Blotechnol. Pfw. 1003, 9, 317-322 317

Growth of Plant Root Cultures in Liquid- and Gas-Dispersed Reactor Environments

Scott A. McKelvey, Jason A. Gehrig, Kathryn A. Hollar, and Wayne R. Curtis* Department of Chemical Engineering/Biotechnology Institute, The Pennsylvania State University, University Park, Pennsylvania 16802-4400

The growth of Agrobacterium transformed “hairy root” cultures of Hyoscyamus muticus was examined in various liquid- and gas-dispersed bioreactor configurations. Reactor runs were replicated to provide statistical comparisons of nutrient availability on culture performance. Accumulated tissue mass in submerged air-sparged reactors was 31 9% of gyratory shake-flask controls. Experiments demonstrate that poor performance of sparged reactors is not due to bubble shear damage, carbon dioxide stripping, settling, or flotation of roots. Impaired oxygen transfer due to channeling and stagnation of the liquid phase are the apparent causes of poor growth. Roots grown on a medium-perfused inclined plane grew at 48 % of gyratory controls. This demonstrates the ability of cultures to partially compensate for poor liquid distribution through vascular transport of nutrients. A reactor configuration in which the medium is sprayed over the roots and permitted to drain down through the root tissue was able to provide growth rates which are statistically indistinguishable (95 9% T-test) from gyratory shake-flask controls. In this type of spray/trickle-bed configuration, i t is shown that distribution of the roots becomes a key factor in controlling the rate of growth. Implications of these results regarding design and scale-up of bioreactors to produce fine chemicals from root cultures are discussed.

Introduction The ability to grow plant roots in culture provides the

opportunity to utilize the catalytic capabilities of plants to produce biologically active chemicals in a controlled reactor environment. There are numerous reviews which describe the wide range of secondary metabolites and progress in producing these compounds in plant root culture (Flores et al., 1987; Wilson et al., 1987; Duran, 1989; Charlwood et al., 1990; Payne et al., 1992; Flores and Curtis, 1992). The advantages and rationale for root culture are thoroughly described in these reviews.

Chemicals of particular interest such as taxol (anti- cancer) and tricosanthin (anti-AIDS) are examples of potential products with a large market base. Due to the relatively low productivities of secondary metabolites and the large quantities of material needed for therapeutic pharmaceuticals, the target for production scale must be quite large. Root cultures present unique scale-up prob- lems due to their morphological structure which has not been encountered in suspended biocatalytic systems. The entwined mass of roots can occupy more than 50% of the physical volume of the reactor, which impedes mixing and mass transfer, even on a small scale.

The amount of published information on roots grown in culture is very small. To date, the largest numbers of successful reactor runs in a single publication are six (Taya et al., 1989) and ten (Hilton and Rhodes, 1990). Inter- pretation of this data is difficult since nearly all data represent nonreplicated runs in different reactor config- urations which were operated for different lengths of time before culture harvest. The lack of experimental data is not surprising due to the slow growth of cultures and difficulties in maintaining sterility in complex “prototype”

* Author to whom correspondence should be addressed.

reactor systems (to provide a reasonable number of doublings, reactors must operate under aseptic conditions for several weeks). The lack of replication is particularly troublesome because plant tissue culture is notorious for displaying variable growth, and the tedious procedure of preparing and inoculating roots takes several hours, which introduces stress in the tissue being handled.

The benchmark against which reactors are compared is the gyratory shake-flask. In addition to being the most common method of culture, the Erlenmeyer flask usually gives the best performance when compared to other reactor systems. Studies on a large number of reactor configu- rations and variations have been conducted. In studies of horseradish root cultures, Taya et al. (1989) concluded that growth in a bubble column with immobilized root cultures gave the best performance; however, this reactor was permitted to grow for 31 days in comparison to 25 days for the gyratory shake-flask control. Other reactor systems which have shown reasonably good performance are the bubble column (Shimomura et al., 1991; Toivonen et al., 19901, a rotating drum (Kondo et al., 1989), a mechanically agitated reactor with isolated impeller (Hilton and Rhodes, 1990), and a trickle-bed reactor in which the medium is recycled and sprayed over the top of a column of roots (Wilson et al., 1990; Flores and Curtis, 1992). The objective of most of these studies was to achieve successful cultivation in reactors-as opposed to examining strategies for reactor scale-up.

To examine problems associated with scale-up, it is necessary to study parameters which become limiting in large-scale culture. The difficulty in scale-up is to provide simultaneous availability of nutrients from both the liquid and gas phases. To reflect this, our experimental reactors were constructed to examine the effects of liquid and gas dispersion on the performance of cultures. Since nutrient limitation will ultimately limit scale-up, it is important to

8756-7938/93/3009-0317$04.00/0 0 1993 American Chemical Society and American Institute of Chemical Engineers

318 Blotechno. Rug.., 1993, Vol. 9, No. 3

utilize a rapidly growing culture so that nutrient con- sumption controls the rate of growth. Slow-growing cultures are limited by physiological factors which could obscure the effects of the physical parameters of the reactor environment. For this reason, root cultures of Hyoscyamus muticus were chosen because of their high rate of growth tissue mass increased 50-fold after 2 weeks of culture (Flores and Curtis, 1992).

In this article, we describe experiments which were designed to obtain quantitative information on the effects of reactor design on the growth of cultures; the objective is not the demonstration of novel reactor systems. The emphasis is on the acquisition of replicated data which is treated statistically to provide definitive results. The work encompasses 90+ reactor runs plus more than 40 gyratory shake-flask controls. The results indicate that hybrid operation with initially gas-dispersed, submerged culture followed by a liquid-dispersed or trickle-bed reactor configuration is a very promising technology for scale-up.

Materials and Methods

L

\u Media

Trickle

n - Incline

Culture Maintenance. Agrobacterium transformed root cultures of H. muticus (line A4C17), obtained from Hector Flores (Biotechnology Institute, The Pennsylvania State University) were used in all experiments. Cultures were maintained on B5 medium (Gamborg et al., 1968) by serial subculture every 2 weeks into 50 mL of medium in 125-mL flasks. Cultures were maintained on a gyratory shaker (2-in. stroke) a t 100 rpm and 25 OC.

Reactor Inoculation/Harvest. All reactor configu- rations and controls were designed to utilize 250 mL of medium. Experiments were inoculated with 0.5 g of fresh weight as described previously (Dunlop and Curtis, 1991). Prior to harvest, reactors were checked for contamination by streaking medium on LB plates. Fresh root mass was determined after 18 days of growth by blotting excess medium between paper towels. Dry mass was determined after lyophilization and desiccation.

Reactor Designs. The four basic reactor configurations used in this work are shown in Figure 1. Two of the reactors provide a submerged culture environment: the gas sparged vessels and gyratory shake-flask controls. The other two configurations, trickle and incline, provide a liquid- dispersed gas-phase culture environment. Subsequent work involved the modification of these reactors, as described below and in the results.

Gyratory Control. The “control” culture condition consisted of 1-L Erlenmeyer flasks inoculated with 0.5 g of blotted fresh tissue. Flasks were stoppered with polyurethane plugs and covered with foil. Usually four controls were employed with two inoculated before and after other reactors to eliminate systematic error intro- duced by the time-consuming inoculation procedure for other reactor configurations.

Sparged Reactor. Submerged, sparged culture was initially accomplished in a 6 X 6 X 9 cm polycarbonate tissue culture vessel (Magenta Corp, GA-7). Humidified sterile air was sparged at 39.5 mL/min (0.16 W M ) through a fritted glass sparger (Corning No. 39525-20). To promote better mixing and prevent root settling, sparging was also conducted in 300-mL conical flasks (Kontes) using various glass spargers, as summarized in Table I.

TrickleReactor. In the trickle configuration, medium drained from the bottom of the vessel to a reservoir and was recirculated at a rate of 0.3 mL/min to the top of the reactor via a peristaltic pump. In preliminary designs, humidified air was continuously introduced into the reactor

Sparged Gyratory Figure 1. Schematic diagrams of the liquid-dispersed trickle and incline reactors, the gas-dispersed sparged reactor, and gyratory shake-flask controls. For the liquid-dispersed reactors, medium was continuously recirculated from a reservoir below the drain indicated in the figure. These configurations served as a starting point for subsequent modifications described in the text.

head-space at a rate of 39.5 mL/min. Unless otherwise noted, trickle reactors were constructed from the GA7 polycarbonate box with the roots distributed on Whatman (3MM Chr) chromatography paper supported by a stain- less wire matrix about 2 cm from the bottom. Other designs employed a 12 in. long, 1 in. i.d. conical glass pipe (Corning No. 72710) with stainless steel head- and base-plates.

The degree of liquid dispersion varied depending upon the method of liquid distribution a t the top of the reactor. Initially, liquid was simply dripped through the top of the reactor through a l/ 16 in. i.d. stainless steel tube. To provide better dispersion in subsequent experiments, spraying of the liquid was accomplished by introducing the liquid at 1.14 mL/min into the reactor through a 22 gauge needle simultaneously with 80-100 mL/mm sterile humidified air. Improved liquid distribution in the spray reactor configuration eliminated the need for filter paper on the stainless steel support mesh, and mesh was centered in the vessel about 4 cm from the bottom. Combinations of reactor vessel and liquid dispersion are identified in the Results and Discussion sections.

Inclined Reactor. The inclined reactor configuration was designed specifically to provide nonuniform liquid distribution within the reactor and require the roots to transport nutrients to the growing regions. In this configuration, the GA7 polycarbonate box was placed on a 15’ slant, and roots were grown on filter paper on the lower side of the vessel. Medium recirculated from the reservoir a t 0.3 mL/min was sufficient to wet the paper, but did not cause significant flow over the surface. The reservoirs for this and the trickle configuration were constructed from 250-mL sidearm vacuum filtration flasks fitted with a silicone stopper pierced with stainless steel tubes. In all cases the reactors were operated for 18 days, which corresponds to late logarithmic phase for 1-L gyratory shake-flask culture.

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Table I. Total Drv Weight Accumulation of H. muticus Root Cultures in Various 250-mL Sparged Reactor Configurations'

L

B Q,

2 - \

m v

E m .- s ' - 5 c3

0 -

vessel

n-12

GA7 conical flask conical flask controlsb erlenmeyer (500 mL)b erlenmeyer (lo00 mL)b erlenmeyer (500 mL)b erlenmeyer (lo00 mLlb

sparger Corning No. 39525-20 Corning No. 39533-12 Ace Glass 9435C none Corning No. 39535-30M Corning No. 39535-30M none none

air flow (mL/min) final dry weight (9) 40 0.80 * 0.24 40 0.87 0.08 80 0.97 * 0.30

2.62 * 0.11 40 1.65 & 0.25 40 2.50 * 0.14

0.92 A 0.16 2.17 * 0.28

replicates 14 3 9

10 6 6 6 6

0 Despite modification of vessel, sparger configuration, and gas flow rates, the performance of static sparged reactors was very poor relative to gyratory shake-flask controls. Sparging into gyratory shake-flasks demonstrates that bubble shear and/or gas stripping is not responsible for poor growth. Flasks grown on gryatory shaker (2-in. stroke) at 80 rpm.

Statistics. Reactors were run in triplicate or quadru- plicate for all experiments. Experiments were repeated as many as three to six times. Gyratory shake-flask controls were run simultaneously with all experiments to provide an internal control for potential variability in the inocula. Data from different inocula were pooled only if there was no significant difference in the corresponding control flasks (95% T-test).

Results In the first series of experiments, the sparged, trickle,

and inclined reactor configurations were all constructed from rectangular polycarbonate vessels (see the Materials and Methods section) for comparison to gyratory shake- flask controls. Both the trickle and inclined reactors utilized recirculation rates of 0.3 mL/min. Three separate experiments were run, using four reactors of each type. Statistical analysis showed that all reactor configurations were significantly different from each other. The average dry root mass of the incline, trickle, and sparged reactors were 1.25 f 0.12,1.09 f 0.13, and 0.80 f 0.24g, respectively (Figure 2). The gyratory shake-flask control outperformed these reactors with an average dry mass of 2.62 f 0.11 g.

Not surprisingly, the inclined reactor configuration displayed a reduced rate of growth compared to the gyratory shake-flask as a result of the intentionally poor liquid distribution. However, this configuration performed significantly better than the sparged reactor configuration. This comparison suggests that the roots compensated for poor liquid distribution better than poor gas distribution (which results from gas-phase channeling in sparged culture environments). Since the tissue was only in contact with the medium on the lower side, these results dem- onstrate that transformed roots are capable of nutrient transport rates (over several centimeters) which can sustain relatively high rates of growth. This observation will become important in larger scale reactor systems where provision of uniform liquid distribution becomes increas- ingly difficult.

The trickle reactor performed even worse than the inclined reactor, resulting in only 42 % of the final tissue mass of the gyratory shake-flask controls. While it was expected that this reactor would give better medium contact than the inclined reactor, in fact, very little of the growing root tissue was wetted with the recirculating medium phase. As a result, the primary mode of medium utilization was transport from the filter paper support in the same manner as the inclined reactor. Since the trickle reactor had 30 % less contact area than the inclined reactor, the reduced rate of growth is consistent with a reduced availability of the liquid phase. Improvements in liquid distribution were addressed in subsequent experiments.

An alternative explanation of the poor growth in the inclined and trickle environments is shock associated with

h 3 1 n=10 -

Trickle Incline Sparged Gyratory

Reactor Configuration Figure 2. Accumulated dry tissue of H. muticus roots grown for 18 days from the fresh weight equivalent of about 26 mg of dry tissue inoculated in 250 mL of medium in each of the reactor configurations depicted in Figure 1. Total dry weights are presented to avoid the ambiguity introduced by volumetric calculations based on different reactor geometries. Error bars indicate one standard deviation of replicated reactor runs.

inoculation. Roots are damaged considerably during the cutting and weighing procedure used in the preparation for inoculation, and subsequent exposure to a predomi- nantly gas-phase environment might be more detrimental than a submerged culture environment. Immediately after inoculation, a slight browning of the tissue is observed in these reactors which is not apparent in the sparged or gyratory shake-flasks. Gas-phase inoculation effects were tested by inoculating fresh tissue into gyratory shake- flasks for 0,24, and 48 h prior to transferal to the inclined reactor configuration. (During the 48-h period, there were no visible signs of growth.) By providing a period of acclimation in submerged culture, the stress of gas-phase inoculation could be eliminated and the effects of growth environment could be decoupled from the possible effects of inoculation stress. Submerged incubation did visibly reduce browning of the inoculum; however, there was no improvement in the dry weight yield. The dry root mass was 0.97,0.27, and 0.69 g dry weight (DW), respectively, for incubation times of 0, 24, and 48 h. By comparison, gyratory shake-flask controls yielded an average of 2.44 f 0.07 g DW. Submerged incubation appeared to suppress growth slightly, possibly due to a delay in vascularization of the inoculum. These results strongly suggest that the difference in yield is due to the rate of growth and not due to stress imposed during the inoculation procedure. The reduced rates of growth of the liquid-dispersed reactors is therefore a result of the limited availability of nutrients within the gas-phase environment throughout the culturing period.

The poorest growth rate was observed in the submerged, sparged reactors. The accumulated dry tissue mass was less than 32% of the gyratory controls. This result was

320

unexpected since the tissue was submerged in the nutrient phase, and the rate of mass transfer within a sparged reactor should be much greater than the biological oxygen demand of the small amount of tissue within these reactors (McKelvey, 1992). Others have observed poor growth in air-spraged reactors relative to other configurations as well (Weathersetal., 1989;Tayaetal., 1989). Tayaetal. (1989) attributed poor growth in a sparged reactor to lifting of the root mass out of the medium, which was improved with anchoring the roots and longer cultivation time. Root flotation was not observed in our reactors. Instead, settling of the roots to the bottom of the reactors was more of a problem since the aspect ratio of a GA7 is 1.5. To eliminate settling and provide for better gas dispersion, experiments were conducted in conical boiling flasks, changing spargers and gas flow rate. The results of this work are summarized in Table I. All static-sparged reactor configurations accumulate less than 40% of the mass in the gyratory shake-flask controls.

These experiments strongly suggest that the growth of cultures in submerged, sparged reactors is not limited by the initial distribution of the gas phase. Instead, it appears that the limitation is imposed by the structure of the roots. As the root mass accumulated, the root tissue becomes an entangled stationary mat within the reactor. As the bubbles follow the path of least resistance to the surface, the channeling of air reduces the available interface for oxygen transfer. An additional problem is that transfer is localized in the regions with the lowest tissue density. Because the root tissue is very effective at inhibiting the convective flow of the medium, transport of oxygen into the dense regions of growth is severely impaired. Growth is reduced because the regions of highest oxygen demand correspond to regions with the greatest resistance to flow. On the basis of these results, it can be anticipated that the use of submerged, sparged reactors will be limited to low tissue culture or cultures that have an intrinsic rate of growth which is slow in comparison to convective oxygen transport.

One alternative explanation for the reduced growth rates in the sparged, trickle, and inclined reactors is that introduction of air into the vessel may “strip” volatile components which are essential for high growth rates. Ethylene is produced by nearly all plant tissue cultures under stress, and its presence might inhibit growth. Carbon dioxide is produced by the metabolism of sugar and is known to alleviate the effects of ethylene (Burg and Burg, 1967). In gyratory shake-flask culture, C02 levels within the flask can reach 5 1 0 % . Reduction of C02 by sparging might therefore be detrimental to cultured roots by reducing the COZ levels within the reactor vessel. The effect of COZ stripping was tested by growing roots in triplicate in 500-mL flasks sparged with air and 10% C02 enriched air. The smaller (500 mL) flasks were used (with 250 mL of medium) to facilitate more uniform gas dispersion by providing a greater depth for sparging (Corning No. 39535-30M). Average dry weights were 0.88 f 0.20,0.54 f 0.07, and 0.37 f 0.06 g for air-sparged, 10% C02, and unsparged control, respectively. For H. muticus root cultures, 10% carbon dioxide enrichment did not enhance growth as compared to air sparging.

All flasks in the carbon dioxide enrichment study grew significantly less than control cultures grown in 1-L gyratory shake-flasks. The reduced rate of growth results from reduced convection in the 500-mL flasks. This was confirmed in an experiment in which 1000- and 500-mL flasks were grown in triplicate with and without air sparging. This experiment was repeated twice to provide

Biotechnol. hog., 1993, Vol. 9, No. 3

n-3 t: 0 3 1 n-4

I Spray Control I Tube Control ‘ Reactor Configuration

Figure 3. Comparison of total accumulated dry mass of H. muticus after 18 days of growth in liquid-dispersed reactor configurations and gyratory shake-flask controls. The only difference between the tube and spray reactor configurations was the aspect ratio and surface area available for inoculation. Controls for individual studies represent gyratory shake-flasks inoculated from the same tissue as the corresponding reactor. Controls were not pooled because they are statistically different (95 % T-test, see the Methods and Materials section). The spray reactor configuration resulted in growth rates which are statis- tically indistinguishable from corresponding gyratory shake-flask controls (95% T-test). The tube configuration performed very poorly due to the limited distribution of the inoculation.

six replicates at each condition, all statistically different at the 95% confidence level. Details of the experiment are given in Table I. The average dry weights were 2.50 and 2.17 g for the 1000-mL sparged and unsparged and 1.65 and 0.92 g for the 500-mL sparged and unsparged flasks, respectively. Sparging of air is clearly beneficial for growth, which demonstrates that bubble-induced damage is not responsible for the reduced growth rate observed in the sparged reactor configurations. Some alteration of morphology, including loss of root hairs, was noted in the sparged gyratory shake-flask which was not observed in other sparged reactors. It should be kept in mind that bubble damage may become a consideration for larger scale reactors where the localized intensity of sparging increases.

The dramatic decrease in growth for 500-mL flasks in comparison to 1000-mL flasks demonstrates the impor- tance of convection in providing growth within reactors. This reinforces the experimental observations in the air- sparged reactors, which also grew poorly as a result of poor mixing within the liquid phase. In designing a large- scale reactor, it is critical to provide a relative velocity between the roots and the medium phase to overcome convective transport limitations. Since the root tissue will be stationary at high densities, the only means of providing this relative velocity is to recirculate the liquid phase. At the same time, there must be sufficient contact with the gas phase to provide replenishment of oxygen as it is depleted from the medium. Both of these criteria should be accomplished in a trickle-bed reactor if the liquid-phase and root tissue are adequately distributed within the reactor.

The final series of experiments was designed to improve the liquid distribution in the trickle-bed reactor. In a large-scale reactor this would be accomplished with a spray nozzle; however, this is not possible at the low flow rates and pressure drops utilized in small-scale reactors. The spray reactor configuration was accomplished by intro- ducing both the air and recirculated medium through a needle in the top of the polycarbonate GA7. In Figure 3 it can be seen that the spray reactor provided growth rates

Bbtechnol. hog., 1993, Vol. 9, No. 3

that were statistically indistinguishable from gyratory shake-flask controls (95% T-test). The average dry weights were 2.13 f 0.12 and 1.92 f 0.12 g, respectively, for the spray and the control. To facilitate statistical analysis, these data were obtained from four identical reactors inoculated at the same time from the same inoculum. This was the last of six experiments for the spray configuration. Most of these experiments yielded one or two reactors and/or controls; the remaining rep- licates fell victim to plugged needles, tubing failure, or contamination. When the composite data-consisting of 12 spray configurations and 14 controls-were pooled (ignoring the statistical pooling criterion set forth in the Methods and Materials section), the same result as shown in Figure 2 was observed there is no statistically significant difference between gyratory shake-flasks and the spray reactor configuration, demonstrating that sufficient nu- trient distribution was achieved.

To provide a qualitative assessment of the influence of root distribution on growth rate, the spray apparatus was also fitted to a 1 in. i.d. glass tube. The area for distribution of inoculum in the tube was only 5.1 cm2 in comparison to 36.0 cm2 for the spray configuration carried out in the reactors constructed from the GA7. The results of root growth in this tube reactor (in triplicate) are 0.11 f 0.02 and 2.31 f 0.13 g, respectively, for the tube and control as shown in Figure 3. In this case, the tube reactor configuration accumulated only 5 % of the mass achieved in shake-flask controls. This result is a graphic demon- stration of the tremendous importance of providing initial distribution of roots within the reactor, as noted by many previous researchers who have worked with root cultures in reactors.

321

Discussion The trickle-bed reactor configuration is shown to have

promising characteristics for scale-up, provided the liquid and root tissue are adequately distributed within the reactor. In this study, inoculum distribution was con- strained by the surface area in the bottom of the experimental trickle-bed reactor configuration. In larger scale reactors, distribution must be accomplished through- out the entire volume of the reactor. We have demon- strated in a 2-L prototype reactor that root distribution can be accomplished by initially inoculating the reactor in the submerged phase (Flores and Curtis, 1992). After the roots have attached to a support matrix within the reactor, the reactor can then be drained and operated in the trickle-bed mode with a reasonably uniform distri- bution of roots within the reactor volume. Inoculation in the submerged phase does not cause difficulties because convective problems in submerged reactors do not develop until the root tissues have grown sufficiently to impair medium convection.

I t must be noted that the reactors used in this work are “differential” reactors in that the depth of the reactor is not sufficient to change the nutrient composition or the fluid flow characteristics. As liquid flows down through a large-scale system, the liquid will tend to channel in the same manner that gas channels in a submerged sparged system. As a result, some of the tissue will not be directly exposed to medium. Reduced medium distribution can be offset somewhat by the root’s ability to transport nutrients (as demonstrated in this work with the inclined reactor). The ability to scale up the trickle-bed reactor will therefore be determined by the interaction of medium distribution within the reactor and nutrient transport within the root. To obtain design information for com-

mercial-scale systems, it will be necessary to perform fluid- dynamic and nutrient utilization studies on large scale systems.

Conclusions

(1) H. muticus roots grown in submerged sparged reactors displayed reduced rates of growth compared to roots grown in gyratory shake-flask controls. The reduc- tion in growth rate is very significant (60-75 % ), indicating that bubble column reactors may severely limit growth as a result of gas channeling and impaired liquid mixing. It is shown that neither COZ “stripping” nor bubble shear contributes to this reduced growth rate.

(2) Roots grown on a perfused inclined plane accumu- lated one-half the mass of tissue grown in submerged gyratory shake-flasks. This configuration did, however, perform significantly better than submerged sparged reactors. This indicates that cultured roots are capable of supporting relatively high rates of growth by transport of nutrients with the tissue. The ability to transport nutrients will offset problems of liquid-phase channeling, which occur in large-scale liquid-dispersed reactor systems such as a trickle-bed.

(3) Growth rates comparable to those from the gyratory shake-flask can be obtained in liquid-dispersed reactors provided there is adequate distribution of both the medium and roots within the reactor. The results of the inclined, trickle, and spray configurations used in this study demonstrate that the dimensions of adequate distribution are on the order of several centimeters. Studies to quantify adequate in terms of reactor design principles still need to be conducted.

Acknowledgment

Assistance with the sparged gyratory shake-flasks was provided by William Reed. Michelle Bush assisted during the “debugging” stage of the tube reactors. The work of Michelle Bush and Kathryn Hollar was supported, in part, by the NSF Research Experience for Undergraduate Program (Grant No. EID-9101249). We also thank G. Ramakrishna Reddy for keeping the laboratory running, which allowed Dr. Curtis time to work with all of the undergraduates involved in this project, and Lorrie Curtis who tolerated the hundreds of late nights required to keep the reactors running. Finally, we acknowledge financial support from the National Science Foundation (Grant No. BCS-9110288), the National Institutes of Health (BRSG Program), the Pennsylvania Research Corporation, and the Du Pont Young Faculty Initiation funds.

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Accepted January 21, 1993.