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three-phase fluidized bed bioreactors for cell culture, especially for bioreactors of the airlift or draft tube type (Siegel and Robinson, 1992). Table 16 lists a number of recent cell culture applications for three-phase fluidization. These include production of monoclonal antibodies and therapeutic proteins, such as erythropoietin. Plant cells have also been cultured in airlift reactors (Scragg, 1992).

The combination of three-phase fluidization and cell culture may represent an ideal union of new technology and immediate application; because the high-value products of mammalian cell culture require relatively small production scales (on the order of 100 liters rather than 100,000 liters), it is possible to investigate the usefulness of three-phase fluidization in what would otherwise be considered a pilot scale set up without having to build an expensive, large scale unit.

Table 16. Recent Applications of Three-Phase Biofluidization to Cell Culture

BHK = baby hamster kidney cells

SS = stainless steel

CHO = chinese hamster ovary cells

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5.3Bioparticles

The type of bioparticle chosen for three-phase biofluidization often differs from that used in traditional three-phase fluidization. Small, dense particles, similar to those used in traditional catalytic three-phase fluidization applications, were often used in the initial studies of biofluidization, and for some time it was generally assumed that a bioparticle consisted of a biofilm grown on a small, dense particle (Heijnen et al., 1989). There are presently many applications, however, using cells entrapped in low density gel beads or growing within low density porous particles (Fan, 1989). These low density particles (alginate beads, 1.05 g/cm3; biofilm covered sand, 1.07 g/cm3) cause striking differences in the hydrodynamic characteristics of the biological systems compared to particles traditionally used in three-phase fluidization, such as glass beads (2.5 g/cm3). Furthermore, the density of the particle often changes with time during the bioreaction because of cell growth, again affecting the hydrodynamics of three-phase fluidized bioreactors.

Immobilization Methods and Particle Selection. Many methods of immobilization have been developed; these and their advantages and disadvantages have been well reviewed (Fan, 1989) and will not be discussed in detail here. Immobilization by the natural formation of a biofilm on a solid support and by the entrapment of cells within a polymer such as calcium alginate are the methods most frequently used in biofluidization research. The growth of animal cells on and in microcarriers, such as macroporous sintered glass spheres, is also a widely studied method of immobilization for three-phase biofluidization. Choice of method and particle material are determined by effects of particle characteristics on reactor performance. Desirable particle qualities include ease of handling and preparation for immobilization, reusability, suitability for steam sterilization, and low cost (Gonçalves et al., 1992). Table 17 lists several examples of particles in use in three-phase biofluidization research.

It must be recalled in selecting a particle that cell immobilization of any means can affect biological processes through various physicochemical effects, such as partitioning and diffusivity, and by directly affecting cell physiology and morphology. For instance, the optimal process pH for immobilized cell systems is likely to differ from the optimal pH for suspended cell culture because of the effects of surface phenomena on local pH values (Gonçalves et al., 1992).

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Table 17. Examples of Particle Materials used for Various Three-Phase Fluidized Bioreactor Applications

The particle materials also affect the ability of various organisms to colonize the particle and form an active biofilm through complicated mechanisms of surface charge, roughness, and porosity (Fan, 1989; Grishin and Tuovinen, 1989; Gonçalves et al., 1992). Cells growing within particle pores may be protected from shear effects (Grishin and Tuovinen, 1989; Livingston and Chase, 1991). Colonization ability has been shown to vary with species as well as with particle material (Lazarova et al., 1994). Even in studies where biomass holdup was constant, the support material affected the biomass activity (Ruggeri et al., 1994). Attrition of particles can also be important; because the attrition rate varies with superficial fluid velocity (Nelson and Skaates, 1988), a candidate material must be tested under the range of conditions anticipated in the fermentation. Colonization is also affected by process conditions such as fluid velocities and even influent composition, underscoring the need for particle screening under the expected process conditions (Gonçalves et al., 1992; Mol et al., 1993).

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Biofilm Effects. Particle size and density are especially important in determining the success of biofluidization. Bioparticle size and density are determined by the initial particle properties, the extent of biomass growth, and biofilm density. For a biofilm covered support, the apparent particle density is determined by Eq. (7) (Fan, 1989).

Particle sizes used for aerobic waste treatment generally range from 100– 1000 μm in size, with most being less than 500 μm in diameter, and reported biofilm thicknesses range from 40–1200 μm, with 100–200 μm being typical (Fan, 1989).

The terminal settling velocity of the particle is determined by the particle size and density as shown in Eq. (8) (Kunii and Levenspiel, 1991):

The effect of biofilm volume on the terminal settling velocity can be described by Eq. (9) (Andrews and Przezdziecki, 1986):

This equation is plotted in Fig. 11, showing that for relatively dense support particles, biofilm growth can reduce the settling velocity if the biofilm density is less than that of the biofilm-free particle. As such bioparticles gain biomass, they will rise to the top of the bed and may even elutriate from the reactor (Sreekrishnan et al., 1991; Myška and Švec, 1994), reducing achievable conversion rates. This situation could be resolved by using lower density particles, such as expanded polystyrene or

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amount of biomass from the particles or is undesirable from an operations standpoint, the addition of a draft tube to the reactor may provide the level of turbulence required (Livingston and Chase, 1991). Alternatively, the optimal biofilm thickness can be maintained by removing particles as the biofilm grows thick and stripping or washing some of the biofilm from the particles (Andrews and Przezdziecki, 1986; Tzeng, 1991).

Modeling studies have shown that when the bed is not monosized and the difference in particle sizes is great enough, it is possible that the large particles will not reach a lower settling velocity than the small particles; hence the large particles will remain in the bottom of the bed (Myška and Švec, 1994). If biofilm thickness control is desired, removal of particles at a location other than the free surface would have to be implemented in this case.

Novel Bioparticle Research. Two major thrusts have been seen in recent particle research—the area of density manipulation so that particle density suits the desired fluidization mode, and the development of magnetic particles for use in magnetically stabilized fluidization. Intraparticle mass transfer is also of interest. Table 18 lists several novel particles developed in recent years to address these and other concerns.

Several methods have been suggested for increasing bioparticle density. Stainless steel mesh has been used as a support framework and density modifier for bioflocs for wastewater treatment. The large void volume available in the mesh, the capability of the relatively denser particles to withstand elutriation at high fluid velocities, thereby allowing higher reactor throughput, and the ability of filamentous organisms to be retained within the mesh were some of the advantages cited (Kargi and Toprak, 1994). Entrapment of stainless steel beads in collagen particles upon which mouse hybridoma cells were immobilized (Shirai et al., 1994) and the inclusion of α-alumina in calcium alginate beads (Paz et al., 1993) have also been demonstrated as methods to increase bead density.

Though most particle density manipulation research has focused on increasing density, one report addresses the possibility of decreasing particle density to allow low aspect bioreactors with low liquid velocities required for fluidization of low density bioparticles. Hollow glass spheres were co-immobilized with yeast cells in calcium alginate beads. The final particle density after cell growth could be varied in proportion to the percentage of hollow glass spheres incorporated (Vorlop et al., 1993).

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Table 18. Examples of Recently Devised Novel Bioparticles

Large, light, granular particles that allow biomass growth without alteration of particle physical properties, hence, no change in fluidization characteristics, have been used for wastewater treatment in a three-phase fluidized bed; the particle material was not specified (Roustan et al., 1993). Another approach to avoiding undesired solid stratification is to periodically remove excess biofilm from the particles (Tzeng, 1991; Livingston and Chase, 1991).

Magnetic beads or support particles are used in magnetically stabilized fluidized beds. The advantages of such a bioreactor are discussed in a Sec. 5.10. A method for manufacturing a magnetic pellicular bead by electrostatically depositing alternating layers of silica and polymer onto magnetic core particles, followed by burning of the polymer and sintering of the silica, was developed to allow exploitation of the multiply derivatizable silica for various biological immobilization processes (Goetz et al., 1991). The resulting particle was dense and, thus, had increased settling velocity, allowing increased flexibility in operating conditions for the magnetically stabilized fluidized bed. This particle has been used to immobilize enzymes, but its porosity and material of manufacture suggest that it may also be suitable for microbial immobilization.

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Improvement of intraparticle mass transfer is the goal of some particle research efforts. One novel approach that has been recently tested is the co-immobilization of algae with bacteria; the algae produced oxygen and the bacteria produced the desired product (Chevalier and de la Noüe, 1988). Another method used microporous particles entrapped within alginate bead bioparticles to prevent excess biomass growth that could hinder intraparticle mass transfer (Seki et al., 1993).

5.4Hydrodynamics

Predictions of bioreactor hydrodynamics and individual phase loadings are important to allow the design engineer to specify column dimensions and to size the required pumps and blowers. The hydrodynamics of three-phase fluidized bed reactors have been studied and reviewed (Muroyama and Fan, 1985; Fan, 1989), though additional work is needed to fully understand these complex systems. The necessity for further study is even greater for three-phase fluidized bed bioreactors, the hydrodynamics of which vary greatly from traditional catalytic systems (Davison, 1989; Tang and Fan, 1990; Karamanev et al., 1992; Nore et al., 1992). As described in Sec. 5.3, the low density bioparticles common in three-phase biofluidization have dramatically different hydrodynamic characteristics than do the dense particles traditionally used in three-phase fluidization. Furthermore, the fluid flow rates used in biofluidization are relatively low compared to those found in traditional fluidization, corresponding to relatively slow biological reaction rates and the need to prevent excess erosion of the biofilm or bioparticle (Tang and Fan, 1990).

The hydrodynamic characteristics of a three-phase fluidized bed bioreactor depend on a number of factors, including particle properties, such as size, density, wettability, and roughness; fluid properties, such as flow rates, surface tension, viscosity, presence of surfactants, and electrolyte concentration; and reactor design, including column geometry and gas distributor design. Major differences between the hydrodynamics of low density systems, as typified by many biological processes, and high density systems include the presence of significant axial variation of individual phase holdups in the low density systems that are not apparent in high density particle systems (Tang and Fan, 1989; Tang and Fan, 1990), greater axial dispersion of solids (Bly and Worden, 1990), changes in bubble coalescence characteristics caused by the presence of fermenta-

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tion medium components or by compounds produced by the growing cells (Sun and Furusaki, 1988; Bly and Worden, 1990; Béjar et al., 1992), and change in gas holdup because of biological gas production within the bed (Davison, 1989). These and other differences are further discussed in the following sections, with a view towards assisting the biochemical engineer to avoid pitfalls in designing three-phase fluidized bioreactors. Unless otherwise noted, the hydrodynamics discussed are those of conventional three-phase fluidized bed reactors, as shown in Fig. 9.

Fluidization Regime. As for traditional fluidization applications, the fluidization regime—dispersed bubble, coalesced bubble, or slug- ging—in which a three-phase fluidized bioreactor operates depends strongly on the system parameters and operating conditions. Generally, desirable fluidization is considered to be characterized by stable operation with uniform phase holdups, typical of the dispersed bubble regime. It would be useful to be able to predict what conditions will produce such behavior.

One approach recently suggested is the mapping of satisfactory and unsatisfactory biofluidization regions according to two parameters based on easily measured physical properties of the phases, such as viscosity, density, and surface tension (Béjar et al., 1992). The success of these parameters in predicting satisfactory fluidization has been demonstrated in both a straight, well-mixed, gas-fluidized bed of calcium alginateimmobilized yeast (Béjar et al., 1992; Vorlop et al., 1993) and in a tapered single-pass liquid-fluidized bed of calcium alginate-immobilized bacteria (Davison et al., 1994). Roustan et al. (1993) also applied mapping parameters to high and low density particles to predict fluidization regime with some success; however, separate maps were required for each type of particle because particle density was not included in the parameters. Further research is required to confirm the broad applicability of this approach to the prediction of whether successful fluidization is possible.

Solids Holdup. The active biomass concentration in the threephase fluidized bed bioreactor is determined by the biofilm characteristics and the solids holdup. Because metabolic rates are moderate relative to chemical catalytic rates, solids holdup in biofluidization applications are generally larger than in chemical applications. Reported overall solids holdups in various three-phase fluidized bioprocesses range from 0.02 to 0.6, with most studies in the range of 0.05 to 0.4 (Sun and Furusaki, 1988; Davison, 1989; Kobayashi et al., 1990; Karamanev et al., 1992; Potthoff and Bohnet, 1993; Roustan et al., 1993; Badot et al., 1994). Solids holdup