- Email: [email protected]
Two-liquid-phase bioreactors
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Two-liquid-phase bioreactors H. M. Van Sonsbeek, H. H. Beeftink and J. Tramper Wageningen Agricultural University, F o o d and Bioprocess Engineering Group, D e p a r t m e n t o f F o o d Science, Wageningen, The Netherlands
The application of two liquid phases that are poorly miscible is a fascinating research topic fi~r biocatalytical conversions because of the promising results. Motives for application include an increase of productivity and achievement of continuous processing, but new limitations arise, e.g., interfacial effects such as biocatalyst accumulation and loss of activity, medium component accumulation, and slow coalescence. Centrifuges, membranes, and immobilization are tools that can overcome part of the problems, but more fundamental knowledge about interfaces and coalescence is still necessary for successfid application. For scaleup and further development of processes based on the obtained results, a choice must be made for the configuration of the experimental setup of a bioreactor. Aspects like aeration, shear stress, batch or continuous processing, and immobilization can play an important role. This review article describes these aspects and the proposals that have been made in recent years concerning two-liquid-phase bioreactors. It shows some aduptations to existing bioreactors, such as loop reactors and stirred-tank reactors.
Keywords: two-liquid-phase; bioreactor; loop reactor; organic solvent; oxygen transfer Introduction This review article deals with bioreactors in which two liquid phases are applied. In 1982 Lilly ~ published an article entitled "Two-liquid-phase biocatalytic reactions" and predicted new commercial applications for multiliquid biocatalytic reactors. In 1987 Lilly et al. 2 published "Two-liquid-phase biocatalytic r e a c t o r s , " which mainly gives a short general description of aspects that can play a role when two liquid phases are applied in bioreactors. The present article is about the same topic and gives an overview of what has been achieved with two-liquid-phase bioreactors to date. Before discussing these bioreactors, a short consideration is given about the introduction of two liquid phases in biotechnological production processes. New developments in biotechnology are focused on new products or the introduction of new processes as a substitute to the conventional way, or improvements in performance of existing biotechnological applications. In recent years much attention has been given to the use of two-liquid-phase systems. The application of organic solvents especially has fascinated many researchers and is still considered to be a promising topic,
Address reprint requests to Dr. Van Sonsbeek at the Wageningen Agricultural University, Food and Bioprocess Engineering Group, Department of Food Science, P.O. Box 8129, 6700 EV Wageningen, The Netherlands Received 30 June 1992; revised l0 November 1992 722
since many biocatalytic reactions involve organic components that are poorly water-soluble. For this reason, addition of water-soluble organic solvents in small proportions has been well known for a long time as a technique to improve availability of poorly water-soluble reactants to cells and enzymes. More recent research concerns the use of two immiscible liquid phases in a bioreactor. In most cases the aqueous phase contains the biocatalyst, possibly immobilized. The second liquid phase can be an organic solvent or another water phase. The latter, called an aqueous two-phase system, consists of two polymer solutions or a polymer and a salt solution. Reversed micelles are a completely distinct category of two-liquid-phase systems and can be considered as pools of water stabilized in an organic solvent by a surfactant. This category will not be discussed here. In this article, research developments concerning two-liquid-phase bioreactors will be discussed with the aim to give a clear comprehension of the motives of their use and to give an overview of the state of the art. The review is focused on the cases where two free liquid phases are present. Systems where one of the phases is only a hydration liquid are not highlighted. Emulsion systems are briefly discussed. First, some general aspects of conventional bioreactors will be highlighted, such as aeration, shear stress, continuous or batch processing, and immobilization. These aspects are discussed because they can play a major role in the performance of two-liquid-phase bioreactors. Therefore these aspects will return in the subsequent
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~ 1993Butterworth-Heinemann
Two-liquid-phase bioreactors: H. M. Van Sonsbeek et al. section where bioreactors are discussed in which an organic solvent and a catalytic aqueous phase are present. The organic solvent can be the continuous or the dispersed phase and it can be the reactant or a solvent in which part of the reactant and/or product is dissolved. In addition, the suitability of aqueous twophase systems as a biological medium and some research examples are given. Finally, some conclusions are drawn and future perspectives are given.
Conventional bioreactors Biotechnological possibilities are often shown on a laboratory scale in flasks or tubes. Further development of such processes means that the same or better performance is pursued on a larger scale, which implies that bioreactor technology becomes important. A great variety of bioreactor types exist, of which the stirredtank reactor (STR) and the bubble column (BC) are the simplest and most widely used. Many biotechnological processes require oxygen that is transferred from air to the culture broth. In stirred-tank reactors air bubbles brought into the reactor are dispersed by the stirrer to obtain a large exchange area for mass transfer. Therefore high oxygen-transfer rates can be achieved, which is important because this is often the limiting factor in aerobic bioconversions. Bubble columns are attractive, even on a small scale, because of their simple construction. On a large scale this advantage becomes even more important because aeration at high energy input rates is much simpler than stirring. Besides these two basic types, other bioreactors have been developed, such as membrane, packed-bed, trickle-bed, or fluidized-bed bioreactors and bioscrubbers. These types will not be discussed in detail here. Because the oxygen-transfer rate is often the ratelimiting step, many biotechnological studies have focused on this topic. The limited transfer rate is the consequence of the contradiction between the need for oxygen of many biocatalysts and the low solubility of oxygen in aqueous environments, which are most often used. Closely related to this aspect is the issue of the cultivation of shear-sensitive cells, such as plant, animal, and insect cells. Stirring enhances oxygen-transfer rates to a great extent, but is only sparingly possible in these cultures, because of damaging effects. Therefore different kinds of reactors, such as airlift loop reactors (ALRs), have been proposed to be suitable for these purposes. 3 But even aeration by sparging air has been found to have a detrimental effect on ceils, 4-6 and various kinds of aeration methods have been developed, e.g., bubble-free aeration using silicon tubing. 7 The described case of obtaining appropriate oxygen supply shows the importance of the choice of the reactor type and adjustments of process conditions to the specific needs of the application. The introduction of nonconventional media can be regarded as such an adjustment that improves processes. Examples of nonconventional media are organic solvents, supercritical solvents, or aqueous two-phase systems. The bioreactors in which these media are applied can be the stan-
dard-type bioreactors or standard types that are adjusted for use with nonconventional media. Examples can be found in the next sections.
Organic solvents in bioreactors The topic of organic solvents as biocatalytic reaction media has been reviewed many times 1.8-17 and needs no further explanation here beyond a short summary of the arguments that are used to indicate the usefulness of this method. The argument that is relevant most often is better solubility of substrates and/or products; an example is steroid transformation. ~8 Another argument is that reverse hydrolytic reactions, which are not feasible in pure aqueous systems, can become feasible; an example is the esterification of a fatty acid and an alcohol.19 In some cases product inhibition can be reduced because of the removal of the product into the organic phase. Transfer of products to the organic phase can shift the apparent reaction equilibria calculated on an overall volume basis. 2° If the product concentration is increased due to better solubility in the organic phase, product recovery becomes easier. There are also a few aspects of organic solvents that give rise to new problems or less attractive phenomena such as inhibition or biocatalyst inactivation. Furthermore, in extractive fermentations, unintentional formation of emulsions and removal of protein from the fermentation broth can occur (protein stripping). A great number of solvent types can be applied in bioreactors, such as alkanes, alkenes, esters, alcohols, ethers, perfluorocarbons, etc. Only the group of the perfluorocarbons will shortly be highlighted, because they are relatively unknown. Mattiasson and Adlercreutz 21 and King e t a l . 2"- describe the use of perfluorocarbons in biotechnology. Perfluorocarbons are ring or straight-chain hydrocarbons in which hydrogen atoms have been replaced by fluorine. Perfluorocarbons are both stable and virtually chemically inert due to the presence of the strong carbon/fluorine bonds (ca. 487 kJ mol-~). In addition, the fluorine atoms offer steric protection to the carbon groups. Because of the low interactions between perfluorocarbon molecules themselves, gas molecules can easily occupy intramolecular spaces and large volumes of gases can thus dissolve. There is no chemical attraction of oxygen or carbon dioxide to perfluorocarbons, which implies that Henry's law for dissolved gas is applicable. Perfluorochemicals should not be confused with a related and currently, from the point of view of environmental care, somewhat controversial group of compounds, chlorofluorocarbons. The choice of the organic solvent depends on the purpose of their use. In case of extractive biocatalysis, for instance, the product should dissolve preferentially in the solvent. Besides product solubility, toxicity of a solvent for the biocatalyst is of general importance, and much research is carried out on this topic. In general, a positive correlation is found between hydrophobicity of solvents and nontoxicity for biocatalysts. A measure for hydrophobicity that was found very suit-
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Review
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able for characterization of solvents, is the log P value, which is the logarithm of the partition coefficient of a solvent in a water-octanol two-phase system. Solvents with a log P value above 4 are very hydrophobic and generally show no toxic effects on biocatalysts. Bruce and Daugulis 23 recently presented solvent selection strategies for extractive biocatalysis, and concluded that it is possible to compose mixtures of solvents that show good extraction properties as well as good biocompatibility, even at relatively high concentrations of toxic solvent. For the application of organic solvents, it is obvious that existing standard-type bioreactors can be used, such as stirred-tank, fluidized-bed, and column reactors. Small adjustments in the experimental setup can often be sufficient to operate a two-liquidphase biocatalytical process. Examples of these adjusted reactors for research purposes are described in the literature and given here. In many investigations of two-liquid-phase biocatalysis, stirred-tank reactors are used. 24 In Figures 1 and 2 the two basic experimental setups for extractive fermentation with stirred-tank reactors are given. 25 The first setup is the simpler one, but it can lead to a rather stable emulsion that requires a long settling time. In that case an external extraction column is more favorable because emulsion formation is less vigorous. An external extraction column is also more effective be-
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cause it is a multistage contactor, whereas for direct solvent addition, only one equilibrium stage can be achieved. Within the group of the authors, Brink and Tramper 26"27investigated the application of organic solvents in an immobilized-cell fluidized-bed reactor and used the epoxidation of propene by immobilized M y c o b a c t e r i u m cells as a model system. Concerning column-type bioreactors, a few proposals can be found in the literature for the configuration with an organic solvent as one of the two liquid phases. Chibata et a/. 28 describe in a patent a bioreactor system with external aeration of hydrocarbons or perfluorocarbons. Pumping these air-saturated solvents via a liquid sparger in the bioreactor, instead of air sparging, enhances oxygen-transfer rates. The background and literature concerning this aspect are described in Aeration of two-liquid-phase systems. Tramper et al. 29 introduced the liquid-impelled loop reactor (LLR), which is schematically drawn in Figure 3, and Van den Tweel e t a / . 3° showed an experiment in which this reactor was used for the conversion of benzene to cis-benzene glycol. Furthermore, Buitelaar et a/. 3~ have been working on the use of the LLR for the cultivation of plant cells, and at present the conversion oftetralin to tetralol by A c i n e t o b a c t e r is investigated. Gianetto et al.32 introduced a continuous extraction loop reactor, which will be discussed later in Batch or continuous. Finally, the membrane reactor is a bioreactor in which two immiscible liquid phases can be applied and must therefore be mentioned here. An essential difference from the other types is that the two liquid phases are not in direct contact, but separated by a membrane. The membrane also serves as immobilization support for the biocatalyst, usually an enzyme. Van der Padt et al.~9 demonstrated the suitability of a membrane biorcactor for the enzymatic esterification of glycerol and fatty acids with the enzyme lipase immobilized on a hollow-fiber membrane (Figure 4). The membrane keeps the polar and apolar liquid phases separated, and this gives the possibility of selective adsorption of monoesters with an adsorption column in the apolar
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Liquid-impelled loop reactor (LLR) (Tramper et aL 29)
Two-fiquid-phase bioreactors: H. IV~. Van Sonsbeek et al. of aqueous two-phase systems, which are at this moment still in a rather experimental stage because of the lack of fundamental knowledge of partitioning.
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circuit. Other examples of research concerning twoliquid-phase membrane bioreactors are the work of Knez and Leitgeb, 33 Koizimu et al., 34 Pronk et al., 35 Steinmeyer and Schuler, 36 and Yamane e t a / . 37"38
Aqueous two-phase systems Besides the use of an organic solvent in combination with an aqueous phase, the aqueous two-phase system is another possibility of using two liquid phases. This is a system that consists of two immiscible aqueous phases, which are polymer solutions or a polymer and a salt solution. The phenomenon has long been known, and a good overview of the principle is given by Albertsson. 39In the last decade the interest in applications of aqueous two-phase systems in biotechnology has increased, due to the combination of two attractive properties. As in other two-liquid-phase systems, partitioning of components and biocatalysts between the two phases occurs, which is important for downstream processing and biocatalyst retention. The second attractive aspect in aqueous two-phase systems is the very low interfacial tension (0.0001-0. l mN m-~) compared to that of organic solvent/water systems (0.5-50 mN m-I). This keeps proteins in solution, and unfolding at the interface is prevented. Due to this property, research on biotechnological applications of aqueous two-phase systems is often focused on isolation of proteins, cells, or organelles from the broth.4°'4~ The partition coefficient for these components in aqueous twophase systems is normally in the order of magnitude of 0.l-10, but in some cases it can be improved by affinity interactions. However, this application as an isolation method is not considered here as a replacement of existing techniques, but as complementary to more selective isolation methods. An attractive feature of this system, as compared to other isolation methods, is that the scaleup seems easy. Another way in which aqueous two-phase systems can be applied for biotechnological purposes is to use them as medium for extractive biocatalysis. Usually the bioconversion preferentially takes place in one of the two phases, and the product can be extracted in the other p h a s e : °'42'43 Andersson and Hahn-H/~gerda142gave an extensive review of accomplishments and prospects for the application
In spite of the consistent claims of the advantages of continuous processing, such as a higher productivity and a more uniform product, most biocatalytical processes are still carried out as batch processes, especially in the food industry. The enormous amount of work that has been done through the years to achieve continuous processing for several applications has not yet proved the suitability that is often claimed. An example of a continuous process that failed in practice is found in the history of beer brewing. The complexity of a continuous brewing process required the close attention of skilled staff, and labor costs turned out to be too high. 44 On the other hand, developments are still going on, which is illustrated by the recent presentation of a continuous process for the production of soy sauce. 45 Improving the feasibility of continuous processing is often used as an argument for the use of two-liquidphase systems. Although it seems difficult to set up a continuous process with two-liquid-phase systems, several articles have been published on extractive biocatalysis.2328'32'46-56 This is a type of continuous biological production process in which the process is not disturbed, and sometimes is even improved, by in situ recovery of the product. Ethanol production is an especially popular topic in this field. 32'47"5°'53'54 A recent example is the work of Gianetto et al. 32 who developed and used a continuous extraction loop reactor (CELR), which can be seen in F i g u r e 5. This reactor is an external-loop reactor, in which liquid circulation is induced by a gas flow in one of the vertical tubes and ethanol is extracted by a countercurrent extraction in the other tube. The experiments concern glucose fermentation by S a c c h a r o m y c e s cerevisiae entrapped in calciumalginate spheres. The authors observed problems with the calcium-alginate matrix, such as low mechanical
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Review resistance, a leakage of viable cells, a significant internal diffusion limitation, and a decrease in viability of the entrapped microorganisms. They emphasize, however, that the reactor is still in a preliminary stage. Mattiasson 43described a classical example of a product-inhibited biocatalytic process, the production of acetone and butanol from glucose using Clostridium acetobutylicum. From batch experiments in aqueous two-phase systems, it was concluded that butanol production should preferably be performed in a continuous process in which the product is removed and fresh substrate added. In general, most two-liquid-phase research is carried out in batch experiments, and not much attention is given to the application. Claimed advantages are often not proven, and processes still need to be designed. Although application has not yet been very successful, there is still faith that the introduction of two-liquidphase systems is suitable for the old ideal of continuous processing.
Immobilization An extensive overview of immobilization of biocatalysts is given by Rosevear. 57 Black 58and Venkatasubramanian et al. ~9 give good descriptions of the applications of immobilized biocatalysts and the reactors that can be involved. There are a few valid motives for immobilization ofbiocatalysts. The most important one for growing cells is the increase of productivity, because it is possible to work with dilution rates above washout conditions. ~8'59 Furthermore, facilitated rec o v e r y of the biocatalyst and protection of the biocatalyst against a damaging turbulent environment can play a role as well. Also, because of diffusion limitations, it is possible that an immobilized biocatalyst will not be exposed to an inhibiting substrate concentration. In two-liquid-phase systems, gel entrapment of biocatalysts can protect them from denaturation at the liquid/ liquid interface. Brink and T r a m p e r 6° found that, by immobilization, physical p h e n o m e n a such as cell aggregation at the water/organic interface and formation of biomass clots could be eliminated and therewith reduce loss of catalytic activity caused by exposure of mycobacteria to organic solvents. This was also found by Buitelaar et al. 61 who applied plant cells of Tagetes minuta in a two-liquid-phase system. For these applications it is of importance to realize that immobilization used as a technique to prevent free biocatalysts in the solution can only be successful if it concerns nongrowing biocatalysts or a high dilution rate.
Aeration of two-liquid-phase systems In many aerobic bioprocesses, the oxygen supply rate to the aqueous phase is the limiting factor. The use of two liquid phases can contribute in two different ways to improve this. First, it is possible to aerate one liquid phase and lead it through the other that contains the biocatalyst. Second, an emulsion can be created that forms the medium and that is aerated by sparging air or oxygen-enriched air. 726
Damiano and Wang 62 presented a method in which a perfluorocarbon was used as an oxygen carrier, c o n tinuously removed from the bioreactor after coalescence of the droplets at the bottom. One circuit of perfluorocarbon was pumped back in the bioreactor through a nozzle at the top. A second circuit of perfluorocarbon was reaerated and then pumped back in the bioreactor. Oxygen was transferred by means of liquid/ liquid contact. Escherichia coli was cultivated, and it was shown that operation of a column with externally aerated perfluorocarbon was capable of supplying oxygen at significantly higher rates than the best run of a bubble column. This is ascribed to a much larger surface area available for mass transfer, which is possible for three reasons: (I) at equal work inputs, drops will be smaller than bubbles due to lower surface tension: (2) bubblcs generally coalesce into larger bubbles much more rapidly than do drops: (3) liquid holdups can be fixed at levels much higher than typical gas holdups. The process described by Damiano and Wang 62 was limited by the coalescence rate of the solvent droplets at the bottom of the reactor. Cho and Wang 63 also used a second liquid phase as an oxygen carrier instead of the direct sparging of a gas into a hybridoma cell culture to increase the oxygentransfer rate. Overall volumetric mass-transfer coefficients were measured up to five times higher than in a directly aerated system. It was concluded that the interfacial area was made larger quite easily. No harmful effects of the perfluorocarbon on the hybridoma cells were detected. Unfortunately, no positive results on the application could bc reported due to cell aggregation at the interface of the medium and the perfluorocarbon. In our laboratory a distinction was made between thc mass-transfer coefficient and the available exchange area of a liquid/liquid system. Mass-transfer coefficients were measured that are lower than in gas/ liquid systems. ~ H o w e v e r , because of a higher specific exchange area in the experimental setup, the masstransfer rate of the liquid/liquid system was favorable, when compared with gas/liquid systems at equal dispersed-phase flow rates. Because of slow coalescence, the maximum dispersed-phase flow rate was limited, rcsulting in a severe restriction for the maximum oxygen-transfer rate in liquid/liquid systems. Ju et a l Y emphasize in their work that in the case of application to low-shear, gently mixed systems, the general efficiency of exclusively externally aerated perfluorocarbons as oxygen-transfer enhancers is unacceptably low. They also point out the problem of protein stripping from the growth medium by the circulated perttuorocarbons. The second way to improve the oxygen-transfer rate by applying two liquid phases is as an emulsion. It has been reported many times that very small droplets of hydrocarbons or perfluorocarbons can have an enhancing effect on the oxygen-transfer rate. 65-75 Linek and Benes 72 studied the mechanism of gas absorption into organic solvent water emulsions. T h e y distinguished organic solvents on the basis of their spreading coefficient:
Enzyme Microb. Technol., 1993, vol. 15, September
Two-liquid-phase bioreactors: H. M. Van Sonsbeek et al. Sow = crwA - (O'oA + Crow)
(I)
where o- is the interfacial tension and the subscripts O, W, and A stand for organic solvent, water, and air, respectively. Organic solvents with a negative spreading coefficient showed no influence on the mass-transfer coefficient, but had a negative influence on the specific exchange area. Organic solvents with a positive spreading coefficient gave positive effects on both the mass-transfer coefficient and the specific exchange area. Hassan and Robinson 67 found results that were in agreement with the data of Linek and Benes. 72 The increase of the specific exchange area in the case of a positive spreading coefficient is assumed to be caused by spreading of the organic solvent as a thin film on the gas/aqueous interface, lowering the surface tension, and thereby increasing the specific interracial area of the gas dispersion. The decrease in the case of a negative spreading coefficient is assumed to be caused by static accumulation of droplets at the interface, thus blocking part of the surface area available for oxygen mass transfer. Rols et al. 73 concluded that the effect of organic-solvent droplets on the specific gas-emulsion exchange area is usually of minor importance compared to the enhancement effect on the mass-transfer coefficient, and denoted the principle as oxygen vector. Several mechanisms of the increased mass-transfer coefficients for organic solvents with a positive spreading coefficient have been proposed, 65-67'72'73 but there remains much uncertainty. It is most likely that in some way there is a regularly occurring interchange of droplets between the air/aqueous interface and the bulk region. Some examples in the literature are found that clearly demonstrate the positive effect of organic-solvent droplets on volumetric transfer coefficients. Rols and Goma 75 found a positive effect of soybean oil on the volumetric oxygen-transfer coefficient during cultivation of A e r o b a c t e r a e r o g e n e s . Ho et al. 6s found an enhanced oxygen supply by adding hexadecane to the oxygen-limited penicillin fermentations with Penicillium c h r y s o g e n u m in shake flasks and in a stirred tank. They denote the transfer-enhancement effect as a higher effective oxygen solubility of the fermentation medium. Ju et al. 65 used E s c h e r i c h i a coli to demonstrate the mass-transfer enhancement effect, when instead of an aqueous phase perfluorocarbon emulsions are used in a stirred-tank reactor. Up to a fivefold increase of the oxygen-transfer coefficient is reported in the literature. TM Therefore, the strategy of adding small droplets of an organic solvent to a fermentation medium seems to be very useful for improving the oxygen-transfer process.
Conclusions and future perspectives
success is still limited. The overoptimistic view, given by many authors with respect to applications of twoliquid-phase systems, suits the sobering view on biotechnology in general, given by Moses and Cape 76 in their book B i o t e c h n o l o g y , The S c i e n c e a n d the B u s i n e s s . There is a world of difference between laboratory achievements and development of processes for commercial purposes. Interfacial effects play the key role in two-liquidphase bioreactors. These effects can be slow coalescence, cell aggregation, or biomass clotting at the interface with loss of activity of the biocatalyst, and accumulation of medium components, especially proteins, at the interface. Slow coalescence makes continuous processing difficult. So far most laboratory biocatalytic reactions in two-liquid-phase systems have been restricted to batch processes. Centrifuges or membranes can be a great help in overcoming this problem. Also many physical methods, such as heating and packing materials, and chemical agents are used for speedup of coalescence. These solutions are always of a practical nature and thus without basic knowledge of the mechanisms. It only works for the process it was developed for. The problem of negative effects of the interface on the biocatalyst can be reduced by immobilization of the biocatalyst. In general, more fundamental knowledge about interfacial effects and the coalescence process is necessary to improve possibilities and process design when two-liquid-phase bioreactors are involved. A few concepts of two-liquid-phase bioreactors exist, but these are still in a rather experimental stage: the continuous extraction loop reactor of Gianetto et al., 32 the liquid-impelled loop reactor of Tramper et a[., 29 and the stirred-tank reactor. 9
References 1 2
3 4 5
6
The number of publications about research work on biocatalysis in nonaqueous media is enormous, and even the review articles on this topic are numerous. In spite of all the advantages that the researchers come up with, serious applications are still scarce. It is good to realize that from a scientific point of view tremendous progress has been made, but that commercial
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Lilly, M. D. Two-liquid-phase biocatalytic reactions. J. Chem. Tech. Biotechnol. 1982, 32, 162-169 Lilly, M. D., Harbron, S. and Narendranathan, T. J. Twoliquid-phase biocatalytic reactors, in Methods in Enzymo/ogy. Vol. 136: Immobilized Enzymes and Cells. Part C (Mosbach, K., ed.) Academic Press, Orlando, FL, 1987, pp. 138-149 Cherry, R. S. and Papoutsakis, E. T. Hydrodynamic effects on cells in agitated tissue culture reactors. Bioproc. Eng. 1986, 1, 29-41 Chalmers, J. J. and Bavarian, F. Microscopic visualization of insect cell-bubble interactions II: The bubble film and bubble rupture. Biotechnol. Prog. 1991, 7, 151-158 Handa, A., Emery, A. N. and Spier, R. E. On the evaluation of gas-liquid interfacial effects on hybridoma viability in bubble column bioreactors. 7th General Meeting of ESACT on advances in animal cell technology: cell engineering, evaluation and exploitation Baden, Austria, 1985. Dev. Biol. Stand. 1987, 66, 241-253 Handa-Corrigan, A., Emery, A. N. and Spier, R. E. Effect of gas-liquid interfaces on the growth of suspended mammalian cells: mechanisms of cell damage by bubbles. Enzyme Microb. Techno/. 1989, 11, 230-235 Mano, T., Kimura, T., lijima, S., Takahashi, K., Takeuchi, H. and Kobayashi, T. Comparison of oxygen supply methods for cultures of shear stress sensitive organisms including animal cell culture. J. Chem. Tech. Biotechnol. 1990, 47, 259-271 Adlercreutz, P. and Mattiasson, B. Aspects of biocatalyst stability in organic solvents. Biocatalysis, 1987, 1, 99-108
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Brink, L. E. S., Tramper, J., Luyben, K. Ch. A. M. and Van 't Riet, K. Biocatalysis in organic media. Enzyme Microb. Technol. 1988, 10, 736-743 Carrea, G. Biocatalysis in water-organic solvent two-phase systems. Trends Biotechnol. 1984, 2, 102-106 Dickinson, M. and Fletcher, P. D. I. Enzymes in organic solvents. Enzyme Microb. Technol. 1989, 11, 55-56 Hailing, P. J. Organic liquids and biocatalysts: theory and practice. Trends Biotechnol. 1989, 7, 50-52 Khmelnitsky, Y. L., Levashov, A. V., Klyachko, N. L. and Martinek, K. Enzyme Microb. Techno/. 1988, 10, 710-724 Klibanov, A. M. Enzymatic catalysis in anhydrous organic solvents. Trends Biochem. 1989, 14, 141-144 Laane, C. Medium-engineering for bio-organic synthesis. Bincatalysis 1987, 1, 17-22 Lilly, M. D., Brazier, A. J., Hocknull, M. D., Williams. A. C. and Woodley, J. M. Biological conversions involving water-insoluble organic compounds. In Bioeatalysis in Organic Media (Laane, C., Tramper, J. and Lilly, M. D., eds.) Elsevier Science Publishers, Amsterdam, 1986, pp. 3-17 Luisi, P. and Laane, C. Solubilization of enzymes in apolar solvents via reverse micelles. Trends Biotechno/. 1986, 4, 153-160 Carrea, G. and Cremonesi, P. Enzyme-catalyzed steroid transformations in water-organic solvent two-phase systems, in Methods in Enzymo/ogy. Vol. 136: Immobilized Enzymes and Cells. Part C (Mosbach, K., ed.) Academic Press, Orlando, FL, 1987, pp. 151-157 Van der Padt, A., Keurentjes, J. T. F., Sewalt, J. J. W., Van Dam, E. M., Van Dorp, L. J. and Van't Riet, K. Enzymatic synthesis of monoglycerides in a membrane bioreactor with an in-line adsorption column. J. Am. Oil Chem. Soc. 1992, 6g, 748-754 Eggers, D. K., Blanch, H. W. and Prausnitz, J. M. Extractive catalysis: solvent effects on equilibria of enzymatic reactions in two-phase systems. Enzyme Microb. Technol. 1989, 11, 84-89 Mattiasson, B. and Adlercreutz, P. Perfluorochemicals in biotechnology. Trends Biotechno/. 1987, 5, 250-254 King, A. T., Mulligan, B. J. and Lowe, K. G. Perfluorochemicals and cell culture. Bio/Technology 1989, 7, 1037-1042 Bruce, L. J. and Daugulis, A. J. Solvent selection strategies for extractive Biocatalysis. Biotechno/. Prog. 1991,7, 116-124 Woodley, J. M., Cunnah, P. J. and Lilly, M. D. Stirred tank two-liquid phase biocatalytic reactor studies: kinetics, evaluation and modelling of substrate mass transfer. BiocatalysL~ 1991, 5, 1-12 Roffler, S. R., Randolph, T. W., Miller, D. A., Blanch, H. W. and Prausnitz, J. M. Extractive bioconversions with non-aqueous solvents. In Extractive Bioconversions (Mattiason, B. and Hoist O., eds.) Marcel Dekker, New York, 1992, pp. 133-171 Brink, L. E. S. and Tramper, J. Modelling the effects of mass transfer on kinetics of propene epoxidation of immobilized Mycobacterium cells: Pseudo-one-substrate conditions and negligible product inhibition. Enzyme Microb. Techno/. 1986,
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Brink, L. E. S. and Tramper, J. Modelling the effects of mass transfer on kinetics of propene epoxidation of immobilized Mycobacterium cells: product inhibition. Enzyme Microb. Technol. 1986, 8, 334-340 Chibata, 1., Yamada, S., Wada, M., Izuo, N. and Yamaguchi, T. U.S. Patent 3,850,753, 1974 Tramper, J., Wolters, I. and Verlaan, P. The liquid-impelled loop reactor: a new type of density-difference-mixed bioreactor. In Biocatalysis in Organic Media (Laane, C., Tramper, J. and Lilly, M. D., eds.) Elsevier Science Publishers, Amsterdam, 1987, pp. 311-316 Van den Tweel, W. J. J., Marsman, E. H., Vorage, M. J. A. W., Tramper, J. and De Bont, J. A. M. The application of organic solvents for the bioconversion of benzene to cisbenzeneglycol. International conference on bioreactors and biotransformations, Gleneagles, Scotland, UK, 1987 Buitelaar, R. M., Susaeta, I. and Tramper, J. Application of the liquid-impelled loop reactor for the production ofanthraquinones by plant cell cultures, in Progress in Plant Cellular and Molecular Biology (Nijkamp, H. J. J., Van Der Plas, L. H. V.
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and Van Aartrijk, J., eds.) Kluwer Academic Publishers, 1990, pp. 694-699 Gianetto, A., Ruggeri, B., Specchia, V., Sassi, G. and Forna. R. Continuous extraction loop reactor (CELR): alcoholic fermentation by fluidized entrapped biomass. Chem. Eng. Sci. 1988, 43, 1891-1896 Knez, Z. and Leitgeb, M. Membrane reactors for ester synthesis. Chem. Biochem. Eng. 1988, 2, 233-234 Koizimu, Y., Mukai, K., Murakawa, K. and Yamane, T. Scaleup of microporous hydrophobic membrane bioreactor with the respect to the continuous glycerolysis of fat by lipase. J. Jpn Oil Chem Soc. 1987, 36, 561-564 Pronk, W., Kerkhof, P. J. A. M., van Helden, C. and Van "t Riet, K. The hydrolysis of triglycerides by immobilized lipase in a hydrophilic membrane reactor. Biotechnol. Bioeng., 1988, 32, 512-518 Steinmeyer0 D. E. and Shuler, M. L. Mathematic modeling and simulations of membrane bioreactor extractive fermentations. Biotechno/. Prog. 1990, 6, 362-369 Yamane, T., Rhee, J. S., Ohta, Y. and Shimizusome, S. Characteristics of continuous glycerolysis of fat by lipase with microporous membrane bioreactor. J. Jpn. Oil Chem. Soc. 1987, 36, 474-479 Yamane, T., Hoq, M. M., Itoh, S. and Shimizu, S. Continuous glycerolysis of fat by lipase in microporous hydrophobic membrane bioreactor. J. Jpn Oil Chem. Soc. 1986, 35, 52-56 AIbertsson, P. A. Partitioning o f Cell Particles and Macromolecules. John Wiley & Sons, New York, 1986 Mattiason, B. Application of aqueous two-phase systems in biotechnology. Trends Biotechnol. 1983, 1, 16-20 Sikdar, S. K., Cole, K. D., Stewart, R. M., Szlag, D. C., Todd. P. and Cabezas, H. Aqueous two-phase extraction in hioseparations: an assessment. Biotechnology 1991,9, 253-257 Andersson, E. and Hahn-H~igerdal, B. Bioconversions in aqueous two-phase systems. Enzyme Microb. Techno/. 1990, 12, 242-254 Mattiasson, B. Bioconversions in aqueous two-phase systems: an alternative to conventional immobilization. In Methods in Enzymology. Vo/. 137: Immobilized Enzymes and Cells. Part D (Mosbach, K.. ed.) Academic Press, San Diego, CA, 1988, pp. 657-667 Springham, D. G. The established Industries. In Biotechnology, The Science and the Business (Moses, V. and Cape, R. E., eds.) Harvard Academic Publishers, 1991, pp. 249-296 Hamada, T., Sugishita, M., Fukushima, Y., Fukase, T. and Motai, H. Continuous production of soy sauce by a bioreactor system. Process Biochem. 1991, 26, 39-45 Aires Barros, M. R., Cabral, J. M. S. and Novais, J. M. Production of ethanol by Immobilized Saccharomyces Bayanus in an extractive fermentation system. Biotechno/. Bioeng. 1987, 29, 1097-1104 Bar, R. and Gainer, J. L. Acid fermentation in water-organic solvent two-liquid phase systems. Biotechnol. Prog. 1987, 3, 109-114 Daugulis, A. J., Swaine, D. E., Kollerup, F. and Groom, C. A. Extractive fermentation-integrated reaction and product recovery. Biotechnol. Lett. 1987, 9, 425-430 Evans, P. J. and Wang, H. Y. Effects of extractive fermentation on butyric acid production by CIostridium acetobutylicum. Appl. Microbiol. Biotechno/. 1990, 32, 393-397 Eiteman, M. A. and Gainer, J. L. In situ extraction versus the use of an external column in fermentation. Appl. Microbiol. Biotechno/. 1989, 30, 614-618 Fourier, R. L. Mathematical model of extractive fermentation: application to the production of ethanol. Biotechno/. Bioeng. 1986, 28, 1206-1212 Park, Y. S., Chang, H. N. and Kim, B. H. Adaptation of Saccharomyces cerevisiae to solvents used in extractive fermentation. Biotechno/. Left. 1988, 10, 261-266 Ramelmeier, R. A. and Blanch, H. W. Mass transfer and cholesterol oxidase kinetics in a liquid-liquid two-phase system. Biocatalysis 1989, 2, 97-120 Roffler, S., Blanch, H. W. and Wilke, C. R. Extractive fermentation of acetone and butanol: process design and economic evaluation. Biotechnol. Prog. 1987, 3, 131-140
Two-liquid-phase bioreactors: H. IV~. Van Sonsbeek et al. 55 56
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Roffler, S. R., Wilke, C. R. and Blanch, H. W. Design and mathematical description of differential contactors used in extractive fermentations. Biotechnol. Bioeng. 1988, 32, 192-204 Sola, C., Casas, C., Godia, F., Poch, M. and Serra, A. Continuous ethanol production by immobilized yeast cells and ethanol recovery by liquid/liquid extraction. Biotechnol. Bioeng. Syrup. 1986, 17, 519-533 Rosevear, A. Immobilised Biocatalysts--a critical review. J. Chem. Tech. Biotechnol. 1984, 34B, 127-150 Black, G. M. Characteristics and performance of immobilised cell reactors. In Process Engineering Aspects of lmmobilised Cell Systems (Webb, C., Black, G. M. and Atkinson, B., eds.) The Institution of Chemical Engineers, Warwickskire, 1986, pp. 75-86 Venkatasubramanian, K., Karkare, S. B. and Vieth, W. R. Chemical engineering analysis of immobilized-cell systems. Appl. Biochem Bioeng. 1983, 4, 311-349 Brink, L. E. S. and Tramper, J. Optimization and modelling of the microbial epoxidation of propene in an organic liquidphase/immobilized-cell system. In Modelling and Control of Biotechnological Processes (Johnson, A., ed.) Pergamon Press, Oxford, 1985, pp. 11 I - I 17 Buitelaar, R. M., Vermu~, M. H., Schlatmann, J. E. and Tramper, J. The influence of various organic solvents on the respiration of free and immobilized cells of Tagetes Minuta. Biotech. Techn. 1990, 4, 415-418 Damiano, D. and Wang, S. S. Novel use of a pertluorocarbon for supplying oxygen to aerobic submerged cultures. Biotechnol. Lett. 1985, 7, 81-86 Cho, M. H. and Wang, S. S. Enhancement of oxygen transfer in hybridoma cell culture by using a perfluorocarbon as an oxygen carrier. Biotechno/. Lett. 1988, 10, 855-860 Van Sonsbeek, H. M., De Blank, H. and Tramper, J. Oxygen transfer in liquid-impelled loop reactors using perfluorocarbon liquids. Biotechnol. Bioeng. 1992, 40, 713-718 Ju, L.-K., Lee, J. F. and Armiger, W. B. Enhancing oxygen transfer in bioreactors by perfluorocarbon emulsions. Biotechno/. Prog. 1991, 7, 323-329
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Bruining, W. J., Joosten, G. E. H., Beenackers, A. A. C. M. and Hofman, H. Enhancement of gas-liquid mass transfer by a dispersed second liquid phase. Chem. Eng. Sci. 1986, 41, 1873-1877 Hassan, 1. T. M. and Robinson, C. W. Oxygen transfer in mechanically agitated aqueous systems containing dispersed hydrocarbon. Biotechnol. Bioeng. 1977, 19, 661-682 Ho, C. S., Ju, L. and Baddour, R. F. Enhancing Penicillin fermentations by increased oxygen solubility through the addition of n-hexadecane. Biotechnol. Bioeng. 1990, 36, 1110--1118 Ju, L.-K., Lee, J. F. and Armiger, W. B. Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions. Biotechnol. Bioeng. 1991, 37, 505-511 Junker, B. H., Hatton, T. A. and Wang, D. I. C. Oxygen transfer enhancement in aqueous/perfluorocarbon fermentation systems: I. Experimental observations. Biotechnol. Bioeng. 1990, 35, 578-585 Junker, B. H., Hatton, T. A. and Wang, D. I. C. Oxygen transfer enhancement in aqueous/perfluorocarbon fermentation systems: II. Theoretical analysis. Biotechnol. Bioeng. 1990, 35, 578-585 Linek, V. and Benes, P. A study of the mechanism of gas absorption into oil-water emulsions. Chem. Eng. Sci. 1976, 31, 1037-1046 Rols, J. L., Condoret, J. S., Fonade, C. and Goma, G. Mechanism of enhanced oxygen transfer in fermentation using emulsified oxygen-vectors. Biotechno/. Bioeng. 1990, 35, 427-435 Rols, J. L. and Goma, G. Enhancement of oxygen transfer rates in fermentation using oxygen-vectors. Biotech. Adv. 1989, 7, 1-14 Rols, J. L. and Goma, G. Enhanced oxygen transfer rates in fermentation using soybean oil-in-water dispersions. Biotechnol. Lett. 1991, 13, 7-12 Moses, V. and Cape, R. E. Biotechnology. The Science and the Business. Harvard Academic Publishers, 1991
Enzyme Microb. Technol., 1993, vol. 15, September
729
Two-liquid-phase bioreactors H. M. Van Sonsbeek, H. H. Beeftink and J. Tramper Wageningen Agricultural University, F o o d and Bioprocess Engineering Group, D e p a r t m e n t o f F o o d Science, Wageningen, The Netherlands
The application of two liquid phases that are poorly miscible is a fascinating research topic fi~r biocatalytical conversions because of the promising results. Motives for application include an increase of productivity and achievement of continuous processing, but new limitations arise, e.g., interfacial effects such as biocatalyst accumulation and loss of activity, medium component accumulation, and slow coalescence. Centrifuges, membranes, and immobilization are tools that can overcome part of the problems, but more fundamental knowledge about interfaces and coalescence is still necessary for successfid application. For scaleup and further development of processes based on the obtained results, a choice must be made for the configuration of the experimental setup of a bioreactor. Aspects like aeration, shear stress, batch or continuous processing, and immobilization can play an important role. This review article describes these aspects and the proposals that have been made in recent years concerning two-liquid-phase bioreactors. It shows some aduptations to existing bioreactors, such as loop reactors and stirred-tank reactors.
Keywords: two-liquid-phase; bioreactor; loop reactor; organic solvent; oxygen transfer Introduction This review article deals with bioreactors in which two liquid phases are applied. In 1982 Lilly ~ published an article entitled "Two-liquid-phase biocatalytic reactions" and predicted new commercial applications for multiliquid biocatalytic reactors. In 1987 Lilly et al. 2 published "Two-liquid-phase biocatalytic r e a c t o r s , " which mainly gives a short general description of aspects that can play a role when two liquid phases are applied in bioreactors. The present article is about the same topic and gives an overview of what has been achieved with two-liquid-phase bioreactors to date. Before discussing these bioreactors, a short consideration is given about the introduction of two liquid phases in biotechnological production processes. New developments in biotechnology are focused on new products or the introduction of new processes as a substitute to the conventional way, or improvements in performance of existing biotechnological applications. In recent years much attention has been given to the use of two-liquid-phase systems. The application of organic solvents especially has fascinated many researchers and is still considered to be a promising topic,
Address reprint requests to Dr. Van Sonsbeek at the Wageningen Agricultural University, Food and Bioprocess Engineering Group, Department of Food Science, P.O. Box 8129, 6700 EV Wageningen, The Netherlands Received 30 June 1992; revised l0 November 1992 722
since many biocatalytic reactions involve organic components that are poorly water-soluble. For this reason, addition of water-soluble organic solvents in small proportions has been well known for a long time as a technique to improve availability of poorly water-soluble reactants to cells and enzymes. More recent research concerns the use of two immiscible liquid phases in a bioreactor. In most cases the aqueous phase contains the biocatalyst, possibly immobilized. The second liquid phase can be an organic solvent or another water phase. The latter, called an aqueous two-phase system, consists of two polymer solutions or a polymer and a salt solution. Reversed micelles are a completely distinct category of two-liquid-phase systems and can be considered as pools of water stabilized in an organic solvent by a surfactant. This category will not be discussed here. In this article, research developments concerning two-liquid-phase bioreactors will be discussed with the aim to give a clear comprehension of the motives of their use and to give an overview of the state of the art. The review is focused on the cases where two free liquid phases are present. Systems where one of the phases is only a hydration liquid are not highlighted. Emulsion systems are briefly discussed. First, some general aspects of conventional bioreactors will be highlighted, such as aeration, shear stress, continuous or batch processing, and immobilization. These aspects are discussed because they can play a major role in the performance of two-liquid-phase bioreactors. Therefore these aspects will return in the subsequent
Enzyme Microb. Technol., 1993, vol. 15, September
~ 1993Butterworth-Heinemann
Two-liquid-phase bioreactors: H. M. Van Sonsbeek et al. section where bioreactors are discussed in which an organic solvent and a catalytic aqueous phase are present. The organic solvent can be the continuous or the dispersed phase and it can be the reactant or a solvent in which part of the reactant and/or product is dissolved. In addition, the suitability of aqueous twophase systems as a biological medium and some research examples are given. Finally, some conclusions are drawn and future perspectives are given.
Conventional bioreactors Biotechnological possibilities are often shown on a laboratory scale in flasks or tubes. Further development of such processes means that the same or better performance is pursued on a larger scale, which implies that bioreactor technology becomes important. A great variety of bioreactor types exist, of which the stirredtank reactor (STR) and the bubble column (BC) are the simplest and most widely used. Many biotechnological processes require oxygen that is transferred from air to the culture broth. In stirred-tank reactors air bubbles brought into the reactor are dispersed by the stirrer to obtain a large exchange area for mass transfer. Therefore high oxygen-transfer rates can be achieved, which is important because this is often the limiting factor in aerobic bioconversions. Bubble columns are attractive, even on a small scale, because of their simple construction. On a large scale this advantage becomes even more important because aeration at high energy input rates is much simpler than stirring. Besides these two basic types, other bioreactors have been developed, such as membrane, packed-bed, trickle-bed, or fluidized-bed bioreactors and bioscrubbers. These types will not be discussed in detail here. Because the oxygen-transfer rate is often the ratelimiting step, many biotechnological studies have focused on this topic. The limited transfer rate is the consequence of the contradiction between the need for oxygen of many biocatalysts and the low solubility of oxygen in aqueous environments, which are most often used. Closely related to this aspect is the issue of the cultivation of shear-sensitive cells, such as plant, animal, and insect cells. Stirring enhances oxygen-transfer rates to a great extent, but is only sparingly possible in these cultures, because of damaging effects. Therefore different kinds of reactors, such as airlift loop reactors (ALRs), have been proposed to be suitable for these purposes. 3 But even aeration by sparging air has been found to have a detrimental effect on ceils, 4-6 and various kinds of aeration methods have been developed, e.g., bubble-free aeration using silicon tubing. 7 The described case of obtaining appropriate oxygen supply shows the importance of the choice of the reactor type and adjustments of process conditions to the specific needs of the application. The introduction of nonconventional media can be regarded as such an adjustment that improves processes. Examples of nonconventional media are organic solvents, supercritical solvents, or aqueous two-phase systems. The bioreactors in which these media are applied can be the stan-
dard-type bioreactors or standard types that are adjusted for use with nonconventional media. Examples can be found in the next sections.
Organic solvents in bioreactors The topic of organic solvents as biocatalytic reaction media has been reviewed many times 1.8-17 and needs no further explanation here beyond a short summary of the arguments that are used to indicate the usefulness of this method. The argument that is relevant most often is better solubility of substrates and/or products; an example is steroid transformation. ~8 Another argument is that reverse hydrolytic reactions, which are not feasible in pure aqueous systems, can become feasible; an example is the esterification of a fatty acid and an alcohol.19 In some cases product inhibition can be reduced because of the removal of the product into the organic phase. Transfer of products to the organic phase can shift the apparent reaction equilibria calculated on an overall volume basis. 2° If the product concentration is increased due to better solubility in the organic phase, product recovery becomes easier. There are also a few aspects of organic solvents that give rise to new problems or less attractive phenomena such as inhibition or biocatalyst inactivation. Furthermore, in extractive fermentations, unintentional formation of emulsions and removal of protein from the fermentation broth can occur (protein stripping). A great number of solvent types can be applied in bioreactors, such as alkanes, alkenes, esters, alcohols, ethers, perfluorocarbons, etc. Only the group of the perfluorocarbons will shortly be highlighted, because they are relatively unknown. Mattiasson and Adlercreutz 21 and King e t a l . 2"- describe the use of perfluorocarbons in biotechnology. Perfluorocarbons are ring or straight-chain hydrocarbons in which hydrogen atoms have been replaced by fluorine. Perfluorocarbons are both stable and virtually chemically inert due to the presence of the strong carbon/fluorine bonds (ca. 487 kJ mol-~). In addition, the fluorine atoms offer steric protection to the carbon groups. Because of the low interactions between perfluorocarbon molecules themselves, gas molecules can easily occupy intramolecular spaces and large volumes of gases can thus dissolve. There is no chemical attraction of oxygen or carbon dioxide to perfluorocarbons, which implies that Henry's law for dissolved gas is applicable. Perfluorochemicals should not be confused with a related and currently, from the point of view of environmental care, somewhat controversial group of compounds, chlorofluorocarbons. The choice of the organic solvent depends on the purpose of their use. In case of extractive biocatalysis, for instance, the product should dissolve preferentially in the solvent. Besides product solubility, toxicity of a solvent for the biocatalyst is of general importance, and much research is carried out on this topic. In general, a positive correlation is found between hydrophobicity of solvents and nontoxicity for biocatalysts. A measure for hydrophobicity that was found very suit-
Enzyme Microb. Technol., 1993, vol. 15, September
723
Review
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able for characterization of solvents, is the log P value, which is the logarithm of the partition coefficient of a solvent in a water-octanol two-phase system. Solvents with a log P value above 4 are very hydrophobic and generally show no toxic effects on biocatalysts. Bruce and Daugulis 23 recently presented solvent selection strategies for extractive biocatalysis, and concluded that it is possible to compose mixtures of solvents that show good extraction properties as well as good biocompatibility, even at relatively high concentrations of toxic solvent. For the application of organic solvents, it is obvious that existing standard-type bioreactors can be used, such as stirred-tank, fluidized-bed, and column reactors. Small adjustments in the experimental setup can often be sufficient to operate a two-liquidphase biocatalytical process. Examples of these adjusted reactors for research purposes are described in the literature and given here. In many investigations of two-liquid-phase biocatalysis, stirred-tank reactors are used. 24 In Figures 1 and 2 the two basic experimental setups for extractive fermentation with stirred-tank reactors are given. 25 The first setup is the simpler one, but it can lead to a rather stable emulsion that requires a long settling time. In that case an external extraction column is more favorable because emulsion formation is less vigorous. An external extraction column is also more effective be-
724
cause it is a multistage contactor, whereas for direct solvent addition, only one equilibrium stage can be achieved. Within the group of the authors, Brink and Tramper 26"27investigated the application of organic solvents in an immobilized-cell fluidized-bed reactor and used the epoxidation of propene by immobilized M y c o b a c t e r i u m cells as a model system. Concerning column-type bioreactors, a few proposals can be found in the literature for the configuration with an organic solvent as one of the two liquid phases. Chibata et a/. 28 describe in a patent a bioreactor system with external aeration of hydrocarbons or perfluorocarbons. Pumping these air-saturated solvents via a liquid sparger in the bioreactor, instead of air sparging, enhances oxygen-transfer rates. The background and literature concerning this aspect are described in Aeration of two-liquid-phase systems. Tramper et al. 29 introduced the liquid-impelled loop reactor (LLR), which is schematically drawn in Figure 3, and Van den Tweel e t a / . 3° showed an experiment in which this reactor was used for the conversion of benzene to cis-benzene glycol. Furthermore, Buitelaar et a/. 3~ have been working on the use of the LLR for the cultivation of plant cells, and at present the conversion oftetralin to tetralol by A c i n e t o b a c t e r is investigated. Gianetto et al.32 introduced a continuous extraction loop reactor, which will be discussed later in Batch or continuous. Finally, the membrane reactor is a bioreactor in which two immiscible liquid phases can be applied and must therefore be mentioned here. An essential difference from the other types is that the two liquid phases are not in direct contact, but separated by a membrane. The membrane also serves as immobilization support for the biocatalyst, usually an enzyme. Van der Padt et al.~9 demonstrated the suitability of a membrane biorcactor for the enzymatic esterification of glycerol and fatty acids with the enzyme lipase immobilized on a hollow-fiber membrane (Figure 4). The membrane keeps the polar and apolar liquid phases separated, and this gives the possibility of selective adsorption of monoesters with an adsorption column in the apolar
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Enzyme Microb. Technol., 1993, vol. 15, September
Liquid-impelled loop reactor (LLR) (Tramper et aL 29)
Two-fiquid-phase bioreactors: H. IV~. Van Sonsbeek et al. of aqueous two-phase systems, which are at this moment still in a rather experimental stage because of the lack of fundamental knowledge of partitioning.
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circuit. Other examples of research concerning twoliquid-phase membrane bioreactors are the work of Knez and Leitgeb, 33 Koizimu et al., 34 Pronk et al., 35 Steinmeyer and Schuler, 36 and Yamane e t a / . 37"38
Aqueous two-phase systems Besides the use of an organic solvent in combination with an aqueous phase, the aqueous two-phase system is another possibility of using two liquid phases. This is a system that consists of two immiscible aqueous phases, which are polymer solutions or a polymer and a salt solution. The phenomenon has long been known, and a good overview of the principle is given by Albertsson. 39In the last decade the interest in applications of aqueous two-phase systems in biotechnology has increased, due to the combination of two attractive properties. As in other two-liquid-phase systems, partitioning of components and biocatalysts between the two phases occurs, which is important for downstream processing and biocatalyst retention. The second attractive aspect in aqueous two-phase systems is the very low interfacial tension (0.0001-0. l mN m-~) compared to that of organic solvent/water systems (0.5-50 mN m-I). This keeps proteins in solution, and unfolding at the interface is prevented. Due to this property, research on biotechnological applications of aqueous two-phase systems is often focused on isolation of proteins, cells, or organelles from the broth.4°'4~ The partition coefficient for these components in aqueous twophase systems is normally in the order of magnitude of 0.l-10, but in some cases it can be improved by affinity interactions. However, this application as an isolation method is not considered here as a replacement of existing techniques, but as complementary to more selective isolation methods. An attractive feature of this system, as compared to other isolation methods, is that the scaleup seems easy. Another way in which aqueous two-phase systems can be applied for biotechnological purposes is to use them as medium for extractive biocatalysis. Usually the bioconversion preferentially takes place in one of the two phases, and the product can be extracted in the other p h a s e : °'42'43 Andersson and Hahn-H/~gerda142gave an extensive review of accomplishments and prospects for the application
In spite of the consistent claims of the advantages of continuous processing, such as a higher productivity and a more uniform product, most biocatalytical processes are still carried out as batch processes, especially in the food industry. The enormous amount of work that has been done through the years to achieve continuous processing for several applications has not yet proved the suitability that is often claimed. An example of a continuous process that failed in practice is found in the history of beer brewing. The complexity of a continuous brewing process required the close attention of skilled staff, and labor costs turned out to be too high. 44 On the other hand, developments are still going on, which is illustrated by the recent presentation of a continuous process for the production of soy sauce. 45 Improving the feasibility of continuous processing is often used as an argument for the use of two-liquidphase systems. Although it seems difficult to set up a continuous process with two-liquid-phase systems, several articles have been published on extractive biocatalysis.2328'32'46-56 This is a type of continuous biological production process in which the process is not disturbed, and sometimes is even improved, by in situ recovery of the product. Ethanol production is an especially popular topic in this field. 32'47"5°'53'54 A recent example is the work of Gianetto et al. 32 who developed and used a continuous extraction loop reactor (CELR), which can be seen in F i g u r e 5. This reactor is an external-loop reactor, in which liquid circulation is induced by a gas flow in one of the vertical tubes and ethanol is extracted by a countercurrent extraction in the other tube. The experiments concern glucose fermentation by S a c c h a r o m y c e s cerevisiae entrapped in calciumalginate spheres. The authors observed problems with the calcium-alginate matrix, such as low mechanical
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substrate in
lass
sparger Figure § Continuous extraction loop reactor (CELR) (adapted from Gianetto et al. 32)
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725
Review resistance, a leakage of viable cells, a significant internal diffusion limitation, and a decrease in viability of the entrapped microorganisms. They emphasize, however, that the reactor is still in a preliminary stage. Mattiasson 43described a classical example of a product-inhibited biocatalytic process, the production of acetone and butanol from glucose using Clostridium acetobutylicum. From batch experiments in aqueous two-phase systems, it was concluded that butanol production should preferably be performed in a continuous process in which the product is removed and fresh substrate added. In general, most two-liquid-phase research is carried out in batch experiments, and not much attention is given to the application. Claimed advantages are often not proven, and processes still need to be designed. Although application has not yet been very successful, there is still faith that the introduction of two-liquidphase systems is suitable for the old ideal of continuous processing.
Immobilization An extensive overview of immobilization of biocatalysts is given by Rosevear. 57 Black 58and Venkatasubramanian et al. ~9 give good descriptions of the applications of immobilized biocatalysts and the reactors that can be involved. There are a few valid motives for immobilization ofbiocatalysts. The most important one for growing cells is the increase of productivity, because it is possible to work with dilution rates above washout conditions. ~8'59 Furthermore, facilitated rec o v e r y of the biocatalyst and protection of the biocatalyst against a damaging turbulent environment can play a role as well. Also, because of diffusion limitations, it is possible that an immobilized biocatalyst will not be exposed to an inhibiting substrate concentration. In two-liquid-phase systems, gel entrapment of biocatalysts can protect them from denaturation at the liquid/ liquid interface. Brink and T r a m p e r 6° found that, by immobilization, physical p h e n o m e n a such as cell aggregation at the water/organic interface and formation of biomass clots could be eliminated and therewith reduce loss of catalytic activity caused by exposure of mycobacteria to organic solvents. This was also found by Buitelaar et al. 61 who applied plant cells of Tagetes minuta in a two-liquid-phase system. For these applications it is of importance to realize that immobilization used as a technique to prevent free biocatalysts in the solution can only be successful if it concerns nongrowing biocatalysts or a high dilution rate.
Aeration of two-liquid-phase systems In many aerobic bioprocesses, the oxygen supply rate to the aqueous phase is the limiting factor. The use of two liquid phases can contribute in two different ways to improve this. First, it is possible to aerate one liquid phase and lead it through the other that contains the biocatalyst. Second, an emulsion can be created that forms the medium and that is aerated by sparging air or oxygen-enriched air. 726
Damiano and Wang 62 presented a method in which a perfluorocarbon was used as an oxygen carrier, c o n tinuously removed from the bioreactor after coalescence of the droplets at the bottom. One circuit of perfluorocarbon was pumped back in the bioreactor through a nozzle at the top. A second circuit of perfluorocarbon was reaerated and then pumped back in the bioreactor. Oxygen was transferred by means of liquid/ liquid contact. Escherichia coli was cultivated, and it was shown that operation of a column with externally aerated perfluorocarbon was capable of supplying oxygen at significantly higher rates than the best run of a bubble column. This is ascribed to a much larger surface area available for mass transfer, which is possible for three reasons: (I) at equal work inputs, drops will be smaller than bubbles due to lower surface tension: (2) bubblcs generally coalesce into larger bubbles much more rapidly than do drops: (3) liquid holdups can be fixed at levels much higher than typical gas holdups. The process described by Damiano and Wang 62 was limited by the coalescence rate of the solvent droplets at the bottom of the reactor. Cho and Wang 63 also used a second liquid phase as an oxygen carrier instead of the direct sparging of a gas into a hybridoma cell culture to increase the oxygentransfer rate. Overall volumetric mass-transfer coefficients were measured up to five times higher than in a directly aerated system. It was concluded that the interfacial area was made larger quite easily. No harmful effects of the perfluorocarbon on the hybridoma cells were detected. Unfortunately, no positive results on the application could bc reported due to cell aggregation at the interface of the medium and the perfluorocarbon. In our laboratory a distinction was made between thc mass-transfer coefficient and the available exchange area of a liquid/liquid system. Mass-transfer coefficients were measured that are lower than in gas/ liquid systems. ~ H o w e v e r , because of a higher specific exchange area in the experimental setup, the masstransfer rate of the liquid/liquid system was favorable, when compared with gas/liquid systems at equal dispersed-phase flow rates. Because of slow coalescence, the maximum dispersed-phase flow rate was limited, rcsulting in a severe restriction for the maximum oxygen-transfer rate in liquid/liquid systems. Ju et a l Y emphasize in their work that in the case of application to low-shear, gently mixed systems, the general efficiency of exclusively externally aerated perfluorocarbons as oxygen-transfer enhancers is unacceptably low. They also point out the problem of protein stripping from the growth medium by the circulated perttuorocarbons. The second way to improve the oxygen-transfer rate by applying two liquid phases is as an emulsion. It has been reported many times that very small droplets of hydrocarbons or perfluorocarbons can have an enhancing effect on the oxygen-transfer rate. 65-75 Linek and Benes 72 studied the mechanism of gas absorption into organic solvent water emulsions. T h e y distinguished organic solvents on the basis of their spreading coefficient:
Enzyme Microb. Technol., 1993, vol. 15, September
Two-liquid-phase bioreactors: H. M. Van Sonsbeek et al. Sow = crwA - (O'oA + Crow)
(I)
where o- is the interfacial tension and the subscripts O, W, and A stand for organic solvent, water, and air, respectively. Organic solvents with a negative spreading coefficient showed no influence on the mass-transfer coefficient, but had a negative influence on the specific exchange area. Organic solvents with a positive spreading coefficient gave positive effects on both the mass-transfer coefficient and the specific exchange area. Hassan and Robinson 67 found results that were in agreement with the data of Linek and Benes. 72 The increase of the specific exchange area in the case of a positive spreading coefficient is assumed to be caused by spreading of the organic solvent as a thin film on the gas/aqueous interface, lowering the surface tension, and thereby increasing the specific interracial area of the gas dispersion. The decrease in the case of a negative spreading coefficient is assumed to be caused by static accumulation of droplets at the interface, thus blocking part of the surface area available for oxygen mass transfer. Rols et al. 73 concluded that the effect of organic-solvent droplets on the specific gas-emulsion exchange area is usually of minor importance compared to the enhancement effect on the mass-transfer coefficient, and denoted the principle as oxygen vector. Several mechanisms of the increased mass-transfer coefficients for organic solvents with a positive spreading coefficient have been proposed, 65-67'72'73 but there remains much uncertainty. It is most likely that in some way there is a regularly occurring interchange of droplets between the air/aqueous interface and the bulk region. Some examples in the literature are found that clearly demonstrate the positive effect of organic-solvent droplets on volumetric transfer coefficients. Rols and Goma 75 found a positive effect of soybean oil on the volumetric oxygen-transfer coefficient during cultivation of A e r o b a c t e r a e r o g e n e s . Ho et al. 6s found an enhanced oxygen supply by adding hexadecane to the oxygen-limited penicillin fermentations with Penicillium c h r y s o g e n u m in shake flasks and in a stirred tank. They denote the transfer-enhancement effect as a higher effective oxygen solubility of the fermentation medium. Ju et al. 65 used E s c h e r i c h i a coli to demonstrate the mass-transfer enhancement effect, when instead of an aqueous phase perfluorocarbon emulsions are used in a stirred-tank reactor. Up to a fivefold increase of the oxygen-transfer coefficient is reported in the literature. TM Therefore, the strategy of adding small droplets of an organic solvent to a fermentation medium seems to be very useful for improving the oxygen-transfer process.
Conclusions and future perspectives
success is still limited. The overoptimistic view, given by many authors with respect to applications of twoliquid-phase systems, suits the sobering view on biotechnology in general, given by Moses and Cape 76 in their book B i o t e c h n o l o g y , The S c i e n c e a n d the B u s i n e s s . There is a world of difference between laboratory achievements and development of processes for commercial purposes. Interfacial effects play the key role in two-liquidphase bioreactors. These effects can be slow coalescence, cell aggregation, or biomass clotting at the interface with loss of activity of the biocatalyst, and accumulation of medium components, especially proteins, at the interface. Slow coalescence makes continuous processing difficult. So far most laboratory biocatalytic reactions in two-liquid-phase systems have been restricted to batch processes. Centrifuges or membranes can be a great help in overcoming this problem. Also many physical methods, such as heating and packing materials, and chemical agents are used for speedup of coalescence. These solutions are always of a practical nature and thus without basic knowledge of the mechanisms. It only works for the process it was developed for. The problem of negative effects of the interface on the biocatalyst can be reduced by immobilization of the biocatalyst. In general, more fundamental knowledge about interfacial effects and the coalescence process is necessary to improve possibilities and process design when two-liquid-phase bioreactors are involved. A few concepts of two-liquid-phase bioreactors exist, but these are still in a rather experimental stage: the continuous extraction loop reactor of Gianetto et al., 32 the liquid-impelled loop reactor of Tramper et a[., 29 and the stirred-tank reactor. 9
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