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The Application of Biotechnological Methods For The Synthesis of Biodiesel
The Application of Biotechnological Methods For The Synthesis of Biodiesel
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
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
801
Kinematic viscosity
at 407C [mm2/s]
Cetan no.
Lower heating
value [MJ/kg]
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
802
M. Adamczak et al.
803
Table 2. Synthesis of methyl esters of fatty acids by immobilized lipases from different oils
Lipase/producer
Substrate/solvent
Yield
[%]
Time
[h]
References
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
92
90
80
76
69.7
84
97
,100
.98
60
90
105
24
12
8
24
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M. Adamczak et al.
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]
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].
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.
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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
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M. Adamczak et al.
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
[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
.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
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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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