Cleaner enzymatic production of biodiesel with easy separation procedures triggered by a biocompatible hydrophilic ionic liquid

Article abstract of DOI:10.1039/c9gc03796a

The great challenges of modern industry and the environment make it important to develop sustainable energy resources with low cost. In this work, a cleaner enzymatic procedure for biodiesel production was developed through the utilization of a biocompatible and hydrophilic ionic liquid [Choline][H₂PO₄]. This ionic liquid can be synthesized from cheap raw materials through simple neutralization procedures, and it has been proved to be well biocompatible. The utilization of this ionic liquid in Novozym 435 catalyzed biodiesel production makes the reaction and work-up procedures very simple, because its hydrophilicity can lead to the implementation of a pseudo homogeneous reaction and then heterogeneous separation. Various oil resources such as triolein, sunflower oil and castor oil can all be converted to biodiesels with high yields. After the completion of reaction, both the ionic liquid and Novozym 435 can be recycled and reutilized for at least five cycles without a significant activity decrease. In addition, this reaction system can be conveniently scaled up to the multi-gram level with high efficiency and feasible separation. Overall, the above mentioned benefits make this ionic liquid based enzymatic system cleaner for the production of biodiesel and promising for further industrial applications.

Full text of DOI:10.1039/c9gc03796a

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Page 1 of 17
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DOI: 10.1039/C9GC03796A
Cleaner enzymatic production of biodiesel with easy separation procedures
triggered by a biocompatible hydrophilic ionic liquid
Anlian Zhu,* Wanlu Feng, Zhiyong Li, Shuang Cheng, Qianhan Chen, Dongshuang
Fan, Yuanyang Guo, Lingjun Li,* Jianji Wang*
School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical
Media and Reactions, Ministry of Education, Collaborative Innovation Center of
Henan Province for Green Manufacturing of Fine Chemicals, Henan Normal
University, Xinxiang, Henan 453007, P. R. China
alzhuchem@126.com; lingjunlee@htu.cn; jwang@htu.cn
Abstract: The great challenges of modern industry and environment make it important
to develop sustainable energy resources with low cost. In this work, a cleaner enzymatic
procedure for biodiesel production was developed through the utilization of a
biocompatible and hydrophilic ionic liquid [Choline][H2PO4]. This ionic liquid can be
synthesized from cheap raw materials through simple neutralization procedures, and
itself has been proven to be well biocompatible. The utilization of this ionic liquid in
Novozym 435 catalyzed biodiesel production makes the reaction and work-up
procedures very simple, because its hydrophilicity can lead to the implement of pseudo
homogeneous reaction and then heterogeneous separation. Various oil resources such as
triolein, sunflower oil and castor oil can all be converted to biodiesels with high yields.
After the completion of reaction, both of the ionic liquid and Novozym 435 can be
recycled and reutilized for at least five cycles without significant activity decrease. In
addition, this reaction system can be convenient scaled up to multi-gram level with high
efficiency and feasible separation. Overall, the above mentioned benefits make this ionic
liquid based enzymatic system cleaner for the production of biodiesel and promising for
the further industrial applications.
Keywords: biodiesel production; enzyme; hydrophilicity; ionic liquid; reusability
Introduction
The shortage of fossil resources and the ever increasing demands for energy have
made it urgent to develop alternative energy resources with renewability, sustainability
and minimum emission of greenhouse gases as compared to petroleum based diesel.1, 2
As a promising green substitute for petroleum diesel, biodiesel is the mixture of methyl
esters of long chain fatty acids (FAMEs) which is usually produced by the esterification

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of fatty acids or the transesterification of triglycerides with methanol from edible oVriew Article Online
DOI: 10.1039/C9GC03796A
non-edible oils and animal fats.3-7 Compared with time and energetic consuming
chemical production of biodiesel catalyzed by acid8-10 or alkaline11-13, enzymatic
production method has received growing attention in recent years due to the mild
reaction conditions, low alcohol oil ratio, no needs of pretreatment of raw materials and
easy workup procedures.14-20
As an alternative for volatile organic solvents, ionic liquids have found extensive
applications in homogeneous, heterogeneous and enzymatic catalysis procedures.21 In
biodiesel production, ionic liquids can not only efficiently overcome the inactivation of
most lipases caused by methanol, but also increase the stability and activity of enzyme
through suitable selection of their cations and anions.22 The presented researches
revealed that hydrophobic ionic liquids are much more favorable than hydrophilic ones
because the latter may alter the conformation of protein and stripe off the essential water
from lipase.16 So, imidazolium-based ionic liquids with longer alkyl chain and
hydrophobic anions such as [C16mim][NTf2] and [C18mim][NTf2] showed much higher
catalytic activity compared with those having shorter alkyl chain and hydrophilic
anions.23 In addition, this hydrophobic ionic liquid with long alkyl chains have been
believed to behave as sponge-like systems and then facilitate the reaction and separation
procedures for the enzymatic production of biodiesel.24 However, the increase in
hydrophobicity of ionic liquid has significant negative effect towards aquatic
environment.25 Thus it is still a great challenge to develop cleaner reaction systems for
the biodiesel production with easy operation and separation.
Very recently, Ou et al26 found that the introduction of hydroxyalkyl moity into the
structure of ionic liquids can efficiently increase the stability and activity of enzyme in
ionic liquids, although their hydrophilicity is also increased. Some cholinium-based
ionic liquids, which have hydroxyethyl group on the cation, have also been proved to
be able to stabilize various proteins and DNA, and the existence of hydroxyethyl moity
can decrease the toxicity of ionic liquids.27 Some deep eutectic solvents (DESs) based
on choline chloride have also been utilized for the production of biodiesel, but the
reusability of DES and enzyme is still need to be optimized due to the relative instability
of DESs in complex reaction systems.17, 28 Therefore, in order to find excellent reusable
ionic liquid reaction systems with high enzymatic activity and low ionic liquid toxicity,
a series of ionic liquids composed with various cations with and without hydroxyethyl
groups and anions with different hydrophobicities have been synthesized (Scheme1),

Page 3 of 17
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DOI: 10.1039/C9GC03796A
and their performances in the production of biodiesel catalytzed by Novozym 435 are
investigated in this work. It is shown that both the cations and anions have significant
influences on the enzymatic activity. The hydrophobic anions have no positive effect
when they are combined with cations having hydroxyl groups, while the ionic liquid
with nucleophilic [Cl] as anion exhibits moderate promoting effect, although the
nucleophilic anions are often believed to be the inhibitor of enzyme.29 Using the
optimized ionic liquid cholinium dihydrogen phosphate, triolein and different oils such
as edible sunflower oil and non-edible castor oil can all reach excellent biodesel yields
under the catalysis of Novozym 435. In addition, due to the hydrophilicity of ionic
liquid, the biodiesel product can be separated by simple decantation from the ionic
liquid and enzyme, which makes the work-up procedure very simple, and the catalytic
system including enzyme and ionic liquid can be reused as a whole for at least five
cycles without significant activity decrease.
OH
OH
OH
OH
HO
OH
OH
HN
H2N
HN
NH
N
NH
DMEOA
OH
OH
OH
OH
DEOA
Choline
DEEOA
MDEOA
TEOA
OH
H
N
OH
H
N
H
N
NH
N
H2
N
O
H3N
EOA
DIPEA
DMEA
Betaine
TEA
MEOA
TMA
CF3SO3
,
,
PF6 NTf2 HSO4 H2PO4
, .
,
,
Cl
I , NO3
CF COO
BF4
,
,
,
,
Ac
3
Scheme 1. The cations and anions of ionic liquids
Materials and methods
Materials
Immobilized lipase from Candida Antarctica (Novozym 435) and triolein were
purchased from Sigma-Aldrich. Ethyl decanoate, methyl palmitate, methyl oleate,
methyl linoleate, methyl stearate, and methyl ricinoleate were purchased from Aladdin
Reagents Company. Hexane and ethyl acetate used for GC analysis were
chromatographically pure and purchased from Macklin Reagents Company. N,N-
dimethylethanolamine, choline, diethanolamine, N,N-diethylethanolamine, N-
methyldiethanolamine, betaine, 2-(methylamino)ethanol, monoethanolamine,
triethanolamine, triethylamine, N-ethyldimethylamine, N,N-diisopropylethylamine,

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trimethylamine, acetic acid, trifluoromethanesulfonic acid, trifluoroacetDicOI: 1a0c.10id39,/C9GC03796A
hexafluorophosphoric acid, tetrafluoroboric acid, bis(trifluoromethane sulfonimide),
choline chloride, choline iodide, nitric acid, sulfuric acid, and phosphoric acid with AR
grade were purchased from Aladdin Reagents Company. Methanol and aqueous
solution for choline hydroxide were purchased from Sinopharm Chemical Reagent Co..
Sunflower oil was purchased from local supermarket. Castor oil was obtained from
Macklin Reagents Company.
Synthesis of ionic liquids
Synthesis of protic ionic liquids. The protic ionic liquids were synthesized through
the protonation of corresponding amines and acids 30 (Scheme 2, path (a)). In a typical
reaction for the synthesis of [DMEOA][H2PO4], 0.1 mol N, N-dimethylethanolamine
(DMEOA) dissolved in 10 ml methanol was slowly added to equal molar phosphoric
acid under stirring in an ice bath for 2 h, and then the mixture was reacted at room
temperature for further 24 h. The solvent was removed at 50 oC by rotary evaporation
in reduced pressure and then dried under vacuum overnight to give the target protic
ionic liquids. The other protic ionic liquids including [DEEOA][H2PO4],
[DMEA][H2PO4],
[MDEA][H2PO4],
[DEOA][H2PO4],
[Betaine][H2PO4],
[TEOA][H2PO4],
[EOA][H2PO4],
[MMEA][H2PO4],
[DIPEA][H2PO4],
[TEA][H2PO4], [TMA][H2PO4], [DMEOA][HSO4], [DMEA][HSO4], [DEOA][HSO4],
[TEOA][HSO4], [DMEOA][Ac], [DEEOA][Ac], and [MMEA][Ac] were synthesized
in the same way.
Synthesis of aprotic ionic liquids. The cholinium-based aprotic ionic liquids were
synthesized by the neutrilization of choline hydroxide with corresponding acids30b
(Scheme 2, path (b)). Typically, 0.1 mol acid in 10ml water was droply added to 0.1mol
choline hydroxide in aqueous solution under stirring in an ice bath for 2 hours, and then
stirred at room temperature overnight. The solvent was then distilled by rotary
evaporation under reduced pressure and then dried in vacuum to give the target aprotic
ionic liquids.

Page 5 of 17
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DOI: 10.1039/C9GC03796A
OH
OH
OH
methanol
ethanol
HX
(a)
(b)
NH
H2PO4
N
OH
HX
+ H2O
N
N
X
OH
X = H2PO4, HPO4, HSO4, Cl, I, Ac, NO3, BF4,
PF6, NTf2, CF3SO3,CF3COO
Scheme 2. Synthesis of the protic and aprotic ionic liquids.
Lipase-catalyzed biodiesel production
The transesterification reactions were performed in 10 ml reaction bottles placed in
an oil bath at assigned reaction temperature. Typically, triolein (0.5 mmol, 443 mg), IL
(10-100%, w/w, based on oil weight), Novozym 435 (0-15%, w/w, based on oil weight)
and methanol (molar ratio of methanol to oil (M:O) 4:1-14:1) were mixed and stirred
at a given temperature. After a specified reaction time (3, 6, 9, 12, 18 or 24 h), the
mixture was cooled to room temperature. A certain amount of internal standard(ethyl
decanoate) was added to the reaction mixture, and then 5 ml ethyl acetate was added as
a solvent. After the fully mixed, 2 ml mixture was taken from the mixture and
centrifuged, 50 ul solution was taken from the upper layer and then diluted to 1 ml using
ethyl acetate and then, 1 l was injected for quantitative analysis using GC-MS system.
GC-MS determination of the biodiesel yield
GC-MS analysis was carried out by using a GC-Trace 1300 (Thermo Scientifc)
instrument coupled with an MS-ISQ (Thermo Scientifc) system. For the gas
chromatographic analysis of sunflower oil, an Innowax column (30 m × 0.25 mm ×
0.25 μm, Thermo Fisher) was used. For FAMEs identification, the following conditions
were used: carrier gas (He) at 1 mL/min; inlet split ratio, 1:50; temperature program:
70 oC, 1 min, 30 oC /min; 170 ◦ C, 100 oC /min; and 230oC, 7 min; MS source ionization
energy, 70 eV; the scan time was 0.2 s, covering a mass range of 50-500 amu. For the
analysis of castor oil, the GC was equipped with an HP-5MS column (30 m × 0.25 mm
× 0.25 μm, Thermo Fisher). For FAMEs identification, the following conditions were
used: carrier gas (He) at 1 mL/min; inlet split ratio, 1:50; temperature program: 80 oC,
◦
o
o
1 min, 60 oC /min; 180 C, 8 min, 30 C /min; 250 C, 3 min; MS source ionization
energy, 70 eV; the scan time was 0.2 s, covering a mass range of 50-500 amu.
The reutilization of enzyme and ionic liquid

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In the optimized reaction system, the produced biodiesel could be separatedDfOroI:m10.1t0h3e9/C9GC03796A
catalytic system by simple centrifugation and decantation when the reaction was
completed. The enzyme and ionic liquid as a whole together with the un-reacted
methanol and by-product glycerinum were washed with ethyl acetate for three times.
The enzyme was dried at room temperature for 12 h, the ionic liquid was dried at 50 oC
for 12 h, and then they could be reutilized for the next runs.
Results and discussion
Influence of the ionic liquids on the biodiesel production
The transesterification reaction between triolein and methanol was chosen to study
the influence of ionic liquids on the enzymatic production of biodiesel. The results
collected in Table 1 showed different ionic liquid effect compared with those reported
in literatures.16,23,29 When the anion of ionic liquids was [H2PO4], the ionic liquids with
cations such as TMA, TEA, TEOA, EOA, MDEOA, MEOA, and betainium showed
negative effect (entries 2-8, Table 1) towards the production of biodiesel compared to
that with no ionic liquid was added (entry 1, Table 1), the ionic liquids with DEEOA,
DMEA, DEOA and DIPEA cations showed slightly promotion for the enzyme activity
(entries 9-12, Table 1), while the ionic liquids with DMEOA and Choline as cations
exhibited excellent promotion effect with the biodiesel yields above 90% (entries 13,
14, Table 1). When [HPO4] was used as anion, the effect of ionic liquids
[Choline]2[HPO4] was negative but [DMEOA]2[HPO4] was a little positive (entries 15,
16, Table 1). A more acidic anion [HSO4] (entries 17-21, Table 1) and a more basic
anion [Ac] (entries 22-26, Table 1) showed different cation dependences, only ionic
liquid [MEOA][Ac] was positive for the enzymatic performance. Then the other anion
effects on the enzymatic production of biodiesel was also investigated using Choline as
the cation (entries 27-34, Table 1), the results showed that more hydrophobic anions
such as [PF6] and [NTf2] even have negative effect on the performances of enzyme
when they were combined with cholinium cation, but the nucleophilic anion such as
[Cl] and [NO3] could give moderate promotion for the production of biodesel. The
above findings fall out of the traditional relationship between ionic liquid structure and
enzyme activity. The possible reason was that the ionic liquids effect towards this
enzymatic reaction was performed through the synergetic effects of the cation and the
anion as a whole, but not through the influences of a single ion. So, the combination of
the cation and the anion of ionic liquids was superior important. The ionic liquid

Page 7 of 17
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[Choline][H2PO4] which is hydrophilic and biocompatible have been provedDtOoI: s10h.1o0w39/C9GC03796A
the highest performance in promoting this enzymatic procedure. In addition, this ionic
liquid is insoluble in the produced biodiesel. So, the biodiesel can be separated by
simple centrifugation and decantation after the reaction, the enzyme and the ionic liquid
can be reused for next cycles by simple washing and drying procedures.
Table 1. The chemical structure of the ionic liquids on the biodiesel yield
Entrya
1
IL
Yield %b Entrya
IL
Yield %b
n.d.
n.d.
30
blank
51
40
19
9
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35 c
[DEOA][HSO4]
[TEOA][HSO4]
[DMEA][HSO4]
[Choline][Ac]
[DMEOA][Ac]
[DEEOA][Ac]
[MEOA][Ac]
2
[TMA][H2PO4]
[TEA][H2PO4]
3
4
[TEOA][H2PO4]
[EOA][H2PO4]
[MDEOA][H2PO4]
[MEOA][H2PO4]
[betaine][H2PO4]
[DEEOA][H2PO4]
[DMEA][H2PO4]
[DEOA][H2PO4]
[DIPEA][H2PO4]
[DMEOA][H2PO4]
[Choline][H2PO4]
[Choline]2[HPO4]
[DMEOA]2[HPO4]
[Choline][HSO4]
[DMEOA][HSO4]
7
5
13
21
46
30
80
70
73
73
90
95
40
73
3
n.d.
5
6
7
71
8
[TEOA][Ac]
16
9
[Choline][BF4]
[Choline][PF6]
[Choline][NTf2]
[Choline][NO3]
[Choline][Cl]
13
10
11
12
13
14
15
16
17
18
7
5
64
76
[Choline][I]
n.d.
54
[Choline][CF3SO3]
[Choline][CF3COO]
[Choline][H2PO4]
5
n.d.
n.d.
areaction conditions: triolein, 443 mg; ILs, 443 mg; lipase, 22 mg; methanol /oil molar
ratio,10:1; reaction time, 24 h, 40 oC. bGC yield. cnon-enzyme.
Influence of temperature on the biodiesel production
It is well recognized that the enzymatic reaction is often temperature sensitive. So,
the influence of temperature on the enzymatic promoted biodiesel production was
investigated from 25 to 100 oC using the optimized ionic liquid [Choline][H2PO4] and
Novozyme 435 as catalyst. The results in Fig. 1 showed that at the temperature of 60
oC, the biodiesel from triolein could reach to quantitative yields, but further increase of

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temperature led to the deactivation of enzyme.
99
100
80
60
40
20
0
95
78
56
48
25
40
60
80
100
Temperature (oC)
Fig. 1 Influence of the temperature on the biodiesel yielda.
aReaction conditions: triolein, 0.5 mmol, 443 mg; [Choline][H2PO4], 100 w%, 443 mg;
lipase, 5 w%, 22 mg; methanol/oil molar ratio,10:1; 24 h.
Influence of the reaction time on the biodiesel production
The effect of the reaction time on catalysis performance was investigated at 60oC and
the results were illustrated in Fig. 2. It can be seen that the yield of biodiesel could reach
to 99 % and the further extension of reaction time had no significant influence. So, 12
h was chosen as the optimal reaction time for the subsequent studies.
100
90
80
70
60
50
0
5
10
15
20
25
Time (h)
Fig. 2 Influence of the reaction time on the biodiesel yielda

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aReaction conditions: triolein, 0.5 mmol, 443 mg; [Choline][H2PO4], 100 w%,D4O4I:310m.10g39;/C9GC03796A
lipase, 5w%, 22 mg; methanol/oil molar ratio,10:1; temperature 60 oC.
Influence of IL dosage on the biodiesel production
The influence of the dosage of ionic liquid on biodiesel production was investigated
using the optimal ionic liquid at 60 oC and the results were collected in Table 2. It can
be seen that when weight ratio of the IL to triolein was 50 %, the target biodiesel could
reach 99 % yield within 12 h. Therefore, 50 wt% choline dihydrogen phosphate based
on triolein weight was optimized for the further investigation.
Table 2. Influence of the dosage of IL on the biodiesel yield
Entrya
Ratio/%
10
Yieldb/%
1
2
3
4
49
90
99
99
20
50
100
aReaction conditions: triolein, 0.5 mmol, 443 mg; [Choline][H2PO4]; lipase, 5 w%, 22
mg; methanol/oil molar ratio, 10:1; temperature 60 oC; reaction time, 12 h. bGC yield.
Influence of the methanol/oil molar ratio on the biodiesel yield
In the enzymatic production of biodiesel, the molar ratio of methanol/oil had a great
influence on the yield of biodiesel. It is evident from Table 3 that the biodiesel yield
could reach the highest level when the methanol/oil molar ratio was 10:1 under the
optimized reaction temperature and ionic liquid dosage, but the further increase of
molar ratio of methanol to oil decreased the yield.
Table 3. Influence of the methanol/oil molar ratio (M: O) on the biodiesel yield
Entrya
M: O
4:1
Yieldb/%
1
2
3
4
5
6
85
77
93
99
94
83
6:1
8:1
10:1
12:1
14:1
aReaction conditions: triolein, 0.5 mmol, 443 mg; [Choline][H2PO4], 50 w%, 222 mg;
lipase, 5 w%, 22 mg; methanol; temperature 60 oC; reaction time, 12 h. bGC yield.

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Influence of the lipase dosage on the biodiesel production
The influence of the dosage of lipase on the biodiesel production was also
investigated and the results were collected in Table 4. It is shown that the maxium yield
was reached at the weight ratio of 5 % (lipase to triolein).
Table 4. Influence of the lipase dosage on the biodiesel yield
Entrya
Ratio/%
Yieldb/%
1
2
3
4
5
6
7
8
0
1
0
40
90
97
99
76
85
85
2
3
5
7
10
15
aReaction conditions: triolein, 0.5 mmol, 443 mg; [Choline][H2PO4], 50 w%, 222 mg;
lipase; methanol/oil molar ratio, 10:1; temperature 60 ◦C; reaction time, 12 h.bGC yield.
Influence of the water content on the biodiesel yield
After drying, water content in the ionic liquid [Choline][H2PO4] was 0.2 %, as
detected by Karl-Fisher titration. So, the influence of water content on the reaction was
investigated in the range from 0.2 % to 5.2 % using the optimized ionic liquid
[Choline][H2PO4], and the results were collected in Table 5. It was shown that the
enzymatic effect had no significant variation within the water content of 0.2-3.2 %,
suggesting that this reaction system had great tolerance to water or moisture.
Table 5 Influence of the water content on the biodiesel yield
Entrya
Ratio/%
0.2
Yieldb/%
1
2
3
4
99
98
98
85
1.2
3.2
5.2
aReaction conditions: triolein, 0.5 mmol, 443 mg; [Choline][H2PO4], 50 w%, 222 mg;
lipase, 5 w%, 22 mg; methanol/oil molar ratio, 10:1; temperature 60 oC; reaction time,
12 h. bGC yield.

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Reutilization of [Choline][H2PO4] and lipase
Considering the relative high cost of enzyme, it is intriguing to reutilize it with simple
operation. In our reaction systems, the biodiesel could reach almost quantitative yield
under the optimized reaction conditions. Meanwhile, due to the hydrophilic property,
the ionic liquid could be automatically separated with the produced biodiesel after the
completion of reaction. After simple centrifugation and decantation, the ionic liquid
and Novozym 435 could be recovered as a whole, after dried separately they can be
used for the next cycle after washing and drying. The reutilization results in Fig. 3
suggested that both the ionic liquid and Novozym 435 could be reused with high
catalytic activity. With the adequate prolonged reaction time to 17 h, the yield of
biodiesel could still reach to 99 % at the 6th cycles.
99
99
100
80
60
40
20
0
94
94
94
91
1
2
3
4
5
6
Runs
Fig. 3 Reutilization of [Choline][H2PO4] and lipase
Reaction conditions: triolein, 0.5 mmol, 443 mg; [Choline][H2PO4], 50 w%, 222 mg;
lipase, 5 w%, 22 mg; methanol/oil molar ratio, 10:1; temperature 60 ◦ C; reaction time,
runs 1-5 were 12 h and run 6 was 17h.
Utilization of oils from different resources
Based on the excellent performance of this enzymatic production system, its
utilization in the biodiesel production from different oils like edible sunflower oil and
non-edible castor oil was also investigated and the results were collected in Fig.4 and

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Table 6. It is clearly indicated that by using these two types of oils, biodiesel yield waVsiew Article Online
DOI: 10.1039/C9GC03796A
as high as 96 % as calculated by equation 1. This result suggests that this reaction
system is great intriguing in promoting biodiesel production from various oil resources.
total weight of FAME
Biodiesel yield = total weight of sunflower oil or castor oil×100% (1)
Fig. 4 The gas phase diagram of sunflower oil (a) and castor oil (b)
Table 6. Utilization of oils from different resources
Composition
FAME
FAME Yield/%
sunflower oil
castor oil
0.6
C16:0
methyl palmitate
5

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C18:0
C18:1
C18:2
C18:1
Total
methyl stearate
2
36
53
-
0.4
methyl oleate
methyl linoleate
methyl ricinoleate
1
2
92
96
96
aReaction conditions: sunflower oil or castor oil, 0.5 mmol, 443 mg; [Choline][H2PO4],
50 w%, 222 mg; lipase, 5 w%, 22 mg; methanol/oil molar ratio, 10:1; temperature 60 o
C; reaction time, 12 h. bGC yield.
Scale-up experiment
In view of the excellent catalytic activity, simple separation procedure, reusability of
the ionic liquid and enzyme, this reaction system has great potential for industrial
applications. So, the scale-up experiment to multi-gram scale using non-edible castor
oil as resource was also investigated and the results showed that the target biodiesel
yield could reach 92 % under the optimized conditions. After the reaction was
completed, the biodiesel could be separated from ionic liquids and enzymes
automatically as an insoluble phase (Fig. 5), which made it possible to feasibly separate
biodiesel product from the catalytic system and reutilize the ionic liquid and enzyme
simply and efficiently. These results further supported the potential of this catalytic
system in industrial applications.
a. In
reaction
b. After
reaction
c.
Biodiesel
product
Fig.5 Pictures of biodiesel production process
(a. in reaction, the mixture was stirred under magnetic stirring; b. after the reaction,
two phases were formed and the biodiesel was on the upper layer; c. after

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DOI: 10.1039/C9GC03796A
centrifugation, the isolated biodiesel product.)
Conclusions
The studies in this work revealed the influences of a series of ionic liquids on the
enzymatic production of biodiesel. The results showed that a biocompatible hydrophilic
ionic liquid [Choline][H2PO4] could efficiently trigger a cleaner biodiesel production
procedure. Various oil resources including triolein, sunflower oil and even castor oil
could be efficiently converted to biodiesel with simplified operation and work-up
procedures, because the hydrophilicity of ionic liquid could lead to the biodiesel
production systems conducted at homogeneous reaction and heterogeneous separation.
After the completion of the transformation, both of the ionic liquid and enzyme could
be recovered and reused for at least five cycles without significant activity decrease,
and the reaction system could be easily scaled up to multigram. All of the above made
this enzymatic biodiesel production procedure meet the criteria of green chemistry and
promising for the further industrial applications. In addition, this work also gave a
successful example for the utilization of hydrophilic ionic liquid in enzymatic
transesterifications, which will indicate a new viewpoint on the influence of ionic
liquids on the activity of enzyme.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of
China (No. 21373079 and 21733011) and S&T Research Foundation of Education
Department of Henan Province (No. 20A150022).
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Graphic Abstracts
A biocompatible and hydrophilic ionic liquid was found to efficiently promote the
enzymatic production of biodiesel with excellent yields from different oil resources.
This ionic liquid is easy to be prepared from cheap raw materials. This reaction
system was easy to be conducted, the work-up procedure was very simple, and both
the ionic liquid and enzyme could be reused for at least five cycles without significant
activity decrease.