800

Eur. J. Lipid Sci. Technol. 2009, 111, 808813

Review Article
The application of biotechnological methods for the synthesis
of biodiesel
Marek Adamczak1, Uwe T. Bornscheuer2 and Wodzimierz Bednarski1
1
2

Department of Food Biotechnology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Greifswald, Germany

Synthesis of biodiesel is performed mainly by chemical catalysis, but can also be performed by enzymatic
or microbial methods, and these might play an important role in future substitution of petroleum-based
diesel. To discover sustainable, economically attractive biotechnological processes for biodiesel synthesis,
close cooperation between different disciplines is needed. Currently, lipases are the enzymes of choice for
the synthesis of fatty acid esters (FAE) from fats and oils, yielding biodiesel with the methyl esters (FAME)
as the most important product. More recently, the direct production of FAE using engineered whole cell
microorganism has also been described (MicroDiesel). Current enzymatic processes are still hampered by
the high costs of the biocatalyst, but significant progress has recently been made leading to the first
industrial enzymatic biodiesel production. Enzymatic biodiesel production is mostly attractive because of
the starting materials (waste frying oils, oils with high water content, etc.), for which conventional chemical
interesterification can hardly be applied.
Keywords: Biocatalysis / Biodiesel / Lipase / Microdiesel / Mycodiesel

Received: April 7, 2009; accepted: July 14, 2009

DOI 10.1002/ejlt.200900078

1 Introduction
According to the US Standard Specification for Biodiesel
(ASTM 6751-02), biodiesel is defined as a fuel comprised of
mono-alkyl esters of long-chain fatty acids derived from
vegetable oils or animal fats. This definition is also acceptable
in the European Union specification concerning biodiesel
(EN 14214). Stricter regulations define biodiesel as fatty acid
methyl esters (FAME), but current discussions might extend
this definition to fatty acid ethyl esters, which can be obtained
using bioethanol as alcohol donor. Lois presented legitimate
comments concerning methodology and accuracy of biodiesel
research [1].
Biodiesel has been reported as a possible substitute or
extender for conventional petrol-based diesel. It is compatible
with conventional diesel and can be used in its pure form or
blended in any proportion with diesel fuel. The properties of

Correspondence: Marek Adamczak, Department of Food Biotechnology, University of Warmia and Mazury in Olsztyn, J. Heweliusz St. 1, 10718 Olsztyn, Poland.
E-mail: marek.adamczak@uwm.edu.pl

2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

biodiesel obtained from different substrates and diesel fuels

are shown in Table 1. This biofuel offers several interesting
and attractive properties, e.g., biodegradability and non-toxicity, compared to petroleum-based diesel. The most important advantage of biodiesel, as a renewable material, is in
maintaining a balanced carbon dioxide cycle. Additionally,
biodiesel combustion results in reduced emission of carbon
monoxide, sulfur, aromatic hydrocarbons and soot particles
[2].
On the other hand, biodiesel synthesis is in conflict with
the utilization of oils as a food (and feed) compound, and its
use is hampered by the acreage required for oilseed crop production. Based on Nielsen [3] enzymatic biodiesel production
is not the answer to the worlds needs for sustainable fuel, but
rather a piece in the puzzle of future energy supply. Moreover,
production of substitutes for petroleum diesel requires the
utilization of readily available, bulk substrates such as cellulose. The production of biodiesel has a number of limitations,
e.g., it is restricted to particular seasons and areas producing
oilseed feedstock (just the plant seed oil is used for biodiesel
production and not the major part of the plant biomass, which
is in contrast to technologies such as biomass-to-liquid or
biogas production), biodiesel crystallizes at low temperatures,
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801

Table 1. Properties of biodiesel from different oils [4, 5]

Vegetable oil
methyl esters

Kinematic viscosity
at 407C [mm2/s]

Cetan no.

Lower heating
value [MJ/kg]

Cloud point [7C]

Flash point [7C]

Density [kg/L]

Rapeseed
Peanut
Soya bean
Babassu
Palm
Sunflower
Tallow
Diesel
Biodiesel blend (20%)

4.15-5.83
4.90
4.50
3.60
5.70
4.60

3.06
3.20

49.8-50.4
54.0
45.0
63.0
62.0
49.0

50.0
51.0

33.6
33.5
31.8
33.5
33.5

43.8
43.2

3-0
5
1
4
13
1
12

150-180
176
178
127
164
183
96
76
128

0.880-0.886
0.883
0.885
0.875
0.880
0.860

0.855
0.859

methanol is a toxic and hazardous chemical that is not

obtained from renewable sources, but is cheaper than (bio)ethanol.
Based on data from the European Biodiesel Board, the
production of biodiesel in 2007 (5.7 million tons) increased
by 16.8% compared to 2006. The capacity of biodiesel production on 1 July 2008 was estimated at 16 million tons (world
capacity 32.6 million tons). The main producer in Europe is
Germany (Figs. 1A and B).
Biodiesel can be synthesized by chemical, enzymatic
(Fig. 2A) and microbial methods (Figs. 2B and C). Alkalibased esterification is the most often applied approach [6].
However, there are some drawbacks such as problems in the
recovery of glycerol, the need to remove the catalyst, the
requirement to use oils without free fatty acids and high
energy consumption. A high level of interest from industry
initiated research concerning non-catalytic biodiesel synthesis
from rapeseed oil in supercritical methanol, which has a conversion of 80% (3507C, 4565 MPa, 240 s) [7]. Recently, the
application of ultrasound and hydrodynamic cavitation has
also been reported to significantly intensify the synthesis of
biodiesel [8, 9]. It was demonstrated that hydrodynamic cavitation was about 40-times more effective than acoustic methods [10].
The most important parameters that influence the transesterification reaction are: reaction temperature, ratio of alcohol to vegetable oil, type of acyl donor and acceptor, type and
amount of catalyst, mixing intensity, quality (purity, free fatty
acid content) of starting materials and water content [11].
With respect to enzymatic synthesis of biodiesel (and
equivalents), a number of reviews concerning different
aspects of enzymatic biodiesel synthesis have been published
recently [1216]. The first report about the enzymatic alcoholysis of sunflower oil, i.e., synthesis of methyl and ethyl
esters of fatty acids, was presented by Mittelbach [17]. The
application of lipases (acylglycerol hydrolases; EC 3.1.1.3)
allowed synthesis of biodiesel from rapeseed, soybean or palm
oil (the most important sources of vegetable oil in the world),
but also from raw materials of poorer quality, such as animal
2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Capacity and production of biodiesel in (A) selected EU

countries [1, Germany, 2, France, 3, Italy (included additional
capacities of hydrodiesel), 4, Austria, 5, UK, 6, Belgium, 7, The
Netherlands, 8, Greece, 9, Poland, 10, Portugal, 11, Bulgaria,
12, Sweden, 13, Slovakia] (http://www.ebb-eu.org/stats.php,
July 2nd, 2009) and (B) worldwide (Biodiesel 2020: A Global Market
Survey, 2nd Edition, http://www.emerging-markets.com/biodiesel/
default.asp).
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Eur. J. Lipid Sci. Technol. 2009, 111, 808813

Figure 2. Possible methods for biodiesel synthesis by (A) chemical or

enzymatic reaction using oils from
plants, microorganisms or algae; (B)
engineered microorganisms, and (C)
the newly discovered strain Gliocladium roseum (left: colorized environmental scanning electron microscope
photo; right: culture plate) (http://
plantsciences.montana.edu/ facultyorstaff/ faculty/strobel/strobel.html), an
endophytic fungus that produces
mycodiesel hydrocarbons.

fats, recycled restaurant oils, waste products and materials that

cannot be used as food [1821] (Table 2).
The current production scale of rapeseed oil and other
vegetable oils is not sufficient to satisfy industrial needs for
biodiesel synthesis, especially as these oils mostly serve as
basic material for food industry (edible oils, margarine production, etc.). The application of used rapeseed oil and other
waste plant oils for production of biodiesel could be possible,
and attractive, if proper procedures for oil purification are
applied, e.g., with carbon-based adsorbents, oxidized active
carbon, magnesium silicate [22, 23]. The high cost of feedstock, which equals more than 80% of the production cost of
biodiesel, is driving new technologies to use lower cost starting
materials. The previously mentioned substrate purification
procedures, and the application of heterogeneous catalysts
and lipases can change the economics of biodiesel synthesis
[24].
2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Another alternative for biodiesel synthesis would be the

use of non-conventional substrates, e.g., microbial oil [25, 26]
or microalgae oil [27], as their fatty acid composition matches
the parameters of biodiesel [28, 29]. The fatty acid composition of the substrate influences the viscosity of biodiesel; this
can be predicted and represents one of the most important
parameter influencing the atomization process [30]. Although
biodiesel cannot completely replace diesel fuel, there are several factors that have stimulated its utilization: higher production of vegetable oils and animal fats as well as waste fat utilization is possible; biodiesel can decrease a nations dependence on imported petroleum; biodiesel does not contribute to
global warming; and when added to the diesel fuel in amount
of 12%, it can result in ultra-low sulfur diesel [6].
This review aims to present the biotechnological methods
that can improve the availability of starting materials and
conditions of biodiesel synthesis.
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Application of biotechnological methods

803

Table 2. Synthesis of methyl esters of fatty acids by immobilized lipases from different oils
Lipase/producer

Substrate/solvent

Yield
[%]

Time
[h]

References

Lipozyme TL IM and Novozym 435

Lipozyme TL, Thermomyces lanuginosa
Novozym 435
Pseudomonas cepacia (protein-coated microcrystals,
PCMC)
Lipozyme IM-77
PS30 (Burkholderia cepacia)
PS30 (Burkholderia cepacia)
Novozym 435
Novozym 435
Novozym 435

Rapeseed oil/tert-butanol
Canola oil/
Palm oil/
Madhuca indica oil/

95
90
97
99

12
24
16

[54]
[55]
[56]
[57]

Soybean oil/
Palm kernel oil/
Coconut oil/
Soybean and rapeseed oil/Degummed soybean oil/
Cotton seed oil/tert-butanol

92.2
15
Trace
98.4
93.8
97

6.3
8
8
24
24
24

[58]
[59]
[59]
[35]
[60]
[46]

Chromobacterium viscosum
Novozym 435
Rhizopus oryzae (immobilized-whole cell)
Novozym 435
Lipozyme TL IM
Rhizopus oryzae (immobilized-whole cell)
Candida cylindracea
Alcaligenes sp.
Novozym 435

Jatropha oil /
Waste edible oil/
Jatropha oil/
Jatropha oil/
Waste cooking oil/
Acidified rapeseed oil/tert-butanol
Activated bleaching earth/diesel oil or kerosene
Activated bleaching earth/
Acid oil (by-product in rapeseed oil refining)/

[61]
[62]
[63]
[63]
[64]
[65]
[53]
[66]
[67]

Edible oils

Waste products/non-edible oils

2 Biocatalytic methods in the synthesis of

biodiesel
As biodiesel is essentially a fatty acid (methyl) ester
obtained from triglycerides (mostly plant oils), lipases (EC
3.1.1.3, triacylglycerol hydrolases) are the enzymes of
choice for the biocatalytic synthesis of this biofuel. Hence,
lipases are the most popular enzymes for the production of
biodiesel, but are also used in other areas involving lipid
modification such as the synthesis of structured lipids [31,
32]. The application of lipases is not primarily because
triglycerides are the natural substrates of these biocatalysts,
but related to the high stability of lipases under process
conditions and the availability of various enzymes on a
commercial scale at modest prices. Nevertheless, their use
in industrial production of biodiesel is challenging because
the FAME (biodiesel) must be very cheap and the margins
are rather small. Consequently, the lipase must show very
high stability and/or must be recycled many times to
reduce the costs of the catalyst, and hence the overall process, significantly to compete with chemical biodiesel production. Although lipases have been shown to allow transesterifications in organic solvents for months without significant loss of activity, the biodiesel synthesis is especially
challenging due to the strong inactivation effect caused by
methanol (see Section 2.1).
2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

92
90
80
76
69.7
84
97
,100
.98

60
90
105
24
12
8
24

2.1 Strategies of lipase-catalyzed biodiesel synthesis

Transesterification reactions catalyzed by lipases (Fig. 2A)
can be performed under mild conditions (30407C). The
small amounts of water and free fatty acids in raw substrates
do not influence on the progress of the reaction; also, the
recovery of glycerol is easy, and purification of the FAME is
usually not required. Additionally, the separation of the product and enzyme is facilitated.
The synthesis of fatty acid esters is usually performed in
low-water medium to avoid hydrolysis of the ester formed.
However, it has also been demonstrated that methyl esters of
oleic acid can be produced in aqueous medium at high water
activity (aw.0.9) in the presence of an enzymatic extract from
Candida deformans at yields of 5882% depending on the
substrate (oleic acid or triolein) used [33].
The transesterification reaction is a rather complicated
process and consists of three steps, each of which is reversible.
The catalysts of the reaction of transesterification are, for
example, lipases obtained from: Rhizomucor miehei, Rhizopus
oryzae, Candida antarctica, Candida rugosa, Pseudomonas
cepacia and Thermomyces lanuginosa, but the commercial
immobilized lipase B from C. antarctica (Novozym 435) is the
most commonly used enzyme (Tables 25). Unfortunately,
this lipase is probably the most expensive one available on the
market.
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Eur. J. Lipid Sci. Technol. 2009, 111, 808813

Table 3. Influence of acyl acceptors on the yield of alkyl esters

synthesis in reactions catalyzed by Candida antarctica lipase B
(Novozym 435)
Used acyl acceptor

Used oil

Yield [%]

References

Dimethyl carbonate
Ethyl acetate
Propanol-2-ol
Methyl acetate
Ethyl acetate

Cottonseed oil
Crude sunflower oil
Crude sunflower oil
Refined soybean oil
Soybean oil

96.4
92.7
93.4
92
63.3

[37]
[70]
[71]
[72]
[73]

Shimada et al. [34] confirmed that methanol added to the

fatty acids in a molar proportion higher than 0.5 (methanol:fatty acids, mol:mol) quickly inactivates immobilized
lipase B from C. antarctica (Novozym 435). A higher level of
methanol is not soluble in oil and is present in the form of
droplets in the oil. However, most of the reactions are performed under conditions in which alcohol is added in more
than stoichiometric amounts, which causes a surplus of alcohol and the risk of enzyme inactivation. This problem has
been successfully overcome by the stepwise addition of small
amounts of alcohol [3436]. The addition of an organic solvent can improve the yield of the reaction, but from an industrial point of view this reaction needs to be performed in a
solvent-free medium.
Instead of using methanol, the lipase-catalyzed synthesis of
FAME can also be performed using alternative alcohol donors
such as methyl (alkyl) acetate or dimethyl carbonate (Fig. 3,
Table 3). Dimethyl carbonate is an alternative acyl acceptor that
is neutral, cheap, and non-toxic. The process of such a biodiesel
synthesis is irreversible because the intermediate compound
(carbonic acid monoacyl ester) immediately decomposes to
carbon dioxide and an alcohol (Fig. 3A) [37]. The application
of methyl acetate as acyl acceptor enhances the stability of
lipase, because triacetin, instead of glycerol, is produced, and its
influence on the activity of lipase is less negative than free glycerol (Fig. 3B) [38, 39]. Moreover, triacetin is an important byproduct with a higher value than glycerol, and can be applied in
the synthesis of alkyl esters. However, this reaction requires a
higher amount of lipase (three times more than alcoholysis) and
an excessive amount of methyl acetate (1:12 of oil:methyl ace-

tate). It has been shown that after 100 batches there was almost
no loss of activity of Novozym 435 [40].
Nevertheless, according to the definition of biodiesel,
alcohols other than methanol can be used for the synthesis of
biofuel. The most popular is ethanol, but other primary and
secondary alcohol, e.g., propanol, butanol, 2-butanol, isopropanol [41] are also used. Lee et al. [42] indicated that
branched-chain esters reduced the crystallization temperature
of biodiesel. In this respect, research should concentrate on
the selection of lipases specific to branched-chain alcohols
[43] or branched-chain derivatives of esters [44].
Improvement of the enzymatic synthesis of biodiesel can
be achieved through the application of tert-butanol as a solvent
for enzymatic reaction or washing with a solvent for enzyme
regeneration [4548]. It has been found that, with the application of tert-butanol as reaction solvent, Lipozyme TL IM
could be used without loss of lipase activity for 200 batches
[49] (Table 4). The worlds first large-scale biodiesel plant
using enzyme technology is operating in China with a capacity
of 20 000 metric tons per year using tert-butanol as co-solvent,
which protects the enzyme and enables very high productivity
[3].

2.2 Selected examples of lipase-catalyzed biodiesel

synthesis
Lee et al. [50] indicated that the problem of biodiesel synthesis
is connected with lipase selectivity. When they used lipase
from R. oryzae (a sn1,3-regioselective lipase) or C. rugosa
(non-selective) in separate experiments, the yield reached at
maximum 70%. A combination of both enzymes, however,
enabled conversions of .99% within in 21 h.
The enzymes used for biodiesel synthesis can be extra- or
intracellular and mostly immobilized biocatalysts are used.
However, free lipase NS81006 from a genetically modified
Aspergillus oryzae was utilized as the catalyst for the effective
synthesis (yield .94 6 1%) of biodiesel from soybean oil [51].
Non-immobilized lipase from Candida cylindracea (now C.
rugosa) showed the highest conversion, 78 6 6%, in n-hexane
with waste vegetable oil absorbed in activated bleaching earth
[52] and 97% when diesel oil or kerosene was used as a solvent
[53].

Figure 3. Synthesis of biodiesel (fatty acid esters) by transesterification of a triglyceride with (A) dimethyl carbonate, and (B) methyl acetate as an acyl acceptor. Note that the reaction with dimethyl carbonate requires water and yields methanol, CO2 and glycerol as byproducts, whereas with methyl acetate triacetin is formed. In case (A), the equilibrium can be shifted more easily to product formation, but
in case (B) this is less facilicated.

2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Application of biotechnological methods

805

Table 4. Influence of the solvent on the yield of alkyl esters synthesis

Enzyme

Solvent

Substrate

Novozym 435
Lipozyme TL IM 1 Novozym 435
Lipozyme TL IM
Novozym 435
Porcine pancreatic lipase (free enzyme)
Porcine pancreatic lipase
(immobilized enzyme)
Candida sp. 99-125
Chirazyme L-2
Lipozyme IM TL1 Novozym 435
Novozym 435
Pseudomonas cepacia
(immobilized enzyme)

t-Butanol
t-Butanol
t-Butanol
Tetrahydrofuran
n-Hexane
n-Hexane

Cottonseed oil
Rapeseed oil
Rapeseed oil
Palm oil
Salicornia oil
Salicornia oil

n-Hexane
t-Butanol
-

Lard
Lard (after fractionation with acetone)
Lard
Degummed soybean oil
Restaurant grease

Temperature
[7C]
50
35
40
25
25

Yield
[%]
97
95
,80
97
45
55

References
[46]
[45]
[49]
[80]
[81]
[81]

40
30
50
30
40

88
74a)
97.2
93.8
60-97

[82]
[83]
[84]
[85]
[86]

a) Conversion

The application of liquids that are ionic at room temperature liquids as a substitute for volatile and toxic organic solvents could make the process of biodiesel synthesis more
environmentally friendly. Ha et al. [68] presented, for the first
time, the application of ionic liquids as a solvent in the production of biodiesel. The highest FAME synthesis by Novozym 435 was achieved in [Emim][TfO], and the yield of the
reaction was eight times higher than in a solvent-free system
and about 15% higher than in a system with tert-butanol.
Recently, Caballero et al. [69] published the preparation of
mixtures of FAMEs and monoacylglycerols (2:1, mol:mol), in
a reaction catalyzed by free or immobilized pig pancreatic
lipase without formation of free glycerol. The authors believe
that this novel biofuel denoted as Ecodiesel-100 has similar
physicochemical properties to petrodiesel or conventional
biodiesel. However, the properties of this new product have to
be confirmed and the low efficiency of the immobilized enzyme needs to be improved.
In some cases, different pretreatment procedures of
lipases affected the progress of methanolysis, e.g., pre-incubation of Novozym 435 in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h resulted in .97% yield
within 3.5 h [74].
The application of intracellular lipases may considerably
reduce the purchase costs of enzymes, and whole-cell lipase
from R. oryzae is frequently used [75, 76]. Whole-cell immobilized lipase can be obtained by simple separation of the cells
and permeabilization by different procedures, e.g., air drying,
acetone treatment [15] or in situ immobilization and acetone
treatment [77]. The activity of whole-cell biocatalysts depend
on the fatty acid composition of the cell-wall membrane, and
in whole-cell biocatalysts there may be different lipases bound
to the cell wall or membrane [78]. The stability of a complex
biocatalyst can be improved by the method used to obtain
active immobilized biocatalysts, but the yield of biodiesel synthesis was 70% to over 90% [15, 79].
2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Besides the progress in enzymatic synthesis of biodiesel

presented in many publications, some issues still remain
unsolved, e.g., there is evidence that the stability of lipases in
oils should be improved, but there is no correlation between
the stability of lipases and the results of parameters conventionally used to characterize oil quality, e.g., peroxide
value, acid value, etc. [87, 88]. To eliminate the so-called
detection effect in establishing enzymatic reaction conditions
for biodiesel synthesis, high-throughput screening methods
for determination of fat quality [89] and a unique method for
determination of synthetic activity of hydrolases [90] can be
used.
On the other hand, many statistical methods [84, 91, 92],
kinetic analysis, and modeling [9396] to optimize the process
of enzymatic synthesis of biodiesel have been published, but
only a few provide information concerning reactors design
and operation conditions, especially for enzymatic reactions
(Fig. 4).
There are traditional, conventional methods for improving
lipase stability by immobilization including using nanoparticles (Table 5) and the application of reaction medium
engineering methods [97, 98], but current molecular biology
methods represent the most important methodology available
to obtain tailor-made enzymes [99, 100]. Two approaches are
possible for obtaining better biocatalysts: rational protein
design and directed evolution methods [101103]. New
enzymes suitable for application in biodiesel synthesis can be
discovered using metagenome approaches. Metagenomics is
the culture-independent genomic analysis of microbial communities, and isolated environmental DNA can be used for
cloning and expression of enzymes and metabolites that cannot be obtained by traditional methods of cultivation [104,
105]. Unfortunately, there are only a few examples of the
application of these methods for obtaining lipases that are
attractive for biodiesel synthesis. Tanino et al. [106] constructed yeast whole cells displaying C. antarctica CBS 6678
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Eur. J. Lipid Sci. Technol. 2009, 111, 808813

Figure 4. Examples of the reactors designed for synthesis of biodiesel: (A) continuous biodiesel reactor (http://www.ascof.com/BiomassBriquette-Machine/Continuous-Biodiesel-Reactor-for-Biodiesel-Production-Equipment.html); (B) oscillatory flow reactor (OFR) at University of Cambridge (http://www.cheng.cam.ac.uk/research/groups/polymer/RMP/arjan/ biofuels.html), (C) reactor designed for batch
and continuous enzymatic synthesis of biodiesel (Department of Food Biotechnology, Olsztyn, Poland).
Table 5. Examples of application of immobilized lipases designed for synthesis of alkyl esters
Lipase

Immobilization method

Yield [%]

References

Thermomyces lanoginosus (Lipozyme TL)

Porcine pancreatic

Covalent binding to magnetic Fe2O4 nanoparticles

Covalent binding to chitosan beads

[107]
[81]

Pseudomonas cepacia
Rhizopus oryzae
Candida antarctica or Burkholderia cepacia
Rhizopus oryza cells
Candida rugosa
Palatase 20000L (NOVO)
Candida sp. 99-125
Candida sp. 99-125

Phyllosilicate sol-gel matrix

Cross-linked polystyrene with N-methylglucamine
Encapsultion in silica areogel
Biomass support particle
Chitosan
Encapsulation by sol-gel method
Immobilization on textile cloth
Immobilization on fiber cloth

Thermomyces lanuginosus (Lipozyme TL)

Microporous polymeric matrix

(powdered)

.90
55
(45 free enzyme)
60-97
2.5 times more active than free enzyme
56a)
80b)
64
77 (productivity 6 times higher than free enzyme)
91
83 (transesterification)
92 (esterification)
97

[86]
[97]
[98]
[108]
[109]
[110]
[111]
[112]
[113]

a) Conversion.
b) Methyl esters content.

lipase B that was more active in ester synthesis (biodiesel efficiency was not analyzed) than the well-known commercialized
C. antarctica lipase B (GenBank Z30645).
Enzymatic methods of biodiesel synthesis can be also
applied in combination with chemical methods to improve the
yield of the reaction and to avoid enzyme inactivation. Haas
and Scott [114] applied a two-step process, which involved (i)
chemical transesterification of fatty acids linked to glycerol
and phosphoglycerols, and (ii) enzymatic esterification of free
fatty acids, for synthesis of alkyl esters from soapstock. The
2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

yields were 90 and 81%, respectively. The best enzyme for

esterification was Novozym 435. The most important problems that have to be solved to improve the reaction yield are
the high water content in soapstock (dehydratation was
required) and the viscosity of the reaction mixture. This was
applied in a hybrid process consisting of enzymatic hydrolysis
of soybean oil in the first step, and an acid-catalyzed esterification in the second step [115]. Biodiesel formation was
99% after 12 h at 507C, but the reaction required a high
methanol excess (1:15, mol:mol).
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Eur. J. Lipid Sci. Technol. 2009, 111, 808813

3 Non-conventional methods and starting

materials for biodiesel synthesis
Many microorganisms, e.g., Cryptococcus curvatus, Lipomyces
lipofera, Lipomyces starkeyi, Rhodotorula glutinis, Yarrowia
lipolytica, and bacteria belonging to the actinomycetes group
such as species of Mycobacterium, Rhodococcus, and Nocardia,
are able to synthesize intracellular triacylglycerides [25, 26,
116]. The content of fat in these oleaginous microorganisms
can reach over 80% of the cellular dry weight [117]. A wide
range of substrates (carbon source) are utilized by microorganisms for triacylglyceride synthesis, e.g., sugars, organic
acids, alcohols, oils, and different waste products, such as
whey and agro-industrial waste (hydrocarbons, plant oils,
sugar-cane molasses, wheat bran, vegetable and fruit waste,
etc.) [118]. The utilization of these substrates makes the process very attractive and possibly economically acceptable
(Fig. 2A). Genetic engineering is also applied to improve fatty
acids or biodiesel synthesis in E. coli by improving the acetylCoA biosynthetic pathway (i.e., by cloning of acetyl-CoA
carboxylase genes, malonyl-CoA synthetase, a malonate
transporter from Rhizobium, and thioesterase enzyme from
plant) [119].

Application of biotechnological methods

807

Based on the results of the analysis of triacylglycerides and

synthesis of wax esters by microorganisms, an alternative for
synthesizing ethyl esters of fatty acids (called microdiesel) by
microorganisms was discovered by the Steinbchel group
[120] (Fig. 2B). They identified a new bifunctional enzyme
with a broad specificity towards fatty acids and alcohols,
exhibiting acyl-CoA:fatty alcohol acyltransferase (wax ester
synthase, WS) and acyl-CoA:diacylglycerol acyltransferase
(DGAT) activity. This bifunctional enzyme, WS/DGAT, is
essential for the synthesis of storage lipids in Acinetobacter
calcoaceticus ADP1 [121]. By heterologous expression of pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis and acyltransferase from Acinetobacter baylyi
ADP1 (cloned in the plasmid, pmicrodiesel), a metabolically
engineered E. coli strain was obtained (Fig. 5) [122]. Ethanol
formation was followed by its esterification with acyl moieties
of CoA thioesters of fatty acids under aerobic conditions in the
presence of glucose and oleic acid. Although they could
demonstrate that this new principle works, the fatty acid ethyl
ester content of only 1.28 g/L requires substantial further
improvement to be economically competitive to chemical or
biocatalytic routes for biofuel production. This includes
beside productivity enhancement issues such as intracellular

Figure 5. Pathway of fatty acid ethyl esters (microdiesel) synthesis

in recombinant E. coli. PdcZm, pyruvate decarboxylase; AdhZm,
alcohol dehydrogenase, both from Zymomonas mobilis; WS/
DGATAb, broad specific acyltransferase from Acinetobacter baylyi;
CoA, coenzyme A [120].

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M. Adamczak et al.

microdiesel separation and product isolation. The synthesis of

fatty acids de novo from sugars by genetically engineered E.
coli has also been suggested [123].
Last year, the Strobel group [124] described a newly isolated Gliocladium roseum NRRL 50072 strain [125] that produces a mixture of hydrocarbons (named mycodiesel) identical to those found in diesel fuel (Fig. 2C). The strain G.
roseum was isolated as an endophyte of Eucryphia cordifolia
(ulmo) from Northern Patagonia (Chile) during the search
for natural producers of volatile antibiotics. The synthesis of
hydrocarbons was possible under microaerophilic conditions
on the oatmeal- or cellulose-based medium. An important
finding was that G. roseum was able to utilize cellulose a
renewable resource for the production of hydrocarbons.
Unfortunately, the yield of hydrocarbon synthesis was lower
than those found on the oatmeal medium, probably because of
the common problems with low rate of cellulose digestion.
This new microorganism could be a valuable source of information for genetic or metabolic engineers to improve or
transfer the knowledge to other more efficient microorganisms.
Microalgae present a more mature alternative. They are
photosynthetic microorganisms that convert sunlight, water
and carbon dioxide into algal biomass. Many of them are rich
in oil content, which can exceed 80% of the dry weight of algae
biomass [126] (Fig. 6). Dismukes et al. [127] extended the
substrates for biodiesel synthesis to aquatic microbial oxygenic photoautotrophs (AMPOs) and added to this group
beside algae, cyanobacteria and diatoms. As in the case of the
microbial product, the oil synthesized by algae is often rich in
polyenoic fatty acids, which makes these oils an attractive
source of fatty acids rather for human nutrition than for biodiesel production due to the inherent low stability of poly-

Eur. J. Lipid Sci. Technol. 2009, 111, 808813

unsaturated fatty acids. To improve the oxidative stability of

this biodiesel, the addition of antioxidants [128], e.g., BHA or
TBHQ was recommended (loadings up to 3000 ppm) for
safeguarding biodiesel from effects of autoxidation during
storage [129].
Recently, Chisti [27, 130, 131] initiated a discussion on the
application of algal oil for biodiesel production. In his opinion,
microalgal biodiesel is the only renewable biodiesel because of
the high productivity (in a tropical zone it can reach 1.535 kg
m3 d1), high oil yield and high biomass doubling time
(3.5 h). To fulfill transport requirements in United States for
biodiesel, about 111 million hectars would have to be used for
growing oil palm but just 5.4 million hectars for microalgae. Li
et al. [132] indicated that it is feasible to expand heterotrophic
Chlorella fermentation for biodiesel production on an industrial scale. The biodiesel production rate ranged from 7.02 g/L
to 6.24 g/L in 5-L and 11 000-L bioreactors, respectively.
Heterotrophic algae are easy to cultivate, but they require
cheap organic carbon source for oil accumulation. Based on
the analysis of the biomass growth, oil content and oil yield
(106.5 g/L, 67.5%, and 71.89 g/L, respectively), Rhodosporidium toruloides Y4 was found to be a very attractive
triacylglyceride producer for biodiesel synthesis [25].

4 Conclusions
In general, research concerning biofuels will be aimed at
improving biomass utilization and discovering the best fuel
molecule(s). The fuel of the future in many opinions will
probably be: liquid, inexpensive, highly combustible and
energetic, stable over long-term storage and transportable by
pipeline. The utilization and substitution of diesel by biodiesel

Figure 6. Algae farms: (A) PetroSun facility opened in April 2008 (Rio Hondo, Texas, Picture from Google Earth) (http://www.petrosuninc.com/algae-biofuels.html). (B) An open pond for growing algae in Israel (Seambiotic) (http://news.cnet.com/8301-11128_39973649-54.html?tag=mncol;title).

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Eur. J. Lipid Sci. Technol. 2009, 111, 808813

requires cooperation in the area of biotechnology, chemistry,

engineering, etc. An exchange of knowledge as proposed by
Wackett could help in cooperation and updating the research
(http://www.biofuelsdatabase.org) [133]. The progress in
biodiesel synthesis requires, among others, improved catalyst
activity and stability, application and improvement of the
conditions of non-edible substrate application, and better
methods of utilizing by-products and glycerol obtained after
biodiesel synthesis. Combined processes of biodiesel synthesis
and production of 1,3-propanodiol by Klebsiella pneumoniae
from glycerol [134] or the utilization of glycerol by algae
for polyunsaturated fatty acids synthesis [135] have been
described. On the other hand, it seems that the research concerning biodiesel does not fully takes advantage of what has
been already done, e.g. in the subject of enzyme immobilization [136138]. More systematic work should be performed to
solve the problems in biodiesel synthesis enabling further
improvements in this area.
There is also the option to utilize solid residues, from
rapeseed after biodiesel production to obtain second generation biofuels to establish integrated process conditions [139].
The application of biotechnology in the synthesis of biodiesel
should cover the improvement of microdiesel synthesis and
medium engineering as well as the application of protein
engineering, directed evolution, and metagenome resources
for further improvement of process efficiency [99, 105]. Further progress and standardization of methods of biodiesel
analysis is also expected [140143].
The initiation of a large project last year by Novozymes A/
S for the enzymatic synthesis of biodiesel is a signal that points
in this direction (http://www.biodieselmagazine. com/article.jsp?article_id=2416).
In addition to the use of vegetable oil production and waste
fat utilization, research efforts should also be intensified into
the use plant materials such as starch, lignin, cellulose and
hemicellulose.

Conflict of interest statement

The authors have declared no conflict of interest.

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