COMMUNICATION TO THE EDITOR

Room-Temperature Ionic Liquids as

Replacements for Organic Solvents in
Multiphase Bioprocess Operations
S.G. Cull,
1
J.D. Holbrey,
2
V. Vargas-Mora,
1
K.R. Seddon,
2
G.J. Lye
1
1
The Advanced Centre for Biochemical Engineering, Department of
Biochemical Engineering, University College London, Torrington Place,
London WC1E 7JE, United Kingdom; e-mail: g.lye@ucl.ac.uk
2
The QUILL Centre, The Queens University of Belfast, Stranmillis Road,
Belfast, BT9 5AG, Northern Ireland
Received 21 July 1999; accepted 25 January 2000
Abstract: Organic solvents are widely used in a range of
multiphase bioprocess operations including the liquid
liquid extraction of antibiotics and two-phase biotrans-
formation reactions. There are, however, considerable
problems associated with the safe handling of these sol-
vents which relate to their toxic and flammable nature. In
this work we have shown for the first time that room-
temperature ionic liquids, such as 1-butyl-3-methylimi-
dazolium hexafluorophosphate, [bmim][PF
6
], can be suc-
cessfully used in place of conventional solvents for the
liquidliquid extraction of erythromycin-A and for the
Rhodococcus R312 catalyzed biotransformation of 1,3-
dicyanobenzene (1,3-DCB) in a liquidliquid, two-phase
system. Extraction of erythromycin with either butyl ac-
etate or [bmim][PF
6
] showed that values of the equilib-
rium partition coefficient, K, up to 2025 could be ob-
tained for both extractants. The variation of K with the
extraction pH was also similar in the pH range 59
though differed significantly at higher pH values. Bio-
transformation of 1,3-DCB in both watertoluene and wa-
ter[bmim][PF
6
] systems showed similar profiles for the
conversion of 1,3-DCB initially to 3-cyanobenzamide and
then 3-cyanobenzoic acid. The initial rate of 3-cyanoben-
zamide production in the water-[bmim][PF
6
] system was
somewhat lower, however, due to the reduced rate of
1,3-DCB mass transfer from the more viscous [bmim]
[PF
6
] phase. It was also shown that the specific activity of
the biocatalyst in the water-[bmim] [PF
6
] system was al-
most an order of magnitude greater than in the water
toluene system which suggests that the rate of 3-cyano-
benzamide production was limited by substrate mass
transfer rather than the activity of the biocatalyst. 2000
John Wiley & Sons, Inc. Biotechnol Bioeng 69: 227233, 2000.
Keywords: multiphase biotransformation; room-tem-
perature ionic liquid; nitrile hydratase; erythromycin ex-
traction
INTRODUCTION
Multiphase processes employing binary mixtures of immis-
cible, or partly miscible, aqueous, and organic phases are
widely used throughout the bioprocessing industry. The
largest and most well-established applications, with respect
to the volume of organic solvents to be processed, are for
the recovery of antibiotics such as penicillin and erythro-
mycin. Recently, however, there has also been considerable
interest in two-phase biotransformation processes involving
the selective modification of poorly water soluble and/or
toxic substrates (Collins et al., 1995; Lilly, 1982; Van Sons-
beek et al., 1993). In these processes, an organic phase is
mixed with the aqueous biomedium; this acts as a reservoir
for the hydrophobic substrate and also physically separates
it from the vicinity of the biocatalyst to which it would be
inhibitory or toxic at high concentrations. Industrial appli-
cations of two-phase biotransformation processes include
epoxidation reactions, ester hydrolysis, steroid dehydroge-
nation and the biodesulfurization of fossil fuels.
The use of organic solvents in bioprocess operations,
however, possesses a number of further problems. The main
concerns are the toxicity of the solvents to both the process
operators and the environment, and the volatile and flam-
mable nature of these solvents which make them a potential
explosion hazard (Schmid et al., 1998). Organic solvents
have also been shown to cause progressive damage to the
cell walls of bacteria reducing their long term operational
stability as a biocatalyst (Osborne et al., 1990; Vermue et
al., 1993; Wilkinson et al., 1994), and considerable effort
has been directed towards understanding solvent-cell wall
interactions and the discovery of solvent-tolerant bacteria
(de Bont, 1998).
Recently, room-temperature ionic liquids have emerged
as a potential replacement for organic solvents in catalytic
processes on both a laboratory and industrial scale (Holbrey
and Seddon, 1999b). A wide range of reactions have been
reported, including advances in alkylation reactions (Earle
Correspondence to: G. J. Lye
Contract grant sponsors: Biotechnology and Biological Sciences Re-
search Council; Esso; Royal Academy of Engineering; Nuffield Founda-
tion; ERDF Technology Development Programme; QUESTOR Centre,
QUB; EPSRC
2000 John Wiley & Sons, Inc.
et al., 1998), Diels-Alder cyclizations (Earle et al., 1999;
Jaeger and Tucker, 1989), hydrogenation of aromatics (Ad-
ams et al., 1999), transition metal catalyzed hydrogenation
(Chauvin et al., 1995), Heck-coupling chemistry (Carmi-
chael et al., 1999; Herrmann and Bohm, 1999), and the
development of commercially competitive processes for
dimerization, oligomerization, and polymerization of ole-
fins (Abdul-Sada et al., 1995a; 1995b; Ambler et al., 1996;
Chauvin et al., 1988; 1989). Ionic liquids have, among a
unique set of chemical and physical properties (Chauvin,
1996; Chauvin and Olivier-Bourbigou, 1995; Seddon,
1997), effectively no measurable vapor pressure, which
lends them ideally as replacements for volatile, conven-
tional organic solvents. The wide and readily accessible
range of room-temperature ionic liquids with corresponding
variation in physical properties, prepared by simple struc-
tural modifications to the cations (Gordon et al., 1998; Hol-
brey and Seddon, 1999a) or changes in anion (Bonhote et
al., 1996; Wilkes and Zaworotko, 1992), offers the oppor-
tunity to design an ionic liquid-solvent system optimized for
a particular processes; in other words, these can be consid-
ered as designer solvents (Freemantle, 1998).
In this work we aim to evaluate the potential of room-
temperature ionic liquids as a direct replacement for organic
solvents in multiphase bioprocessing operations. As an ex-
ample of a two-phase biotransformation reaction, we will
consider the Rhodococcus R312 catalyzed transformation of
1,3-dicyanobenzene (1,3-DCB) to 3-cyanobenzamide and
3-cyanobenzoic acid. The versatility of the Rhodococci for
the biotransformation of nitriles (Bunch, 1998) and the syn-
thetic applications of this enzyme system (Crosby et al.,
1994; Maddrell et al., 1996) are well known. As an example
of a liquidliquid extraction process, we will consider the
extraction of the polyketide antibiotic erythromycin-A. The
polyketides are a large and diverse class of naturally occur-
ring compounds exhibiting antimicrobial, anticancer, and
immunosuppressant effects which have annual sales in the
region of $10 billion (Carreras and Santi, 1998).
MATERIALS AND METHODS
Chemicals and Microorganism
The organic solvents, toluene and butyl acetate, and all
other chemicals and biochemicals were purchased from
Sigma-Aldrich Chemical Co. (Dorset, UK) and were of the
highest purity available.
The microorganism used, Rhodococcus R312, was kindly
supplied by Professor Nick Turner (University of Edin-
burgh, UK) and was stored on nitrile-metabolizing agar at
4C (Kerridge, 1995). Cells used in the biotransformation
experiments were prepared in shake-flask fermentations us-
ing a medium containing 200 mL deionized water, 1 g L
1
special peptone, 0.6 g L
1
malt extract, 0.6 g L
1
yeast
extract, 20 g L
1
glucose, and 0.002 g L
1
FeSO
4
7H
2
O.
The fermentations were carried out in 2 L flasks in a shak-
ing incubator operated at 200 rpm and 30C. Cells were
subsequently harvested by centrifugation at the end of the
exponential growth phase (this was typically 2 days after
inoculation at an OD
600
of 1.0).
Synthesis and Purification of Ionic
Liquid [bmim][PF
6
]
1-Butyl-3-Methylimidazolium Chloride, [bmim]Cl
An autoclave (3 L) was charged with 1-methylimidazole
(1030 g, 12.54 mol, 1000 mL, freshly distilled from CaH
2
)
and 1-chlorobutane (1205 g, 13.02 mol, 1360 mL) and
heated at 75C for 48 h under 3 bar pressure of dinitrogen.
The reactor contents were transferred to a round-bottomed
flask and the excess chlorobutane was removed under re-
duced pressure with heating. The pale yellow molten prod-
uct was transferred to a dry-box and poured into a shallow
tray, where it crystallized on cooling as an off-white solid
(yield 2200 g, 99%).
1-Butyl-3-Methylimidazolium
Hexafluorophosphate, [bmim][PF
6
]
A stirred solution of [bmim]Cl (313 g, 1.764 mol) in H
2
O
(250 mL) was cooled to 0C in an ice-bath, and hexafluo-
rophosphoric acid (264 mL, 40% aqueous solution) was
added slowly with rapid stirring. The resulting biphasic
mixture was stirred for 2 h, then allowed to come to room
temperature, then the upper, aqueous phase was decanted.
The lower, ionic liquid phase was washed with water (5
500 mL), and saturated aqueous NaHCO
3
solution (2 500
mL) to ensure neutrality, and then extracted into dichloro-
methane (400 mL). The organic phase was dried over
MgSO
4
, filtered, the solvent removed under reduced pres-
sure and the ionic liquid dried for 6 h at 70C in vacuo.
Yield 400 g, (1.41 mol, 80%) as a colorless liquid (Calcu-
lated for C
8
H
15
N
2
PF
6
: C, 33.81; H, 5.32; N, 9.86. Found: C,
33.62; H, 5.37; N, 9.90%).
The structure of the ionic liquid is shown in Figure 1.
Two-Phase Biotransformation of 1,3-DCB
The substrate-containing phase, either toluene or [bmim]
[PF
6
] (2 mL), containing 1 g L
1
1,3-DCB was added to 8
mL of 0.1M potassium phosphate buffer, pH 7, containing
100 g L
1
(wet weight) of the biocatalyst, Rhodococcus
R312, in a 25 mL shake flask. The flask was then placed in
a shaking incubator operated at 200 rpm and 30C. Samples
of the mixture (200 L) were subsequently removed at
designated time intervals and immediately added to 50 L
of 10M HCl
(aq)
in a 2 mL Eppendorf tube to quench the
transformation (Kerridge, 1995). The samples were then
centrifuged at high speed for 10 min to separate the two
liquid phases after which 100 L of the aqueous phas was
removed, filtered through a 0.2 m syringe filter (What-
man, UK) and analyzed for substrate and product con-
228 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 69, NO. 2, JULY 20, 2000
centrations by HPLC. All experiments were performed in
triplicate.
Activity of Rhodococcus R312 Cell After Exposure
to a Second Phase
The activity of the Rhodococcus R312 cells was also deter-
mined during prolonged agitation in either water-toluene or
water-[bmim][PF
6
] two-phase systems. Experiments were
performed as described above except that the concentration
of Rhodococcus R312 in the aqueous phase was 50 g L
1
and no substrate was present. The residual specific activity
of the cells was then determined by diluting 200 L of an
aqueous-phase sample to 5 g L
1
with 800 L 0.1M potas-
sium phosphate buffer, pH7, and 1 mL of the same buffer
containing 0.2 g L
1
1,3-DCB (final concentration 0.1 g
L
1
). Samples of this reaction mixture were then removed at
20-s intervals and the specific activity of the cells was de-
termined by monitoring the initial rate of amide formation
by HPLC. One unit of activity was defined as the production
of 1 M 3-cyanobenzamide per minute and was expressed
as units per mg of dry weight of bacterial mass (U/mg
bio
).
All experiments were performed in triplicate.
Equilibrium Partitioning of Erythromycin
Equilibrium partition coefficients of erythromycin-A (struc-
ture shown in Fig. 1) between either aqueous-butyl acetate
phases (K = C
org
/C
aq
) or water-[bmim] [PF
6
] phases (K =
C
il
/C
aq
) (where C is the erythromycin concentration in each
phase) were determined as a function of the aqueous phase
pH. Samples (1 mL) of 50 mM buffered aqueous solutions
over the pH range 511, each containing 0.5 g L
1
eryth-
romycin, were contacted with an equal volume of the ex-
tracting phase (either a widely used organic solvent, butyl
acetate, or the [bmim][PF
6
] ionic liquid) in tightly sealed
vials. After vigorous mixing followed by phase separation
under gravity for 24 h, samples from each of the aqueous
phases were removed and the erythromycin concentration
determined. Control experiments had previously shown that
this was sufficient time for extraction equilibrium to be
attained in both biphasic systems. The concentrations of
erythromycin in the extracting phases, C
org
or C
il
, were
calculated by mass balance; no interfacial precipitate,
change in the volume of the phases, or change in the aque-
ous-phase pH was observed in any of the experiments. All
experiments were performed in duplicate.
Analytical Techniques
The substrate, 1,3-DCB, and products of the biotransforma-
tion, 3-cyanobenzamide and 3-cyanbenzoic acid, were sepa-
rated and quantified using a Dionex HPLC system fitted
with a 4.6 mm 25 cm (5 m) Zorbax Rx-C8 reverse-phase
column. The mobile phase was a 1:1 (v/v) acetronile:phos-
phorylated water mixture pumped isocratically at a flow rate
of 1 mL min
1
. The concentration of erythromycin in aque-
ous solutions was quantified by the reaction of the antibiotic
with 9M aqueous H
2
SO
4
according to the method of Daniel-
son et al. (1993). Control experiments showed that the
[bmim][PF
6
] ionic liquid did not intefere with either the
HPLC or the H
2
SO
4
assays.
The density of the ionic liquid was determined by gravi-
metirc analysis while the viscosity was determined using a
Contraves Rheomat 115 viscometer over a range of shear
rates. All determinations were performed in triplicate at
30C.
RESULTS AND DISCUSSION
Two-Phase Biotransformation
of 1,3-Dicyanobenzene
The hydration of 1,3-DCB by the nitrile hydratase Rho-
dococcus 312 is a reaction of scientific and industrial inter-
esta number of illustrations already exist in the literature
(e.g., Crosby et al., 1994; Maddrell et al., 1996; Martinkova
et al., 1995). The substrate, however, is very poorly water
soluble and the desired product 3-cyanobenzamide can be
further hydrated by the second enzyme in the nitrile me-
tabolism pathway, amidase, to yield the corresponding acid.
Currently, we are investigating the optimization of 3-cya-
nobenzamide production in a water-toluene, two-phase sys-
tem where the organic phase acts as a reservoir for the
substrate greatly increasing the productivity of the reactor.
Toluene was selected as the organic phase, after initially
screening a wide range of solvents, because it has a high
saturation concentration of 1,3-DCB, a desirable substrate
partition coefficient (C
aq
/C
org
), is unchlorinated and has an
acceptable log
10
(P) of 2.9 (Laane et al., 1987). Like most
Fig. 1. Structure of (A) [bmim][PF
6
] and (B) erythromycin-A.
COMMUNICATION TO THE EDITOR 229
organic solvents, however, toluene has a damaging effect on
the cell wall of the biological catalyst and is hazardous to
work with, particularly at large scale, because of its toxicity
and flammability. The potential replacement of toluene with
a room-temperature ionic liquid such as [bmim][PF
6
] would
be both scientifically interesting, in relation to cell wall-
ionic liquid interactions, and highly desirable from an in-
dustrial perspective.
Before performing biotransformation experiments it was
necessary to first determine the relative solubility of the
substrate in each of the two-phase systems. Table I shows
the experimentally determined saturation concentrations of
1,3-DCB in toluene and [bmim][PF
6
] phases, together with
equilibrium distribution coefficients of the substrate and the
physical properties of the liquids. The saturation concentra-
tion of 1,3-DCB in toluene is seen to be considerably higher
than that for the ionic liquid though both are still greater
than the saturation concentration of 1,3-DCB in the aqueous
biotransformation buffer which is only 0.34 g L
1
. The equi-
librium partition coefficients are similar in the two systems
and of an order of magnitude which will facilitate the con-
tinuous delivery of low levels of the substrate, from either
the toluene or [bmim][PF
6
] phase, into the conjugate aque-
ous phase during the biotransformation. One of the future
advantages of using ionic liquids, however, is the potential
to be able to rationally control both the solubility and par-
titioning of substrate molecules. This can be achieved by
variation of the cations (Gordon et al., 1998) and anions
(Wilkes and Zaworotko, 1992) for example.
Table I also shows that the ionic liquid is more dense (
1190 kg m
3
) than both the toluene and the aqueous
phase ( 1000 kg m
3
) and will, therefore, be present as
the lower phase in most two-phase systems. The viscosity of
the ionic liquid ( 0.293 kg m
1
s
1
) over a range of
shear rates from 24 to 877 s
1
) was also found to be nearly
300 times that of water ( 0.001 kg m
1
s
1
) and nearly
600 times greater than that of toluene ( 5.1 10
4
kg
m
1
s
1
). Both the ionic liquid and toluene displayed a
Newtonian rheology over the range of shear rates investi-
gated. Simple mixing trials, in which equal volumes of the
two phases were mixed on an orbital shaker at 200 rpm in
the absence of cells, showed that the majority of phase
separation occurred in the water-[bmim][PF
6
] system after
approximately 10 min. This was somewhat slower than in
the water-toluene system where the majority of phase sepa-
ration occurred in approximately half this time.
Aqueous-phase reaction profiles for the Rhodococcus
R312 catalyzed transformation of 1,3-DCB in water-toluene
and water-[bmim][PF
6
] two-phase systems are shown in
Figures 2 and 3, respectively. Both figures show the ex-
pected conversion of 1,3-DCB first to 3-cyanobenzamide
and then 3-cyanobenzoic acid. The slower initial rates of
both amide and acid production in the water-[bmim][PF
6
]
system could be due either to the slower rate of 1,3-DCB
mass transfer from the more viscous [bmim][PF
6
] phase
(see also respective K
1,3-DCB
values in Table I) or inhibitory/
toxic effects of the ionic liquid towards the Rhodococcus
R312 cells. To test the latter possibility the residual activity
of the cells was measured as a function of time during an
extended period of mixing in each of the two-phase systems.
Figure 4 shows the retention of nitrile hydratase activity in
aqueous-toluene and water-[bmim][PF
6
] systems relative to
a single aqueous-phase control sample (the large error bars
are associated with the difficulty of removing a representa-
tive sample of cells during mixing of the two-phase sys-
tems). It is apparent from Figure 4 that toluene has a more
detrimental effect on the activity of the cells compared to
[bmim][PF
6
] where, during the early period of mixing, the
specific activity of the biocatalyst is an order of magnitude
larger. This would suggest, then, that the slower initial rate
of amide production observed in Figure 3, for the
[bmim][PF
6
] system, is due to the reduced rate of substrate
mass transfer between the two phases rather than any in-
hibitory/toxic effects of the ionic liquid. The rapid initial
decline in the aqueous-phase concentration of 1,3-DCB also
suggests that the rate of amide production is limited by
substrate mass transfer rather than cellular activity (c.f. Fig.
2 where there is an approximately constant level of 1,3-
DCB maintained in the aqueous phase due to its continuous
supply from the conjugate toluene phase). Even though the
initial rate of 3-cyanobenzamide production was lower in
Table I. Physical and equilibrium properties of toluene and [bmim][PF
6
]
systems (aqueous phase consisted of 0.1M phosphate buffer, pH 7).
Property Toluene [bmim][PF
6
]
[1,3-DCB]
sat
(g L
1
) 30 1.0
K
1,3-DCB
( C
aq
/C
tol
or C
aq
/C
il
) 0.025 0.013
Density (kg m
3
) 867 1190
Rheological behavior Newtonian Newtonian
Viscosity (kg m
1
s
1
) 5.1 10
4
0.293
Fig. 2. Rhodococcus R312 catalyzed transformation of 1,3-DCB in a
water-toluene two-phase system; () 1,3-DCB, () 3-CB, () 3-CA.
Toluene phase ratio 0.2 v/v, initial [1,3-DCB] in toluene 1 g L
1
.
230 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 69, NO. 2, JULY 20, 2000
the water-[bmim][PF
6
] system, the final yield of product in
the aqueous phase was slightly higher.
Visual observations, after settling of the phases under
gravity, showed that in the water-toluene system the cells
were aggregated together and that the majority were closely
associated with the liquidliquid interface. This had the ef-
fect of stabilizing the oil-in-water emulsion formed during
the course of the biotransformation which resulted in the
formation of a stable interphase in which the majority of
toluene was located. In the water-[bmim][PF
6
] system,
however, the cells showed little aggregation and were more
evenly dispersed throughout the bulk of the aqueous phase.
This has obvious benefits for the subsequent downstream
processing of the reactor phases, where product recovery
and breakage of the emulsion can be difficult (Schroen and
Woodley, 1997), and for the recycling and reuse of both the
[bmim][PF
6
] phase and, potentially, the biocatalyst.
LiquidLiquid Extraction of Erythromycin-A
Liquidliquid extraction is a process widely used industri-
ally for the recovery and purification of both antibiotics and
biotransformation products. Desirable criteria for the design
of such processes include favorable equilibrium partition
coefficients (typically K values > 10) and the ability to
manipulate solute partitioning through the control of param-
eters such as the pH and ionic strength of the aqueous feed
stream.
Figure 5 shows the equilibrium partitioning of the mac-
rolide antibiotic erythromycin-A (pK
a
8.6) between conven-
tional aqueous-butyl acetate phases and a novel water-
[bmim][PF
6
] two-phase system. The magnitude of the
erythromycin partition coefficients in the pH range 59 are
seen to be similar for both the butyl acetate and ionic liquid
systems although the mechanism of erythromycin extraction
in the pH range 1011 appears to be somewhat different.
Our erythromycin experiments with [bmim][PF
6
] in the pH
range 1011 were subsequently repeated using a number of
different aqueous buffer solutions to check for specific ion
effects though all gave similar results.
In the butyl acetate system, the highest K values for eryth-
romycin are observed when pH > pK where the molecule is
unprotonated and hence, most soluble in the organic phase.
Clearly this is not the case for erythromycin extraction with
the ionic liquid although Huddleston et al. (1998) have re-
cently reported the extraction of a range of simple substi-
tuted benzene derivatives, such as aniline and benzoic acid,
Fig. 4. Retention of Rhodococcus R312 nitrile hydratase activity in ()
water-toluene and () water-[bmim][PF
6
] two-phase systems; ( ) ac-
tivity of single aqueous-phase control. One unit of activity was defined as
the production 1 M 3-cyanobenzamide per minute and is expressed as
units per mg of dry weight of biomass.
Figure 5. Effect of pH on equilibrium erythromycin partition coefficients
in () water-butyl acetate and () water-[bmim][PF
6
] two-phase systems.
Fig. 3. Rhodococcus R312 catalyzed transformation of 1,3-DCB in a
water-[bmim][PF
6
] two-phase system; () 1,3-DCB, () 3-CB, ()
3-CA. [bmim][PF
6
] phase ratio 0.2 v/v, initial [1,3-DCB] in
[bmim][PF
6
] 1 g L
1
.
COMMUNICATION TO THE EDITOR 231
to be greater at pH values where the molecules are un-
charged. Nevertheless, the reduced solubility of erythromy-
cin in the ionic liquid phase at pH values greater than 9
might facilitate its recovery by a liquidliquid stripping pro-
cess (erythromycin transfer from [bmim][PF
6
] to a second
aqueous phase) carried out at high pH values.
CONCLUSIONS
In this study we have demonstrated for the first time that
room-temperature ionic liquids, such as [bmim][PF
6
], can
be considered as a direct replacement for conventional or-
ganic solvents in multiphase bioprocess operations. Ex-
ample applications include the liquidliquid extraction of
antibiotics and two-phase biotransformation processes. The
replacement of organic solvents with these ionic liquids
would enable major process design and economic con-
straints associated with the toxicity and flammability of or-
ganic solvents, to be overcome. Further research is clearly
required to explore the process implications of using ionic
liquids and the rational selection or design of ionic liquids
for particular applications.
University College London (UCL) hosts the Biotechnology and
Biological Sciences Research Councils sponsored Advanced
Centre for Biochemical Engineering and the Councils support is
gratefully acknowledged. SGC would also like to thank the BB-
SRC for the provision of a studentship. GJL would like to thank
Esso and the Royal Academy of Engineering for the award of an
Engineering Fellowship and the Nuffield Foundation for finan-
cial support (NUF-NAL). JDH would like to thank the ERDF
Technology Development Programme and the QUESTOR Cen-
tre, QUB, for financial support. KRS would like to thank the
EPSRC and Royal Academy of Engineering for the award of a
Clean Technology Fellowship.
NOMENCLATURE
[bmim][PF
6
] 1-butyl-3-methylimidazolium hexafluorophosphate
C concentration (g L
1
)
3-CA 3-cyanobenzoic acid
3-CB 3-cyanobenzamide
1,3-DCB 1,3-dicyanobenzene
K equilibrium partition coefficient (K C
org
/C
aq
, or C
il
/
C
aq
)
Greek letters
density (kg m
3
)
viscosity (kg m
1
s
1
)
Subscripts
aq aqueous
bio biomass (dry weight)
il ionic liquid
org organic
sat saturation
tol toluene
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COMMUNICATION TO THE EDITOR 233