Novel biocompatible cholinium-based ionic liquids - Toxicity and biodegradability

Article abstract of DOI:10.1039/b922247b

The synthesis, characterisation and toxicological assessment of a new group of environmentally friendly ionic liquids are presented. Focussing on the toxic effect of the anion, the ionic liquids were designed by combining the benign cholinium cation, [NMe₃(CH₂CH₂OH)]⁺, with a range of linear alkanoate anions ([C_nH_2n+1CO ₂]^-, n = 1-9), as well as two structural isomers (n = 3 or 4). The toxicity of these ionic liquids was evaluated using filamentous fungi as model eukaryotic organisms. Surprisingly, most of the tested species showed active growth in media containing extremely high ionic liquid concentrations, up to molar ranges in some cases. The biodegradability of these ionic liquids was assessed, and new biotechnological applications for them are proposed, e.g. as solvents for biopolymers. This study leads to the better understanding of the anion influence on the ionic liquid toxicity, but its core is the recognition that conscious design of ionic liquids can be used to deliver truly biocompatible salts without adversely affecting one of the most striking of their properties - their outstanding solvent ability.

Full text of DOI:10.1039/b922247b

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PAPER
www.rsc.org/greenchem | Green Chemistry
Novel biocompatible cholinium-based ionic liquids—toxicity and
biodegradability†
a
b
b
a
a
Marija Petkovic, Jamie L. Ferguson, H. Q. Nimal Gunaratne, Rui Ferreira, Maria C. Leit a˜ o,
a,b
a
a,c
Kenneth R. Seddon, Lu ´ı s Paulo N. Rebelo and Cristina Silva Pereira*
Received 28th October 2009, Accepted 8th January 2010
First published as an Advance Article on the web 9th February 2010
DOI: 10.1039/b922247b
The synthesis, characterisation and toxicological assessment of a new group of environmentally
friendly ionic liquids are presented. Focussing on the toxic effect of the anion, the ionic liquids
+
were designed by combining the benign cholinium cation, [NMe
3
(CH
2
CH
2
OH)] , with a range of
-
linear alkanoate anions ([C
n
H
2n+1CO
2
] , n = 1-9), as well as two structural isomers (n = 3 or 4).
The toxicity of these ionic liquids was evaluated using filamentous fungi as model eukaryotic
organisms. Surprisingly, most of the tested species showed active growth in media containing
extremely high ionic liquid concentrations, up to molar ranges in some cases. The biodegradability
of these ionic liquids was assessed, and new biotechnological applications for them are proposed,
e.g. as solvents for biopolymers. This study leads to the better understanding of the anion
influence on the ionic liquid toxicity, but its core is the recognition that conscious design of ionic
liquids can be used to deliver truly biocompatible salts without adversely affecting one of the most
striking of their properties—their outstanding solvent ability.
Introduction
In the expanding studies on the toxicity and biodegradability
of ionic liquids, they were not a priori accepted as environ-
The chemistry of ionic liquids has developed dramatically during
the last decade. Their generic, yet not universal, properties,
such as negligible vapour pressure, nonflammability, chemical
and thermal stability, and outstanding solvation ability, enabled
4
mentally benign. It is widely accepted that the head group of
5,6
the cation has a deciding role in toxicity, that longer side
7
chains have a more severe impact on living cells, and that
incorporation of an ester group significantly increases their
1
rapid advance in numerous applications. The potential of ionic
8
biodegradability. The anion plays an essential role in the ionic
liquids is further emphasised because their physical and chemical
properties may be finely tuned by varying the cation and the
2
liquid physicochemical properties, and it is known to contribute
9
to the overall toxicity, but its effect is usually neglected. The
2
anion. Their dual nature is, relative to conventional molecular
predictive value of certain trends, both for the cation and the an-
ion, is unquestionable; however, generalisations should be taken
cautiously, given the current limited understanding of the modes
of toxicity of ionic liquids, their biodegradation pathways, and
their behaviour concerning biosorption, bioaccumulation, etc.
This complexity can be simplified if ionic liquids are synthesised
from carefully selected naturally-derived materials, which are
organic solvents, a clear advantage.
In order to improve old, or to create novel, ionic liquid
based processes, aiming at cost-efficiency and sustainability, an
interdisciplinary approach is essential. So-called risk-conscious
design and “thinking in terms of structure–activity relation-
ships” should be applied before implementing any new materials
on a large scale. Thus, a better, structure-based, understanding of
10
safer, carrying reduced toxicity and enhanced biodegradability.
3
the environmental fate of ionic liquids is critical. Unfortunately,
Although a wide range of cholinium ionic liquids have
this is a complex equation which crosses numerous unknown
abiotic and biotic factors. The enormous diversity of ionic
liquids is also a major concern from the ecotoxicological point
of view, since testing such a vast number of ionic liquids and
organisms is inconceivable.
11-17
been known for some time,
the alkanoates have been
largely ignored. We present here a new group of cholinium
ionic liquids. Cholinium, a quaternary ammonium cation,
+
18
[
NMe
3
(CH
2
CH OH)] , which is an essential micronutrient,
2
was chosen as the benign cation and combined with alkanoates
of systematically elongated chains. In order to better understand
the anion influence on ionic liquid toxicity, the toxicities of the
obtained ionic liquids were evaluated using filamentous fungi—
a
19
Instituto de Tecnologia Qu ´ı mica e Biol o´ gica, Universidade Nova de
previously demonstrated to be an excellent test system.
Lisboa, Av. da Rep u´ blica, 2780-157, Oeiras, Portugal
b
The Queen’s University Ionic Liquid Laboratories, QUILL, The Queen’s
University of Belfast, Belfast, BT9 5AG, UK
Experimental section
c
Instituto de Biologia Experimental e Tecnol o´ gica (IBET), Apartado 12,
2
781-901, Oeiras, Portugal. E-mail: spereira@itqb.unl.pt
Chemicals
†
Electronic supplementary information (ESI) available: The spectro-
1
13
The materials used in the ionic liquid syntheses, including
source and grade, were as follows: cholinium hydrogencarbonate
scopic data of the ionic liquids ( H, C NMR and ESI-MS) are provided.
See DOI: 10.1039/b922247b
This journal is © The Royal Society of Chemistry 2010
Green Chem., 2010, 12, 643–649 | 643

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(
1
(
Aldrich, 75 wt.% in H
00% puriss), propanoic acid (Aldrich, 99.5%), butanoic acid
Lancaster, 99+%), 2-methylpropanoic acid (Aldrich, 99%),
pentanoic acid (Aldrich, 99%), 2,2-dimethylpropanoic acid
2
O), ethanoic acid (Aldrich, 99-
Table 1 The thermal properties of the ionic liquids: onset (Tonset), de-
m g
composition (Tdec), melting (T ) and glass transition (T ) temperatures.
a
◦
a
◦
◦
◦
g
Ionic liquid
Tonset / C Tdec / C T / C T / C
m
(
(
Aldrich, 99%), hexanoic acid (Alfa Aesar, 99%), octanoic acid
Sigma, 99%) and decanoic acid (Aldrich, 96%). Sodium salts of
cholinium ethanoate
cholinium propanoate
cholinium butanoate
cholinium 2-methylpropanoate
cholinium pentanoate
169
113
97
110
165
210
172
166
172
203
177
169
166
168
80
n.d.
45
35
31
57
52
26
50
-74
the corresponding carboxylic acids were prepared via addition
of the acid to sodium methoxide (Sigma Aldrich 25 wt.% in
methanol), vacuum-filtration, and washing the precipitated salt
with cold methanol, followed by drying under high vacuum
(
cholinium 2,2-dimethylpropanoate 112
cholinium hexanoate
cholinium octanoate
cholinium decanoate
106
107
116
◦
80 C, >14 h).
Other chemicals used in the study were: cholinium chloride
choline; Sigma, ≥98%), sodium dodecyl sulfate (SDS, Amer-
a
T
onset and Tdec defined as the temperatures at which the baseline slope
(
changes during the heating, and at 50% weight loss, respectively. Please
note these are from scanning TGA, and do not represent isothermal
stabilities.
sham Biosciences, >99%), benzalkonium chloride (BAC, pre-
dominantly [C12 (CH )]Cl, but also contains
14 and C16 homologues, Aldrich), ethanol (Panreac, absolute
H
25N(CH
3
)
2
2
C
6
H
5
C
PA) and propanone (Fisher Scientific, analytical grade).
(
IBETPeE). They had previously been isolated from cork
20,21
samples purchased from several Portuguese cork industries.
Ionic liquids
Toxicity tests
The ionic liquids were prepared by dropwise addition of the
corresponding acid (1 : 1) to aqueous cholinium hydrogencar-
bonate, stirring at ambient temperature and pressure. Water
was then removed under reduced pressure, first using a rotary
The toxicity of ionic liquids to the fungal isolates was evaluated
by determining their minimal inhibitory concentrations (MIC)
and minimal fungicidal concentrations (MFC), distinguishing
between growth inhibition and death, respectively.
◦
evaporator (e.g. 70 C, 30 min), and then stir-heating in vacuo
◦
(
65-70 C, 24 h, ca. 0.01 mbar).
-1
The minimal culture medium containing glucose (1.0 g l ),
1
13
The ionic liquids were characterised by H and C NMR
-1
-1
-1
K
2
HPO
4
(1.0 g l ), NaNO
3
(3.0 g l ), ZnSO
4
·7H
2
O, (0.01 g l ),
(
2
Bruker Avance III spectrometer, 400 MHz) spectroscopy at
5 C. Their purity was confirmed by electrospray ionisation
-1
-1
CuSO
4
·5H
2
-1
O (0.005 g l ), MgSO
4
-1
·7H
2
O (0.5 g l ), FeSO
4
·7H
2
O
◦
(0.01 g l ) and KCl (0.5 g l ), was dissolved in distilled
mass spectrometry (ESI-MS) (Waters LCT Premier fitted with
electrospray). The spectroscopic analyses are provided as Sup-
porting Information.† The water contents, determined by Karl-
Fischer titration, were below 0.1 wt%. The obtained salts fulfilled
the requirements of the present study.
◦
water, sterilised in an autoclave (20 min; 121 C), and finally
supplemented with the testing compounds. Final concentrations
of ionic liquids and control compounds in growth media were
in the range from 2.5 mM up to 2 M (distributed stepwise from
0
.5 mM to 0.1 M).
3
Each liquid medium (1 cm ) was inoculated with a suspension
of fungal spores, prepared as previously described, in order
to obtain the final concentration of 10 spores per cm , and
divided into four wells (0.25 cm each) of a 96-well microtitre
Thermal properties analysis
22
5
3
The thermal stabilities and decomposition temperatures of the
ionic liquids were measured using a thermal gravimetric analyser
3
◦
plate. Cultures were incubated in the dark, at 25 C, for 14 days.
(
TGA Q5000 V3.10 Build 258). All samples were recorded in
Fungal growth (or lack thereof) was followed by measuring
the absorbance (600 nm) of the medium and, when necessary,
at the end of incubation gauging by eye the formation of
mycelium (turbidity) and/or spores. The lowest concentration
that inhibited the formation of mycelium was taken as the MIC.
Additionally, all the samples where no active growth was
detected were used as inocula and spread, with a ~2 ml loop,
onto malt extract agar medium (Oxoid, UK). The plates were
aluminium pans under a dinitrogen atmosphere. Samples were
heated up to 300 C at a heating rate of 5 C min until complete
thermal degradation was achieved. Universal Analysis, version
◦
◦
-1
4
.4A software, was used to determine the onset (Tonset) and the
decomposition (Tdec) temperatures, as the temperatures at which
the baseline slope changes during the heating, and at which
5
0% of weight loss was observed, respectively. Additionally,
their melting (T ) and glass transition temperatures (T ) were
m
g
◦
incubated in the dark, at 25 C, for 7 days. The lowest
determined by differential scanning calorimetry (DSC) (DSC
Q2000 V24.2 Build 107). Heating and cooling cycles were
concentration of the test compound which results in unviable
spores was taken as MFC. MIC and MFC values should not be
interpreted as absolute ones, but rather as an indication of the
inhibitory and the fungicidal upper concentrations limits.
The control samples were incubated under the same con-
ditions: negative control (ionic liquid free medium), cationic
surfactant control (BAC), anionic surfactant control (SDS),
anion effect control (sodium salts), common organic solvents
(ethanol and propanone) and blank samples (non-inoculated).
Cholinium chloride (choline) was also tested to measure the
cation effect.
◦
-1
conducted at the rate of 5 C min . Results are presented in
Table 1.
Fungal isolates
The following fungal isolates were used, all belonging to the
Instituto de Biologia Experimental e Tecnol o´ gica (IBET) culture
collection: Penicillium brevicompactum Dierckx (IBETPeA),
P. glandicola (Oudem) Seifert and Samson (IBETPeB), P. cory-
lophilum Dierckx (IBETF6), and P. diversum Raper and Fennell
6
44 | Green Chem., 2010, 12, 643–649
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Biodegradability assessment of the ionic liquids
and their response to the ionic liquid environment was also
19
inferred. One representative fungal isolate from each suscepti-
bility group was therefore selected for this work. They were, set
by order of tolerance: P. brevicompactum, P. glandicola, P. cory-
lophilum and P. diversum. These fungal isolates were able to grow
in media containing relatively high concentrations of cholinium
alkanoates (Table 2), and yet showed very distinct behaviours,
e.g. MIC values varied from 2.5 mM to 1.5 M (Fig. 2). The fungi
tolerance ranking defined here is consistent with that previously
Penicillium corylophilum was chosen for studies on ionic liquid
biodegradability. Concentrations of tested compounds used in
this assay were below the previously determined MICs. Fungal
3
cultures of 20 cm were inoculated as described above, and
◦
incubated in the dark, at 25 C, under agitation (90 rpm), for
3
2
8 days. The aliquot of 1 cm was taken from the cultures,
filtered (0.2 mm), freeze-dried to remove water, dissolved in
3
1
0
.7 cm of deuteriated water, and analysed by H NMR
19
reported, further highlighting the robustness and the predictive
value of the testing system, indifferent to the chemical nature of
the salt.
spectroscopy. Ionic liquid biodegradability in the static fungal
1
cultures (toxicity tests) was analysed by H NMR spectroscopy,
and in some cases the anion biodegradability confirmed by liquid
23
chromatography.
Results and discussion
In the present work, a new group of ionic liquids, composed
18
by the benign cholinium cation and alkanoate anions, was
investigated (Fig. 1). The physical and thermal properties
of cholinium ethanoate and propanoate have been reported
24
previously, but these salts are seldom studied. The linear
chains in the alkanoate anions were systematically elongated,
from ethanoate to decanoate, and two branched isomers, 2-
methylpropanoate and 2,2-dimethylpropanoate, were also anal-
19
ysed. This constitutes, pace cholinium ethanoate, the very first
systematic study on their toxicity.
Previously, it was demonstrated that several phylogenetically
closely related fungal isolates, all belonging to the Penicillium
genus, were distinctively grouped according to their growth
susceptibility in media containing ionic liquids. Importantly, a
high degree of correlation between the species genetic proximity
Fig. 2 Minimal inhibitory concentrations (MIC) of the cholinium
alkanoates ([NMe
3
(CH
2
CH
2
OH)][C
n
H
2n+1CO
2
], n = 1-9 linear chain
length): comparison of log10(MIC) values of cholinium alkanoates
in four fungal isolates, Penicillium brevicompactum, P. glandicola,
P. corylophilum, P. diversum. MIC values plotted in logarithmic scale.
19
Fig. 1 Chemical route for obtaining the cholinium alkanoate ionic liquids, [NMe
the anions used: (a) ethanoate, (b) propanoate, (c) butanoate, (d) pentanoate, (e) hexanoate, (f) octanoate, (g) decanoate, (h) 2-methylpropanoate,
i) 2,2-dimethylpropanoate.
3
(CH
2
CH
2
OH)][C
n
H
2n+1CO
2
] (n = 1-9) and chemical structures of
(
This journal is © The Royal Society of Chemistry 2010
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Table 2 Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) of cholinium alkanoates (M) in four fungal isolates
(
Penicillium brevicompactum, P. glandicola, P. corylophilum, P. diversum). Results obtained for control compounds (sodium salts of corresponding
carboxylic acids, cholinium chloride, SDS, BAC, ethanol and propanone) are also presented.
Fungal species
P. brevicompactum
P. glandicola
P. corylophilum
P. diversum
Test compounds
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
cholinium ethanoate
cholinium propanoate
cholinium butanoate
cholinium 2-methylpropanoate
cholinium pentanoate
cholinium 2,2-dimethylpropanoate
cholinium hexanoate
cholinium octanoate
cholinium decanoate
sodium ethanoate
sodium propanoate
sodium butanoate
sodium 2-methylpropanoate
sodium pentanoate
sodium 2,2-dimethylpropanoate
sodium hexanoate
sodium octanoate
sodium decanoate
cholinium chloride
SDS
>1.5
0.6
0.5
>2
0.6
1
1.5
n.d.
1.5
0.5
0.03
0.01
1.5
1.25
>1
>1
n.d.
1
0.15
0.025
0.02
>2
<0.001
<0.0025
1
0.8
0.5
0.5
0.6
0.4
0.4
0.2
0.01
0.005
0.75
0.6
0.4
0.4
n.d.
0.25
0.05
0.0075
0.001
1.25
<0.0001
<0.00025
n.d.
n.d.
0.9
0.7
0.75
0.75
0.6
0.7
0.2
0.02
0.0075
0.75
0.75
0.6
0.45
0.7
0.15
0.25
0.3
0.75
0.0625
0.01
0.0025
0.4
0.3
0.4
0.4
0.1
0.25
0.05
0.02
0.01
1.25
<0.0001
0.0025
2
0.5
0.7
0.15
0.25
0.3
0.45
0.25
0.075
0.1
0.025
0.075
0.01
0.0025
0.001
0.2
0.075
0.05
0.075
0.03
0.05
0.5
0.375
0.075
0.2
0.05
0.15
0.025
0.005
0.0025
0.5
0.15
0.075
0.15
0.5
n.d.
0.75
0.4
0.03
0.01
1.25
1
0.25
0.75
n.d.
0.5
1.5
0.07
0.03
0.005
0.5
0.4
0.5
0.6
n.d.
0.4
0.4
0.1
0.05
0.25
0.05
0.03
0.015
>2
0.125
0.005
2
0.075
0.025
0.005
0.005
>2
<0.0001
<0.001
1
0.1
0.05
0.02
0.005
>2
<0.0001
<0.00025
n.d.
n.d.
0.01
0.02
0.015
>2
<0.001
<0.0025
1
0.0025
0.005
0.8
<0.0001
<0.001
0.5
BAC
Ethanol
propanone
>4
>4
>4
>4
>4
>4
n.d. not determined. MIC and MFC concentrations (M) were reproducible in the four replicates. Data consistency was further confirmed re-testing,
randomly, ~20% of the fungal cultures. The concentrations ranged from 2.5 mM to 2 M and were distributed stepwise increasing from 0.5 mM to
0
.1 M.
The selection of tested compounds enabled us to investigate
between their toxicities and that of propanoate (equal linear
chain length).
the specific influence of the anion on the overall toxicity of
the ionic liquid. It becomes apparent that the anion toxicity is
defined by the length of the linear chain and can be ranked as
follows:
The toxicity of cholinium alkanoates, as a function of the
anion, may be partially understood by QSAR analyses using
different molecular descriptors, such as the 1-octanol/water
26
partition coefficient, log10(Kow). In studies with ionic liquids
which are not hydrophobic, log10(Kow) can be measured from
ethanoate < propanoate < butanoate < pentanoate <
hexanoate < octanoate < decanoate
27
thermodynamic data or indirectly determined by chromato-
graphic methods if the anion is a constant (the anion governs
A similar effect was reported for the cation, where high
correlation between toxicity and the side chain in the cation
28
miscibility with the HPLC mobile phase). Most studies have
focussed on the cation, leaving the effect of the anion neglected.
Nevertheless, a correlation between toxicity and lipophilicity
25
has been observed. The dodecanoate anion was apparently
slightly less toxic than decanoate (data not shown), but was
not considered in the present study because it has induced the
formation of a precipitate.
29
of the cation or the anion has been observed elsewhere. In
the present study, the log10(Kow) values of the isolated anions
30
MIC and MFC values followed the same trend (Table 2).
Fungal ionic liquid susceptibility versus the length of the linear
chain in the anion is depicted in Fig. 3 for P. brevicompactum
and P. diversum (the most and least tolerant fungal isolates,
respectively). In more than 40% of the cases, the reported MIC
and MFC values were very similar, suggesting that growth
inhibition and the spores’ viability are somehow correlated
events.
were predicted using algorithms available on the ChemSpi-
der website, www.chemspider.com. These values established a
relative scale of lipophilicity for the anions studied, increasing
linearly, from -0.285 ± 0.184 to 3.965 ± 0.185, with the increase
of the number of carbon atoms in the linear anions. The
branched isomers constitute an outlier group, reporting, relative
to their linear isomers, slightly lower log10(Kow) values. The
lipophilic trend shown by this set of data describes perfectly
the observation that toxicity increases with the elongation of
the linear chain in the anion. In addition, branching, which
generally reduces lipophilicity, resulted in lower toxicity. This
interpretation thus constitutes a reasonable view of the ionic
liquids’ toxicity, but experimental standardised methods, and
probably other molecular descriptors, are necessary for better
understanding of the specificity of their toxicity mechanisms.
2
-Methylpropanoate and 2,2-dimethylpropanoate are
branched isomers of butanoate and pentanoate, respectively.
They exhibited, relative to the linear isomers, lower inhibitory
and fungicidal effects, especially evident in the latter isomer
31
(
Table 2). Although MIC and MFC values in the non-linear
isomers were in good agreement for the same species, no trend
could be defined. Likewise, no correlation can be observed
6
46 | Green Chem., 2010, 12, 643–649
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Fig. 4 Comparison of the toxic effects, log10(MIC) (minimal in-
hibitory concentration, MIC) of the tested cholinium alkanoates
[NMe (CH CH OH)][C
(
3
2
2
n
H
2n+1CO
2
], n = 1-9 linear chain length) and
the sodium alkanoates in Penicillium glandicola. MIC values plotted in
logarithmic scale.
explained exclusively by the properties of the individual counter
ions.
The biodegradation of ionic liquids in P. corylophilum
cultures, after four weeks of incubation, was monitored by
NMR spectroscopy (Table 3). The peaks attributed to the
cholinium cation were conserved in the spectral analyses, thus
suggesting that it was only partially degraded (Fig. 5). Under
aerobic conditions, choline has been previously reported to
32
undergo almost complete biodegradation (93%). The anions
demonstrated distinct biodegradability potentials, which were
confirmed by liquid chromatography (data not shown). Very effi-
cient degradation was observed in the longer linear chain anions,
butanoate, pentanoate, hexanoate and octanoate, shown by the
disappearance of the anion peaks in the spectra. In contrast, the
short linear chain anions (ethanoate and propanoate) were not
fully degraded, most probably due to their high concentration
in media (0.375 and 0.5 M). This was supported by the chro-
matographic analyses of P. corylophilum static cultures (toxicity
tests). The anion biodegradability was observed to be highly
concentration dependent, e.g. ethanoate, 0.125 and 0.25 M,
was degraded after two weeks up to 52% and 16%, respec-
tively. The branched chain anions, 2,2-dimethylpropanoate and
2-methylpropanoate, both less toxic than their corresponding
linear isomers, were more resistant to fungal attack, leading
to null or partial degradation. Chain branching is generally
Fig. 3 Minimal inhibitory and fungicidal concentrations (MIC and
MFC, respectively) of the cholinium alkanoates ([NMe (CH CH OH)]
3
2
2
[
C
n
H
2n+1CO
2
], n = 1-9 linear chain length): comparison of log10(MIC)
and log10(MFC) values of cholinium alkanoates for the most (A) and
the least (B) tolerant fungal isolates, P. brevicompactum and P. diversum,
respectively. MIC and MFC values plotted in logarithmic scale.
The cationic and anionic surfactant controls, SDS and BAC,
and the common organic solvents, ethanol and propanone, were
either extremely more toxic or less toxic, respectively (Table 2).
The sodium salts used as controls showed the same trend, but
were slightly more toxic than the ionic liquids, reiterating the
biocompatible nature of cholinium in this test system (Table 2).
Fungal susceptibility to the ionic liquids and the salt controls
versus the length of the linear chain in the anion is depicted
in Fig. 4 for P. glandicola. In comparison, cholinium chloride,
which measured individually the cation contribution, showed
the lowest ionic liquid toxicity. This reinforces the statement that
ionic liquid toxicity is a very complex equation that cannot be
32
considered to increase resistance to aerobic biodegradation.
The distinctive biodegradability potential of isomers is well
proven while comparing the biodegradation levels of butanoate
and 2-methylpropanoate in media containing the same ionic
Table 3 Biodegradation assessment of ionic liquids using fungal isolate Penicillium corylophilum. Concentrations of tested compounds used in this
assay were below the previously determined minimal inhibitory concentrations (MIC) of ionic liquids
1
Ionic liquid
MIC/M
Tested conc/M
Anion degradation ( H NMR spectral analyses)
cholinium ethanoate
cholinium propanoate
cholinium butanoate
cholinium 2-methylpropanoate
cholinium pentanoate
cholinium 2,2-dimethylpropanoate
cholinium hexanoate
0.45
0.7
0.15
0.25
0.3
0.75
0.0625
0.01
0.375
0.5
0.1
0.1
0.1
0.5
0.05
0.01
not detected
not detected
complete degradation
partial degradation
complete degradation
not detected
complete degradation
complete degradation
cholinium octanoate
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Green Chem., 2010, 12, 643–649 | 647

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uation, using filamentous fungi as model eukaryotic organisms,
demonstrated that fungi can actively grow in media containing,
in some cases, concentrations up to molar range. The cholinium
alkanoates were less toxic than their corresponding sodium salts.
It becomes apparent that the anion toxicity is defined by its
lipophilicity, as a consequence of the length of the linear chain
and its branching. The cation was not completely mineralised by
the fungi, and the isomers demonstrated also distinct biodegrad-
ability potentials, with branched isomers being more resistant
to P. corylophilum attack. The methodology used for toxicity
assessment has proven to be a rather robust one. Nevertheless,
advanced studies on the ionic liquids’ interaction with the spore
coats and other cellular boundary structures are necessary to
further predict their environmental risks. Cholinium alkanoates
are not only environmentally benign and biodegradable, but
also extraordinarily good solvents for some very recalcitrant
plant biocomposites, in some cases reaching dissolution yields
23
of 60 wt%. There are no doubts that this family of ionic
liquids, which promises high value for future biotechnological
applications, will be harnessed further in future.
Acknowledgements
M.P. is grateful to FC&T for the fellowship SFRH/BD/
3
1451/2006. The work was partially supported by a grant
from Iceland, Liechtenstein and Norway through the EEA
financial mechanism (Project PT015) and by FC&T (Projects:
REDE/1504/REM/2005 and PTDC/QUI/71331/2006). The
authors thank M Carmo Bas ´ı lio and Dr Vit o´ ria San Rom a˜ o for
providing the fungal strains used in this study, and Dr Helena
Matias for providing data from the NMR facility - Centro
de Resson aˆ ncia Magn e´ tica Ant o´ nio Xavier at ITQB-UNL,
Oeiras, Portugal. J.L.F. and K.R.S. thank the QUILL Industrial
Advisory Board, and the EPSRC (Portfolio Partnership Scheme,
grant number EP/D029538/1), for their continuing support.
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