Abstract
Isoamyl acetate synthesis employing aqueous Candida antarctica lipase B (CaLB) solution was performed in a two-phase solvent system comprising hydrophilic ionic liquid 1-butyl-3-methylpyridinium dicyanamide and n-heptane. An X-junction glass microfluidic chip was used to obtain uniform microdroplets of n-heptane within a continuous phase of ionic liquid with dissolved enzyme and reactants, namely, isoamyl alcohol and acetic anhydride. A developed flow pattern resulted in a very large specific interfacial area for the reaction with amphiphilic CaLB and simultaneous extraction of isoamyl acetate in n-heptane. Biotransformation was performed within a continuously operated microfluidic system consisting of an X-junction chip and a silanized tube of a submillimeter diameter, which was further integrated with a microseparator based on a hydrophobic membrane. Efficient separation of n-heptane with product from ionic liquid phase containing enzyme and remaining substrates and products was achieved, enabling reuse of CaLB together with ionic liquid phase. More than 80% of productivity was preserved in each of the eight consecutive biotransformations within the integrated microfluidic system with reused lipase B in the ionic liquid phase. Subsequent ionic liquid regeneration accomplished by vacuum distillation enabled efficient reuse of this solvent in esterification with fresh enzyme.
1 Introduction
Short-chain esters are important natural flavor compounds widely used in the food [1], cosmetic [2], and pharmaceutical [3] industries. With increasing orientation toward their “natural” production, employment of enzymes has been gaining importance, and lipases (triacylglycerol lipases, EC 3.1.1.3) continue to be the most widely used enzymes in biotechnology [4].
Since the pioneer work of Klibanov in the early 1980s, it was made clear that enzymes, especially many lipases and proteases, can be used in hydrophobic organic solvents without severe loss in their activity [5]. There are plentiful biotransformations employing lipases in organic solvents [6, 7], solvent-free systems [8–10], supercritical fluids [11, 12], microemulsions [13, 14], deep eutectic solvents [15], and in ionic liquids [16–18]. One unique characteristic of lipases is the phenomenon of interfacial catalysis [19]. Advantages of lipase-catalyzed reactions in two-liquid phase systems include increase in substrate and/or product solubility, shift of reaction equilibrium in the direction of synthesis rather than hydrolysis, and increase in selectivity toward a desired product, taking advantage of the conformational role of solvents upon lipases [20]. Two-phase reaction/separation systems allow for dramatic reduction in the number of downstream steps [21]. Process streams are usually applied in batch or semibatch operational modes, although continuous process operations are known to offer several advantages [21–23].
Miniaturization, as a method for process intensification, allows for the harnessing of the high surface-to-volume ratio in an effort to achieve higher yields over shorter periods of time, higher product purity, and better process control along with the reduction in cost and equipment associated with downstream processing [24, 25]. A multiphase flow is formed when two or more partially or nonmiscible fluids are brought in contact and are subjected to a pressure gradient. The flow behavior in microfluidic devices depends on the relative flow rates of the fluid phases involved; the resulting interactions between gravitational, interfacial, inertial, and viscous forces; and the wetting behavior of the channel walls [24, 26].
Enzymatic microreactors could operate as homogeneous systems with multiphase parallel [27–29] or segmented [30–32] flow and as a heterogeneous system with immobilized enzymes [17, 33, 34]. The first report on an enzymatic reaction in an ionic-liquid (IL)/organic solvent two-phase system within a microfluidic device was published by Pohar et al. [31], where a continuously operated Ψ-shaped microreactor was used for lipase-catalyzed synthesis of isoamyl acetate in the 1-butyl-3-methylpyridinium dicyanamide ([bmpyr][dca])/n-heptane two-phase system. The chosen solvent system with dissolved Candida antarctica lipase B (CaLB), which was attached to the ionic liquid/n-heptane interfacial area due to its amphiphilic properties, was shown to be highly efficient and enabled simultaneous esterification and product removal [31]. Because ionic liquids are still very expensive, processes involving these solvents are competitive only if the ionic liquid phase is recycled and reused as many times as possible [22]. Such protocols were already tested in several batch processes [14, 22, 23, 35, 36], while, to the best of our knowledge, the reuse of solvents within microfluidic devices employing biocatalysts has not yet been described.
The aim of this work was to develop a miniaturized reactor system that would provide a very high interfacial area for the reaction and simultaneous product separation, integrated with a phase separator that would enable reuse of the solvent along with the dissolved enzyme. Lipase-catalyzed isoamyl acetate synthesis was performed in a two-phase solvent system comprising water-miscible ionic liquid [bmpyr][dca] and n-heptane. An X-junction glass microfluidic chip was used to obtain uniform microdroplets of n-heptane within the continuous phase of ionic liquid with aqueous CaLB solution and both reactants, isoamyl alcohol and acetic anhydride. Biotransformation was performed within a continuously operated microfluidic system consisting of an X-junction glass chip and a silanized polymer-based tube of a submillimeter diameter, which was further integrated with a microseparator based on a hydrophobic membrane. Ionic liquid phase with biocatalyst was reused in several consecutive biotransformations performed within an integrated microfluidic system. Finally, ionic liquid was regenerated by vacuum distillation and reused for isoamyl acetate synthesis with fresh enzyme.
2 Materials and methods
2.1 Materials
Aqueous solution of lipase B from C. antarctica with a declared lipase activity of minimum 5000 LU/g of liquid was purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Water-miscible ionic liquid [bmpyr][dca] (purity >98%) was purchased from Merck KGaA (Darmstadt, Germany). Isoamyl alcohol, isoamyl acetate, acetic anhydride, n-heptane, and (3-Aminopropyl)triethoxysilane (APTES) were all purchased from Sigma-Aldrich Chemie GmbH and were of analytical grade. Perfluoroalkoxy (PFA) tubes with 0.5 mm of i.d. were purchased from VICI (Schenkon, Switzerland).
2.2 Batch biotransformations
Batch experiments were carried out at room temperature (22°C) in test tubes, which were mixed at 1500 rpm on a vortex mixer. Esterification was performed in 2 ml of n-heptane and 2 ml of [bmpyr][dca] ionic liquid with dissolved reactants (1.5 M isoamyl alcohol with 0.5 M acetic anhydride). The reaction was started by the addition of 10% (v/v of IL) aqueous enzyme solution to give a final volume of 4 ml. At different reaction times, the two phases were separated with a centrifuge and 50 µl of the n-heptane phase was extracted and analyzed by a gas chromatograph as detailed in Section 2.7.
2.3 Microreactor setup and integration with a membrane microseparator
The whole experimental setup is shown in Figure 1. It comprises three parts: fluid delivery section (syringe pumps, microfluidic chip, and PTFE tube), flow visualization system (digital microscope), and phase separation system (membrane microseparator). A commercially available glass microreactor from Dolomite Microfluidics (Hertfordshire, UK) with a rectangular X-junction microfluidic chip was used for the experiments and was connected to high-performance syringe pumps (Harvard Apparatus, Holliston, MA, USA), which ensured highly controllable flow rates. Three inlets joined in an X-shaped junction with dimensions of 190 µm and 195 µm regarding depth and width, respectively, and further connected to the outlet channel with dimensions 190 µm, 390 µm, and 11.25 mm regarding depth, width, and length, respectively. At the outlet of the glass X-junction microfluidic chip, a silanized PFA tube of different lengths (from 0.4 to 7 m) and with 0.5 mm i.d. was fitted. For the experiments with reused ionic liquid phase, a 3.7-m-long silanized PFA tube of 0.5 mm i.d. was employed.
Scheme of the experimental setup of the integrated process with ionic liquid and enzyme reuse (dashed lines represent manual transfer).
For the silanization of the microchannel surface, PFA tubes were initially washed with deionized water and ethanol and cleaned with 4 M NaOH at 60°C for 1 h. After washing with deionized water, microchannel inner surface was treated with 10% (v/v) APTES aqueous solution for 24 h and then rinsed with deionized water.
The microchannel system was further integrated with PTFE membrane-based microseparator (Dolomite Microfluidics, Hertfordshire, UK) with an internal volume of 14 µl. At the outlets of the separator, phases were collected in closed tubes before further analysis.
2.4 Biotransformation in a continuously operated integrated microreactor system
Ionic liquid phase with 10% (v/v IL) of CaLB aqueous solution was placed in two syringes. In the first one, acetic anhydride was added to reach 0.5 M final concentration, and in the other syringe, isoamyl alcohol was added to reach 1.5 M final concentration within this phase. Ionic liquid phase was pumped through two separate inlets that were perpendicular to the inlet with n-heptane. Flow rates through each inlet were set to 20 µl/min. Biotransformations were performed at room temperature (22°C) within the previously described miniaturized microreactor system. After the separation and collection of both phases at the membrane separator outlets, n-heptane phase was analyzed for reactant and product concentrations as described in Section 2.7.
Ionic liquid phase with dissolved enzyme was refilled in syringes. Fresh isoamyl alcohol and acetic anhydride were added to reach inlet concentrations set in the first cycle. The amount of additional reactants was calculated from the produced isoamyl acetate and eight consecutive biotransformations were performed within a continuous microreactor system.
2.5 Regeneration of ionic liquid
Ionic liquid with dissolved lipase B, which was reused 8 times in a continuously operated esterification process with the separation of phases, was further regenerated using vacuum distillation. The distillation was performed at a temperature of 60°C under minimal pressure (p<500 Pa) over a duration of 4 h. A regenerated ionic liquid was reused as a solvent in a batch and in a continuous process as described in Sections 2.2. and 2.3. The regeneration of ionic liquid was repeated and the solvent was again tested in biotransformation.
2.6 Fluid flow analysis
The flow inside the microfluidic device was observed with an Aigo GE-5 digital microscope (Beijing Huaqi Information Digital Technology Co., Beijing, China). The obtained pictures were later processed with the freeware version of ImageJ 1.47b to determine the average diameter of the formed n-heptane microdroplets. This diameter and the corresponding n-heptane fluid flow rate were further used to define the volume and number of produced microdroplets per second in continuous ionic liquid phase. Furthermore, a specific surface area was evaluated from the surface of n-heptane droplets per total volume of fluid within the segment of the reactor.
2.7 Analysis of reactants and products
Isoamyl acetate, acetic anhydride, and isoamyl alcohol concentrations in ionic liquid phase after extraction in n-heptane, as well as in n-heptane phase, were determined by a gas chromatograph HP 6890 (Hewlett-Packard, Palo Alto, CA, USA) equipped with a hydrogen flame ionization detector and an HP-INNOWAX column (30 m×0.25 mm i.d.×0.25 µm). Detailed description of the method has been previously published [31]. Retention times for isoamyl acetate, isoamyl alcohol, and acetic anhydride were 1.49, 1.7, and 2.57 min, respectively.
3 Results and discussion
3.1 Fluid flow in a microreactor
A parallel flow of the n-heptane and [bmpyr][dca] within microchannel with Y-shaped inlets and outlets by which the straightforward separation of the two phases at the exit of the microreactor was ensured gave almost no conversion within the residence times tested in our earlier studies [31]. Therefore, a Ψ-shaped microreactor was employed, which resulted in a unique flow regime, consisting of long slugs of n-heptane and very small droplets constantly circulating inside the microchannel, providing a large interfacial area for the reaction and simultaneous product extraction [31].
In this work, a commercial hydrophilic microdroplet formation X-junction microfluidic device was employed to obtain liquid-liquid flow in the form of uniform microdroplets providing a large interfacial area with a defined interfacial area. Based on our previous studies (data not published), X-configuration of junction chip with hydrophilic surface was selected, while the fluid flow rates of the chosen solvents through each inlet channel were set to 20 µl/min. The formation of 85 microdroplets/s was achieved, where the estimated diameter of droplets, shown in Figure 2A, was 190 µm, corresponding to the depth of X-shaped junction and the main part of the glass channel. Specific interfacial area of droplets, evaluated from Figure 2A, was cca. 1×104/m, which is within the range reported for slug flow of an organic/aqueous two-phase system in a capillary microreactor [37] and two magnitudes of order higher than in conventional batch reactors with liquid-liquid systems [27].
Flow pattern within a microfluidic device: (A) microdroplet formation in an X-junction chip; (B) stable densely packed microdroplets in a PFA microreactor.
To ensure necessary retention time for the reaction, the silanized PTFE tube was employed as a hold-up section, where microdroplets of n-heptane in water miscible ionic liquid [bmpyr][dca] with 10% (v/v) of aqueous CaLB solution were preserved and no coalescence of microdroplets inside the 3.7-m-long PFA tube was obtained (Figure 2B).
3.2 Integration with the membrane microseparator
A chosen solvent system consisting of a hydrophilic [bmpyr][dca] and hydrophobic n-heptane phase enabled in situ product extraction to n-heptane phase, while the reactant stayed mostly in the ionic liquid phase. These findings are based on our previous studies on isoamyl acetate, isoamyl alcohol, acetic anhydride, and acetic acid extraction from [bmpyr][dca], which revealed that only isoamyl acetate had an 80% yield extracted into n-heptane, while all reactants had very low partitioning coefficients in this system (0.01 for acetic anhydride, below detectable value for acetic acid, and 0.44 for isoamyl alcohol) [31]. However, the separation of phases at the exit of the microreactor section was necessary to isolate the product and to reuse the ionic liquid with the enzyme, which was not achieved previously.
As shown in Figure 3, a miniaturized separator employing hydrophobic PTFE membrane with a total volume of 14 μl was connected with the PFA tube in order to continuously separate both phases. Flow rates employed (total flow rate was 60 µl/min) also ensured that there was enough time for separation of phases to occur. Analysis of separated n-heptane phase revealed the presence of isoamyl acetate and isoamyl alcohol, as expected from the previously mentioned partitioning coefficients, while the ionic liquid phase contained an aqueous solution of CaLB together with unreacted isoamyl alcohol, acetic acid, and nonextracted isoamyl acetate.
Integration of a microreactor section with a membrane microseparator.
3.3 Biotransformation in a continuously operated integrated system
As evident from Figure 4, a developed microreactor system enabled efficient lipase-catalyzed esterification. By the increasing residence time within the miniaturized integrated system resulting from the changing of PFA tube length and by keeping the same flow rates through the inlets in order to ensure equal flow pattern, a rise in isoamyl acetate concentration was observed, reaching a concentration of 0.51 M in the n-heptane phase at the exit of the microseparator after 12.3 min (Figure 4). Considering 0.32 M of isoamyl acetate present in the ionic liquid phase at the exit of the microseparator and the fluid flow ratio of both phases, 57.5% practical yield (115% for acetic anhydride inlet concentration) was obtained comprising a two-step reaction, yielding 2 moles of isoamyl acetate from 1 mole of acetic anhydride [6]. This is lower than obtained in our previous study with the same substrates and solvent system using the Ψ-shaped microchannel system, where 75% yield (150% for acetic anhydride inlet concentration) was obtained within 33 min [31]. In our previous work, where isoamyl acetate synthesis applying the water/n-hexane system with acetic acid as the acyl donor was performed in a microreactor with Y-shaped inlet and outlet with parallel flow, only 35% yield could be achieved within a single pass through the microreactor due to the presence of water, which had a negative effect on the thermodynamic balance and shifted the equilibrium toward hydrolysis [28].
![Figure 4 The influence of residence time in integrated microreactor system with different lengths of PFA tubes on isoamyl acetate concentration in n-heptane phase at the exit of the membrane microseparator. Esterification conditions: flow rates of all phases, 20 µl/min, 22°C; inlet concentrations: 1.5 M isoamyl alcohol, 0.5 M acetic anhydride, 10% (v/v) of CaLB aqueous solution in [bmpyr][dca].](/document/doi/10.1515/gps-2013-0082/asset/graphic/gps-2013-0082_fig4.jpg)
The influence of residence time in integrated microreactor system with different lengths of PFA tubes on isoamyl acetate concentration in n-heptane phase at the exit of the membrane microseparator. Esterification conditions: flow rates of all phases, 20 µl/min, 22°C; inlet concentrations: 1.5 M isoamyl alcohol, 0.5 M acetic anhydride, 10% (v/v) of CaLB aqueous solution in [bmpyr][dca].
A volumetric productivity of 66.1 g/(m3 s) was calculated from the concentration of synthesized product in both phases exiting the microseparator after 12.3 min, while 29.8 g/(m3 s) of product could be obtained from the n-heptane phase. In a continuous microfluidic-based process using the [bmpyr][dca]/n-heptane two-phase flow in the form of long concave-tail droplets mixed with fine droplets dispersion [31], productivity was lower (24.2 g/(m3 s), calculated from product concentration and flow rate of the n-heptane phase). It should be noted that both systems differ in enzyme concentration and, thereby, water content in the ionic liquid phase, which was 10% (v/v) in this study, while 20% (v/v) was used in a Ψ-shaped microchannel system [31], as well as in total substrate concentration, which was 32% higher in this study, so direct comparison of both reaction systems is not possible. Furthermore, the ionic liquid/n-heptane inlet flow ratio in this study was 2 times higher and flow rates applied at the conditions used for productivity calculations were 12 times higher as compared with the study of Pohar et al. (60 and 5 μl/min total flow rate, respectively) [31]. This influenced in situ product extraction to the n-heptane phase and, thereby, reaction rate. A recent study of the influence of fluid flow ratio of an aqueous/organic two-phase system on enzyme-catalyzed conversion of 1-heptaldehyde to 1-heptanol using segmented flow microreactor also revealed a huge impact of this parameter on volumetric productivity [30]. A detailed investigation of the effect of [bmpyr][dca] and n-heptane flow rates and their ratio on fluid flow pattern, as well as substrate and enzyme concentration on product synthesis within microfluidic system, is foreseen in order to optimize the integrated biotransformation process.
3.4 Biotransformation with reused enzyme and ionic liquid
The possibility of reusing ionic liquid with an enzyme was tested in a continuously operated microfluidic system with the integrated membrane separator allowing for complete separation of phases. As previously discussed, the reused ionic liquid phase contained an aqueous solution of CaLB together with unreacted isoamyl alcohol, acetic acid, and nonextracted isoamyl acetate. Therefore, acetic anhydride was added in same amounts as in the first biotransformation cycle, while isoamyl alcohol addition was adjusted to reach inlet concentrations set in the first cycle.
Results represented in Figure 5 revealed that more than 80% of the productivity was preserved in eight consecutive semicontinuous biotransformations within the integrated microfluidic system with reused CaLB in the ionic liquid phase, which confirmed previous findings that enzyme could retain its activity in several imidazolium-based ionic liquids [38, 39]. The drop in productivity might be attributed to the presence of products in the ionic liquid inlet flow, which resulted in the reversed reaction to form acetic acid. The latter has an inhibitory effect on CaLB, caused partially by the pH drop in the biocatalyst aqueous microenvironment [6]. On the contrary, isoamyl alcohol and isoamyl acetate revealed no negative effect on CaLB, used also in this study.
Preserved esterification yield considering both phases at the exit of the membrane microseparator at a retention time of 12.3 min after reuse of ionic liquid phase with dissolved CaLB. Process conditions were the same as described in Figure 4.
Isoamyl acetate concentrations evaluated in the ionic liquid and n-heptane phases at the exit of the microseparator revealed 61% extraction efficiency at the first cycle and around 72% in further cycles. This shows that extraction was not complete within the 12.3 min residence time regarding previously defined partitioning coefficient for isoamyl acetate in this solvent system, which would give 80% of extraction yield reached at equilibrium [31]. Further optimization of operating conditions leading to better product extraction is therefore necessary.
Our results are in accordance with the work of Itoh et al. [35], where in repeated recycling of the enzyme in 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim][PF6], monophasic transesterification reaction in a batch mode was proven to be possible, although the reaction rate gradually dropped with repetition of the reaction process when vinyl acetate was used as acyl donor. Recyclability of ionic liquid together with the immobilized CaLB (Novozym 435) in isoamyl acetate synthesis from acetic acid and isoamyl alcohol was tested by Feher et al. [36], and their results revealed the same level of lipase B activity within 10 cycles. Pavlidis et al. [14] also developed a simple procedure suitable for ester separation and enzyme reuse in a batch process. Lipase B-catalyzed reactions were performed in water-in-ionic liquid microemulsion systems stabilized by nonionic surfactants. After 10 cycles, the activity of the lipase B was still 90% due to the microenvironment, which offered excellent protection to the entrapped enzyme after repeated use [14].
In order to continuously operate a developed integrated microreactor system, further integration of recycling pumps and additional system for continuous supply of fresh reactants into reused ionic liquid phase should be considered. This concept seems to be very promising regarding its environment-friendly character considering solvent recycle and biodegradable catalyst operating at mild conditions.
3.5 Regeneration of ionic liquid
Ionic liquid, which was reused 8 times in a semicontinuously operated esterification process, was further regenerated using vacuum distillation and tested in a batch process employing fresh enzyme and reactants. As evident from Figure 6, regenerated ionic liquid showed no significant difference in the isoamyl acetate synthesis compared with the fresh solvent. The regeneration of ionic liquid was repeated and the solvent was again tested for biotransformation, revealing no effect on esterification yield (Figure 6). The regenerated ionic liquid was tested also in a microreactor system, confirming data from the batch process (data not shown).
![Figure 6 Test of regenerated ionic liquids (ILs) for CaLB catalyzed isoamyl acetate synthesis in batch process with 10% (v/v) of CaLB solution in ionic liquid, 1500/min, 22°C, and the following initial concentrations: 1.5 M isoamyl alcohol, 0.5 M acetic anhydride; volume ratio of n-heptane: [bmpyr][dca] was 1:1. Yield considers product in both phases.](/document/doi/10.1515/gps-2013-0082/asset/graphic/gps-2013-0082_fig6.jpg)
Test of regenerated ionic liquids (ILs) for CaLB catalyzed isoamyl acetate synthesis in batch process with 10% (v/v) of CaLB solution in ionic liquid, 1500/min, 22°C, and the following initial concentrations: 1.5 M isoamyl alcohol, 0.5 M acetic anhydride; volume ratio of n-heptane: [bmpyr][dca] was 1:1. Yield considers product in both phases.
Our results are in accordance with studies done by Dennewald et al. [22], where 25 batch biotransformations were followed by phase separation via centrifugation, product isolation through distillation, and reuse of the ionic liquid with no decrease in conversion observed during the 25 subsequent batch processes. The 1H NMR analysis of ionic liquid phase samples after its regeneration with vacuum distillation revealed no compounds resulting from the possible degradation of cation or anion due to the repeated use [22].
After distillation, a precipitation of protein from ionic liquid was observed. Similar results were reported in the literature, where the biocatalyst had to be replaced after the regeneration due to its denaturation [9, 36].
4 Conclusion
Lipase-catalyzed isoamyl acetate synthesis was performed in a two-phase solvent system comprising water-miscible ionic liquid [bmpyr][dca] and n-heptane. A miniaturized reactor system based on an X-junction glass microfluidic chip providing a very large interfacial area for the reaction and simultaneous product separation, integrated with a phase separator enabling reuse of solvent along with the dissolved enzyme, was developed. Ionic liquid phase with biocatalyst was reused in several consecutive biotransformations performed within the integrated microfluidic system. Finally, ionic liquid was regenerated by vacuum distillation and reused for isoamyl acetate synthesis with fresh enzyme. Further optimization of the integrated biotransformation process based on investigation of fluid flow rates and their ratio on fluid flow pattern, as well as of substrate and enzyme concentration on product synthesis and its extraction in n-heptane phase within a microfluidic system, is foreseen.
About the authors
Uroš Novak studied chemical engineering at the Faculty of Chemistry and Chemical Technology at University of Ljubljana, Ljubljana, Slovenia, where he obtained his BS in 2010. He is currently pursuing a PhD in chemical engineering under the mentorship of Assoc. Prof. Polona Žnidaršič-Plazl, working with downstream processes within microstructured devices.
Polona Žnidaršič-Plazl graduated with a degree in chemical engineering at the University of Ljubljana, where she also obtained her master’s degree in biochemistry and her PhD. Since 1989, she has been employed at the Faculty of Chemistry and Chemical Technology of the University of Ljubljana, Slovenia, where she currently works as an associate professor. Her major research interests are biotransformations, the application of microreactor technology in biotechnology, and microbial production of enzymes. She is a member of the Scientific Committee of the EFB Section on Applied Biocatalysis (ESAB) and of the editorial board of the journal Chemical and Biochemical Engineering Quarterly.
The financial support of the Ministry of Education, Science, and Sport of the Republic of Slovenia through Grant P2-0191 and PhD Grant 1000-10-310199 (U. Novak) is gratefully acknowledged. P. Žnidaršič-Plazl was partly supported by the European Union FP7 Project BIOINTENSE–Mastering Bioprocess integration and intensification across scales (Grant Agreement Number 312148). The authors express their gratitude to J. Pavšič and A. Pajntar for their help in experimental work and to M. Cvjetičanin for help in providing photos of microdroplet formation.
References
[1] Aravindan R, Anbumathi P, Viruthagiri T. Indian J. Biotechnol. 2007, 6, 141–158.Search in Google Scholar
[2] Hasan F, Ali Shah A, Hameed A. Enzyme Microbial Technol. 2010, 39, 235–251.Search in Google Scholar
[3] Krings U, Berger RG. Appl. Microbiol. Biotechnol. 1998, 49, 1–8.Search in Google Scholar
[4] Hult K, Berglund P. Trends Biotechnol. 2007, 25, 231–238.Search in Google Scholar
[5] Zaks A, Klibanov AM. Proc. Natl. Acad. Sci. 1985, 82, 3192–3196.Search in Google Scholar
[6] Romero MD, Calvo L, Alba C, Daneshfar A. J. Biotechnol. 2007, 127, 269–277.Search in Google Scholar
[7] Hari Krishna S, Divakar S, Prapulla SG, Karanth NG. J. Biotechnol. 2001, 87, 193–201.Search in Google Scholar
[8] Macedo GA, Pastore GM, Rodrigues MI. J. Food Agric. Environ. 2003, 1, 59–63.Search in Google Scholar
[9] Ghamgui H, Karra-Chaabouni M, Bezzine S, Miled N, Gargouri Y. Enzyme Microbial Technol. 2006, 38, 788–794.Search in Google Scholar
[10] Denčić I, de Vaan S, Noel T, Meuldijk J, de Croon M, Hessel V. Ind. Eng. Chem. Res. 2013, 52, 10951−10960.Search in Google Scholar
[11] Natalia D, Greinera L, Leitnera W, Marion B. J. Supercrit. Fluids 2012, 62, 173–177.10.1016/j.supflu.2011.11.025Search in Google Scholar
[12] Gariani RA, Fernando A, Luengo GA, Vale LS, Bazito RC, Comasseto JV. Tetrahedron Lett. 2011, 52, 3336–3338.Search in Google Scholar
[13] Pavlidis IV, Tzafestas K, Stamatis H. Biotechnol. J. 2010, 5, 805–812.Search in Google Scholar
[14] Pavlidis IV, Gournis D, Papadopoulos GK, Stamatis H. J. Mol. Catal. B: Enzymatic 2009, 60, 50–56.10.1016/j.molcatb.2009.03.007Search in Google Scholar
[15] Durand E, Lecomte J, Barea B, Piombo G, Dubreucq E, Villeneuve P. Process Biochem. 2012, 47, 2081–2089.Search in Google Scholar
[16] Sheldon RA, Lau RM, Sorgedrager MJ, van Rantwijk F, Seddon KR. Green Chem. 2002, 4, 147–151.Search in Google Scholar
[17] Deetlefs M, Seddon KR. Chem. Today 2006, 24, 16–23.10.1130/1052-5173(2006)16[23:EE]2.0.CO;2Search in Google Scholar
[18] Cvjetko M, Vorkapic-Furac J, Znidarsic-Plazl P. Process Biochem. 2012, 47, 1344–1350.Search in Google Scholar
[19] Reis P, Holmberg K, Watzke H, Leser ME, Miller R. Adv. Colloid Interface Sci. 2009, 147–148, 237–250.Search in Google Scholar
[20] Faber K. Biotransformations in Organic Chemistry: A Textbook, Springer-Verlag: Berlin, 2011.10.1007/978-3-642-17393-6Search in Google Scholar
[21] Paiva AL, Malcata FX. J. Mol. Catal. B: Enzymatic 1996, 3, 99–109.10.1016/S1381-1177(96)00057-4Search in Google Scholar
[22] Dennewald D, Pitner WR, Weuster-Botz D. Process Biochem. 2011, 46, 1132–1137.Search in Google Scholar
[23] De Diego T, Manjón A, Lozano P, Iborra JL. Bioresour. Technol. 2011, 102, 6336–6339.Search in Google Scholar
[24] Hessel V, Schouten JC, Renken A, Yoshida JI, Eds., Handbook of Micro Reactors, 1st ed., Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2009.Search in Google Scholar
[25] Hessel V, Vural GI, Wang Q, Nöel T, Lang J. Chem. Eng. Technol. 2012, 35, 1184–1204.Search in Google Scholar
[26] Günther A, Jensen KF. Lab Chip 2006, 6, 1487–1503.10.1039/B609851GSearch in Google Scholar
[27] Maruyama T, Uchida J, Ohkawa T, Futami T, Katayama K, Nishizawa K, Sotowa K, Kubota F, Noriho K, Goto M. Lab Chip 2003, 3, 308–312.10.1039/b309982bSearch in Google Scholar
[28] Žnidaršič-Plazl P, Plazl I. Process Biochem. 2009, 44, 1115–1121.Search in Google Scholar
[29] Swarts JW, Vossenberg P, Meerman MH, Janssen AE, Boom RM. Biotechnol. Bioeng. 2008, 1, 855–861.Search in Google Scholar
[30] Karande R, Schmid A, Buehler K. Adv. Synth. Catal. 2011, 353, 2511–2521.Search in Google Scholar
[31] Pohar A, Plazl I, Žnidaršič-Plazl P. Lab Chip 2009, 9, 3385–3390.10.1039/b915151fSearch in Google Scholar
[32] Pohar A, Žnidaršič-Plazl P, Plazl I. Chem. Eng. J. 2012, 189–190, 376–382.Search in Google Scholar
[33] Mohr S, Fisher K, Scrutton NS, Goddarda NJ, Fielden PR. Lab Chip 2010, 10, 1929–1936.10.1039/c003561kSearch in Google Scholar
[34] Lloret L, Eibes G, Moreira MT, Feijoo G, Lema JM, Miyazaki M. Chem. Eng. J. doi: http://dx.doi.org/10.1016/j.cej.2013.03.018.10.1016/j.cej.2013.03.018Search in Google Scholar
[35] Itoh T, Nishimura Y, Ouchi N, Hayase S. J. Mol. Catal. B: Enzymatic 2003, 26, 41–45.10.1016/S1381-1177(03)00147-4Search in Google Scholar
[36] Feher E, Illeová V, Kelemen-Horváth I, Bélafi-Bakó K, Polakovič M, Gubicza L. J. Mol. Catal. B: Enzymatic 2008, 50, 28–32.10.1016/j.molcatb.2007.09.019Search in Google Scholar
[37] Jovanović J, Rebrov EV, Nijhuis TA, Kreutzer MT, Hessel V, Schouten JC. Ind. Eng. Chem. Res. 2012, 51, 1015–1026.Search in Google Scholar
[38] Lozano P, De Diego T, Carrie D, Vaultier M, Iborra JL. J. Mol. Catal. B: Enzymatic 2003, 21, 9–13.10.1016/S1381-1177(02)00128-5Search in Google Scholar
[39] Roosen C, Müller C, Greiner L. Appl. Microbiol. Biotechnol. 2008, 81, 607–614.Search in Google Scholar
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