Immobilized cells: a review of recent literature

C3

Immobilized cells: a review of recent literature C H A R L E S D. S C O T T Chemical Technology Division, Oak Ridge National Laboratory,* Oak Ridge, Tennessee 37831, U S A

Summary. Advanced biocatalytic systems using immobilized cells have potential utility in various bioprocessing schemes and for biomedical and analytical purposes. Important advances continue to be made in the techniques of immobilization, the characterization of the resulting biocatalyst and potential applications. This review examines the recent literature in the area of cellular immobilization; it covers applications ranging from wastewater treatment to the production of therapeutics. New immobilization materials, several different bioreactor concepts and the properties of many specific systems are discussed. Anticipated future research trends are also outlined.

Keywords: Immobilized cells; bioreactor systems; process applications; fermentation products; biomedical products; wastewater treatment

Introduction Many advanced bioreactor systems require that the bigcatalyst (e.g. microorganisms, plant or animal cells) be immobilized into or onto a solid support material to reduce cell washout, thus significantly increasing the biocatalyst concentration while providing for optimum contact with the substrate. There are also important therapeutic and analytical applications for immobilized organisms. Some microorganisms and other cellular materials have a natural inclination to adhere to surfaces and, in this way, become immobilized. It has been suggested that the earliest purposeful use of immobilized cells in the beginning of the 19th century took advantage of this type of microbial immobilization with a biological film in a trickle-filter system for producing acetic acid. 1'2 Since that time, similar approaches have been developed for wastewater treatment. During the past few years, many new techniques have been developed in which cells are immobilized by entrapment or encapsulation. The advent of the series of biannual meetings sponsored by the Engineering Foundation entitled 'Enzyme Engineering' (the eighth in the series was recently held in Denmark) established a formal focus on the immobilization of biocatalysts, including some consideration of cellular systems. The most recently published proceedings of that series also includes excellent papers on cellular immobilization. 3'4 Some useful collections of papers on microbial immobilization began to appear in the late 1970s, 5'6 and additional collections of papers have recently been published, including those that range * Operated by Martin Marietta Energy Systems Inc., for the US Department of Energy under Contract No. DE-ACO5-840R21400

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from the general work edited by Chibata and Wingard 7 to a more specific collection of articles on microbial adhesion a and a series of papers that emphasizes practical approaches. 9 As the field has matured, some comprehensive reviews were also published. In fact, these have proliferated during recent years and they range from rather general coverage of a portion of the field to discussions of specific applications or techniques (Table 1). Although few of them attempt to cover the field broadly, they provide, when taken in totality, a good historical description of cellular immobilization up to recent times. While this paper is essentially an extension of the previous reviews, it covers only the research reported throughout the past three years (1983-1985), found in over 50 journals and books. The material is presented in a somewhat more encompassing way than some of the earlier reviews in that a much broader area of application is addressed. Although the immobilization of enzymes and other cell fragments represents similar techniques, that research is referenced only when it has a direct impact on cell immobilization. It is almost impossible to ensure completeness for such a task, and the author makes no such claim. However, it is felt that most of the important recent research approaches are represented here, especially those appearing in publications using English.

Immobilization techniques The immobilization of microorganisms can be defined as any technique that limits the free migration of cells. Table 1

Recent reviews of cellular immobilization

Subject

References

General reviews Techniques and characterization Methods of immobilization Microbial adhesion Microbial carriers Process engineering Transport properties Applications Beverage production Biomedical products Biosensors Food manufacturing Fuel production Industrial chemicals Mammalian cells Photosynthetic processes Plant cells Wastewater treatment

1, 10-22 9, 23-31 32 33 34-36 23, 24, 37, 38 39 40-45 46-49 50 51-53 2, 54, 55 28 56 29, 57-61 62-65

0141-0229/87/020066-08 $03.00 © 1987 Butterworth & Co. (Publishers) Ltd

Immobilized cells: a review of recent literature: C. D. Scott Table 2

Research on immobilization techniques

Techniques

Attachment Aggregation Adhesion to surfaces Entrapment Encapsulation Alginate Carrageenan Other natural gels Polyacrylamide Radiation polymerization General and miscellaneous Membranes Porous material

References

66-71 32, 62, 72-104 38, 73, 105-143 116, 144-152 38, 115, 128, 137, 140, 153-155 26, 41, 42, 105, 122, 149, 1 56-161 27, 162, 163 121, 1 56, 164-175 46, 48, 176-179 62, 121, 180-185

Cell mobility can be restricted by aggregating the cells or by confining them into, or attaching them to, a solid support. It is not necessary formally to catalogue immobilization techniques in order to discuss this subject, but a degree of characterization is convenient. Various terms have been used to describe different types of immobilization, but these terms do not seem to be accepted universally. There are two broad types of immobilization: (1) attachment, where the microorganisms adhere to surface or other organisms by selfadhesion or chemical bonding; and (2) entrapment, where the organisms are caught in the interstices of fibrous or porous materials or are physically restrained within or by a solid or porous matrix such as a stabilized gel or a membrane. There are also subcategories within each of these types of immobilization, and both approaches may be used for some applications. In such cases, the dominant mechanism will be chosen for identification purposes. Table 2 presents a compilation of recent publications categorized by immobilization technique.

Attachment to surfaces The natural tendency of many types of microorganisms to adhere to solid surfaces can sometimes cause problems as in the case of marine fouling or dental plaque; however, it. has also presented opportunities for bioprocessing applications, especially in the area of environmental control technology where naturally occurring microbial films are effectively used in fixed-film bioreactors. 62-65 Anchorage-dependent mammalian cells that are used to produce important therapeutic biochemicals or for biochemical monitoring can also be induced to attach to surfaces that can be retained in a bioreactor environment. 2a' 77, 94.96.100.103 Sometimes, microorganisms that do not normally adhere to surfaces may be induced to attach by altering the physical or chemical properties of the cells or the surface,SO, 93, 98 by ionic attraction 75' 82, 92 or by chemical bonding. 24' 77, 82, 1 0 l

Mechanisms of attachment Research into the mechanisms involved in microbial attachment has been somewhat limited. This phenomenon has not been completely elucidated, but it is apparent that some types of organisms secrete macromolecules such as polymucosaccharides that act as a 'glue' to initiate microbial-surface interactions. 32 It has been suggested that the 'adhesives' used by some marine shellfish are similar to the material used to initiate microbial attachment.X05,1a6 Some types of organisms do not effectively attach to surfaces on their own but can rely on the symbiotic actions of other attachment organisms that may exist in a mixed culture, even though the latter do not contribute to the biological reaction of interest. This phenomenon is seen in some wastewater treatment systems using attached microbial films of mixed cultures. 81

Attachment of cells Many cells have the ability to adhere to other organisms (aggregate) or to solid surfaces. This attachment, which may be either natural or induced, can frequently form the basis for an inexpensive but effective immobilization technique.

Aogregation Some organisms tend to form aggregates or floc particles, particularly in instances where they are in suspension culture at high concentration for extended periods. This occurs with various yeast strains, including Saecharomyces cerevisiae, where the aggregates can be used in continuous t o w e r r e a c t o r s , 6 7 ' 7 ° ' 7 1 and even some bacteria (e.g. certain strains of Zymomonas mobilis) form natural floc particles that are relatively stable even when exposed to high shear fields in columnar bioreactors such as fluidized beds. 66 These types of biomass particles can be very stable within a columnar bioreactor, allowing extremely high biomass loadings and resulting in high bioconversion rates. Microbial aggregation can also be induced under some environmental conditions. For example, polyelectrolytes can be used to enhance the aggregation of certain bacteria systems just as they are used to cause aggregation of some colloidal particulates by covalent bonding. 6s In a few cases, even the action of aeration will induce microbial aggregation. 69

Entrapment of cells Entrapment of cells represents a more definite means of immobilization that does not have a significant dependence on cellular properties. In this case, the cells are held either within the interstices of porous materials, such as a sponge or fibrous substance, or by the physical restraints of membranes or encapsulizing gel matrices. This approach, which is by far the most popular for cellular immobilization, was used by the majority of researchers whose publications are reviewed here (see Table 2).

Encapsulation Physical entrapment of organisms inside a polymeric matrix is one of the most widely used techniques for cellular immobilization. The resulting material must have sufficient porosity to allow the transport of substrate in and product out while restraining the cells. Hydrocolloidal gels are used more frequently than any other material for this application, with the natural gels such as alginate and carrageenan being the most favoured. However, other polymeric networks can also be used, including the most popular synthetic material, polyacrylamide. The gel is generally formed into useful biocatalyst beads by first adding the cells as a suspension to an

Enzyme Microb. Technol., 1987, vol. 9, February

67

Review aqueous solution of the gelling material. This material is then formed into droplets either by forcing it dropwise through a nozzle or orifice or by dispersing it into a noninteracting liquid medium. The droplets are subsequently stabilized into biocatalyst beads with entrapped organisms via polymerization or other types of crosslinking. For example, alginate droplets can be stabilized with divalent ions such as Ca 2 +, and carrageenan droplets are crosslinked typically with K ÷. Both of these materials interact reversibly with the cation and tend to disintegrate when it is removed. In a different approach, the original calcium cation used to stabilize alginate has been replaced with aluminium ion after the beads were formed. 1°9 Alginate beads can also be predried to improve stability, 133 and they can be formed by internal gelation in areas where calcium ion is released chemically. 137 Various types of mixed gels have also been investigated. 158' 168 In a somewhat different way, the acrylamide monomer is polymerized into a stable bead matrix. A catalyst must be used in this process. This type of material produces a very stable entrapment matrix, but at high polymer concentrations it may be toxic to the immobilized organism. Prepolymerization of some synthetic materials before crosslinking can also form a stable biocatalyst matrix. 16°'172 A more complete description of these materials and processes is given in some of the review papers (e.g. refs 9 and 24). A number of other media are also being studied. These include, for example, natural gel materials such as chitosan, 38 egg white, 156 and locust bean gum, 168 mixed gels and polymers 158' 168 and material that can be polymerized by radiation. 27' 164, 165 Techniques that will permit the large-scale production of biocatalyst beads by forced flow of the gelling material through multiple nozzles are being developed 23' 29 and it has also been shown that the imposition of vibrational energy to the bead formation process will allow the production of monodispersed beads. 175 Bead formation by suspension of the m o n o m e r solution in a hydrophobic phase may prove to be an important technique in the future.125,155.17o Recent studies have shown that noninteracting material can be added to the gel matrix to effect desired gel properties 1°7' 1~7 and various techniques are being studied to reduce cell leakage from the biocatalyst beads.130,133

Membranes Cells can be restrained by semipermeable membrane materials that isolate the organisms from the bulk liquid. The cells can be immobilized into the membrane (a technique frequently used for the fabrication of biosensors),46, 48, 49 or they can be allowed to propagate into a void that is enclosed by the membrane (a technique used with membrane reactor systems). 177 179 The membrane allows the molecular transport of soluble material to and from the immobilized cells while confining and protecting the enclosed organisms. Hollow-fibre bioreactors, with the organisms confined to one side of the porous fibre and the soluble substrate and products on the other side, seem to be the most practical.177.178 However, growth must be controlled to prevent an excessive build-up of biomass since it could cause pressure that would rupture the membrane.180

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Porous materials Some porous materials have voids that allow organisms to penetrate and grow into large colonies. Apparently, these voids must be sufficiently large to allow easy penetration by the microorganisms but not larger than a few cellular diameters. 6 This can result in a relatively stable biocatalyst system in which the microorganisms tend to be isolated within the interstices of the medium. Such materials can include mats of fibres such as cotton or nylon mesh or cloth, 122' 184 metallic mesh 181 and various types of foam or sponge.62,181,183,185

Characteristics of immobilization As the immobilization of cells becomes relatively commonplace, emphasis is beginning to be focused on the definition and characterization of the immobilized material. For example, the Working Party on Immobilized Biocatalysts (now the Working Party on Applied Biocatalysis) within the European Federation of Technology has recently established preliminary guidelines for the characterization of immobilized biocatalysts. 187 The members of that group suggest that a proper definition of such material should include: (1) the quantity of biocatalyst in the matrix; (2) dimensions of the particles; and (3) diffusional limitations of the biological reaction. Innovative research is now being increasingly directed toward the study of the biocatalyst material itself(see Table 3).

Biofilm kinetics Many studies have been carried out on the dynamics and kinetics of microbial films. The results of a workshop on the subject indicated that there is an initial microbial attraction to the surface followed by the excretion of adhesion polymers that attach the cells firmly to the surface. 32 Thereafter, additional excretions and microbial interactions create a film. There is undoubtedly an optimum film thickness 88 that, hopefully, can be predicted by various models of the biofilm system.62, 78,85,188 Specialized models have also been developed, including one for the film on rotating-disc contactors. 86

Mass transfer studies Immobilized whole-cell systems have been examined from a mass transfer perspective in an excellent review. 37 Some important studies have been initiated into the study of internal diffusion in immobilized systems. Researchers have shown that alginate gels without microorganisms have glucose diffusion rates that approach those in water. 189 However, such material does have a diffusional resistance for larger molecules, although some gel formulations have apparent pore sizes that are sufficiently large to allow the excretion of produced enzymes. 11° A decrease in the Table 3

Characteristics of immobilized cellular systems

Characteristic

References

Guidelines Biofilm kinetics Mass transport Morphology Physiochemical environment Process modelling

187 32, 62, 78, 85, 86, 88, 188 23, 37, 38, 86, 110, 150, 189-191 136, 138, 171, 176 95, 116, 143, 192 38, 67, 78, 90, 153, 193

Immobilized ceils: a review of recent fiterature : C. D. Scott

diffusion rate is also observed for various formulations and for situations where the gel material is loaded with microorganisms.2a, as, 150 Mathematical modelling of mass transport in immobilized biocatalysts is also of interest. Recent work has included a correlation of the effects of physical and chemical properties on diffusion coefficients and mass transfer in general, as' 150,193 In addition, methods for the estimation of the Damkohler and Thiele Modulus have been developed. 2a' 190

Morphology Some attention has been given to the physical environment of the cells in the immobilized state, particularly with organisms encapsulated in biocatalyst beads. Light and electron microscopy and scanning electron microscopy have been the primary tools for such studies. 171'176 Cell concentration has been found to increase near the outer surface of the beads, at least for large beads, is6' 138 In some cases, the cells appear to propagate in small vacuoles within the bead, ultimately filling up the available space. 1as

Physiochemical environment The microenvironment of entrapped organisms will affect the efficiency of the system. Various approaches are being used to study these effects. For example, poly(ethylene glycol), in the presence of a suspension culture, has been used to simulate the reduction of water activity in the immobilized state. 192 The degree of water adsorption was found to affect microbial attachment. 95 Both respiration and growth rates of immobilized organisms have been shown to decrease with increased cell concentration, 143 and general microbial metabolism has been found to decrease upon immobilization. 116

Process modelling The research activity relative to the modelling of bioreactor systems is increasing, in general. Many of these efforts are focused on stirred-tank bioreactors with cells in free suspension. The modelling of bioreactors with immobilized cells is primarily oriented toward columnar reactors, which include tower reactors 67 as well as packed_bed 7s. 90.153, 193 and fluidized-bed systems.3 s In some cases, staged bioreactors have been modelled.as, 153 Most of these modelling efforts have been developed with an empirical approach, but mechanisms are also beginning to be incorporated.

Table 4

Application of immobilized cellular system

Product or application Biomedical Steroids Other uses Biosensors Chemicals Ethanol

Gaseous fuels Macromolecules Other chemicals Food and beverages Wastewater treatment Other applications

References 41, 83, 106, 123, 160, 163, 172 42, 127, 139, 160, 169 48, 49 38, 66, 67, 69-71, 75, 80, 87, 91, 93, 101, 107, 109, 111-114, 117, 118, 126, 128, 131, 132, 137, 140, 149, 151-153, 155, 158, 160, 168, 172, 176, 193, 194 51, 76, 121,145, 1 54 99, 138, 177 38, 73, 74, 108, 111, 119, 122, 124, 135, 141, 147, 157, 173, 174, 178, 180 50, 124, 134, 142, 161 51, 62, 64, 68, 72, 76, 78, 79, 81, 84, 85, 90, 95, 97, 102, 105, 115, 144, 154, 163 61, 94, 118, 129, 140, 141, 184

it is possible that the immobilized agents themselves could have therapeutic use. 127, 139

Biosensors Enzyme catalysed reactions have been effectively used to measure the concentrations of various solutes. Microbial systems are a natural extension of this technique. Membrane immobilized organisms, in conjunction with a measurement electrode, are being investigated for this application. Both amperometric and potentiometric measurements can be used for a variety of different determinations. 4s' 49

Chemicals Immobilized cells have been historically used for the production of commodity chemicals, and it is likely that they will have an increasing utility in the future for both commodity and specialty chemicals. The investigation of immobilized organisms for the production of a variety of chemicals is under way. This is currently the most popular research application for this field.

Biomedical

Ethanol. The fermentation of carbohydrates to ethanol has been a much studied immobilized cell processing system. This concept appears to be ready for full commercialization in the near future. 114'12s Most work, by far, deals with the well understood yeast Saccharomyces cerevisiae; however, other yeasts have also been studied, and some bacterial systems have been shown to have significant potential, especially Zymomonas mobilis. 66' 132.151 This organism has been found to have significant activity, even at alcohol concentrations as high as 12.6%. 132 While glucose has usually been the feed material of choice, other sugars such as xylose are also being considered; 193 even waste materials such as cheese whey 126 have been shown to be an adequate feed material. Fermentation systems can also produce multiple products that include ethanol such as isopropanol-butanolethanol. 111

The major potential use for immobilized cells in the biomedical area is for the production of pharmaceuticals and reagents. Most recent research has been for the production or transformation of steroids.41, sa, 106,123,160, 162,172 In addition, immobilized systems are also being studied for the production of antibiotics arid other therapeutics. 42' 160, 169 Of course.

Gaseous fuels. Interest is still high for the use of immobilized cellular systems to produce gaseous fuels. Methane and hydrogen can be produced from waste materials,51, 76,154. but hydrogen is the primary product in processing systems that utilize photobiochemistry.121,145

Applications Immobilized cells are being used or investigated for many different applications (see Table 4). The most popular of these is for the production of ethanol from carbohydrates.

Enzyme Microb. Technol., 1987, vol. 9, February 69

Review

M acromolecules Proteins and polypeptides represent an important class of biologically derived products. There is continuing interest in the use of immobilized microorganisms to obtain such materials. Recent research emphasis has been centred on the production of enzymes, including cellulolytic enzymes 99 and fl-lactamase.las' 177

Other chemicals Various other commodity chemicals, such as 2,3butadiol, 122 ethylene oxide, 73 phenolics 178 and other oxychemicalslOS. ~ t . 119.179 can be produced with immobilized organisms. Organic acids and their salts are also potential productsJ 24' 174, 18o Various carbohydrates, including high-fructose syrup, can be obtained as well. 74' 1 3 5 . 1 5 7 Higher value chemicals are of increasing interest as products of immobilized organisms processes. Not only do these include materials with therapeutic and diagnostic value in medical treatment (previously described), but also amino acids.38.141, 14.7.173 Even gaseous organics such as ethylene oxide can be produced by fermentation in 'dry' packed beds. 73

Food and beverages Immobilized cells are being evaluated for several applications in the food and beverage industries. Packed-bed systems are being used to provide flavours for addition to cheese 134 and immobilized organisms are being tested for the prefermentation of milk, a necessary first step in cheese manufacturing. ~24 Other systems are being examined for use in the alcoholic beverage industry, that is, for wine treatment 161 and for beer brewing. 142

Wastewater treatment

mammalian cells can be attached or detached from microcarriers, depending on the chemical state of the system. 94 The use of enzymes coimmobilized or used in sequence with intact cells allows interesting new processing techniques to be developedJ is' 14o, 141 Another potentially promising use of immobilization is the reinoculation of bioreactor systems by microorganisms entrapped in cotton cloth, ls4

Bioreactor systems Much of the past research on immobilized cells has concentrated on immobilization techniques and the characterization of immobilized systems. Since specific applications are becoming the centre of attention, the bioreactor concepts involved in carrying out these applications have gained increased importance. Most bioreactor systems now being studied for immobilized cells are continuous columnar systems such as packedbed or fluidized-bed systems (Table 5). In fact, such systems demand that the organisms be immobilized to prevent washout at the relatively high flow rates that are used. To a lesser degree, other concepts are also being investigated.

Fluidized-bed bioreactors Bioprocessing concepts that require relatively short reactor residence times can effectively use fluidized-bed systems. In general, small biocatalyst particles must be used to ensure fluidization, and they must be sufficiently stable to withstand significant shear forces for long periods of time. Relatively large systems are currently being tested as pilot plants prior to commercialization; 128 however, innovations continue to be made, including the use of cascading multiple reactors)76

Wastewater treatment continues to be one of the most significant areas of application for immobilizedcell processes. Most concepts are based on relatively undefined microbial systems with mixed cultures formed as a biological film on solid surfaces that are retained in packed-bed or fluidized-bed bioreactors. However, encapsulated-cell systems with well defined microbial populations are also being studiedJ 15,163 Much of the recent research has been directed toward nitrification 62"115'144 and denitrification of wastewater,64, 68.54. 105 but there has also been significant interest in the removal of dissolved organic materials.TZ, 51, 55.97 The production of methane while removing organic contaminants from wastewater by anaerobic processes is of continuing interest; 76'97' 154 and specialized approaches, such as the use of immobilized microorganisms as adsorbents for the removal of dissolved heavy metals, 163 are also being investigated.

Bioreactors that utilize membranes for the retention of cells are used by a small number of researchers. The majority of the work in this area is carried out with

Other applications

Table 6

Immobilization techniques have also been used for other applications. For example, a technique is under development for using microencapsulation of suspended microorganisms as a means of measuring microbial concentration. 129 Immobilization of plant and mammalian cells is important not only for enhancing cellular concentration in a flow system, but entrapment of these cells also serves as a means of protection from convective shear forces. 61 In addition, it has been shown that

Type of bioreactor

References

Fluidized bed

38, 62, 66, 81, 84, 88, 102, 114, 117, 120, 128, 138, 148, 167, 176, 181 18, 42, 58, 72-76, 78, 79, 85, 90-92, 97, 111-115, 118, 120-122, 124, 135, 136, 140, 142, 146, 147, 149, 153, 158, 161, 193, 194 177-180 65, 67, 69, 78, 82, 86, 107, 117, 126, 134, 183, 184, 194

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Packed-bed bioreactors Packed-bed bioreactors are being studied for immobilized cellular processes more than any other bioreactor configuration. In general, such systems are appropriate when relatively long retention times are required and external biomass build-up is minimal. There has been some innovation in the design and operation of such bioreactor concepts, including the use of a horizontal packed bed, 113 a dry or gas-phase system, 73 and multiple columns in sequence. 76' 147 Gas sparging in a packed bed has been tested to remove the fermentation product, ethanol. 91

Membrane bioreactors

Bioreactor concepts for immobilized cells

Packed bed

Membrane Others

Immobilized cells: a review of recent literature: C. D. Scott

hollow-fibre reactors, ~7' 178,180 but other membrane systems such as ultrafiltration cells are also used. 179 Most of the applications are for cellular systems that have relatively low growth rates since a significant increase in immobilized biomass could result in rupture of the membrane. This phenomenon will probably continue to restrict the general use of such reactor concepts.

Other concepts

10 11 12 13 14 15

To a minor degree, the stirred-tank bioreactor (the historical configuration) continues to be investigated for immobilized cell systems. 7s' s2,126.134, 184 Tower reactors (one of the original columnar concepts) are also being studied. 67' 69 Other innovations, including crossflow bioreactors,117 rotating-disc systems, 65, a6. t s 3 and sieve-plate reactors, 1°7 continue to be developed; and the use of multiple liquid phases, including organic solvents, 195 is being examined.

16

Future research

24 25 26 27 28

Many techniques for cell immobilization are becoming well developed and are now generally available to researchers. While the study of new approaches and materials will undoubtedly continue, the research trend will probably be directed towards the more effective utilization of existing techniques. Specialized use of immobilization techniques for mammalian and plant cells should continue to represent a small, but important, area of research; however, concepts for the production of commodity and specialized chemicals should still represent the major portion of the effort. Additional work on large-scale production of the immobilized biocatalyst is still needed, and a further characterization of such materials, especially with high cell loadings, would allow better predictions for potential production systems. The study of advanced bioreactor systems for the utilization of immobilized cells will probably become increasingly important, with the primary focus continuing to be centred on columnar systems. The immobilization of cells is now sufficiently well developed to allow serious consideration of several new processing concepts. Thus, the major emphasis is likely to be on process development, including the operation of pilot plants. This emphasis should result in an increasing use of such systems in the industrial sector.

References 1 Chibata, I. and Tosa, T. Appl. Biochem. Bioeng. 1983, 4, 1-9 2 Linko, P. and Linko, Y.-Y. Appl. Biochem. Bioeng. 1983, 4, 53151 3 Chibata, I., Fukui, S. and Wingard, L. B. (eds) Enzyme Engineering Plenum Press, New York, 1982, vol. 6 4 Laskin, A. I., Tsao, G. T. and Wingard, L. B. (eds) Ann. N.Y. Acad. Sci. 1984, 434 5 Buchholz, K. (ed.) Characterization of Immobilized Biocatalysts D E C H E M A Monograph 84, Verlag Chemie, Weinheim, 1979 6 Venkatasubramanian, K. (ed.) Immobilized Microbial Cells, ACS Symposium Series no. 106, American Chemical Society, Washington, 1979 7 Chibata, I. and Wingard, L. B. (eds) Applied Biochemistry and Bioengineering Academic Press, New York, 1983, vol. 4 8 Marshall, K. C. (ed.) Microbial Adhesion and Aggregation Springer-Verlag, Berlin, 1984 9 Woodward, J. (ed.) Immobilised Cells and Enzymes: a Practical Approach IRL Press, Oxford, 1985

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29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

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