Fatty acid alkyl esters as feedstocks for the enzymatic synthesis of alkyl methacrylates and polystyrene-co-alkyl methacrylates for use as cold flow improvers in diesel fuels

Article abstract of DOI:10.1007/s11746-011-1834-8

The enzymatic transesterifications of fatty acid methyl esters (FAME) with hydroxyethyl methacrylate (HEMA) were carried out using the Candida antarctica lipase B immobilized within a porous polymethacrylate resin. The enzymatic activity in the transesterification reaction of FAME with HEMA depended on the polarity of the solvent and the highest yield was obtained in toluene (non-polar). The molar ratio of 1:4 (for methyl laurate: HEMA) and 1:2 (for methyl oleate:HEMA) was most favorable for the transesterification yield. The reaction condition (at 60 °C/24 h), and the enzyme concentration of 5% (w/w) for methyl laurate with HEMA, 2% (w/w) for methyl oleate with HEMA resulted in the highest final yield. Under these conditions, the maximum yields for the transesterification of methyl laurate with HEMA, methyl oleate with HEMA were 97 ± 5.4% and 91 ± 4.7%, respectively. After ten batches of transesterification of FAME with HEMA, enzyme activity was retained at the level of 88 ± 2.6% and 76 ± 2.3%, respectively, compared with their initial activity. Also, alkyl methacrylate/ styrene copolymers were synthesized by radical polymerization of HEMA-LMA (or HEMA-OMA) and styrene. The prepared copolymers have average molecular weights from 2.6 × 10⁴ to 5.5 × 10⁴. Especially, the poly(styrene-co-alkyl methacrylate)s (PStmHAMAn) led to a reduction in the pour point in ultra low sulfur diesel (ULSD) treated with 200-1,000 ppm of poly(styrene-co-alkyl methacrylate). Diesel fuel containing 1,000 ppm of the copolymer (PSt2HLMA8) showed a 15 ± 1.25 °C reduction in its pour point. AOCS 2011.

Full text of DOI:10.1007/s11746-011-1834-8

J Am Oil Chem Soc (2011) 88:1727–1736
DOI 10.1007/s11746-011-1834-8
ORIGINAL PAPER
Fatty Acid Alkyl Esters as Feedstocks for the Enzymatic Synthesis
of Alkyl Methacrylates and Polystyrene-co-alkyl Methacrylates
for use as Cold Flow Improvers in Diesel Fuels
•
Young-Wun Kim Gyeong Tae Eom
•
•
Jin-Sook Hong Keun-Wo Chung
Received: 10 August 2010 / Revised: 5 April 2011 / Accepted: 7 April 2011 / Published online: 27 April 2011
Ó AOCS 2011
Abstract The enzymatic transesterifications of fatty acid
methyl esters (FAME) with hydroxyethyl methacrylate
(HEMA) were carried out using the Candida antarctica
lipase B immobilized within a porous polymethacrylate
resin. The enzymatic activity in the transesterification
reaction of FAME with HEMA depended on the polarity of
the solvent and the highest yield was obtained in toluene
(non-polar). The molar ratio of 1:4 (for methyl lau-
rate:HEMA) and 1:2 (for methyl oleate:HEMA) was most
favorable for the transesterification yield. The reaction
condition (at 60 °C/24 h), and the enzyme concentration of
5% (w/w) for methyl laurate with HEMA, 2% (w/w) for
methyl oleate with HEMA resulted in the highest final
yield. Under these conditions, the maximum yields for the
transesterification of methyl laurate with HEMA, methyl
oleate with HEMA were 97 5.4% and 91 4.7%,
respectively. After ten batches of transesterification of
FAME with HEMA, enzyme activity was retained at the
level of 88 2.6% and 76 2.3%, respectively, com-
pared with their initial activity. Also, alkyl methacrylate/
styrene copolymers were synthesized by radical polymer-
ization of HEMA-LMA (or HEMA-OMA) and styrene.
The prepared copolymers have average molecular weights
from 2.6 9 104 to 5.5 9 104. Especially, the poly(styrene-
co-alkyl methacrylate)s (PStmHAMAn) led to a reduction
in the pour point in ultra low sulfur diesel (ULSD) treated
with 200–1,000 ppm of poly(styrene-co-alkyl methacry-
late). Diesel fuel containing 1,000 ppm of the copolymer
(PSt2HLMA8) showed a 15 1.25 °C reduction in its
pour point.
Keywords Enzymatic synthesis ꢀ Fatty acid methyl
esters ꢀ Hydroxy ethyl methacrylate ꢀ Candida antarctica
lipase B ꢀ Poly(styrene-co-alkyl methacrylate)s ꢀ
Pour point depressant
Introduction
It is known that ultra low sulfur diesel (ULSD) may contain
substantial amounts of waxy materials. When these mate-
rials are subjected to temperatures below their cloud points,
these tend to deposit on cold surfaces or crystallize and
agglomerate into a solid gel [1, 2]. The gelling or stiffening
of ULSD introduces rheological problems that interfere
with oil production or pipeline transportation and cause
handling problems [3]. The high wax crude oils are char-
acterized by high pour point, high viscosity, high gel
strength, and abundant wax deposits. Several techniques
such as the wintering process [4], blending process [5, 6],
and additive addition [7] have been used to minimize the
problems caused by wax deposition in diesel fuel. Among
them, a few polymers having long alkyl side chains, so-
called ‘‘comb-like’’ polymers, are known to decrease the
rate of wax formation. Polymeric modifier such as poly
(ethylene-co-vinyl acetate) (EVA) [8–10], poly(styrene-co-
alkyl methacrylates) [11, 12], poly(fumarate-co-vinyl ace-
tate) [13], poly(alkyl methacrylate-maleic anhydride) [14],
polymethylmethacrylate [15–18], and their acid amide
derivatives [19–21] have been known for several decades
as commercial flow improvers and wax inhibitors of diesel
fuel, but none of them are sufficiently effective in diesel
fuel. Thus, it has become necessary to develop new cold
Y.-W. Kim (&) ꢀ G. T. Eom ꢀ J.-S. Hong ꢀ K.-W. Chung
Chemical Biotechnology Research Center, Korea Research
Institute of Chemical Technology, P.O. Box 107,
Yuseong-gu, Daejeon 305-600, Korea
e-mail: ywkim@krict.re.kr
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J Am Oil Chem Soc (2011) 88:1727–1736
flow improver for ULSD. Moreover, considering cost and
environmental issues, it is necessary to synthesize cold
flow improvers based on biodiesel-like materials, fatty acid
methyl esters (FAME), and enzymatic processes.
p-nitrophenol product was monitored by measuring the
absorbance at 405 nm with a spectrophotometer. One unit
of lipase activity was defined as the amount of enzyme
releasing 1 lmol of p-nitrophenol per min.
In order to prepare the compounds which can satisfy the
above criteria of the monomers for synthesis of pour-point
depressants, 2-(methacryloyloxy)ethyl alkyl esters were
selected as the monomeric compounds, which could be
prepared by lipase-catalyzed transesterification of fatty
acid methyl esters (FAME) with hydroxyethyl methacry-
late (HEMA). HEMA has a vinyl group that can be poly-
merized. FAME have various length of hydrocarbons.
Therefore, 2-(methacryloyloxy)ethyl alkyl esters are
expected to be efficient monomers for the synthesis of
pour-point depressants. In this study, a lipase was used as
catalyst for the transesterification reaction, because lipase-
catalyzed transesterification reaction has advantages over
conventional chemical-methods owing to mild reaction
conditions and energy-saving process [22–24].
Immobilization of Lipase
Lipase immobilization was performed as previously
described [25]. The macroporous polymethacrylate resin
(30 g) was washed with 1 L of 25% methanol (3 times)
sequentially prior to use. After removal of methanol by
filtration, the resin was washed with 1 L of 50 mM Tris-
HCl buffer (pH 7.5). The supernatant was removed and
replaced with 1 L of fresh 50 mM Tris-HCl buffer (pH
7.5). Seven hundred ml of the commercial CalB solution
The culture broth (the solution of CalB prepared from the
culture supernatant of A. oryzae) [26] containing CalB
(700 mL) was added and mixed at 150 rpm for 6 h at 25 °C
by mechanical stirrer. The immobilized CalB was filtered,
washed twice with 1 L of 50 mM Tris-HCl buffer (pH 7.5)
for 15 min, and then dried at room temperature for 20 h.
The activity of the immobilized CalB was 0.23 U/g dry
resin (pNPP unit).
We investigated the reaction conditions such as the
effect of methanol removal in the reaction system, molar
ratio of substrates, enzyme amount, and reaction tempera-
ture for the transesterification of FAME with HEMA. And
the copolymerization of these monomers with styrene was
also performed.
Enzymatic Transesterification
The transesterification reactions were performed following
the same procedure published in the literature as follows
[24]. Into a 100-mL round-bottomed flask equipped with a
magnetic stirrer, thermometer, and a water cooled con-
denser, 16.0 mmol of FAME (methyl laurate and methyl
oleate) and 32.0 mmol of HEMA was added along with 5%
(based on FAME weight) of the immobilized CalB in
15 mL of solvent such as toluene, acetonitrile, and 1,4-
dioxane. The transesterification reactions were carried out
at the temperatures in the range of 25–80 °C with vigorous
stirring for 24 h with removing methanol through N2 purge.
To optimize conditions, the reactions were performed
under various conditions by changing substrate ratios of
FAME and HEMA, enzyme concentrations. After the
transesterification reaction, the immobilized CalB was
separated from the reaction medium by filtration. The
reaction mixture was concentrated under reduced pressure
of 30 mmHg at 50 °C and the crude mixture was separated
on a silica gel column (100–200 mesh) using ethyl acetate/
hexane (4/1) as the eluent. Solvent was removed under
reduced pressure.
Materials and Methods
Materials
HEMA, methyl laurate (99.5%), methyl oleate (99%), sty-
rene, and 2,20-azobisisobutyronitrile (AIBN) were pur-
chased from the Sigma Chemical Co. (St. Louis, MO, USA).
Silica gel 60 (Merck Co.) was used for separation of product.
Toluene, acetonitrile, and 1,4-dioxane were obtained form
Merck Co. (Darmstadt, Germany). All other chemicals such
as methanol, ethyl acetate, hexane, chloroform-d6, and TMS
were analytical grade. Macroporous polymethacrylate resin
(VPOC 1600, Lewatit, Germany), tris-HCl buffer (Sigma,
St. Louis, MO, USA), Cal-B (Novozymes A/S, Bagsvaerd,
Denmark) for immobilization of lipase were used.
Assay of Lipase Activity
Lipase activity was measured by spectrophotometric
method using p-nitrophenyl palmitate (pNPP) as a sub-
strate. The 10 mM pNPP dissolved in acetonitrile was
mixed with ethanol and 50 mM sodium phosphate buffer
(pH 7.0) to meet the final ratio of acetonitrile: ethanol:
sodium phosphate buffer to 1:4:95 (v/v/v). The reaction
was started by adding an appropriate amount of enzyme to
1,000 ll of reaction mixture at 45 °C and the release of
Batch Operational Stability Tests
After each transesterification reaction, the lipase (1 g) was
recovered by filtration and washed with a mixture
(100 mL) of hexane and ethyl acetate (10:2 vol/vol) to get
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J Am Oil Chem Soc (2011) 88:1727–1736
1729
rid of the synthetic products from the lipase. After washing,
the enzyme was dried for 24 h at 25 °C in a vacuum oven
(1 mmHg). After that, the enzyme was reused under the
enzymatic transesterification conditions as described
above.
the electron impact (EI) mode at 70 eV with an ion
source temperature of 230 °C, a quadrupole temperature
of 150 °C, and a translating line temperature of 270 °C.
The mass scan ranged from 50 to 550 m/z with an Em
voltage, 1,035 V.
GC-MS Analysis
Copolymerization and Analysis
The transesterification reactions of FAME with HEMA
were monitored by gas chromatography (HP 5890
Model). A DB-Wax capillary column with dimensions of
0.25 mm 9 30 m i.d. 9 0.25 lm film thickness (Agilent
Technologies, Palo Alto, CA, USA) was used for the
separation of the products. The initial temperature of 115
°C was raised to 230 °C at a rate of 20 °C/min, and kept
at 230 °C for 20 min. The split ratio was 1:50, and helium
was used as the carrier gas with a flow rate of 1 mL/min.
The injector and detector temperatures were 200 and 230
°C, respectively. The mass spectrometer was operated in
As shown in Fig. 1a, the structures of synthetic monomers
were confirmed by 1H-NMR and FT-IR spectrum analyses.
1H-NMR spectra were obtained with 300 MHz BRUKER
DPX-300 spectrometer using CDCl3. FT-IR spectra were
measured on a Bio-RAD FTS165 spectrometer. Also, the
detailed procedures to synthesize copolymers (Fig. 1b) are
described as follows: a synthesized monomer such as
HEMA-LMA (or HEMA-OMA) and styrene with different
monomer feed ratios (mol% HEMA-LMA/mol% Styrene
viz. 40/60, 50/50, 60/40, 70/30, and 80/20) was dissolved
in 100 mL toluene and the solution was poured into a
Fig. 1 Synthesis of monomers
(a) and copolymers (b) for cold
flow improvers and synthetic
route of by-product a and b (c)
A
CH3
CH3
O
Lipase
+
CH2
C
CH2
C
R C OCH3
-CH3OH
C=O
C=O
O
CH2CH2 OC R
O CH2CH2 OH
O
HEMA-LMA : R=(CH2)10CH3
HEMA-OMA : R=(CH2)7CH=CH(CH2)7CH3
B
CH3
CH3
H
Copolymerization
+
CH
CH2
C
CH2
CH2
C
CH2 C
n
m
C=O
C=O
O
O
CH2CH2 OC R
CH2CH2 OC
R
O
O
PStmHLMAn : R=(CH2)10CH3
PStmHOMAn : R=(CH2)7CH=CH(CH2)7CH3
C
CH3
CH3
CH3
C
Lipase
-CH3OH
CH2
C
+
CH2
C
CH2
C=O
C=O
C=O
CH3
CH2
O
CH2CH2 OC
O
R
O CH2CH2 OC C
O CH2CH2 OH
O
by-product A
HEMA-LMA : R=(CH2)10CH3
HEMA-OMA : R=(CH2)7CH=CH(CH2)7CH3
+
CH3
CH2
HO CH2CH2 OC C
O
by-product B
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J Am Oil Chem Soc (2011) 88:1727–1736
round-bottom flask under N2 atmosphere. The monomers
were mixed together with AIBN initiator 0.2% (w/w). The
solution was then heated to 60 °C and was reacted for 24 h.
After the completion of copolymerization, the reaction
mixtures were poured into excess methanol with stirring.
The obtained precipitate was filtered and dried in vacuum
(1 mmHg) at 60 °C to a constant weight.
be assigned as the fragment of [CH3(CH2)10C(=O)O]?.
The compound with retention time of 13.49 min was
identified as the product (HEMA-LMA) of [M? = m/e
312]. This compound shows 100% intensity at m/z 113,
[CH2=C(CH3)C(=O)OCH2CH2]?, 80% intensity at m/z 69,
[CH2=C(CH3)C=O]?, and 50% intensity at m/z 183,
[CH3(CH2)10C(=O)O]?. From these results, we concluded
that the compound with retention time of 8.03 min was
identified as the compound B, CH2=C(CH3)C(=O)OCH2
CH2O(O=)C(CH3)C=CH2, and the compound with reten-
tion time of 12.97 min was identified as the compound A,
CH3(CH2)10C(=O)OCH2CH2OH, judging from the fact
that the higher molecular weight compounds have longer
retention time.
The prepared copolymers were analyzed using 1H-NMR
spectroscopic technique for determining the chemical
structure of the copolymers. 1H-NMR spectra were
obtained at 300 MHz on a BRUKER DPX-300 spectrom-
eter using CDCl3 as a solvent and TMS as an internal
reference. The molecular weights of copolymers were
determined using a size exclusion chromatograph consist-
ing of a Waters 2690 HPLC pump, three Ultrastyragel
The analysis of the reaction product for synthesizing
HEMA-OMA was similar to that of HEMA-LMA.
The structure of the prepared monomers was confirmed
by 1H-NMR spectral and FT-IR spectroscopic analysis.
1H-NMR spectra of HEMA-LMA and HEMA-OMA
monomers are shown in Fig. 2. As can be seen in Fig. 2A
(HEMA-LMA), peaks at 6.2 and 5.6 ppm were assigned
as the protons of the CH= in vinyl group of HEMA. The
methylene proton adjacent to the oxygen atom of HEMA
shows a chemical shift of 4.3 ppm; the proton of the
methylene adjacent to carbonyl group of laurate shows a
chemical shift of 2.3 ppm; the proton in the methyl group
of HEMA exhibits a chemical shift of 1.95 ppm, and the
chemical shifts of the methylene of lauryl unit are ranged
within 1.6–1.25 ppm; the proton in the methyl group of
lauryl shows a chemical shift of 0.87 ppm. This indicates
that there exists an alkyl chain moiety of laurate and a
methacrylate moiety of HEMA in the prepared monomer,
HEMA-LMA. As seen in Fig. 2b (HEMA-OMA), the
vinyl proton of the oleyl unit shows a chemical shift of
5.6 ppm; the chemical shifts of the methylene of oleyl unit
are ranged within 2.0–1.25 ppm; the other protons in
HEMA-OMA show similar chemical shift as seen in
HEMA-LMA.
˚
columns (two mixed-B and one 500-A column), and a
Waters refractive index detector, using THF as solvent at a
flow rate of 1 mL/min, and narrow disperse polystyrene
(PS-1, Mw: 580–7,800,000 g/mol, Part No 2010-0501, PL,
UK) as calibration standards. Cold flow properties of
copolymers were characterized by measuring pour point of
diesel fuel containing copolymers through ASTM D97
method using Automated Pour Point tester (CPP 5Gs, ILS
Co, France) [27].
Results and Discussion
Enzymatic Synthesis of 2-(Methacryloyloxy)Ethyl
Alkyl Esters
The monomers, HEMA-LMA and HEMA-OMA, for cold
flow improvers were synthesized as shown in Fig. 1a.
2-(Methacryloyloxy)ethyl alkyl esters (HEMA-LMA and
HEMA-OMA) were selected as the monomeric com-
pounds, which could be prepared by lipase-catalyzed
transesterification of fatty acid methyl esters (FAME-L and
FAME-O) with hydroxyethyl methacrylate (HEMA). GC-
MS was used to identify the reaction products. In the gas
chromatogram of the reaction to synthesize HEMA-LMA,
methyl laurate and HEMA presented retention times of
7.76 and 7.83 min, respectively. Three new peaks appeared
at 8.03, 12.97, and 13.49 min. According to GC-MS
analysis, the compound with a retention time of 8.03 min
was identified as compound of [M? = m/e 198]. This
compound shows 100% intensity at m/z 69, [CH2=
C(CH3)C=O]?. The compound with a retention time of
12.97 min was identified as compound of [M? = m/e 244].
This compound shows 100% intensity both at m/z 104 and
m/z 183. The peak at m/z 104 could be assigned as
[CH2C(=O)OCH2CH2OH]? and the peak at m/z 183 could
FT-IR spectra of HEMA-LMA and HEMA-OMA
monomers are shown in Fig. 3. CH3 and CH2 absorption
peaks of long-chain alkyl at 2,923 and 2,853 cm-1; C=O
stretching peaks of methacrylate at 1,732 cm-1; C=C
stretching vibration peaks of methacrylate at 1,639 cm-1
.
This also indicates that there exists an alkyl chain fragment
of FAME and a methacrylate fragment of HEMA in the
prepared monomers, HEMA-LMA and HEMA-OMA.
2-(Methacryloyloxy)ethyl Lauryl Ester (HEMA-LMA)
1H-NMR (CDCl3): d = 6.2 (1H, s, =CH), 5.6 (1H, s, =CH),
4.3 (4H, t, 2OCH2), 2.3(2H, t, C(=O)CH2), 1.95 (3H, s, CH3C=),
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J Am Oil Chem Soc (2011) 88:1727–1736
1731
Fig. 2 1H-NMR spectra of
HEMA-LMA (a) and HEMA-
OMA (b)
A
b
f
O
e
c
O
O
g
e
d
a
O
b
e
g
23.27
d
a
4.40
f
4.11
f
3.00
2.78
2.76
1.03
1.00
c
B
b
b
f
O
j
h
e
i
c
O
O
a
g
d
e
j
i
h
O
b
g
e
a
c, j
20.53
d
i
7.06
f
f
h
3.89
3.71
3.29
2.00
2.01
0.93
0.93
7
6
5
4
3
2
1
0
(ppm)
2-(Methacryloyloxy)Ethyl Oleyl Ester (HEMA-OMA)
HEMA-LMA
HEMA-OMA
1H-NMR (CDCl3): d = 6.1 (1H, s, =CH), 5.6 (1H, s,
=CH), 5.3 (2H, m, 2CH=), 4.3 (4H, t, 2OCH2), 2.3 (2H, t,
C(=O)CH2), 2.0 (4H, m, 2CH2), 1.95 (3H, s, CH3C=), 1.6
(6H, m, CH2 ? 2CH2), 1.2 (16H, m, 8CH2), 0.8 (3H, t,
CH3).
FT-IR (KBr) 2,923 (C–H stretching), 1,732 (C=O),
1,639 (C=C), 1,080 (C–O), cm-1
.
Effects of Solvents in Transesterification
The effect of solvents in the transesterification of FAME
with HEMA was investigated (reaction condition: FAME
(1 mol), HEMA (2 mol), enzyme (5 wt%), 24 h, 50 °C).
We selected transesterification solvents such as toluene
(log P = 2.5), acetonitrile (log P = 0.3), and 1,4-dioxane
(log P = -0.3), all of which have higher boiling point than
that of methanol (log P = -0.75) [28]. In the transesteri-
fication reaction of FAME-L with HEMA, the yields of
HEMA-LMA were 89.6 4.5, 87.5 3.9 and 83.5
3.3% in toluene, acetonitrile, and 1,4-dioxane, respectively
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers(cm-1)
Fig. 3 FT-IR spectrum of HEMA-LMA and HEMA-OMA
1.6 (2H, m, CH2), 1.25 (16H, m, 8CH2), 0.87 (3H, t,
CH3).
FT-IR (KBr) 2,923 (C-H stretching), 1,732 (C=O),
1,639 (C=C), 1,080 (C–O), cm-1
.
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J Am Oil Chem Soc (2011) 88:1727–1736
(Fig. 4). The yields of the by-product were 8.2 0.3,
1.4 0.1 and 1.0 0.05% in toluene, acetonitrile, and
1,4-dioxane, respectively. In the transesterification reaction
of FAME-O with HEMA, the reaction yields seem to be
solvent dependent compared with FAME-L with HEMA.
The yields of HEMA-OMA were 83.9 3.4, 70.1 2.1,
and 29.8 0.3% in toluene, acetonitrile, and 1,4-dioxane,
respectively. The yields of the by-product were 7.6 0.2,
15.8 0.6, and 0.7 0.02% in toluene, acetonitrile and
1,4-dioxane, respectively. From these results, we could
conclude that the transesterification reaction yields of
FAME-L with HEMA did not largely depend on solvent,
whereas that of FAME-O with HEMA largely depended on
the polarity of the solvent. Transesterification reaction
yields in toluene (relatively nonpolar) were higher than
those in acetonitrile and 1,4-dioxane, suggesting that non-
polar solvent is more favorable and effective for transe-
sterification of more lipophilic HEMA-OMA, compared
with that of HEMA-LMA.
Effect of the Molar Ratio of the Substrate
The effect of the molar ratio was investigated in the
transesterification reaction of FAME with HEMA (reaction
condition: toluene, enzyme (5 wt%), 24 h, 50 °C), we tried
FAME:HEMA molar ratios from 1:1 to 1:5 (Fig. 5). In the
range of 1:2 and 1:5, the yields are considered to be sta-
tistically the same since the p values (the significance
level = 0.05) calculated from analysis of variance
(ANOVA) were found to be 0.76 and 0.15 for the product
(HEMA-LMA) and by-product, respectively, by the use of
one-way ANOVA using Microsoft Excel 2007. The aver-
age yields of product (HEMA-LMA) and by-product in
transesterification of FAME-L with HEMA were obtained
in 91.1 4.4 and 8.4 0.4%, respectively, (Fig. 5a). In
case of the synthesis of HEMA-OMA, the yields were
statistically the same at ratios of methyl oleate to HEMA
between at least 1:2 to 1:5.
HEMA-LMA
100
HEMA-OMA
Effect of Reaction Temperature
80
To determine the effect of temperature on the transesteri-
fication of FAME with HEMA, reactions were performed
at 40, 50, 60, 70 and 80 °C, respectively. The transesteri-
fication reactions below 40 °C were not carried out,
because methanol was not removed in the reaction system
below 40 °C. In transesterification of both HEMA-LMA
and HEMA-OMA, the best yields were obtained at 60 °C in
97 5.4 and 89 4.1%, respectively (Fig. 6). At 70 °C,
catalytic activity decreased presumably due to thermal
deactivation of the biocatalysts and polymerization of
HEMA.
60
40
20
0
Toluene
Acetonitrile
1,4-Dioxane
Fig. 4 Effect of solvents in the transesterification of FAME with
HEMA
B
A
HEMA-LMA
Side Product
HEMA-OMA
Side Product
100
100
80
60
40
20
0
80
60
40
20
0
1 : 1
1 : 2
1 : 3
1 : 4
1 : 5
1 : 1
1 : 2
1 : 3
1 : 4
1 : 5
FAME-L : HEMA (Mole Ratio)
FAME-O : HEMA (Mole Ratio)
Fig. 5 Effect of molar ratios of substrate in the transesterification of FAME-L (a) and FAME-O (b) with HEMA
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J Am Oil Chem Soc (2011) 88:1727–1736
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120
HEMA-LMA
HEMA-OMA
100
80
60
40
20
0
100
80
60
40
20
0
HEMA-LMA
HEMA-OMA
1
2
3
4
5
6
7
8
9
10
40
50
60
70
Number of Batch
Temperature, oC
Fig. 8 Enzyme stability in the transesterification reaction of FAME
with HEMA
Fig. 6 Effect of reaction temperature in the transesterification of
FAME with HEMA
Effect of Enzyme Concentration
Enzyme Stability
The effect of enzyme concentration on the catalytic activity
of the immobilized lipase in the transesterification of
FAME with HEMA was studied. Enzyme concentration on
the basis of the mass of substrates varied in range from 1 to
7 wt%, with 1 wt% increments at the same condition
selected in the previous experiment. As shown in Fig. 7,
the highest yield of HEMA-LMA and HEMA-OMA was in
97.4 5.4% (enzyme 5 wt%) and 91.0 4.7% (enzyme 3
wt%), respectively. Above 5 wt% enzyme concentrations,
the transesterification yields decreased because the product
(HEMA-LMA and HEMA-OMA) could be easily trans-
formed to the by-products (Fig. 1c).
Immobilized enzymes can be recycled, but the activity
eventually decreases due to several factors, such as
desorption, substrate deactivation, and product inhibition.
Therefore, we investigated the stability of the immobilized
lipase after each use. Figure 8 shows the results of the
relative conversion yields of HEMA-LMA and HEMA-
OMA, expressed as a percentage of the yield of the first
batch reaction. After ten times transesterification, enzyme
activity was retained at 88 2.6% (HEMA-LMA) and
76 2.3% (HEMA-OMA), indicating that the immobi-
lized lipase can be recycled at least 10 times.
Synthesis of Copolymers
It is well known that cold flow improvers should have a
hydrophobic character to be soluble in the fuel medium
and a polar character to adhere to the wax crystals and
saturated fatty acid methyl esters. In this study, we syn-
thesized alkyl methacrylate/styrene copolymers by radical
polymerization of HEMA-LMA (or HEMA-OMA) and
styrene (Fig. 1b). The structure of the prepared copoly-
mers was confirmed by 1H-NMR spectral and FT-IR
spectroscopic analysis. Figure 9 shows the 1H-NMR
spectrum of HEMA-LMA/Styrene copolymer. As seen in
120
HEMA-LMA
HEMA-OMA
100
80
60
40
20
0
1
Fig. 9, comparing the H-NMR spectrum with styrene and
HEMA-LMA with the copolymer, the signals at 5.76 and
5.42 ppm disappeared, which are attributable to
CH2=CH–Ar of styrene and the peaks at 6.69 and
5.54 ppm are attributable to CH2=CH- of HEMA-LMA
increased the signals at 1.26 ppm attributable to aliphatic
H of the copolymer.
1
3
5
7
Enzyme Concentration, %
Fig. 7 Effect of enzyme concentration in the transesterification of
FAME with HEMA
123

1734
J Am Oil Chem Soc (2011) 88:1727–1736
CH
CH2
b
a
c
c
a
a
c
b
Styrene
O
b
f
b
g
e
O
O
e
d
h
g
a
O
a
d
e
f
f
HEMA-LMA
c
g
CH3
C
H
C
CH2
CH2
f
b f
32
68
a
C=O
O
PSt6HLMA4
b
g
e
c
d
CH2CH2 OC
i
a
i
O
d h
c
e
(PPM)
7
6
5
4
3
2
1
0
Fig. 9 1H-NMR spectra of (a) styrene, (b) HEMA-LMA and (c) PSt6HLMA4
Cold Flow Properties
The prepared copolymers were characterized by deter-
mining the weight average molecular weight (Mw), num-
ber average molecular weight (Mn), and polydispersity
molecular distribution (PD = Mw/Mn), using GPC analy-
sis, and the results are listed in Table 1. The results indicate
that the prepared copolymers have an average molecular
weight from 2.6 9 104 to 5.5 9 104.
The cold flow property was significantly influenced of
n-paraffin and saturated bio-diesel contained in ULSD,
therefore cold flow low properties might deteriorate due to
an increase in the formation of the n-paraffin and a satu-
rated bio-diesel crystal nucleus. The mechanism of the
Table 1 Synthetic results of styrene-alkyl methacrylate copolymers
Polymers
Reactant (mol %)
St
Compositiona (mol %)
GPC, molar mass
AM
St
AM
Mn
Mw
PD
PSt6HLMA4
PSt5HLMA5
PSt4HLMA6
PSt3HLMA7
PSt2HLMA8
PSt6HOMA4
PSt5HOMA5
PSt4HOMA6
PSt3HOMA7
PSt2HOMA8
a
6
5
4
3
2
6
5
4
3
2
4
5
6
7
8
4
5
6
7
8
67
55
45
35
26
68
57
46
36
25
33
45
55
65
74
32
43
54
64
75
20,200
19,500
20,000
16,300
13,200
16,700
17,000
14,700
15,700
17,500
45,600
55,200
41,500
33,400
26,500
54,000
50,500
43,600
48,600
48,400
2.25
2.83
2.07
2.04
2.00
3.22
2.95
2.95
3.09
2.75
1
Calculated from H-NMR results
123

J Am Oil Chem Soc (2011) 88:1727–1736
1735
molar ratio of FAME:HEMA, the reaction temperature,
and the enzyme concentration. The highest yields,
97 5.4% for methyl laurate/HEMA and 91 4.7% for
methyl oleate/HEMA, were obtained at 60 °C in toluene as
the solvent. The copolymers, polystyrene-co-alkyl meth-
acrylates, were synthesized by radical polymerization of
HEMA-LMA (or HEMA-OMA) and styrene and have
average molecular weight from 2.6 9 104 to 5.5 9 104. The
poly(styrene-co-alkyl methacrylate)s (PStmHAMAn) led
to a reduction in the pour point in ultra low sulfur diesel
(ULSD) and the best one, PSt2HLMA8, showed a
15 1.25 °C reduction in their pour points at 1,000 ppm
concentration.
Table 2 Pour point properties of copolymers in ULSD
Polymers
Pour point depression (°C)
200 ppm
500 ppm
1,000 ppm
PSt6HLMA4
PSt5HLMA5
PSt4HLMA6
PSt3HLMA7
PSt2HLMA8
PSt6HOMA4
PSt5HOMA5
PSt4HOMA6
PSt3HOMA7
PSt2HOMA8
2.5
2.5
5.0
2.5
5.0
0
2.5
5.0
7.5
7.5
7.5
0
5.0
7.5
10.0
12.5
15.0
2.5
0
2.5
2.5
2.5
2.5
2.5
0
2.5
0
5.0
0
5.0
Acknowledgments This work was financially supported by the
MKE by grants (Contract No 10028387) to KRICT. The authors
thank the industrial members of ECO solutions Co. LTD and EMAX
solutions Co. LTD.
formation of the crystal nucleus is unknown, but a variety
of additives applied as an inhibitor to retard the formation
of the crystal nucleus. In this work, synthesized polymers
were added to ULSD at a level of 200, 500 and 1,000 ppm,
respectively. Pour points of ULSD containing synthesized
copolymers were evaluated. The results are listed in
Table 2. It is seen that pour point depression performance
(DPP) of the PStmHLMAn copolymers (m = moles of
styrene and n = moles of HLMA unit) possess better pour
point depression capabilities than that of the copolymer
with HOMA unit (DPP 5.0–15.0 vs. 2.5–5.0 at 1,000 ppm).
Also, DPP of the copolymers with more HLMA showed
lager DPP than that of the copolymers with more styrene
unit. An increasing number of polar HLMA or HOMA
moieties showed the better cold flow properties. Compared
to HOMA, more polar HLMA was much more effective for
the cold flow properties at the same content in the
copolymer. According to the general knowledge of the
pour point depression, the copolymer can co-crystallize
with the wax in the ULSD to possess good cold flow per-
formance if the structure of the copolymer is suitable for
the composition of the wax in the ULSD. It is thus con-
cluded that the HLMA unit is the optimal matching alkyl
chain for preparing the cold flow improvers suitable for the
tested basic fuels.
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