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Biotechnol. Prog. 1990, 6, 452-457
Plant Cell Culture Using a Novel Bioreactor: The Magnetically Stabilized Fluidized Bed Joye L. Bramble, David J. Graves,*and Peter Brodeliust Department of Chemical Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104
A novel bioreactor using magnetically stabilized fluidized bed (MSFB) technology has been developed that has certain advantages for cultivating cells continuously. In this system, the cells are protected from shear and are constrained to move through the fermenter in lock-step fashion by being immobilized in calcium alginate beads. The MSFB permits good mass transfer, minimizes particle collisions, and allows for the production of cells while maintaining a controlled cell residence time. Details of the experimental system are described. In addition, the experimental performance of an MSFB used to grow plant cells in batch mode is compared t o the results obtained in shake flask culture.
Introduction Production of Biochemicals from Plant Cells. In addition to their use as a food source, higher plants have been utilized for pharmaceuticals, fragrances, flavors, and coloring agents ( I ) for many centuries. Over 80% of all known natural products are of plant origin, and production is currently normally carried out by cultivation of the plant followed by extraction of the desired compound. However, this process is labor-intensive and is associated with many limitations including disease, drought, and only periodic availability. Since many of these compounds are complex molecules that cannot be chemically synthesized, alternative means for production of plant-derived products have been receiving considerable attention. Techniques for growing plant cells in culture have developed relatively slowly from a first report about thirty years ago (2). At this point in time, the cost of such processes is so high t h a t researchers interested in commercializing this process are concentrating on cells that produce high-value secondary metabolites such as codeine, digoxin, vanilla, mint, and betacyanins ( 3 ) . However, growth in fermentors in the manner that is familiar for microorganisms is generally not very satisfactory for several reasons: (1)Plant cells grow very slowly (with doubling times of days rather than hours) so that maintaining absolute sterility for long periods of time is a vital concern. Microbial contaminants will quickly overrun a plant cell culture system. ( 2 ) Plant cells are large (30-100 pm), have rigid walls, and tend to grow in aggregates. They are destroyed by impeller speeds as low as 28 rpm ( 4 ) . (3) Plant cell suspensions tend to stick to fermenter surfaces and become very thick (up to 60 g of biomass dry weight/L) as they grow. This characteristic plus the shear sensitivity means that it is often difficult to attain good oxygen transfer with conventional approaches to cell growth. Cultured plant cells normally are grown in a dedifferentiated state where the cells have lost their specific morphology (as, for example, root cells, leaf cells, etc.). However, it has been observed that fast-growing cells that are highly dedifferentiated generally do not produce large quantities of secondary metabolites (5). Once the cells enter the stationary phase, they start to differentiate.
* Author to whom correspondence should be addressed. Permanent address: Department of Plant Biochemistry, University of Lund, S-22007 Lund, Sweden. t
Frequently, this is the time when they begin producing the secondary metabolite products of interest (6). Most of these products are retained in vacuoles within the cell. Most plant cell culture systems designed to produce a bioproduct therefore will have two sequential fermenters, one optimized for growth (the growth fermenter) and one for metabolite production (the production fermenter). Plants are also likely to require equipment for intracellular product recovery. A typical example is the commercial process to produce shikonin, a red dye used medicinally and in cosmetics (7). The net result of these characteristics is that the production fermenter for plant cells frequently must be operated in a batch fashion. Among the other characteristics that constrain one in designing plant cell reactors are the following: (1) Plant cells produce their own hormones and require the presence of such hormones for growth. Therefore, the use of high dilution rates or discarding the “conditioned” nutrient medium from a batch fermentation is generallyundesirable. Likewise, there is a minimal cell inoculum density below which no cell growth occurs (3). ( 2 ) Cell aggregation or artificial confinement in a porous support is frequently either necessary or desirable to stimulate certain metabolic pathways (8). Although this phenomenon is not yet clearly understood, hormones may play an important role in this behavior as well. Substrate depletion or product accumulation near the center of a cell mass may also play a role in some cases. (3) Stressful conditions (such as low oxygen or phosphate levels) often are needed in the production fermenter to induce the formation of the highest levels of secondary metabolite. This is especially crucial with plant cells because of the low levels (milligrams per liter) and low formation rates (milligramsper liter per day) of product often seen. However, exposure to such conditions for too long can kill the cells. Thus, excellent control over cell residence time is desirable so that the cells can be “rescuedn at the appropriate time or discarded if they have died. Properties of the Magnetically Stabilized Fluidized Bed. Fluidized beds have been used for many years to contact solid and fluid phases. Where good heat transfer and transport of the solids are required, they are frequently advantageous. However, when one wishes to prevent mixing of the solids and yet to move them through a system in plug-flow fashion, the ordinary fluidized bed fails badly. If, however, the particles are magnetizable and the bed is
8756-7938/90/3006-0452$02.50/00 1990 American Chemical Society and American Institute of Chemical Engineers
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Biotechnol. frog.., 1990, Vol. 6,No. 6 Spent medium (with extracellular product)
solids Input
Beads with fresh cells
Solids output
Figure 2. Sequential sampling: The above example shows how samples that have been in the column for different periods of time can be removed by shutting down the reactor and removing each discrete element.
Porous distributor plate Liquid recycle at high rate
Beads with spent cells (+intracellular product) Fresh Medium
Addition olox!gen (through bubbling or membrane system1
Figure 1. Schematic drawing of a hypothetical magnetically stabilized fluidized bed system used t o culture cells on a continuous basis.
surrounded by a magnetic field, the particles adhere to one another and a nonmixing plug flow of the solids can be obtained relatively easily. As we have seen, this fixed residence time is just what is often required to produce secondary metabolites in plant cell culture. Such a system is called a magnetically stabilized fluidized bed (MSFB). Early work was done by Hershler (9,lO) in 1961, but the first systematic studies of MSFBs were not conducted until the 1970s by Rosensweig and his colleagues (11-13). Since then, they have been employed in chromatographic and adsorptive separation, including bioseparations in our laboratories and those of others (14). A detailed description of such beds is beyond the scope of this report but can be found in the above-cited work. Plant Cell Culture Using the MSFB. Due to the nature of the MSFB, and in particular its ability to maintain a constant particle transit time, and to the special requirements of plant cells, we considered applying it to the production of plant cell metabolites. Using a hypothetical system such as that shown in Figure I, one would immobilize the plant cells in a gel bead containing magnetically susceptible solids. These beads would then be added to the top of an MSFB column and would move downward in a lock-step fashion, being removed at the appropriate time. The beads would be fluidized with the desired medium, with a high rate of recycle used to provide good mass transfer and reoxygenation without diluting endogenous hormones. Such a system provides high oxygen transfer rates yet maintains the immobilized cells in a low-shearenvironment. Since the particles would be relatively motionless, particle collisions that cause support damage would be eliminated. Immobilization of the cells prevents washout or damage by the pump, simulates the environment of a cellular aggregate, and provides a means to control cell residence time in the system. Cells are readily removed from such beads by treating them with citrate or EDTA. For the
high-value materials usually considered worthy of production by plant cell culture, the added costs of immobilization and resuspension are unlikely t o be prohibitive. Such an MSFB system can be utilized in a variety of ways to aid in the production of secondary metabolites. Cells that are already in the stationary phase can be put in a t the top of the column and irrigated with production medium. When the cells start to lose viability or productivity, they can be removed from the bottom of the column and either revived for recycling (applicable to cases where the product is extracellular) or broken open to recover intracellular products. It is possible with the MSFB to harvest such cells continuously from the bottom of the reactor when they have accumulated maximal amounts of product. The MSFB thus has some of the best features of both the batch and the continuous stirred fermenters with few of their disadvantages. An alternative way in which the MSFB fermenter can be used is to produce biomass. Since excessive cell growth and division tends to split the beads, a fixed reactor residence time is highly desirable in such a case. Here again, the MSFB is extremely useful since the solids flow rate can be set such that the cells are removed just before this bead splitting occurs. This cannot be accomplished with conventional fluidized beds because of the uncontrolled contact time and can be achieved only discontinuously (batchwise) with a fixed bed. Third, virtually instantaneous changes can be made in the growth medium for various purposes. Solvents are sometimes briefly used to “permeabilize” a plant cell and cause it to begin excreting products. Elicitors, specific biochemicals from pathogens such as fungi, can also be employed to stimulate product production. In addition, there are also several interesting types of fundamental studies that can be performed with the MSFB to elicit important information about cell growth and metabolism. Since the cells are immobilized in an artificial aggregate, this system could be used to investigate the effect of such an environment on cell growth and metabolism. Due to the investigator’s ability to remove solids from the bed in a sequential fashion, it is possible to sample from all regions in the bed by rapidly removing all the solids sequentially (Figure 2). If the bed previously had been a t a steady state with continuous bead addition and removal, each successivelayer would contain cells that have been in contact with the medium for a specific period of time. Thus, it is possible to analyze the effects of a given
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&
Bead Addition Flask
0
I
0
CH3
H 7-Methylxan t hine M S F
B
s q
Theobromine
SAH
E
0
z
o
,
,
,
,
, Flask
Figure 3. Illustration of the set-up of the complete reactor system.
medium for many different periods of time in a single experiment. Finally, since the fluid composition.can be altered almost instantly, one can study the effect of medium compositionand growth factors on cell growth and metabolism by a pulse or step response technique, where cell samples are removed sequentially a t fixed time intervals after the disturbance. Other basic studies include varying the dilution rate to analyze the effect of growth factors and to study the effects of excreted toxins on growth rate and product formation. Although these advantages appear attractive, there are several disadvantages in the use of an MSFB fermenter that must also be considered. Such a system is considerably more complex than a stirred tank due to the addition of magnetic coils and of ports for the entering and exiting solids streams and the requirement for a stable, magnetically susceptible support particle. The complexity of this system also provides a potentially greater chance of microbial contamination. Finally, coils designed for high fields can generate heat, which must not be permitted to affect the temperature of the cellular environment. System Description. The goal of this project was to design, construct, and evaluate an MSFB reactor for use with plant cells. For this particular system (Figure 3), we recirculated the liquid in order to maintain a high velocity to fluidize the particles and supply oxygen yet avoid washing out endogenous hormones and wasting medium. We chose to reaerate the liquid stream with a hollow fiber device on each pass. A recycle vessel is used to permit continuous addition and removal of liquid. Coffeaara bica was chosen as the model plant cell system, since the cells are easy to grow and produce easily analyzed extracellular alkaloid products (caffeine and theobromine; see Figure 4 for the synthetic pathway).
Materials and Methods Chemicals. Sodium alginate H F (Protanal highviscosity fraction) was obtained from Protan Scotia Marine Inc. (North Hampton, NH). Murashige and Skoog basal medium was purchased from Flow Laboratories (McLean, VA). Kinetin, thiamine hydrochloride, and (2,4-dichlorophenoxy)acetic acid (2,4-D) were obtained from Gibco Laboratories (Grand Island, NY). Magnetite (FesO&,-325 mesh, was from Aesar (Seabrook,NH). Cysteine, caffeine, theobromine, citric acid, myo-inositol, and glucose assay kit 510-A were purchased from Sigma (St. Louis, MO). All other chemicals were purchased from commercial suppliers.
CH3
Caffeine
Figure 4. Synthetic pathway for theobromine and caffeine. E = methyltransferase enzyme, SAM = S-adenosylmethionine, and SAH = S-adenosylhomocysteine.
Equipment. Alkaloid analysis was conducted by using high-performance liquid chromatography (HPLC). The HPLC system was assembled from components composed of a 10-cm by 4.6-mm Brownlee Labs RP-18 Spherisorb column containing 5-pm particles with a 3-cm guard column, an MSI Model-310 HPLC pump, an ISCO UA-5 absorbance/fluorescence detector with a Type 6 optical unit, and a Hewlett-Packard 3370A integrator. Oxygen measurements were made with a Yellow Springs Instruments Model 58 meter. Suspension cultured cells were cultivated in shaker flasks on a Lab-Line junior orbit shaker. Callus cells were incubated in a Thelco precision incubator. All sterile work was conducted in a Model 868 laminar flow hood from CCI/Forma Scientific. Power was supplied to the four solenoidal coils by a Kepco DC power supply rated at 15 A and 40 V. Aeration was provided with a TAF 12 dialysis cartridge used as a membrane oxygenator (Terumo Corp., Tokyo, Japan). This unit had a surface area of 1.2 m2 and an internal volume of 80 mL. We measured an oxygen Kla value of 62.4 h-l at a liquid circulation rate of 300 mL/min in this type of cartridge. This was 5-10 times the value needed to supply the cells at their maximum possible oxygen demand rate. Each cartridge was used for one experiment and was then discarded. Air was pumped through the hollow fibers in the cartridge by a Virtis Omni Culture air pump. A Masterflex (Cole Parmer, Chicago) peristaltic pump with longlife Norprene tubing was used to circulate medium through the MSFB system. Water was deionized with a Barnstead PCS deionizer and then distilled with a Corning AG-lb still. Spectrophotometric measurements were made with a PyeUnicam Model SP1800 spectrophotometer. Samples were centrifuged in a Clay Adams safety-head centrifuge. Cultivation of Cells. Callus cultures of C. arubica were supplied by Dr. Peter Brodelius, Institut fur Biotechnologie, Zurich, Switzerland, and Dr. Henrik Pedersen, Rutgers University, New Brunswick, NJ. Both cell lines originated with Dr. Thomas Baumann, University of Zurich. Suspension cultures were initiated by adding 8.8 g of cells to 50 mL of Murashige and Skoog medium (15) (with glucose as the carbon source) supplemented with 100 mg/L myo-inositol, 10 mg/L L-cysteine, 1.0 mg/L thiamine hydrochloride, 1.0 mg/L 2,4-D, and 0.2 mg/L kinetin in a 125-mL Erlenmeyer flask. The cells were cultivated on
Biotechnol. Prog., 1990,Vol. 6,No. 6
a gyratory shaker at 150 rpm. The suspension cultures were subcultured every 7-10 days by doubling the volume of the suspension with fresh medium and splitting into two new flasks. Preparation of Beads with Immobilized Cells. The following procedures were carried out under sterile conditions: C . arabica cells that were 8-10 days old (following subculture) were washed with medium on a glass filter. A 50/50 (w/w) mixture of cells to alginate solution was prepared by adding 20 g of cells to 20 g of alginate solution containing 3.0% (w/w) Protanal H F (sodium alginate) and 2.5% (w/w) magnetite, FeaOl, in culture medium. Beads approximately 3-4 mm in diameter were made by placing the cell/alginate suspension in a 50-mL syringe and expelling the suspension dropwise into 300 mL of well-stirred medium supplemented with 50 mM CaC12. The beads were left in the medium for 1 h to allow for complete reaction of alginate with the Ca2+ions and then were washed with normal medium and filtered in a Buchner funnel. Removal of Cells from Beads. Following culture, when alginate-free cells were needed, approximately 1.0 g of beads was suspended in 10 mL of medium containing 0.1 M sodium citrate at pH 6.0. The beads were dissolved by agitating the suspension for 30 min-1 h. After the beads were completely dissolved, the suspension was filtered and washed on a nylon filter (50-pm pore size) in a Buchner funnel. T o allow the cells to recover from the above treatment, we incubated them in fresh medium with mild aeration for 1 h. Analytical Procedures. D r y Weight. Samples were filtered on a nylon filter (50-pm pore size) and then weighed (total fresh weight). A portion of the sample, about 400 mg, was removed, dried overnight a t 50 "C,and reweighed. The dry weight of the original sample was calculated from the size of the aliquot. Glucose Assay. The glucose concentration of the medium was determined by using the Sigma 510-A glucose assay kit. All samples were diluted 10-fold before being tested. Alkaloid Analysis. An isocratic mobile phase of methanol/water (34:66) with 1 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer a t pH 5.8 a t 1 mL/ min was used in the HPLC system to determine alkaloids. A 10-jiL sample was injected, and the column effluent was monitored a t 280-300 nm. Respiration. A 400-mg sample of cells was added to 10 mL of medium in a 20-mL plastic container with a magnetic stirrer and a rubber adapter, which formed an airtight seal against an oxygen probe placed in the container. Before the oxygen probe was inserted, the suspension of cells was aerated lightly for 10 min to saturate the medium with oxygen. Mild mixing was achieved by placing the system on a magnetic stirring plate at a low setting. The change in oxygen concentration in solution was then followed for 20 min, and the respiration rate of the cell sample was determined from the decrease in oxygen concentration with time. Cultivation of Cells Using the MSFB. Apparatus Assembly. Before assembly, the followingequipment was autoclaved: MSFB column, recycle vessel, filter unit, air bubbler, water trap, air filters, and tubing. These items were then assembled under sterile conditions in a laminar flow hood. The hollow fiber aeration cartridge, which was purchased presterilized, was then flushed and filled with medium. In our last and most successful experiment, we first soaked the cartridge in 95% ethanol for 1h, since earlier studies showed signs of contamination after 6-10
455
days of use, which we traced to this item. Second, the recycle vessel was filled with 200 mL of medium. Next, all items but the column were connected, and clamps were attached at the appropriate locations to keep the system closed. The column was then prepared by filling with 1 L of medium. The beads, which were prepared a day in advance and were left on a shaker overnight, were added to the column by using a specially designed bead input vessel to create a bed height of 20 cm (approximately 300 g of beads). The bead input vessel consisted of a 500-mL flask with a ground glass joint at the bottom, which mated with the bead input port on t h e column, and a rubber bulb connected to a stopper placed in the mouth of the flask. By repeatedly squeezing the bulb, we forced the beads to pass from the flask into the column. After the column had been filled with medium and beads, a Plexiglas water jacket was slipped into place. The apparatus assembly was completed after placing the column within the magnetic coils. At that point, two final tubing connections were made, one from the column to the aeration unit and one to the filter unit. In preliminary experiments reported here, no attempt was made to add or remove beads continuously, although bead and medium samples were taken periodically. Batch Mode. A glass column 52 cm long with a 5-cm inside diameter was used for cell cultivation. The column was positioned in the center of the four coils. A magnetic field strength of 200 Oe was generated a t 2 A and 35 V. Using a water flow rate of 100 mL/min in the water jacket, we could remove the heat generated by the magnetic coils, thus maintaining the column a t 25 "C. Medium entered the bottom of the column and flowed through a porous polypropylene liquid distributor, exiting from a port a t the top. The medium was recirculated through the hollow fiber dialyzer for reoxygenation before being returned to the column. The filter-sterilized air flow rate in the dialyzer was 1L/min. To prevent evaporation of medium, the air was bubbled through sterile water before being passed through the cartridge. An empty flask to retain moisture droplets and a second sterile filter were added downstream of the aeration cartridge. The beads were fluidized at a liquid flow rate of 400600 mL/min (a superficial velocity of about 20-30 cm/ min, or an interstitial velocity of about 50-75 cm/min). Because of the large size of the beads (about 50 times as large as those typically used in chromatography), the behavior of the bed was much more like that of a packed bed than an MSFB. Processes such as channeling or bead chaining were never observed, and we believe that because of the high circulation rate all beads were exposed to the culture medium with approximately equal efficiency. Dry weight and cell viability were found for each sample collected (at 4-6-day intervals). A 5-mL liquid sample was collected simultaneously from the liquid stream entering the bead collection vessel; this was analyzed for glucose and alkaloid levels.
Results and Discussion Before attempting to grow cells in beads, either with or without added magnetite, we wanted to determine how readily typical molecules could diffuse into or out of the beads. To do this, we added beads to stirred solutions containing either glucose, caffeine, or bovine serum albumin. The first two molecules are, respectively, a nutrient and a product, and both are relatively small in size. The third is a large molecule intended to probe the pore size of the beads. As one of these molecules pene-
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Table I. Results of Diffusion Experiments diffusing molecule
diffusion coefficient, cm/min X 10-4 in water in 1.5% alginate in 3.0% alginate in 1.5% alginate + 5 YO magnetite in 1.5% alginate + 10% magnetite in 1.5 % alginate + 20 % magnetite in 1.5% alginate + 10% magnetite + 25% cells in 1.5% alginate + 10% magnetite + 33% cells in 1.5% alginate + 10% magnetite + 50% cells a
glucose 4.08a 4.1 4.1 4.1
caffeine 3.40b 3.4 3.4 3.4
bovine serum albumin 0.43a 0 0 0
4.1
3.4
0
3.0
2.5
0
3.0
3.0
0
3.0
3.0
0
3.0
2.5
0
From Tanaka et al. (1984). From Stryer (1981).
trated the beads, its concentration in the solution phase fell with time. The concentration versus time curve for this solution was analyzed to provide diffusion coefficients. The theory for such studies is well developed, and details of the theory as well as the experimental details are given elsewhere (16). In addition to plain beads containing 1.5 or 3.0% alginate, we measured beads with 1.5% alginate and 5, 10 or 20% magnetite. Additional studies were carried out on 1.5% alginate beads containing 10% magnetite and 25,33, and 50% nonviable cells (killed by overnight anoxia). Both the magnetite and the cells were used to assess the diffusionhindrance caused by particulate barriers dispersed in the gel. The results of these studies are given in Table I. Large molecules such as albumin apparently do not penetrate the beads to any appreciable extent. In contrast, there is virtually no difference between the diffusivities of the small moleculss in water and those in the gels until very high concentrations of magnetite or cells are included. Our later experiments were conducted with 3% alginate and 2.5% magnetite, so we are confident that any diffusion problems which existed would have been due to the size of the beads and the speed of any biochemical reactions, not the bead composition. In future work we hope to achieve a somewhat smaller bead size to avoid diffusional problems due to the large bead radius. (Microscopic examination of sectioned beads that had been in the system for a week or more showed that nearly all the cell growth was taking place within a short distance of the bead surface.) In order to evaluate the performance of the MSFB reactor, we conducted a series of experiments comparing the behavior of cells in the MSFB reactor to cells cultivated in a shaker flask. One set consisted of freely suspended cells in shaker flasks, another of beaded cells and magnetite in similar flasks, and the third of similar beaded cells in the MSFB. All experiments were conducted in standard MS medium supplemented with 50 mM calcium chloride. We conducted four partially or totally successful experiments with our MSFB reactor. (Nine others failed for a variety of reasons.) The first three moderately successful experiments were carried out without alcohol sterilization of the cartridge oxygenator. The first two were successful for 6 days and the third for 10 days before contamination appeared. A number of tests indicated strongly that the dialysis cartridge was the source of this problem. Consequently, the fourth successful experiment in the MSFB was carried out with two modifications. The cartridge was alcohol sterilized, and 100 mg/L of the
0
I
I
I
10
20
30
*
FneCells
4
MSFB
Time (days)
Figure 5. Comparison of growth for cells cultivated in the MSFB reactor and in shaker flasks.
antibiotic vancomycin (found to be a nontoxic level) was added to the medium. This experiment was halted after the cells grew to such an extent that the beads had expanded, completely filled the reactor, and were starting to flow out into the recycle vessel. This occurred by day 19. Because of time limitations, we were unable to carry out further experiments to determine whether the alcohol treatment alone would have been sufficient to prevent sepsis. The results for these experiments are presented in Figures 5-7. In Figure 5, we observe that the freely suspended cells and the immobilized cells in shaker flasks exhibit the same total cell growth. The cells cultivated in the MSFB reactor initially lagged behind but by day 12 achieved a dry weight equivalent to or slightly higher than that in shaker flasks. One point to note is that the results prior to day 12 are an average of multiple experiments and the results after day 12 were obtained in only one experiment (the fourth successful one described above). There are many likely reasons for the lag in initial growth that was observed. One possibility is that the unavoidably (slightly) larger ratio of medium to cells in the MSFB system may have caused some dilution of endogenous growth factors. More efficient aeration and trauma due to unfavorable conditions while the column was being assembled are others. If we evaluate the results for the alkaloid production (Figures 6 and 7), we observe that, during the growth phase, all cells produced equivalent quantities of theobromine. However, by the stationary phase t h e cells in free suspension produced the highest (7.0 mg/L) and the cells in the MSFB reactor produced the lowest (5.0 mg/L) amounts of theobromine. The results for the caffeine production (Figure 7) show large differences in caffeine production between the cells in free suspension and those immobilized with magnetite in either the shaker flask or the MSFB. In the stationary phase, the immobilized cells in the shaker flask exhibited somewhat higher levels of caffeine (10 mg/L at day 20) than the cells in the MSFB (5 mg/L at day 19). These results suggested that (a) magnetite inhibits caffeine production [this was confirmed in other experiments (16)] and (b) there are differences in the behavior of the cells grown in the two different types of reactors. Although the differences do not appear in the cell growth, they do appear in the production of caffeine. Similar results have been observed in other studies, where it was found that cells in continuous stirred fermenters produced lower levels of secondary metabolites than the same cells in a shaker flask (17). The authors of this work believed these results were a consequence of overventilation with air, which was displacing volatile components evolving from the plant cells. This volatile component was believed to be either ethylene, a plant
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Acknowledgment This work was partially supported by the National Science Foundation (Grant CBT84-19140 A01). Partial support was also provided by the University of Pennsylvania research foundation. We thank Meeyoung Toh for her assistance in carrying out the bead diffusion experiments.
If" f 0
10
Literature Cited 20
30
Time (days)
Figure 6. Production of theobromine for cells cultivated in the MSFB reactor and in shaker flasks.
0
10
20
30
Time (days)
Figure 7. Production of caffeine for cells cultivated in the MSFB reactor and in shaker flasks.
hormone that is naturally produced by the cells, or COZ. Studies with ethephon (a precursor of ethylene soluble in aqueous solution) in suspension cultures of C. arabica have shown that ethylene does not affect cell growth but greatly enhances the production of theobromine and caffeine (18). Therefore, if we were to displace the naturally occurring ethylene produced by cells in our MSFB reactor, we would expect lowered levels of caffeine. One way to test for this possibility would be to run experiments in the MSFB at a series of lower air flow rates to reduce ethylene washout or to add ethylene to the aeration gas.
Conclusions The magnetically stabilized fluidized bed has been used successfully to grow plant cells (C.arabica) in culture. Although the system was reasonably complex, we learned to operate it for an extended period of time without microbial contamination. A membrane dialysis cartridge used as an oxygenator proved adequate in supplying oxygen. Although the system was not operated with continuous beaded cell addition and removal, no obstacles to such an application are apparent, and this opens the way to a number of significant studies. Differences in behavior between cells in shaker flasks and those in the MSFB system were noted, showing that even small changes in environmentalfactors can affect complex eukaryotic cells profoundly. Magnetite causes large effects as well, suggesting that in future work it would be advisable to protect the magnetic material with an impermeable coating so that any tendency toward dissolution and the release of ions would be prevented.
(1) Sahai, 0.;Knuth, M. Commercializing Plant Tissue Culture Processes: Economics, Problems, and Prospects. Biotechnol. Prog. 1985, 1, 1. (2) Muir, W. H.; Hildebrandt, A. C.; Riker, J. J. Plant Tissue Cultures Produced from Isolated Plant Cells. Science 1954, 119, 877. (3) Brodelius, P.; Deus, B.; Mosbach, K.; Zenk, M. H. Immobilized Plant Cells for the Production and Transformation of Natural Compounds. FEBS Lett. 1979,103,93. (4) Wagner, I.; Vogelmann, H. In Plant Tissue Culture and its Biotechnological Application; Barz, W., Reinhard, E., Zenk, M. H., Eds.; Springer-Verlag: Berlin, 1977; p 245. (5) Lindsay, K.; Yeoman, M. M. The Relationship Between Growth Rate, Differentiation, and Alkaloid Accumulation in Cell Cultures. J. Exp. Bot. 1983, 34, 1055. (6) Hagimori, M. T.;Matsumura, T.; Obi, Y. Effects of Mineral Salts, Initial p H and Precursors on Digitalis. Formation by Shoot-Forming Cultures of Digitalis purpurea L. Grown in Liquid Media. Agric. Biol. Chem. 1983, 47, 565. (7) Curtin, M. E. Harvesting Profitable Products from PlantTissue Culture. BiolTechnology 1983,1, 649. (8) Kinnersley, H. M.; Dougall, D. K. Increase in Anthocyanin Yield from Wild Carrot Cell Cultures by a Selection System Based on Cell Aggregate Size. Planta 1980,149, 200. (9) Hershler, A. Fluid Treating Method and Apparatus. US. Patent 3,219,319, 1965. (10) Hershler, A. Method for the Production and Control of Fluidized Beds. U S . Patent 3,439,899, 1969. (11) Rosensweig,R. E. Fluidization: Hydrodynamic Stabilization with a Magnetic Field. Science 1979, 204, 57. (12) Rosensweig, R. E.; Siegell, J. H.; Lee, W. K.; Mikus, T. Magnetically Stabilized Fluidized Solids. AZChE Symp. Ser. 1981, 77, 8. (13) Siegell, J. H. Liquid-Fluidized Magnetically Stabilized Bed. Powder Technol. 1987,52, 139. (14) Burns, M. Continuous Affinity Chromatography Using a Magnetically Stabilized Fluidized Bed. Ph.D. Thesis, University of Pennsylvania, Philadelphia, PA, 1986. (15) Murashige, T.; Skoog, F. A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Culture. Physi o / . Plant. 1962, 15, 473. (16) Bramble, J. L. Production of Secondary Metabolites from Plant Cells Using a Magnetically Stabilized Fluidized Bed. Ph.D. Thesis, University of Pennsylvania, Philadelphia, PA, 1989. (17) Smart, N. J.; Fowler, M. W. Effect of Aeration on LargeScale Cultures of Plant Cells. Biotechnob Lett. 1981,3,171. (18) Cho, G. H.; Kim, D. I.; Pedersen, H.; Chin, C.-K. Ethephon. Biotechnol. Prog. 1988,4, 184. (19) Tanaka, H. M.; Matsumura, M.; Veliky, I. A. Diffusion Characteristics of Substrates in Ca-Alginate Gel Beads. Biotechnol. Bioeng. 1984,26, 53. (20) Stryer, L. Biochemistry, 2nd ed.; W. H. Freeman and Co.: San Francisco, CA, 1981.
Accepted September 28, 1990.
