Organic Solvents are widely used in a range of multiphase bioprocess operations. Room-temperature ionic liquids, such as 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim][PF 6] can be successfully used in place of conventional solvents.
Organic Solvents are widely used in a range of multiphase bioprocess operations. Room-temperature ionic liquids, such as 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim][PF 6] can be successfully used in place of conventional solvents.
Organic Solvents are widely used in a range of multiphase bioprocess operations. Room-temperature ionic liquids, such as 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim][PF 6] can be successfully used in place of conventional solvents.
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 References Abdul-Sada AK, Ambler PW, Hodgson PKG, Seddon KR, Stewart NJ. 1995a. Ionic liquids. World patent WO95/21871. Abdul-Sada AK, Atkins MP, Ellis B, Hodgson PKG, Morgan MLM, Sed- don KR. 1995b. Alkylation process. World patent WO95/21806. Adams CJ, Earle MJ, Seddon KR. 1999. Stereoselective hydrogenation reactions in chloroaluminate(III) ionic liquids: A new method for the reduction of aromatic compounds. Chem Commun 11:10431044. Ambler PW, Hodgson PKG, Stewart NJ. 1996. Butene polymers. European patent application EP/0558187A0558181. Bonhote P, Dias AP, Papageorgiou N, Kalyanasundaram K, Gratzel M. 1996. Hydrophobic, highly conductive ambient-temperature molten- salts. Inorg Chem 35:11681178. de Bont JAM. 1998. Solvent-tolerant bacteria in biocatalysis. Trends Bio- technol 16:493499. Bunch AW. 1998. Biotransformation of nitriles by Rhodococci. Antonie vanleeuwenhoek 74:8997. Carmichael AJ, Earle MJ, Holbrey JD, McCormac PB, Seddon KR. 1999. The Heck reaction in ionic liquids. Organic Letters 1:9971000. Carreras CW, Santi DV. 1998. Engineering of modular polyketide syn- thases to produce novel polyketides. Curr Opin Biotechnol 9:403411. Chauvin Y. 1996. Two-phase catalysis in nonaqueous ionic liquids. Ac- tualite chimique v:4446. Chauvin Y, Commereuc D, Hirschauer A, Hugues F, Saussine L. 1988. Process and catalyst for the dimeriation or codimerization of olefins. French Patent FR 2,611,700. Chauvin Y, Commereuc D, Hirschauer A, Hughes F, Saussine L. 1989. Process and catalyst for the alkylation of isoparaffins with alkenes. French Patent FR 2,611,572. Chauvin Y, Mussmann L, Olivier H. 1995. A novel class of versatile solvents for two-phase catalysts: Hydrogenation, isomerization, and hydroformylation of alkenes catalyzed by rhodium complexes in liquid 1,3-dialkylimidazolium salts. Angew Chem Int Ed Engl 34: 26982700. Chauvin Y, Olivier-Boubigou H. 1995. Nonaqueous ionic liquids as reac- tion solvents. Chemtech 25:2630. Collins AM, Woodley JM, Lidell JM. 1995. Determination of reactor op- eration for the microbial hydroxylation of toluene in a two-liquid phase process. J Ind Microbiol 14:382388. Crosby J, Moilliet, Parratt JS, Turner NJ. 1994. Regioselective hydrolysis of aromatic dinitriles using a whole cell catalyst. J Chem Soc Perkin Trans I 13:16791687. Danielson ND, Holeman JA, Bristol DC, Kirzner DH. 1993. Simple meth- ods for the qualitative identification and quantitative determination of macrolide antibiotics. J Pharm Biomed Anal 11:121130. Earle MJ, McCormac PB, Seddon KR. 1998. Regioselective alkylation in ionic liquids. Chem Commun 20:22452246. Earle MJ, McCormac PB, Seddon KR. 1999. Diels-Alder reactions in ionic liquids. Green Chem 1:2325. Freemantle M. 1998. Designer solventsIonic liquids may boost clean technology development. Chem Eng News 76:3237. Gordon CM, Holbrey JD, Kennedy A, Seddon KR. 1998. Ionic liquid crystals: Hexafluorophosphate salts. J Mater Chem 8:26272636. Herrmann WA, Bohm VPW. 1999. Heck reaction catalyzed by phospha- palladacycles in non-aqeous ionic liquids. J Organomet Chem 572:141. Holbrey JD, Seddon KR. 1999a. The phase behaviour of 1-alkyl-3- methylimidazolium tetrafluoroborates. J Chem Soc Dalton Trans 13:21332139. Holbrey JD, Seddon KR. 1999b. Ionic liquids. Clean Prod Proc 1:223237. Huddleston JG, Willauer HD, Swatloski RP, Visser AE, Rogers RD. 1998. Room temperature ionic liquids as novel media for clean liquidliq- uid extraction. Chem Commun 16:17651766. Jaeger DA, Tucker CE. 1989. Diels-Alder reactions in ethylammonium nitrate, a low-melting fused salt. Tetrahedron Lett 30:17851788. Kerridge A. 1995. The microbial biotransformation of nitrile compounds. Ph.D. thesis, University of Exeter, United Kingdom. 232 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 69, NO. 2, JULY 20, 2000 Laane C, Boeren S, Vos K, Veeger C. 1987. Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 30:8187. Lilly MD. 1982. Two-liquid-phase biocatalytic reactions. J Chem Tech Biotechnol 32:162169. Maddrell SJ, Turner NJ, Kerridge A, Willetts AJ, Crosby J. 1996. Nitrile hydratase enzymes in organic synthesis: enantioselective synthesis of the lactone moiety of the mevinic acids. Tetrahedron Lett 37:60016004. Martinkova L, Olsovsky P, Prepechalova I, Kren V. 1995. Biotransforma- tions of aromatic dinitriles using Rhodococcus equi cells. Biotechnol Lett 17:12191222. Osborne SJ, Leaver J, Turner MK, Dunnill P. 1990. Correlation of bio- catalytic activity in an organic-aqueous two-liquid phase system with solvent concentration in the cell membrane. Enzyme Microb Technol 12:281291. Schmid A, Kollmer A, Mathys RG, Withot B. 1998. Development toward large-scale bacterial bioprocesses in the presence of bulk amounts of organic solvents. Extremophiles 2:249256. Schroen CGPH, Woodley JM. 1997. Membrane separation for downstream processing of aqueous-organic bioconversions. Biotechnol Progr 13:276283. Seddon KR. 1997. Ionic liquids for clean technology. J Chemical Tech Biotech 68:351356. Van Sonsbeek HM, Beeftink HH, Tramper J. 1993. Two-liquid-phase bio- reactors. Enzyme Microb Technol 15:722729. Vermue M, Sikkema J, Verheul A, Bakker R, Tramper J. 1993. Toxicity of homologous series of organic solvents for the gram-positive bacteria Arthrobacter and Nocardia sp. and the gram-negative bacteria Aci- netobacter and Pseudomonas sp. Biotechnol Bioeng 42:747758. Wilkes JS, Zaworotko MJ. 1992. Air and water stable 1-ethyl-3- methylimidazolium based ionic liquids. J Chem Soc Chem Commun 13:965967. Wilkinson D, Woodley JM, Ward JM. 1994. Process implications of sol- vent interaction with microbial catalysts. In: Proceedings of the 1994 IChemE research event. p 162164. COMMUNICATION TO THE EDITOR 233