Eur. J. Lipid Sci. Technol.

106 (2004) 809814

DOI 10.1002/ejlt.200401051

809

Rudolf Brenneis
Burkhard Baeck
Gerd Kley

Alcoholysis of waste fats with 2-ethyl-1-hexanol

using Candida antarctica lipase A in large-scale tests

Federal Institute for Materials

Research and Testing (BAM),
Berlin, Germany

Commercial native lipase A from Candida antarctica was used to produce alkyl esters
through the alcoholysis of (waste) fats with 2-ethyl-1-hexanol. The process was carried
out in batch stirred tank reactors (from 100 mL up to 3000 L).
The content of alkyl esters in reaction mixtures was determined by gradient HPLC
using an evaporative light scattering detector and the reaction progress was controlled
by determining the ratio of the palmitic acid ester peak area to the oleic acid ester peak
area in HPLC chromatograms.
The results show that alcoholysis is the favoured reaction in presence of excess water
and water-insoluble alcohols in comparison with hydrolysis (fatty acid content ,5%).
The optimum amount of water for the alcoholysis was found to be 80100% of the
amount of fat. In the presence of low quantities of water both alcoholysis and hydrolysis are slow.
Conversion rate increases with increasing temperature to 6570 7C.
Based on these results a large-scale test to produce 3000 L of alkyl ester (to be used as
lubricant coolant) was carried out. The experiments have proved that alcoholysis is
completed after about 710 h depending on temperature.

1 Introduction
The direct conversion of triacylglycerols (TAG) by lipasecatalysed alcoholysis in alkyl esters (AE) and glycerol is of
increasing practical interest. This process can be used to
synthesise value-added products, for instance biodiesel
or biodegradable lubricants (ester oils).
Ester oils have many technological and ecological advantages over petroleum oils in certain fields of (total loss)
lubrication. But the conventional production of these
esters is expensive which makes an extensive use prohibitive. Lower-cost feedstock, such as restaurant grease
or tallow, would be more advantageous to be used.
The goal of the work performed was to develop a biotechnological process to produce ester oils from waste fat
[1]. In [2] we reported the lipase-catalysed alcoholysis of
various model fats with different linear, branched-chain
and cyclic water-insoluble monohydric alcohols by lipases from Candida antarctica, A-component. We also ex-

Correspondence: Rudolf Brenneis, Bundesanstalt fr Materialforschung und -prfung (BAM), Richard-Willsttter-Str. 11,
12489 Berlin, Germany. Phone: 149-30-6392-5849, Fax: 14930-6392-5917; e-mail: rudolf.brenneis@bam.de

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

amined the connections of fatty acid composition and

alcohol structure on the alcoholysis specificity of this
lipase. There is certain specificity for the alcoholysis of
saturated fatty acids and branched-chain alcohols,
especially 2-ethyl-1-hexanol. Furthermore the alkyl esters
of this low-cost water-insoluble alcohol have physical
characteristics (density, viscosity, low-temperature performance) similar to petroleum oil-based lubricant coolants and the evaporation loss of 2-ethyl-1-hexyl fatty acid
esters is very low (flash point about 240 7C) [2].
The substrate specificity of Candida antarctica (A-component) has been used for the analytical assessment of
the state of lipid modification using high-performance
liquid chromatography (HPLC).
Enzymatic alcoholysis of TAG with or without the use of
organic solvent has been reported in a number of publications [311]. Alcoholysis with relatively long-chain and
branched alcohols proceeds efficiently even in organic
solvent-free systems.
In this work effects of different parameters, such as optimum amount of water, on alkyl ester production have
been studied. The common opinion about the influence of
water is that, under natural conditions in absence of water
www.ejlst.de

Research Paper

Keywords: Enzymatic alcoholysis, Candida antarctica lipase A, HPLC analysis, ester

oil, large-scale test.

810

R. Brenneis et al.

Eur. J. Lipid Sci. Technol. 106 (2004) 809814

or its presence in trace quantities, alcoholysis is favoured,

while hydrolysis of TAG occurs in the presence of excess
water. Our investigations have shown that alcoholysis is
the typical lipase-catalysed reaction in aqueous media, in
which lipase A from Candida antarctica is dissolved (in
presence of water insoluble alcohols).
Though alcoholysis is the predominant reaction compared
to hydrolysis, water promotes the unfavourable formation
of fatty acids (,5%).
Recently, a large-scale test has been carried out to produce 3000 L of ester oil for testing it as a lubricant coolant
for metal cutting.

2 Materials and methods

Candida antarctica (A-component). The lipase is nonposition specific, the activity level can vary from batch
to bach, but it will typically range around 5 KLU/g [12].

2.3 Apparatus
Most of the lipase-catalysed alcoholysis experiments
were carried out in 100-mL cylindric glass tubes of an
Apparatus for determination of demulsibility characteristics of petroleum, oils or synthetic fluids (Herschel Stirring Method based on ASTM D 401-DIN
51599-FTM 7913201-ISO 6614) by Petrotest Instruments (Dahlewitz, Germany). In this misused tank
reactor the components were stirred with a blade mixer
(1600 rpm) and their temperature was kept constant by
a water bath.

2.1 Materials
2-Ethyl-1-hexanol was obtained from Merck-Schuchardt
(Hohenbrunn, Germany) and European Oxo GmbH
(Oberhausen, Germany).
Recycled restaurant grease was supplied by Kanzler &
Co. (Groalmerode, Germany) = F1 and by B. Vierhouten
BV (Ermelo, Netherlands) = F2, and model fat (mixture of
vegetable fats on the basis of palm and peanut oils) was
supplied by Gerlicher (Berlin, Germany) = F3. The fatty
acid compositions of these input fats are shown in Tab. 1.
TAG is favourable to use with a low proportion of polyunsaturated chains as starting material, because the
processes of autoxidation and photo-oxidation as well as
polymerisation must be prevented or inhibited since they
lead to the deterioration of ester oil products.
Glycerol was purchased from Merck (Darmstadt, Germany).
Tab. 1. Fatty acid compositions of the input fats.
Fatty acids

F1

F2

Furthermore 1.5-L-, 15-L-, 100-L- and 3000-L-scale

batch stirred tank reactors were used for the lipase-catalysed investigations.

2.4 Preparation tests of AE from the TAG

(investigations in the 100-mL-reactor)
In these investigations the reaction mixture consisted of
TAG:alcohol from 1:31:3.1 (molar ratio) and 1 wt-%
Novozym 868 L in relation to TAG. The amount of water
(e.g. Fig. 2), the amount of glycerol in water (e.g. Fig. 3)
and the temperature (e.g. Fig. 4) were varied.
Lipase preparation (enzyme 1 water) was added to the
substrate solution (TAG 1 alcohol) to start the reaction. At
a certain time the mixer was stopped. The lipase in the
sample was denaturated by heating it to 90 7C for 15 min.
The ester phase (above) is well separated from the
water/glycerol phase (below) during this time and is
suitable for the HPLC-analysis.

F3

[%]
C16:0
C18:0
C18:1
C18:2
C18:3

48.1
1.2
44.2
5.1
0.4

48.4
11.8
35.1
3.5
0.8

54.0
8.7
33.7
2.1
0

2.5 Preparation of AE from the TAG (production

in larger reactors)

Novozym 868 L (new product: Novozym 735) from

Novo Nordisk (Bagsvaerd, Danmark) is an experimental
liquid preparation of a highly thermostable lipase from

Batch reactions were performed at 5055 7C in the

stirred tank reactors. First, a solution of TAG and 2ethyl-1-hexanol (molar ratio 1:31:3.1) was prepared.
Next, the main contingent of water was added. Finally,
when the reaction temperature was achieved, a solution of lipase (1 wt-% in relation to TAG) and residual
water was added to the mixture to start the enzymaticcatalysed conversion. Progress of the alcoholysis was
followed by taking samples for HPLC at selected time
intervals.

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.ejlst.de

2.2 Enzymes

Eur. J. Lipid Sci. Technol. 106 (2004) 809814

Alcoholysis of waste fats

811

2.6 HPLC analysis

3 Results and discussion

The content of AE and TAG as well as di- and monoacylglycerols (DAG, MAG) and free fatty acids (FFA) in
reaction mixtures was determined by gradient HPLC
(degasser DG-1310, liquid chromatograph LC-10AD,
diode array detector SPD-M10A, column oven CTO-10A,
system controller SCL-10A) from Shimadzu (Kyoto,
Japan), dynamic mixing chamber from Knauer (Berlin,
Germany) and a standard column (C18) EC 250/4 Nucleosil 1203 from Macherey-Nagel (Dren, Germany) with an
evaporative light scattering detector Sedex 55 from
S.E.D.E.R.E. (Alfortville, France). Purified air was used as
carrier gas at constant pressure (200 kPa); the column was
held at 35 7C.

Fig. 1A shows the HPLC chromatogram of a fat (recycled

restaurant grease) as an example, with the typical peaks
of TAG (retention times: 2736 min) and small peaks of the
decomposition product DAG (retention times in the range
of 1516 min).
Figs. 1B1D show HPLC chromatograms obtained from
the analysis of the following mixture: 500 g of used recycled grease, 217 g of 2-ethyl-1-hexanol, 500 g of water,
5 g of enzyme depending on the reaction time.

The samples (about 0.03 g) were immediately diluted with

25 mL acetonitrile/dichloromethane (50:50, v/v) from Promochem (Wesel, Germany). Separations were made of a
flow rate of 1.3 mL/min. In Tab. 2 a detailed elution scheme
is shown.
Peak areas were calculated using Cass VP 5 software
from Shimadzu (Duisburg, Germany).
The lipase A from Candida antarctica was shown to convert saturated fatty acids in TGA at a higher rate as compared to unsaturated fatty acids. Palmitic and oleic acids
are the dominant fatty acids in the fatty acid composition
(Tab. 1). Based on the substrate specificity it is possible to
determine the state of lipid modification by the palmitic/
oleic acid value. Palmitic/oleic acid value, simply defined
as the ratio of the palmitic acid ester peak area to the oleic
acid ester peak area in the HPLC chromatogram, was
used as the measure of reaction progress. A low palmitic/
oleic acid value indicates a better conversion rate than a
high palmitic/oleic acid value assuming identical starting
materials.
Additionally, the liberated fatty acids were titrated with
0.1 M KOH in all tests.

Tab. 2. Elution scheme for HPLC investigations.

Time
[min]

Acetonitrile#
[%]

Dichloromethane##
[%]

Methanol##
[%]

0
30
45
60

100
30
10
0

0
35
45
55

0
35
45
45

Acetonitrile (Promochem, Wesel, Germany) 1 0.12%

trifluoroacetic acid (Merck, Darmstadt, Germany).
Dichloromethane and methanol from Promochem
(Wesel, Germany).

Fig. 1. HPLC chromatograms (retention times 540 min) of

the reaction mixture (500 g used recycled restaurant grease
F1, 217 g 2-ethyl-1-hexanol, 500 g water, 5 g enzyme) after
reaction time 0 min = A, 15 min = B, 45 min = C, 270 min = D.
1.5-L stirred tank reactor, 50 7C, 500 rpm, and paddle mixer.
(Retention time: 2-ethyl-1-hexyl oleic acid ester = 11.3 min,
2-ethyl-1-hexyl palmitic acid ester = 11.6 min).

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.ejlst.de

##

812

R. Brenneis et al.

Fig. 1B illustrates the high conversion rate of the enzymatic

alcoholysis after 15 min. Based on the retention times of alkyl
esters (2-ethyl-1-hexyl linoleic acid ester: 8.9 min, 2-ethyl-1hexyl oleic acid ester: 11.3 min, 2-ethyl-1-hexyl palmitic acid
ester: 11.6 min, 2-ethyl-1-hexyl stearic acid ester: 14.4 min)
it is in principle possible to determine the quantities of this
ester components. But as the detection of intermediate DAG
and MAG also takes place in this range of retention time it is
advantageous to determine the palmitic/oleic acid value
(= 4.98). The TAG content was reduced to 5.9%. After 45 min
(Fig. 1C) the palmitic/oleic acid value was decreased to 2.28
and the TAG content was 1.3%. After 150 min the TAG content was 0%, but the palmitic/oleic acid value still keeps
changing. The HPLC chromatogram in Fig. 1D (reaction time
= 270 min) indicates that the amount of TAG is 0% and the
palmitic/oleic acid value (1.08) is constant in comparison
with the palmitic/oleic acid values of the next samples
(300 min, 330 min, 360 min) and with the palmitic/oleic acid
value of the fatty acid composition of the input fat. This
means that the reaction is finished.
2-Ethyl-1-hexanol is a chiral compound that exists in the
form of two enantiomers. The theoretical yield of each
enantiomer never exceeds 50%. Figs. 1AD show a bio-

Eur. J. Lipid Sci. Technol. 106 (2004) 809814

catalytic conversion which leads to the formation of a
product with a nearly 100% theoretical yield from a racemate. As far as the enantioselectivity of Candida antarctica lipase A is concerned the high conversion suggests
no preference towards any of the alcohol enantiomers,
i.e. the high efficiency is a result of the exceptionally high
tolerance of this lipase.
Several factors such as the amount of water and glycerol
or temperature can affect the alkyl ester production.
Fig. 2 demonstrates the importance of a certain water
content on lipase-catalysed alcoholysis. It can clearly be
seen that the reaction rate for hydrolysis (amount of free
fatty acids ,5%) is quite low in comparison with the
alcoholysis.
Alcoholysis is slow in the presence of low quantities of
water. This may be interpreted by a low enzyme mobility
due to the water/enzyme-in-oil/alcohol-emulsion on the
one hand, and an enrichment of the product glycerol at
the enzyme surface on the other, which leads to a blockage of active sites (see Fig. 3). When higher water contents are there it is possible to remove the glycerol from

Fig. 2. TAG and FFA contents and palmitic/

oleic acid value against water content.
Reaction mixture: 30.0 g vegetable fat F3,
13.1 g 2-ethyl-1-hexanol, water, 0.3 g enzyme. 100-mL stirred tank reactor, 50 7C,
1600 rpm, blade mixer, 360 min.

Fig. 3. Palmitic/oleic acid value against

glycerol in water. Reaction mixture: 15.0 g
vegetable fat F3, 6.55 g 2-ethyl-1-hexanol,
15.0 g water 1 glycerol, 0.15 g enzyme.
100-mL stirred tank reactor, 50 7C,
1600 rpm, blade mixer, 60 min.

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.ejlst.de

Eur. J. Lipid Sci. Technol. 106 (2004) 809814

the enzyme by water. The optimum amount of water (low
palmitic/oleic acid value, low amount of fatty acids and
triacylglycerols in Fig. 2) for our alcoholysis process was
found to be 80100% in relation to the amount of fat. This
means in practice (see also Fig. 5), that the amount of
water used in the process should be kept relatively high in
order to advance extensive alcoholysis.
Fig. 3 shows the effect of glycerol content (in the water) on
the formation of alkyl esters. The specified glycerol in
water values refer to the initial mixture. However, the
conversion rate of alcoholysis has a maximum (low palmitic/oleic acid value) in the ranges of 1025% glycerol in
water. No AE formation was found in the presence of 90%
glycerol in water.
The observation that glycerol content has an effect on
enzyme activity is interesting for the purpose of applying
the used water/glycerol/enzyme mixture for the next
batches in the stirred tank reactor. Because of the
increase in glycerol content, it is necessary from time to
time to replace the water/glycerol/enzyme mixture with
new water/enzyme mixture.

Alcoholysis of waste fats

813

The effect of temperature was also studied (Fig. 4). The

conversion rate is increased with increasing temperature
to 6570 7C.
Based on these results we started the production of about
3000 L of alkyl ester from 2100 kg used fat and 916 kg 2ethyl-1-hexanol using enzymatic alcoholysis in a 3000-L
water-heated wall-stirred tank reactor (two batches).
When the first batch was ready, the two phases were discharged after the separation time in separate tanks. The
next day the stirred-tank reactor was filled with fat and 2ethyl-1-hexanol for the second batch. When the reaction
temperature of about 55 7C was reached, the water/glycerol/enzyme mixture from the first batch was added. Due
to a low temperature in the factory building in February the
temperature of this mixture was only 8 7C and thus the
temperature of the complete mixture about 30 7C.
Consequently, the initial conversion rate is very low. With
increasing temperature the reaction rate of enzymatic
alcoholysis increased.
Fig. 5 shows the results (palmitic/oleic acid value versus
time) of the two batches which prove that the conversion
reaction of both batches is completed after a certain time.

Fig. 4. Palmitic/oleic acid value against

temperature. Reaction mixture: 15.0 g
vegetable fat F3, 6.55 g 2-ethyl-1-hexanol,
15.0 g water, 0.15 g enzyme. 100-mL stirred tank reactor, 1600 rpm, blade mixer,
45 min.

Fig. 5. Palmitic/oleic acid value against

time. Reaction mixture (first batch): 1050 kg
used recycled restaurant grease F2, 458 kg
2-ethyl-1-hexanol, 1050 kg water, 10.5 kg
enzyme. Reaction mixture (second batch):
1050 kg used recycled restaurant grease
F2, 458 kg 2-ethyl-1-hexanol, water/glycerol/enzyme mixture from the first batch.
3000-L stirred tank reactor, ,500 rpm,
disk-type agitator, ,55 7C.

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.ejlst.de

814

R. Brenneis et al.

We prepared 2740 kg (,3115 L) of alkyl esters including

an excessive amount of alcohol and ,4.8% of free fatty
acids.
After evaporating the excessive 2-ethyl-1-hexanol the alkyl ester will be tested as a lubricant coolant in a grinding
process.

4 Conclusion
Commercial native lipase (Candida antarctica lipase A) was
used for the synthesis of alkyl esters using alcoholysis of
recycled restaurant grease with 2-ethyl-1-hexanol. Water
has been found to play an important role in this process.
Our studies have shown that the determination of palmitic/oleic acid value by means of HPLC is a suitable method
to get information about the conversion rate of enzymatic
alcoholysis using input fats with dominant contents of
palmitic and oleic acids in the fatty acid composition.

Acknowledgements
This work was financially supported within the programme Innovations Centrum Biocatalysis (ICBio) by the
Deutsche Bundesstiftung Umwelt (DBU).

References
[1] R. Brenneis, P. Kcher, G. Kley, B. Baeck, T. Schwilling:
Untersuchungen zur Herstellung von biologisch abbaubaren
Verlustschmierstoffen aus Altfetten. Mll und Abfall 34 (2002)
244249.

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eur. J. Lipid Sci. Technol. 106 (2004) 809814

[2] J. Hesselbach, C. Herrmann, R. Bock, T. Dettmer, G. Kley, P.
Kcher, R. Brenneis, R. Meyer-Pittroff, O. Falk, A. Hansen:
Khlschmierstoffe aus technischen tierischen Fetten und
Altspeisefetten-Herstellung, Technologie und kobilanzierung. Transkript (Sonderheft Nachhaltige Biokatalyse, Okt.
2003) 2427.
[3] M. Mittelbach: Lipase-catalyzed alcoholysis of sunflower oil.
J. Am. Oil Chem. Soc. 67 (1990) 168170.
[4] J.-F. Show, D.-L. Wang: Lipase-catalyzed ethanolysis and
isopropanolysis of triglycerides with long-chain fatty acids.
Enzyme Microb. Technol. 13 (1991) 544546.
[5] Y.-Y. Linko, M. Lms, A. Huhtala, P. Linko: Lipase-catalyzed
transesterification of rapeseed oil and 2-ethyl-1-hexanol. J.
Am. Oil Chem. Soc. 71 (1994) 14111414.
[6] Y.-Y. Linko, M. Lms, A. Huhtala, O. Rantanen: Lipase biocatalysis in the production of esters. J. Am. Oil Chem. Soc.
72 (1995) 12931299.
[7] L. A. Nelson, T. A. Foglia, W. N. Marmer: Lipase-catalyzed
production of biodiesel. J. Am. Oil Chem. Soc. 73 (1996)
11911195.
[8] B. Selmi, D. Thomas: Immobilized lipase-catalyzed ethanolysis of sunflower oil in a solvent-free medium. J. Am. Oil
Chem. Soc. 75 (1998) 691695.
[9] B. K. De, D. K. Bhattacharyya, C. Bandhu: Enzymatic synthesis of fatty alcohol esters by alcoholysis. J. Am. Oil Chem.
Soc. 76 (1999) 451453.
[10] Y. Watanabe, Y. Shimada, A. Sugihara, Y. Tominaga: Enzymatic conversion of waste edible oil to biodiesel fuel in a
fixed-bed bioreactor. J. Am. Oil Chem. Soc. 78 (2001) 703
707.
[11] M. M. Soumanou, U. T. Bornscheuer: Lipase-catalyzed
alcoholysis of vegetable oils. Eur. J. Lipid Sci. Technol. 105
(2003) 656660.
[12] Novo Nordisk-Description Novozym 868 L.
[Received: July 23, 2004; accepted: September 15, 2004]

www.ejlst.de