between alcohol and raw material should be determined
experimentally considering each process as a new problem. The
possible operation condition is, usually, molar ratio 30:1. The type
of alcohol, as well as the oils, is the same as the one that can be
used in alkali catalyst reaction. The amount of catalyst supposed to
be added to the reactor varies from 0.5 to 1 mol%, and if the typical
value is 1%, some authors prefer to use 3.5 mol% [61,62].
Temperature is another important factor for this kind of reaction
with a range varying from 55 to 80oC. Thus the acid transesterification is a great way to make biodiesel if the sample has relatively
high free fatty acid content. In general, 1 mol% of sulfuric acid is a
good amount for a final conversion of 99% in a time around 50 h.
The acid catalyzed reactions give very high yield in esters,
though the drawback of that method is the long time required for
the reaction to happen since the reaction usually requires longer
than 24 hrs to finish. Several groups have proposed the
esterification of triglycerides in fats and oils in a two-step process
using an acidic catalyst in the first step followed by a second
esterification using an alkaline catalyst. The methods can include
specifications such as a reaction temperature of 60 to 120oC in
order to restrain the interesterification reaction of the fat component
[63]; the use of an acidic cation exchange resin as a solid
esterification catalyst [64]; or the removal of the glycerol after the
first step before the second step is started [65] in order to optimize
the reaction. Nevertheless, Basu et al. and Gupta et al. proposed
single step processes for producing esters from fat or from oil. The
first group presented a process by mixing the feedstock with an
alcohol, and a mixture of calcium acetate and barium acetate as
catalyst, heating the reaction mixture to 200-250oC for about 3
hours and cooling the mixture rapidly [66], while the second group
presented a method based in the use of alkyl Tin oxide as catalyst
where glycerol can be separated from the fatty acid alkyl ester, as
immiscible phase, by decantation, the excess alcohol can be
recovered by evaporation or distillation and, the fatty acid alkyl
esters can be purified by washing with water [67].
Due to the slow diffusion of triglycerides through the catalyst
pores in heterogeneous catalysts such as supported metals, basic
oxides and zeolites this type of transesterification reaction is slow,
so higher alcohol to glyceride molar ratio is required to achieve
appreciable conversion of above 70%.
In order to accelerate the reaction, the reactants can be exposed
to microwaves or radio frequency energy, followed by the transfer
of these reactants over a heterogeneous catalyst at sufficiently high
velocity to produce high shear conditions. Then these emulsified
reactants can be maintained at a pressure above autogeneous
pressure for an improvement of the reaction. Using one or more of
these steps can result in higher process rates, higher conversion
levels, or both [68].

Manzanera et al.

In general, there are two basic disadvantages related to

chemical catalyzed biodiesel. The first one is that during catalysis,
the process is relatively slow and secondly, these methods require
removal of the catalyst. Additionally, and as we have mentioned
before, during basic catalysis as well as purification of the catalyst
product, the by-products of the saponification reaction must be
removed. In conclusion some of the disadvantages of the homogeneous chemical catalysis such as product purification can be
overcome by using heterogeneous catalysts or enzymes as catalysts.
2.3. The Enzymatic Catalyst
In the enzymatic approach, the transesterification is carried out
by a protein called lipase overcoming some drawbacks, such as
difficulty in the recovery of glycerol and the energy-intensive
nature of the process. In contrast to chemically catalysts,
biocatalysts allow synthesis of specific alkyl esters, easy recovery
of glycerol, and transesterification of glycerides with high free fatty
acid content [69]. Lipases are enzymes used to catalyze some
reactions such as hydrolysis of glycerol, alcoholysis and acidolysis,
but it has been discovered that they can be used as catalyst for
transesterification and esterification reactions too. Biocompatibility,
biodegradability and environmental acceptability of this biotechnological procedure are the desired properties in a green technology
such as the biodiesel production. The extracellular and the
intracellular lipases are also able to catalyze the transesterification
of triglycerides effectively. Much work has been done on the lipasecatalyzed transesterification of triglyceride [69-75]. Furthermore,
transesterification using a lipase is carried out in a nonaqueous
system. When waste oil containing a large amount of water is used,
the progress of the reaction would be inhibited. Therefore, facilities
for removing water should be necessary, which increases the cost.
A method for producing a fatty acid alcohol ester in the presence of
water was described in [76] by using lipases. However, in this
method, the enzyme has to be purified, and isolation of the enzyme
takes time and increases the production costs. Thus, in the
transesterification using a lipase, none of the above problems has
been solved so that there is a demand for a method for producing a
fatty acid ester more efficiently.
Foglia et al. proposed a method that utilizes lipases to
transesterify triglyceride-containing substances and to esterify free
fatty acids to alkyl esters using short chain alcohols such as 1butanol and isobutanol. The proposed enzymes included 1,3specific lipase from Mucor miehei and Rhizopus delemar, the acylspecific lipase from Geotrichum candidum, and the nonspecific
lipase from Candida antarctica and Pseudomonas cepacia, for the
production of biodiesel [77]. But still and as mentioned earlier, one
common drawback with the use of enzyme-based processes is the
high cost of the enzyme. In this respect, immobilization of enzymes
has generally been used to obtain reusable enzyme derivatives,
enabling in the recycling of the biocatalyst and hence lowering this
cost. In the case of biocatalysts in nonaqueous media, immobilization is also reported to result in better activity. Thus, many transesterification processes employing lipases have used an
immobilized form of the enzyme [74,75,78-80].
Wu and Chen described a technique to increase and regenerate
the activity of immobilized lipases. The technique deals with the
effect caused by physical factors, such as the immiscibility between
the alcohol and the fatty acid glycerides. When methanol or ethanol
is absorbed into the voids of an immobilized lipase, the entry of
fatty acid glyceride into the voids will be blocked, stopping the
reaction from taking place. To wash the deactivated immobilized
lipase, we need to use a solvent that has to be harmless to the lipase,
and with a good solubility to oil, grease, moisture, and methanol or
ethanol. The proposed solvents were iso-propanol, 2-butanol and
tert-butanol [81-83]. The inventors also discover that the activity of
an immobilized lipase can be significantly increased when such an


ideal solvent is used to perform an immersion pretreatment on an

immobilized lipase.
The enzymatic method has been proposed to make biodiesel
directly from the soapstock generated after the alkali refining
processes. The soapstock contains 10-60% water, but also contains
35-85% of fatty derivatives including partial glycerides. The
combination of this technology with feedstock availability offers an
economic and competitive approach to produce biodiesel, since for
each metric ton of alkali refined vegetable produced by this method,
approximately 30 kg of soapstock is generated too. In this way, the
feedstock (the soaps) can be neutralized and split with strong acids
until pH 2-8 is reached, and then they can be subjected to an
enzymatic esterification using lipases at a temperature of 30 to 45oC
[84]. To reduce the cost of keeping these temperatures, a special
type of lipase has been described for the production of biodiesel at
low temperature. The lipase is produced by Micrococcus antarcticus and has good activity and stability at lower temperatures
saving the cost of warming up the reaction [85].
Nevertheless, there are three major difficulties in using lipases
to produce biodiesel. Still the first difficulty is that the price of
lipase is much higher than the price of alkali even when the lipase is
reused. Secondly, the lipase process requires up to 48 hours to
complete the reaction, which is significantly longer than that for the
base catalysis. The third difficulty with enzyme catalysis is that, for
transesterification with methanol which is the preferred alcohol for
biodiesel preparation of the reaction is extremely sluggish and
proceeds in most cases with only low conversion efficiency, if at
all. In this way, the use of intact microorganisms containing lipases
has been described as well for the esterification of glycerides where
fatty acid esters can be prepared in a simple manner avoiding the
cost of enzyme purification [86]. In contrast, synthesizing biodiesel
through bio-methods has the following advantages: the reaction
conditions are milder, there are no toxic emissions, the reactions are
not affected by free fatty acid nor by small amounts of water in the
renewable oils used as raw materials. On top of that there is a
possibility of regeneration and reuse of the immobilized residue,
because it can be left in the reactor if the reactive flow is kept. The
use of enzymes in reactors allows use of high concentration of them
and that makes for a longer activation of the lipases. Additionally,
the immobilization of lipase could protect it from the solvent that
could be used in the reaction and that will prevent all the enzyme
particles getting together. Finally, the separation of products is
easier using these biocatalysts. Nevertheless further research needs
to be made in order to reduce the high cost for biotechnological
production of biodiesel fuel, specially the use of lipase,
immobilized cells or algae cultivation. Further discussion will be
made in this respect on the current and future developments section.
2.4. The Supercritical Method
Chemically catalyzed triglyceride transesterification requires a
multiple step process with one or more batch reactors. Initially, the
triglycerides and alcohol form two immiscible liquid phases, and as
the reaction proceeds, two separate liquid phases are formed. One
contains the newly formed alkyl esters of the triglyceride and the
other contains the glycerol, with the excess alcohol, catalyst and
feed oil being dispersed into both phases. The reaction time for
each step takes at least several hours and once completed, the liquid
products must be allowed sufficient time to separate phases before
additional processing and separation steps can occur to produce the
final products. It is important to recycle the excess of alcohol and
the unused catalyst must be neutralized.
Even the most efficient of the traditional chemically catalyzed
processes require multiple hours to process each batch of feed.
Additionally, significant problems arise in the separation steps.
Significant quantities of glycerol left in the alkyl esters diminish the
quality of the diesel fuel and likewise contaminated glycerol also
loses much of its value compared to pure uncontaminated glycerol.

Recent Patents on Biotechnology 2008, Vol. 2, No. 1


Finally, the separation procedures necessary to adequately clean the

two product-streams produce large quantities of wastewater thereby
creating additional cost and process complexity therefore affecting
the environment.
Such processes are also costly due to the high cost of catalysts,
heterogenization of catalysts, which is even more remarkable for
the enzymatic transformation. Recently, a non-catalytic transesterification reaction in supercritical methanol has been proposed
[87-94]. A Super Critical Fluid is defined as a substance above its
critical temperature and critical pressure. The critical point
represents the highest temperature and pressure at which the
substance can exist as a vapor and liquid in equilibrium. In order to
reach the super critical state the alcohol and vegetable oil are
pumped into a reactor with the pressure between 20 Bar and 400
Bar and a temperature between 150oC and 450oC. A mixture of
non-reacted alcohol, glycerol, and esters is thereby formed, and is
released via a flow and pressure control valve. The mixture of nonreacted alcohol, glycerol, and esters is then cooled in a cooler, and
an upper phase of non-reacted alcohol is separated from the
intermediate phase of esters and the lower phase of glycerol. The
intermediate phase of esters and the lower phase of glycerol are
then directed to a separation reservoir where the esters are separated
from the glycerol. There are specific protocols for the Super Critical
Method in continuous mode such as the described by Dall et al.
[95] or where the supercritical state is only applied to the alcohol
[96]. However, the main disadvantage of this method is the energy
required to obtain the supercritical state that increases greatly the
production cost of the chemical catalyzed methods.
A summary of the advantages and disadvantages of each
technological possibility to produce biodiesel could be found in
Table 1 (from Marchetti et al. [97]).
Another area of research of interest is the use of different
apparatus and refineries in order to perform reactions easily,
quickly, efficiently, and safely. Both batch and continuous-flow
technologies have been used to set-up and scale-up the reaction [98100]. Although in general biodiesel is produced by the batch-type
process, the disadvantages of the batch-type process are time
consuming for the process, the unreliable production quality and the
high labor cost. Alternatively the preferred method is known as the
Connemann process [101]. In contrast to batch reaction methods
[47], the Connemann process utilizes continuous flow of the
reaction mixture through reactor columns, in which the flow rate is
lower than the sinking rate of glycerol as shown in Fig. (2). This
results in the continuous separation of glycerol from the biodiesel.
The reaction mixture may be processed through further reactor
columns to complete the transesterification process. Residual
alcohol, glycerol, free fatty acids and catalyst may be removed by
aqueous extraction.
In 1996, Krawczyk presented a flow diagram for producing
biodiesel via transesterification on the industrial scale. The process
mainly consisted of a transesterification reactor, an alcohol/glycerol
distillation column and a methyl/ethyl ester distillation column. A
continuous deglycerolization process to produce biodiesel from
refined rapeseed oil by alkali-catalyzed transesterification at
ambient pressure and 65-70 C was introduced by Connemann and
Fischer [102]. In this process, a distillation column was also used to
separate methanol from biodiesel and glycerol. The alcohol was
recycled to the transesterification reactor and multi-stage water
washing was employed to purify the biodiesel product. The above
industrial manufacturing information on biodiesel production
formed the principle basis for the design of the alkali-catalyzed
More recently, researchers have put forward several continuous
production processes using catalytic methods, which can increase
the production efficiency and quality of biodiesel [103-107].

30 Recent Patents on Biotechnology 2008, Vol. 2, No. 1

Table 1.

Manzanera et al.

Comparison of the Different Technologies to Produce Biodiesel (from Marchetti et al., [96])

Alkali catalysis

Lipase catalysis


Acid catalysis

Reaction temperature (oC)





Free fatty acid in raw materials

Saponified products

Methyl esters



Water in raw materials

Interference with reaction

No influence

Yield of methyl esters



Recovery of glycerol




Purification of methyl esters

Repeated washing


Repeated washing

Production cost of catalyst


Relatively expensive

Fig. (2). Simplified flow scheme of biodiesel production plant as described

by Connemann et al. in [101].
Triglycerides are supplied from the oil tank at a rate of 57 l/h through a heat
exchanger, together with the alcohol (1.4-1.6 times) and catalyst (0.240.36% w/w) into a mixer at a rate of 10 l/h. Reaction mixture is passed into
the top of the first reactor, set up as a standing column at a lower rate of
flow than the glycerol sinking rate, being separated off in the direction of
flow. Transesterification of 85-90% already takes place in this column.
Reaction product is then passed into a second stirring reactor, where further
transesterification -up to 95-97%- takes place. Aqueous extraction solution
and separator are used to extract biodiesel. Further purification steps are
repeated in order to improve reaction efficiency and to remove, traces of
soap, alcohol, water and glycerol.

Interference with reaction





Both methods, batch and continuous-flow require an efficient

blending of triglycerides with the alcohol and catalyst together at
the transesterification reactor. Mixing efficiency of the reagents is
of crucial importance and is considered as one of the most
important parameters affecting the transesterification reaction. In
order to guarantee a proper mixing several devices can be used,
such as oscillatory baffled reactors [108-110], where a pulsed
stream of a heterogenous multiphase mixture is passed through a
flow tube. Alternatively, instead of the baffles described above, a
metal helix can be present in the flow tube, obtaining in this way a
much more thorough mixing of the heterogeneous mixture,
increasing in this way the biodiesel yield during the transesterification process [111]. Intensive mixing can also be achieved
using centrifugal pumps, high shear mixers, or motionless mixers
[55,112]. The disadvantage of high intensity mixing is that the
presence of soap materials, partially reacted triglycerides, or excess
alcohol can lead to stable emulsions and very slow separation of the
ester and glycerol phases.
Ultrasonic reactors have been described for chemical activation
via sonochemistry, where reaction is induced by ultrasonic sound
waves. These waves are provided through the energy derived from
the collapse of cavitation bubbles generated within the ultrasonic
reactor. Such energy is derived when the cavitation bubbles
collapse rapidly and violently and, in doing so, generate apparent
temperatures of many thousands of degrees Kelvin and apparent
pressures of several thousand atmospheres within the individual
bubbles. Thus, the bubbles that are formed serve as individual
microreactors operating at high temperature and high pressure
while the liquid region immediately adjacent to the bubbles is at
temperatures ranging generally from about 70oC to 80oC and at
between 1.0 and 5.0 atmospheres of pressure. Through asymmetric
collapse of the bubbles, micro jets are created, leading to intensive
mixing of the liquids [113]. Microwaves are reported to greatly
speed up the hydrolysis and esterification processes, reducing the
reaction time to around one quarter of the time required by the
conventional process and reducing the quantity of required alcohol
to just over the stoichiometric quantity. In order to use microwaves
Pyrex bioreactors can be used [114]. Additionally the reactor can be
preceded by different devices to precondition the reagents in order
to improve the reaction, from vacuum chambers to introduce the
alcohol in gaseous form in the reaction mixture and to keep the
reaction vessel under vacuum (i.e. 15-50 mm Hg) and at
temperatures of 180-200oC [115], or an atomiser (atomization
apparatus) to produce the vaporization of the alcohol with the
chemical catalyst in a mixing chamber prior to entry into the
reaction vessel [116].
Sometimes pretreatment of the feedstock is needed. In order to
pretreat the oil feedstock before it is added to the reactor different


apparatus have been developed. The design of the apparatus

depends on the type and quality of feedstock. In the case of algae
feedstock, there are apparatus for separating algae cells from liquid
medium, for example based on a tangential flow filter device, [117],
or based on a multi-stage loop-flow flotation column where bubbles
are used to adsorb the algal cells that can be separated from the
aqueous medium [118] or based on whirlpool circulation using
special bags [22]. Modified cylindrical screening can be used as
filters when waste oils and fats are used as feedstock [119], or the
above mentioned filtering devices for waste fryer oils based on
vacuuming and heating passing the heated fluid via a strainer
[36,37]. Devices containing particles or polymers that include
amine or imine moieties for filtering contaminants formed upon
heating or cooking with oil are also used [120]. Tysinger et al.
described a process in which free fatty acids and other impurities
may be removed from mechanically extracted soybean oil by
physical refining. In physical refining, the oil is vacuum distilled at
high temperatures to separate more volatile components from the
oil. However, this physical refining is of limited value in refining
solvent extracted soybean oil, due to the higher levels on elemental
phosphorous content, of non-hydratable phospholipids, and the high
temperatures required for physical refining, may suppose an
increase in production cost non affordable for the prerequisites of
this product [121]. In order to reduce the production cost a compact
processor including a vapor recovery system for removing excess
alcohols from the fuel has been developed as well. This vapor
recovery system includes a system for conserving and storing the
excess alcohol for further use in the processing of biodiesel fuel so
the process can be scaled down as well for small refineries [122].
For industrial scale, different technologies are also applied in order
to recover the methanol and methoxide vapor from the reaction
vessel by condensing it [123]. This selectively continuous process
of separating the reaction products eliminates the costly washing
step, and those further steps associated with such processing,
because continuous separation is more efficient than batch
decantation, yielding reaction products of greater purity. No
additional water is introduced into the system since water washing
has been eliminated and because the vacuum system used to
recover the methanol from the FAME and glycerol products is also
dry. This permits the use of a molecular sieve column to eliminate
the minute amounts of water present in the excess methanol,
whether water comes from air leaks into the system or from
moisture in the natural oil feed etc., rather than rectification of the
excess methanol from the wash water using distillation as in the
batch approach. Desiccant columns equipped with molecular sieves
are used to dry the recovered excess methanol. Bam and coworker
designed the technology to not only recover the excess alcohol but
to recover unspent catalyst. Since unspent catalyst is contained in
the by-product alcohol, the recycle of the by-product alcohol also
recycles unspent catalyst, which reduces the amount of fresh
catalyst required. Vapors comprising unreacted alcohol from the
extractive distillation are condensed in condenser located just after
the reactor [124].
Once the reaction has occurred, separation of the alkyl esters is
needed. Stidham et al. [125], Hayafuji [126] and Wimmer [127]
disclosed the processes to wash impurities such as soap particles
from the product alkyl ester. A more recent approach is based on
bringing unpurified biodiesel in contact with cellulose-containing
material by mixing the latter in order to eliminate the glycerol and
catalyst residues, such as potassium soaps, accumulated during the
production of the biodiesel. The system includes an agitated
sedimentation tank with a conduit for delivering purified biodiesel
into de agitated sedimentation tank [128].
Once the reaction has taken place in the reactor an alcohol/
glycerol distillation device has to be included. Alasti has recently
described a refinery for the separation of alcohol, glycerol, esters
and salts performed by volatility [129].

Recent Patents on Biotechnology 2008, Vol. 2, No. 1


4.1. NOx (Nitrogen Oxide) Emissions
If biodiesel improves engine emissions in most categories when
compared to petroleum based diesel fuel, however it is also
responsible for an increase in nitrous oxide emissions. Blends
containing higher concentrations of biodiesel, show a proportional
increase in emissions of nitrogen oxides (NOx). Presently, NOx
emissions could be a significant limitation to the widespread
adoption of biodiesel fuels. NOx is a generic term for mononitrogen oxides. These oxides are produced during combustion, and
are of interest as air pollution. They are believed to aggravate
breathing conditions, to produce ozone at surface heights, which is
also an irritant, and to react with the oxygen present in the air, and
eventually can form nitric acid when dissolved in water [130].
When NOx is dissolved in atmospheric moisture it can result in acid
rain that potentially can damage entire forests. Some researchers
have sought to address the NOx issue by the addition of certain fuel
additives to the biodiesel. For example, Bradin described a fuel
additive composition including fatty acid alkyl esters and glyceryl
ethers. The additive-containing fuel is made by a multi-step process
that includes separation of glycerol from biodiesel, conversion of
glycerol to glycerol ether, and then addition of the glycerol ether
back into the biodiesel fuel [52]. Other researchers describe
controlling engine emission NOx by producing a blending of a
synthetic middle distillate derived from Fischer-Tropsch produced
synthesis gas (syngas) and biodiesel, which generate very low
levels of NOx and sulfur emissions when used in diesel engines
[131]. Addition of water to the fuel, to cool down the combustion
process reduces the formation of NOx as well. However, that
process undesirably lowers fuel BTU (British Thermal Units) value
by replacing fuel with water. A large number of materials have
been found to be catalytically active for selective catalytic reduction
of NOx. By far, the most widely reported in the open literature are
metal-exchanged zeolites, such as Cu-ZSM-5, Co-ZSM-5, and FeZSM-5. However, the zeolite-based materials lose much of their
activity when water (common in exhaust streams) is added to the
exhaust stream. While other catalysts that possess greater water
stability have been found, such as metal-supported oxides, these
tend to lack the desired activity shown by the metal zeolites, and
produce large amounts of N2O (also a major pollutant). Baacke et
al. described a zeolite catalyst and a method for reduction of
nitrogen oxides by mixing the waste gas with ammonia [132].
However, the use of ammonia in the reduction of nitrogen oxide is
less desirable, especially in transportation systems, due to the toxic
nature of the ammonia that must be stored locally, presenting a
On the other hand, Green et al. described a single phase catalyst
obtained by intimately mixing the powders of the oxide binder and
the metal salts. This patent describes the mixing of particles of a
metal exchanged zeolite with an oxide [133,134]. Also Sachtler and
Chen described that the metal can be introduced into the zeolite in
an entirely different way, such as by metal vapor deposition [135].
Recently advances in the use of cerium-oxide, have been described
to hold the potential to nearly eliminate NOx emissions from both
petrodiesel and biodiesel, and diesel fuel additives based on cerium
oxide that can improve fuel consumption by 11% in unmodified
diesel engines [136]. Recently, a catalyst for reducing nitrogen
oxide has been developed to remove NOx and particulate matter
using platinum as activating element [137].
4.3. Oxidation Stability
A different problem in using biodiesel is that since biodiesel has
a higher content of unsaturated fatty acid esters, it easily oxidizes in
the presence of oxygen, UV light, heat, or trace metals, such as iron
or copper. The products formed from this oxidation give rise to
sediment and gum formation within the fuel and lead to corrosion

32 Recent Patents on Biotechnology 2008, Vol. 2, No. 1

and plugging in injection pumps and fuel lines in engines, heaters,

and machines which utilize this fuel source. Consequently, there is
a need for a biodiesel fuel composition with improved oxidation
stability. Asbahr and Bomba described the use of phenyl
compound, and derivatives as an antioxidant for biodiesel [138].
The list of possible antioxidants was extended by Abou-Nemeh
Ibrahim, mainly consisting in aromatic derivatives such as 3,4,5trihydroxybenzoic acid n-propyl ester, 1,2,3-trihydroxybenzene,
butylated hydroxyanisole, 2,6-di-tert-butyl-1-hydroxy-4-methylbenzene, alpha-tocopherol acetate, alpha-tocopherol, gammatocopherol, delta-tocopherol, propyl gallate, dodecyl gallate, gallic
acid, octyl gallate, ascorbyl palmitate, pyrogallol, alpha naphthol,
natural tocopherol, eugenol, or 6-ethoxy-1,2-dihydro-2,2,4trimethylquinoline, although non aromatic compounds are included
as well such as dilauryl thiodipropionate, isopropyl 2-hydroxy-4methylthio butanoate, lecithin, stearyl citrate, palmityl citrate,
chlorophyll, ascorbic acid, citric acid, etc [139]. The use of
aromatic derivatives along with N- and S-containing antioxidants
can undermine the environmentally friendly nature of biodiesel as a
clean fuel by polluting the environment, an important reason for
focusing on the non aromatic and the non N- and S-containing
compounds as antioxidants.
4.3. Glycerol Production
The transesterification of oils and fats results in the generation
of glycerol as a byproduct of biodiesel. The free glycerol formed
must be removed from the mixture of esters, since glycerol can
cause damage to the engine [140]. Free and bonded glycerol, are the
main parameters for defining the quality of biodiesel. During the
transesterification process, free glycerol can easily be removed by
washing steps, whereas a low content of glycerides can only be
achieved by the use of specific catalysts and reaction conditions or
by further distillation of the product. A higher content of free
glycerol may cause problems during storage or in the fuel system,
due to separation of glycerol, or can lead to injector fouling or the
formation of higher aldehyde emissions. A higher content of
glycerides, especially triglycerides, may cause formation of
deposits at the injection nozzles, at the piston and at the valves
[141]. A simple method for the almost complete removal of
glycerol from methanol-free biodiesel streams coming out from
industrial transesterification reactors has recently been described by
Yori et al. The method is posed as a "dry" alternative to the
conventional "wet" methods involving water washing. It is based on
the use of silica beds and relies on the adsorption at room
temperature to retain the small amounts of glycerol dissolved in the
solutions of fatty acid methyl esters and adjust their content to the
quality standards for biodiesel fuel [142]. Nevertheless, the
difficulty for the removal of glycerol as by-product is mainly
depending on the technical process of transesterification (see Table
1). Once glycerol surplus is removed it can be used in different
ways as described previously.
Energy and climate change are the now main subjects among
the scientific community, and modern society. And there is broad
agreement that there is no single solution to these problems of
meeting future demands for energy and managing the
environmental consequences of energy production. The future
developments will consist of set of many different technologies.
Recent scientific advances focus on basic biology and technology
that can be applied to metabolic engineering in order to comply
with these needed energy production.
Biodiesel is a good alternative energy source and a substitute
for petroleum-based diesel fuel with the available technology. The
current scientific and technological knowledge can provide the
means to obtain a biofuel with net energy gain, producing
environmental benefits and at a cheaper cost than that for the

Manzanera et al.

petroleum-based diesel. However, different and combined protocols, apparatus and feedstock would be needed in order to produce
biodiesel in large quantities.
The most promising alternatives in order to obtain an
environmentally friendly product are based on technology with
biological origin especially those methods catalyzed by lipases.
This technology works at the lowest reaction temperature; it is not
influenced by the presence of water or free fatty acid in the
feedstock, hence reducing the production cost, and produces the
highest yields of biodiesel. In addition, the lipase technology allows
an easy recovery of glycerol, without the need of purifying the
methyl esters. It is of paramount importance to reduce the
production cost derived from the protein purification though.
Therefore, the use of whole microorganisms, as biodiesel cell
factories, with the ability to synthesize alcohol esters could save
this enzyme purification cost.
Steinbchel and coworkers have created a fuel-refining
Escherichia coli bacterial strain by modifying it with genes taken
from two other microorganisms, Zymomonas mobilis and
Acinetobacter baylyi [143]. The modified E. coli were cultured in
growth medium containing glucose and oleic acid, which the
bacteria processed into fatty acid ethyl esters, a diesel-substitute
named as "microdiesel". This was achieved by heterologous
expression in E. coli of the Zymomonas mobilis pyruvate
decarboxylase and alcohol dehydrogenase and the unspecific
acyltransferase from Acinetobacter baylyi strain ADP1. By this
approach Steinbchel et al., managed to obtain ethanol formation
and to combine it with a subsequent esterification of the ethanol
with the acyl moieties of coenzyme A thioesters of fatty acids.
Using this transgenic microorganism the obtained concentration of
FAEE was of 1.28 g/L and the FAEE content of the cells was of 26
% of the cellular dry mass, that were achieved by fed-batch
fermentation using renewable carbon sources.
This novel approach might pave the way for industrial
production of biodiesel equivalents from renewable resources by
employing engineered microorganisms, enabling a broader use of
biodiesel-like fuels in the future.
Unlike regular biofuel, microdiesel is produced without toxic
chemicals. Waste oils could allegedly be used as source of fatty
acids, reducing the need to grow crops specifically for biodiesel
production and therefore reducing the production cost of this fuel.
International energy agency
New York mercantile exchange
Intergovernmental panel on climate change
US National renewable energy laboratory
Fatty acid methyl esters
Fatty acid ethyl esters
Dissolved air flotation
Super critical fluid
British termal units
Maximino Manzanera was granted by Programa Ramn y Cajal
(MEC, Spain and ERDF, Euopean Union).

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