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Cultivation of roots in bioreactors
Cultivation of roots in bioreactors Wayne R. Curtis The Pennsylvania State University, U n i v e r s i t y Park, USA Over the past few years, the number of reports of roots cultured in reactors has increased dramatically. This has provided much-needed information on the physical and metabolic requirements of cultured roots, and has established techniques for the manipulation of root tissue. Most of this work, however, is conducted on a relatively small scale (less than 5 I), and many are demonstrations as opposed to systematic studies. The real challenge of scale-up will be encountered when moving to the 100-1000 I scale, while maintaining dry tissue densities in excess of 10-20 (g dry weight) I-1. Achievement of this goal will require small-scale studies, focused on obtaining design and operational information, coupled with measurements of fundamental scale-up parameters in large-scale systems. Current Opinion in Biotechnology 1993, 4:205-210 Introduction Plant roots have received considerable attention as potential production systems for plant-derived chemicals because they exhibit stability, and secondary metabolite productivities comparable to, or greater than, the native root [1"]. The ability to exploit plant root cultures as a source of biologically active chemicals will depend o n the development of suitable reactor systems. The unique structure of roots as biocatalysts presents a tremendous challenge for aseptic reaction engineering. At about 200 g fresh weight (FW) per liter, or 12 g dry weight (DW) per liter, roots form an interlocked mass which becomes stationary within the vessel. In gyratory shake flasks, root tissue can reach densities in excess of 350 g 1-1 FW (20 g 1-1 DW). The root matrix stagnates liquid flow and efficiently coalesces bubbles, which can result in severe mass-transfer limitations, despite the relatively slow rate of growth of roots (doubling times of about two days). The products of interest from root tissues, such as pharmaceuticals, pesticides, flavorings, and dyes, are specialty chemicals which will require a scale of production of 10 000 liters or more. The engineering objective is to provide sufficient oxygen and nutrients to a root ball weighing several tons. While this is routinely achieved by trees, roots within a reactor are far tess organized vascularly and are present at a much higher tissue density. Additionally, the p o w e r input to achieve the necessary rates of mass transfer must be sufficiently gentle to minimize mechanical damage to the roots and root hairs. Although this is a formidable task, the potential gains are tremendous. Chemicals from plants are presently some of our best candidates for the treatment of cancer and AIDS. If we are to continue tapping the
biochemical diversity of plants in the future, w e must develop ways of culturing these tissues in reactors so that w e do not destroy the environments where the native plants are found. In developing such reactor systems, the location of the product, either intracellular or extracellular, will have a tremendous impact o n the reactor design. In the case of intracellular products, the root tissue is a product, and the reactor design must facilitate harvest. If the c o m p o u n d is secreted into the medium, the root is behaving as a catalyst, and recovery of the root tissue is not necessary. For extracellular products, longevity of biocatalytic activity is the primary concern. Because of the impact of metabolite retention on reactor design, a brief review of advances in intracellular metabolite release is presented in the context of reactor design for product recovery.
Current research Roots have been cultivated in a wide range of reactor configurations that vary from completely novel designs to simple modifications of existing reactors. Several designs that have been used for the cultivation of roots are s h o w n schematically in Figure 1. A summary of reactor studies conducted over the past year is presented in Table 1. The results achieved in mechanically agitated reactors are surprisingly good [2-']. This supports the previous findings of Jung and Tepfer [3] w h o have cultured roots in volumes up to 20 liters in mechanically agitated reactors. Final tissue densities will obviously be limited in these systems as a result of mechanical tissue damage [4,5]; however, this alternative should not be disregarded, particularly ff the product is intracellular.
Abbreviations DW-~clry weight; FW fresh weight. © Currrent Biology Ltd ISSN 0958-1669
205
206
Biochemical engineering
May contain draft-tube or immobilization mesh
(a)
(c)
d~
o
o
0
o
0 o
Air
Air
Screen to prevent root-impeller contact
Reversible
Medium
pump
t'ump
reservoir Air
Fig I. Reactor configurations used for the culture of roots: (a) bubble column, (b) isolated impeller, (c) stirred tank, (d) ebb-and-flow, (e) trickle-bed.
While bubble columns can be used successfully to culture roots [2"',6"',7-], their performance is rather poor compared to other systems [2"',6".]. This recent work supports earlier studies showing that bubble columns perform rather poorly in comparison to other reactor systems [4,8]. The most likely causes are either insufficient oxygen because of channeling, or tissue damage caused by sparging. In the case of H y o s c y a m u s m u t i cus root cultures, sparging into gyratory shake flasks did not reduce growth [6"], which supports gas-phase channeling as the primary cause of poor growth. In addition to several previous reports [4,9], studies on the successful use of an isolated impeller reactor are reported for the culture of beet hairy roots [10-.]. Isolated impeller systems are considered separately from simple mechanically agitated systems because the roots b e c o m e immobilized and convective flow is produced externally to the root mass, which results in much less tissue damage. For the culture times used in this work, the final tissue density achieved in this system is similar to that of the stirred tank (about 8-10 g 1-1 DW), particularly if calculations are based on the total medium volume and not just the growth section of the vessel. The ability of impellers to provide sufficiently uniform convection in larger scale systems will ultimately determine the feasibility of this approach. A recent report of an ebb-and-flow reactor appears to be the first reactor in which flow patterns are domi-
nated by external pumping (analogous to a plug-flow reactor) [11"]. At the end of culture, the average liquid velocity can be estimated at 0.53 cm s- 1, which is close to the experimental value of 1 cm s-1 which was required to overcome mass-transfer resistances for roots enclosed in a spherical tea ball [12"]. This reactor supported one of the highest tissue productivities to date, demonstrating the need to overcome convective masstransfer limitations within the root mass. These results are contrary to previous work with an ebb-and-flow reactor [4]; however, in these earlier studies the fill and drain cycle was very slow (average liquid velocity at end of culture of about 0.052 cm s-1), which would probably not be sufficient to induce internal flows in stagnant growth centers within the reactor.
The feasibility of sustaining high rates of growth in reactors in which the medium is sprayed over the tissue has been demonstrated with both nutrient mists [13",14"] and coarse spray [6"']. After initial growth as a bubble column for inoculum distribution, this technique has been used to overcome problems of bubble coalescence in a 2-liter [15"] and 14-liter reactor [16"]. These reactor studies dearly demonstrate that roots can be grown in a variety of reactor configurations. The question that remains unanswered is which of these alternatives can be scaled-up economically?
Cultivation of roots in bioreactors Curtis 207
Table 1. Current research on growth of root cultures in reactors.
Root culture
Culture time (days)
Inoculum (g I- 1DW)
Final density (g I- 1DW)
Emphasis
Reference
1
Tagetes patula
28
-
9.3
Integrated recovery
[2"]
5 0.25 2
Tagetes patula Hyoscyamus rnuticus Lithospermum erythrorhizon
28 18 220
0.11 -
6.8 3.2 -
Integrated recovery Growth study Integrated recovery
[2"] [6"] [7"]
Isolated impeller
1
Beta vulgaris
13
1
12
Product release
[9]
Ebb-and-flow
2
Hyoscyamus muticus
18
0.22
11.7
Growth study
[11 ]
Nutrient mist
1.4 0.28
Beta vulgaris Carthamus tinctorius Hyoscyamusmuticus
7 7 18
0.43 a 0.52 a 0.10 a
1.3 a 1.5 a 6.9 a
Growth study Growth study Growth study
[13",14"] [13",14"] [6-]
2 15
Hyoscyamus muticus Hyoscyamus muticus
30 28
0.22
11.9
Fungal el icitation Residence time distribution
[15"] [16"]
Reactor
Working volume (litres)
Stirred tank Bubble column
1.4
Bubble column and trickle-bed sequence
aReactor density calculated based on 75% volume of culture space. DW, dry weight.
Reactor operation
To address the issue of scale-up, the remainder of this review is organized around the operational phases of inoculation, growth, and product recovery, followed by a brief discussion of problems associated with variability and reproducibility. As a result of the tedious and time-consuming nature of tissue-culture reactor studies, many of the observations described b e l o w were either not replicated, or were observed only for a single species of plant root. Inoculation Much of the heterogeneity in experimental inoculation techniques arises from the conflict between the search for practical or theoretical information. Root tips are often used as the inoculum because they are the center of active growth. While this procedure provides a rapid start for small-scale culture, it is impractical for larger scale reactors, as a result of both the labor involved in dissecting tips and the fact that root meristems represent such a small fraction of the inoculum. Dissected bulk tissue is far more practical, and has b e e n used successfully in m a n y studies [6",13"-15"]. In work with beet root tissue, Dilorio et al. [13"] observed that cultures initiated from root tips grew considerably faster than bulk tissue in shake culture, but there was no difference in performance of tips and bulk tissue inoculated in nutrient mist reactors. In m a n y studies, the source of tissue is not specified. In future w o r k it is important that the source and method of preparation of the inoculum should b e specified to provide insight into inoculum effects.
Manual dissection, e v e n of bulk tissue, is not a practical way of preparing root cultures for inoculation in largescale reactors. We recently demonstrated that several root species (H. muticus, beet, and a proprietary line) can b e briefly h o m o g e n i z e d to facilitate inoculation without adversely affecting growth rates compared to bulk tissue dissection (K Hollar and WR Curtis, unpublished data). We have used this technique to inoculate a 15-liter reactor system [16-]. The breadth of applicability of this technique should be determined because it can be easily adapted to aseptic inoculation of large-scale systems by introducing the root fragments as a slurry [16",17"]. Poor distribution of inoculum within the reactor is another problem that is often avoided in small-scale reactor studies by tediously placing the root tissue in the reactor b y h a n d [4,6-',10"',13",14"]. While uniform distribution enhances reactor performance by minimizing the onset of localized mass-transfer limitations, it does not give a true evaluation of reactor performance. It does, however, permit a quantitative assessment of the m a x i m u m performance of the environmental conditions of a given reactor configuration. Practical distribution can only be achieved on a large scale b y suspending the inoculum in a liquid phase. Additional observations of the detrimental effects of p o o r root distribution have b e e n reported [2"',4,6",17"], but no quantitative analytical studies on the effects of uniformity within reactors have b e e n carried out. In submerged (gas-dispersed) reactor systems, essentially no support is n e e d e d for the roots because of the b u o y a n c y of the surrounding medium. In contrast, liquid-dispersed reactors, such as a trickle-bed,
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Biochemical engineering require physical support, not only for the root tissue, but also for the weight of the entrained liquid which can b e equal to the mass of the roots [15"]. The distance over which support is provided does not vary much, as reported b y different workers: 2.5 cm Pall ring distillation packing [15"], horizontal mesh spaced at 2.5 cm [11",13"], and 13.75 cm Intalox saddle process packing [16"]. Although requirements for support will vary with liquid loading and root type, the influence of support dimensions on root distribution will probably play a more important role. The uniformity of inoculum distribution will b e determined b y the distribution of pinch points (where packing contacts or branches) and flow patterns within the reactor. Growth and maximum tissue density As a result of the severe mass-transfer limitations imposed by dense root structures, the rate of root growth within a reactor must always be evaluated in the context of the final tissue density. The objective of reactor design is not only to support high growth rates, but to sustain those growth rates at high density b y minimizing problems associated with mass-transfer limitations. Sorting out physical versus physiological limitations in growth is complicated b y difficulties in obtaining growth data. Correlation of growth with electrical conductivity is still a c o m m o n w a y of monitoring growth [2"',4,9]. However, this technique is not directly related to mass within the reactor, and can be in error if stress associated with the reactor environment changes the rates of inorganic ion uptake. For example, in the study b y Buitelaar et al. [2..], the average difference b e t w e e n actual final dry weights and those calculated with conductivity correlations indicated that errors greater than 10% were introduced for most reactor runs. While conductivity correlations are an invaluable tool in the control of production systems, they should be used with caution in evaluating reactor performance (particularly if correlations are developed from shake-flask data). Sugar usage is probably a m o r e robust measure of culture performance because it is a direct reflection of metabolism [18]. Measurement of respiration from CO 2 and 02 levels in the gas exiting the reactor is another metabolism-based m e t h o d of monitoring reactor performance; however, to date, this has not b e e n implemented for root cultures. The extent to which carbon use efficiency changes in root cultures under different environmental conditions has not b e e n adequately assessed for its impact on the accuracy of metabolic measurements of growth. Drained liquid weight provides a more direct experimental measurement of mass within the reactor [5], although roots entrain large amounts of m e d i u m which introduce considerable systematic error. Drained weights are likely to be 50-100% higher than blotted fresh weights [15"]. The m e a n residence time of an inert tracer has recently b e e n tested as a measure of tissue mass within the reactor [16"]. This technique m a y provide a more direct, non-invasive physical m e a s u r e m e n t of root mass for large-scale reactor systems. Difficulties in obtaining direct measurements of growth time courses greatly impair the ability to evaluate the
effects of reactor environments on growth. It is clear that with few exceptions, root cultures do not display sustained logarithmic growth. As a result, doubling times and specific growth rates do not have m u c h significance--and can be very misleading. The onset of internal mass transfer limitations within individual growth centers depends on the density of the inoculum and size of the root fragments. Systematic studies on the effect of inoculum density and inoculure fragmentation are needed. As inoculum densities vary b y almost an order of magnitude (Table 1) and inoculum preparation by different researchers varies considerably, comparisons of absolute rates of growth from different studies are not very meaningful. Difficulties in interpreting the effects of physical parameters on the growth of root cultures are c o m p o u n d e d b y the lack of information on the physiological requirements for growth. Information is just beginning to a p p e a r on the effects of carbon sources, inorganic substances, and gas composition. Given the tremendous adaptations exhibited by whole plants, large variations in physiological requirements can be expected. For example, the influence of carbon dioxide in largescale reactor systems is very complicated. The potential effects are beneficial (stress abatement, carbon assimilation), detrimental (reduced oxygen availability, feedb a c k repression of metabolism) and u n k n o w n (pH modulation). In nutrient-mist reactors, 1% CO 2 enhanced the tissue accumulation of beet root and safflower [14"], while sparging of 10% CO2 in shake flasks suppressed the growth of H. m u t i c u s roots [6"]. These differences in response could b e a result of the reactor environment, the plant species, or an interaction of the physiological response within a particular environment. The lack of understanding of root growth is as m u c h a reflection of the complexity of plant physiology as it is of plant root culture. Our general understanding of plant physiology lags substantially behind that of mammalian and microbial physiology, and the situation for root physiology is e v e n worse than that of plants as a whole. Fortunately, understanding is not a prerequisite for applications, and the simplistic approaches to culture used so far have b e e n very successful. Root cultures have p r o v e d to b e sufficiently amenable to culture, so that achieving industrial-scale culture should b e possible through the application of sound engineering principles. Product recovery The nature of product formation, either intracellular or secreted, will profoundly affect reactor design for a particular compound. The desire to facilitate the removal of roots has b e e n the stimulus for n u m e r o u s novel reactor designs. Keeping in mind that the ultimate scale for most secondary products will b e tens of thousands of liters, most designs are impractical. In the case of intracellular products, simple designs such as the mechanically agitated reactor or bubble column, will probably be applicable despite the slower rates of growth and/or lower volumetric productivities. For extracetlular metabolites, the ability to re-use tissue favors much higher density immobilized culture con-
Cultivation of roots in bioreactors Curtis ditions. An advantage of root structure is the ease of implementating an integrated product recovery, particularly for immobilized-culture systems that recycle media. Solid adsorbents and liquid paraffin have been used as metabolite sinks to enhance shikonin recovery from Lithospermum erythrorhizon grown in an airlift reactor [7"]. The recovery of thiophenes from Tagetes patula was enhanced in stirred tanks with biocompatible secondary phases: fluorocarbon (FC40) and hexadecane [2"']. Volatile extractants such as hexane killed the root cultures if added in situ. By contacting media with hexane in an external loop extractor, it has been shown that toxicity resulting from phase contact can be eliminated, and sesquiterpene recovery can be facilitated from root cultures into volatile solvents .to permit economical product isolation [19]. In all cases where integrated recovery has been used, productivity was enhanced, presumably as a result of a reduction in feedback repression of metabolite synthesis. Considerable progress has also been made in facilitating the release of intracellular products from root tissues. Release of intracellular nicotine from root tissue was enhanced by manipulating extracellular pH [20"]. Similarly, oxygen deprivation [9] and heat shock combined with calcium modulation [8] caused the release of pigments from beet root cultures. In these cases of stress-induced product release, it is not clear h o w much tissue death contributes to 'release'. The ability of cultures to continue growing simply indicates that the meristems are not damaged [9]. In terms of production, however, the mechanism by which release is facilitated is inconsequential, provided the reactor regenerates catalytic activity through cyclic growth and production. Such tissue turnover may be advantageous in permitting an essentially indefinite reactor operation as the age distribution within the reactor reaches a constant level. Combining release with secondary-phase trapping is a promising route to the recovery of intracellular products from root culture. In light of this progress, it is likely that high-density immobilized culture systems, such as the trickle-bed reactor, will be applicable to both intracellular and extracellular metabolites.
Problems: reproducibility and variability One of the implicit difficulties in interpreting the results of root growth in bioreactors is reproducibility. In most cases there are no replications, and in cases where runs are repeated, the reproducibility is poor, even by tissue-culture standards. Using stirred tank reactors, Buitelaar et al. [2.'] observed variabilities of 24-32% for stirred tank reactors, and 40-59% for bubble columns. McKelvey [21] observed a variability as low as 4% for shake flask culture, but it rose to as much as 24% for bubble column and trickle-bed configurations. In their work with a nutrient mist bioreactor, DiIorio et al. [13"] found that deviations of replicated final tissue weights were kept within 10%. This enhanced reproducibility could be attributed to their use of preconditioned media. As a result of the observed variability of growth in reactor systems, single-point comparisons of reactor
performance must be interpreted with extreme care. As this research field matures, more replications will have to be performed at the expense of breadth of experimentation.
Conclusion At low tissue density, roots can be cultivated in essentially any bioreactor configuration, particularly on a small scale. In cases where the intended product cannot be released to the medium, bubble columns or mechanically agitated reactors are the most promising alternative. For metabolites that are released to the medium, immobilized systems such as packed-bed or trickle-bed reactors should provide higher scale tissue loadings. Studies focusing on scale-up (with adequate replication) are needed, including the development of improved inoculation and tissue-harvest techniques.
Acknowledgements Part of this work was supported by the National Science Foundation (NSF Grant no. BCS-9110288), a n d the Pennsylvania State University College of Agriculture Inter-College Research Grant, Project Number 3211 (Root reactor design for scaled-up production of pharmaceutical proteins).
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1. •.
PAYNEG, BRINGI V, PRINCE C, SHULER M: P l a n t c e l l a n d T i s s u e c u l t u r e i n Liquid S y s t e m s . New York: Hanser; 1992:227-279. Extensive review of physiology, biochemistry, and reactor engineerhag for root cultures. BUrrELAARRM, LANGENHOFFRAM, TRAMPERJ: G r o w t h a n d Thiophene Production by Hairy Root Cultures of Tagetes p a t u l a in V a r i o u s T w o - L i q u i d - P h a s e Bioreactors. Enzyme Mtcrob Tecbnol 1991, 13:487-494, In sttu extraction for thlophene recovery has been developed. Solvents with logP greater than five were found to be biocompatible. Recovery was implemented in a variety of reactor configurations, including bubble colunm, stirred tank, a n d liquid-impelled loop. Blockage of flow w a s experienced with the liquid-impelled loop. Stirred-tank cultures grew better t h a n bubble column cultures. The greatest recovery of thiophenes was observed in bubble c o l u m n with hexadecane as an extractant. 2. ..
3.
JUNG G, TEPFER D: U s e o f G e n e t i c T r a n s f o r m a t i o n b y t h e Ri T-DNA o f Agrobacterium rhizogenes to Stimulate B i o m a s s a n d T r o p a n e A l k a l o i d P r o d u c t i o n i n Atropa belladonna and Calystegia sepium R o o t s G r o w n in vitro. Plant Sci 1987, 50:145-151.
4.
TAYA M, YOYAMA A, KONDO O, KOBAYASHI T, MATSUI C: G r o w t h C h a r a c t e r i s t i c s o f P l a n t H a i r y R o o t s a n d T h e i r C u l t u r e s i n B i o r e a c t o r s . J Chem Eng Japan 1989, 22:84-89.
5.
WILSONPDG, HILTON MG, MEEHAN PTH, WASPE CR, RHODES M: T h e c u l t i v a t i o n o f T r a n s f o r m e d Roots f r o m Labor a t o r y to Pilot Plant. In Progress in Plant Cellular and Molecular Biology. Edited by Nijkamp HJJ, v a n Der Plas LHW, Van Aartrijk J. Dordrecht, Netherlands: Kluwer Academic Publishers; 1990:700-705.
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MCKELVEYSA, GEHRIGJ, HOLLAR KA, CURTIS WR: G r o w t h o f P l a n t Root Cultures i n Gas- a n d L i q u i d - D i s p e r s e d Reactor E n v i r o n t n e n t s . BiotechnoI Prog 1993, 9:in press. A series of small-scale reactor configurations were developed to provide quantitative information on the effects of liquid and gas dispersion within root bioreactors. Simple sparged reactors s h o w e d the poorest growth rate for H. muticus, even poorer than gas-phase reactors in which the medium was intentionally maldistributed. Reactors in which the medium was sprayed over the roots displayed growth rates comparable to gyrator*/shake-flask controls. The results suggest that roots are more capable of compensating for p o o r liquid dispersion than for p o o r gas dispersion within reactor systems. 7.
SHIMOMURAK, SUDO H, SAGA H, KAMADA H: S h i k o n i n
Production and Secretion b y Hairy Root cultures o f L i t h o s p e r m u m erythrorhixon. Plant Cell Rep 1991, 10:282-285. shikonin was recovered in an external loop for 220 days using XAD2 resin. An ammonium-free medium was required for shikonin production.
8.
of the Biotecb USA Conference, Oct 2-4, San Francisco. San KONDOO, HONDA H, TAYA M, KOBAYASHIT: C o m p a r i s o n o f G r o w t h P r o p e r t i e s o f Carrot Hairy Root i n V a r i o u s Bioreactors. Appl Microbiol Biotechnol 1989, 32:291-294.
10. •.
KINO-OKAM, HONGO Y, TAYA M, TONE S: C u l t u r e o f Red Beet H a i r y R o o t i n Bioreactor and Recovery o f Pigm e n t Released f r o m the Cells b y Repeated O x y g e n Starvation. J Chem Eng Jpn 1992, 25:490--495. Oxygen starvation for u p to 16 hours facilitated release of the pigment betanin. Repeated anoxia combined with absorption of pigments on styrene-divinybenzene copolymer resin permitted pigment recovery for 20 days. The release appeared to occur preferentially from mature tissue, permitting continued growth of meristems. 11.
Society for Agricultural Engineering; 1991. A 2-liter ebb-and-flow bioreactor was used with 1 min fill and drain cycles and 2 rain liquid and gas dwelling times. After 18 days of growth, an inoculum of 8 g FW yielded 459 g FW (23.4 g DW) which represents a higher growth rate than gyratory shake-flask controls. PRINCECL, BRINGI V, SHULERME: CoIlvorztive Mass Transfer i n Large Porous Biocatalysts: Plant O r g a n Culture. Biotechnol Prog 1991, 7:195-199.
Onion root cultures were placed inside a spherical tea ball within a defined flow field to determine the external flow rates required to induce sufficient internal flows to sustain maximal oxygen uptake. The evaluation was conducted at 320 g 1-1 FW. 13.
DIIomo AA, CHEETHAMRD, WEATHERSPJ: G r o w t h o f Tratmformed Roots i n a N u t r i e n t Mist Bioreactor: Reactor Performance and Evaluation. Appl Mtcrobiol Biotechnol
1992, 37:457-462. The authors describe the adaptation of a nutrient mist bioreactor (which was an ultrasonic transducer to produce fine mists) for application with root cultures. Culture perfusion resulted in slower
DIIORIOAA, CHEETHAMRD, WEATHERSeJ: Carbon Dioxide Improves the Growth o f Hairy Roots Culture o n Solid Mediuna and in Nutrient Mists. Appl Microbiol Biotechnol
1992, 37:463-467. Enrichment of nutrient-mist carrier gas with 1% CO 2 resulted in a 15% enhancement in the growth rate of beet (Beta vulgaris) and safflower (Carthamus tinctorius). The effects of carbon dioxide enrichment were m u c h greater in solid culture than in a bioreactor. FLORESHE, CURTIS WR: Approache~ to Understanding and Manipulating the Biosynthetic Potential o f Plant Roots. Proc N Y Acad Sci 1992, 655:188-209. H. muticus roots were grown in a 2-liter trickle-bed and a 2-liter sparged control reactor for four weeks and induced with a ffmgal elicitor. The trickle-bed reactor produces three times more solavetivone. 15.
16.
RAMAKmSHNAND, CURTIS WR: Application of Residence
Time Distribution Studies to Plant Root Culture Bioreactors. American Institute of Chemical Engineers Annual Meeting, Nov. 1-6, Miami, Florida, 1992, paper no. 164g. New York: American Institute of Chemical Engineering; 1992. A residence-time distribution technique was developed for root cultures, using blue-dextran dye as a tracer. The technique was used to correlate cell density in submerged reactor systems, and to determine liquid hold-up in trickle-bed root reactors. 17.
RODRIGUES-MENDIOLAMA, STAFFORD A, CRESSWELLR, ARIASCASTRO C: Bioreactors for Growth o f Plant Roots. Enzyme Microb Technol 1991, 13:697-702. An airlift was operated with a draft tube for TrtgoneUa foenumgraceum and a m e s h tube for Nicotiana rustica. A more uniform distribution was achieved using the mesh. 18.
TOWONEN L, OJALA M, KAUPPINEN V: I n d o l e ~ l k a l o i d
Production b y Hairy Root Cultures o f C a t h a r a n t h u s roseus: G r o w t h Kinetics and Fermentation. Biotechnol Lett 1990, 12:519-524.
CUELLOJL, WALKER PN, CURTIS WR: Ebb-and-Flow Bioreac-
tor for Hairy Root Cultures. American Society of Agricultural Engineering, International Winter Meeting, Chicago, IL, Dec. 17-20, 1991 paper no. 917528. Chicago: American
12.
14.
WEATHERSPJ, DIIORIO A, CHEETHAM RD: A Bioreactor for Differentiated P l a n t Tissues. In Biotech USA: Proceedings Francisco: Conference Management Corp; 1989:247-256.
9.
growth than media recycling, suggesting a media-conditioning effect for root cultures.
19.
CURRYJP: In situ Extraction of Secondary Metabolites from Hyoscyamus muticus in a Continuous Bioreactor. BS Honors Thesis. Pennsylvania: The Pennsylvania State University; 1991.
20.
LARSONWA, HSU JT, HUMPHREYAE: Quantification of Nico-
tine Release from a Nicotiana tabacum Hairy Root Culture. American Institute of Chemical Engineers Annual Meeting, Nov. 1-6, Miami, Florida, 1992, paper no. 154h. New York: American Institute of Chemical Engineering; 1992. Nicotine release behaved as an equilibrium ion-trapping mechanism. In response to pH perturbations, release of nicotine can be described by a two-compartment model. 21.
MCKELVEY SA: Effect of Bioreactor Design
on Growth in Agrobacterium Transformed Hairy Root Cultures of Hyoscyamus muticus. BS Honors Thesis. Pennsylvania: The Pennsylvania State University; 1992.
WR Curtis, Department of Chemical Engineering/Biotechnology Institute, The Pennyslvania State University, University Park, PA 16802, USA.
biochemical diversity of plants in the future, w e must develop ways of culturing these tissues in reactors so that w e do not destroy the environments where the native plants are found. In developing such reactor systems, the location of the product, either intracellular or extracellular, will have a tremendous impact o n the reactor design. In the case of intracellular products, the root tissue is a product, and the reactor design must facilitate harvest. If the c o m p o u n d is secreted into the medium, the root is behaving as a catalyst, and recovery of the root tissue is not necessary. For extracellular products, longevity of biocatalytic activity is the primary concern. Because of the impact of metabolite retention on reactor design, a brief review of advances in intracellular metabolite release is presented in the context of reactor design for product recovery.
Current research Roots have been cultivated in a wide range of reactor configurations that vary from completely novel designs to simple modifications of existing reactors. Several designs that have been used for the cultivation of roots are s h o w n schematically in Figure 1. A summary of reactor studies conducted over the past year is presented in Table 1. The results achieved in mechanically agitated reactors are surprisingly good [2-']. This supports the previous findings of Jung and Tepfer [3] w h o have cultured roots in volumes up to 20 liters in mechanically agitated reactors. Final tissue densities will obviously be limited in these systems as a result of mechanical tissue damage [4,5]; however, this alternative should not be disregarded, particularly ff the product is intracellular.
Abbreviations DW-~clry weight; FW fresh weight. © Currrent Biology Ltd ISSN 0958-1669
205
206
Biochemical engineering
May contain draft-tube or immobilization mesh
(a)
(c)
d~
o
o
0
o
0 o
Air
Air
Screen to prevent root-impeller contact
Reversible
Medium
pump
t'ump
reservoir Air
Fig I. Reactor configurations used for the culture of roots: (a) bubble column, (b) isolated impeller, (c) stirred tank, (d) ebb-and-flow, (e) trickle-bed.
While bubble columns can be used successfully to culture roots [2"',6"',7-], their performance is rather poor compared to other systems [2"',6".]. This recent work supports earlier studies showing that bubble columns perform rather poorly in comparison to other reactor systems [4,8]. The most likely causes are either insufficient oxygen because of channeling, or tissue damage caused by sparging. In the case of H y o s c y a m u s m u t i cus root cultures, sparging into gyratory shake flasks did not reduce growth [6"], which supports gas-phase channeling as the primary cause of poor growth. In addition to several previous reports [4,9], studies on the successful use of an isolated impeller reactor are reported for the culture of beet hairy roots [10-.]. Isolated impeller systems are considered separately from simple mechanically agitated systems because the roots b e c o m e immobilized and convective flow is produced externally to the root mass, which results in much less tissue damage. For the culture times used in this work, the final tissue density achieved in this system is similar to that of the stirred tank (about 8-10 g 1-1 DW), particularly if calculations are based on the total medium volume and not just the growth section of the vessel. The ability of impellers to provide sufficiently uniform convection in larger scale systems will ultimately determine the feasibility of this approach. A recent report of an ebb-and-flow reactor appears to be the first reactor in which flow patterns are domi-
nated by external pumping (analogous to a plug-flow reactor) [11"]. At the end of culture, the average liquid velocity can be estimated at 0.53 cm s- 1, which is close to the experimental value of 1 cm s-1 which was required to overcome mass-transfer resistances for roots enclosed in a spherical tea ball [12"]. This reactor supported one of the highest tissue productivities to date, demonstrating the need to overcome convective masstransfer limitations within the root mass. These results are contrary to previous work with an ebb-and-flow reactor [4]; however, in these earlier studies the fill and drain cycle was very slow (average liquid velocity at end of culture of about 0.052 cm s-1), which would probably not be sufficient to induce internal flows in stagnant growth centers within the reactor.
The feasibility of sustaining high rates of growth in reactors in which the medium is sprayed over the tissue has been demonstrated with both nutrient mists [13",14"] and coarse spray [6"']. After initial growth as a bubble column for inoculum distribution, this technique has been used to overcome problems of bubble coalescence in a 2-liter [15"] and 14-liter reactor [16"]. These reactor studies dearly demonstrate that roots can be grown in a variety of reactor configurations. The question that remains unanswered is which of these alternatives can be scaled-up economically?
Cultivation of roots in bioreactors Curtis 207
Table 1. Current research on growth of root cultures in reactors.
Root culture
Culture time (days)
Inoculum (g I- 1DW)
Final density (g I- 1DW)
Emphasis
Reference
1
Tagetes patula
28
-
9.3
Integrated recovery
[2"]
5 0.25 2
Tagetes patula Hyoscyamus rnuticus Lithospermum erythrorhizon
28 18 220
0.11 -
6.8 3.2 -
Integrated recovery Growth study Integrated recovery
[2"] [6"] [7"]
Isolated impeller
1
Beta vulgaris
13
1
12
Product release
[9]
Ebb-and-flow
2
Hyoscyamus muticus
18
0.22
11.7
Growth study
[11 ]
Nutrient mist
1.4 0.28
Beta vulgaris Carthamus tinctorius Hyoscyamusmuticus
7 7 18
0.43 a 0.52 a 0.10 a
1.3 a 1.5 a 6.9 a
Growth study Growth study Growth study
[13",14"] [13",14"] [6-]
2 15
Hyoscyamus muticus Hyoscyamus muticus
30 28
0.22
11.9
Fungal el icitation Residence time distribution
[15"] [16"]
Reactor
Working volume (litres)
Stirred tank Bubble column
1.4
Bubble column and trickle-bed sequence
aReactor density calculated based on 75% volume of culture space. DW, dry weight.
Reactor operation
To address the issue of scale-up, the remainder of this review is organized around the operational phases of inoculation, growth, and product recovery, followed by a brief discussion of problems associated with variability and reproducibility. As a result of the tedious and time-consuming nature of tissue-culture reactor studies, many of the observations described b e l o w were either not replicated, or were observed only for a single species of plant root. Inoculation Much of the heterogeneity in experimental inoculation techniques arises from the conflict between the search for practical or theoretical information. Root tips are often used as the inoculum because they are the center of active growth. While this procedure provides a rapid start for small-scale culture, it is impractical for larger scale reactors, as a result of both the labor involved in dissecting tips and the fact that root meristems represent such a small fraction of the inoculum. Dissected bulk tissue is far more practical, and has b e e n used successfully in m a n y studies [6",13"-15"]. In work with beet root tissue, Dilorio et al. [13"] observed that cultures initiated from root tips grew considerably faster than bulk tissue in shake culture, but there was no difference in performance of tips and bulk tissue inoculated in nutrient mist reactors. In m a n y studies, the source of tissue is not specified. In future w o r k it is important that the source and method of preparation of the inoculum should b e specified to provide insight into inoculum effects.
Manual dissection, e v e n of bulk tissue, is not a practical way of preparing root cultures for inoculation in largescale reactors. We recently demonstrated that several root species (H. muticus, beet, and a proprietary line) can b e briefly h o m o g e n i z e d to facilitate inoculation without adversely affecting growth rates compared to bulk tissue dissection (K Hollar and WR Curtis, unpublished data). We have used this technique to inoculate a 15-liter reactor system [16-]. The breadth of applicability of this technique should be determined because it can be easily adapted to aseptic inoculation of large-scale systems by introducing the root fragments as a slurry [16",17"]. Poor distribution of inoculum within the reactor is another problem that is often avoided in small-scale reactor studies by tediously placing the root tissue in the reactor b y h a n d [4,6-',10"',13",14"]. While uniform distribution enhances reactor performance by minimizing the onset of localized mass-transfer limitations, it does not give a true evaluation of reactor performance. It does, however, permit a quantitative assessment of the m a x i m u m performance of the environmental conditions of a given reactor configuration. Practical distribution can only be achieved on a large scale b y suspending the inoculum in a liquid phase. Additional observations of the detrimental effects of p o o r root distribution have b e e n reported [2"',4,6",17"], but no quantitative analytical studies on the effects of uniformity within reactors have b e e n carried out. In submerged (gas-dispersed) reactor systems, essentially no support is n e e d e d for the roots because of the b u o y a n c y of the surrounding medium. In contrast, liquid-dispersed reactors, such as a trickle-bed,
208
Biochemical engineering require physical support, not only for the root tissue, but also for the weight of the entrained liquid which can b e equal to the mass of the roots [15"]. The distance over which support is provided does not vary much, as reported b y different workers: 2.5 cm Pall ring distillation packing [15"], horizontal mesh spaced at 2.5 cm [11",13"], and 13.75 cm Intalox saddle process packing [16"]. Although requirements for support will vary with liquid loading and root type, the influence of support dimensions on root distribution will probably play a more important role. The uniformity of inoculum distribution will b e determined b y the distribution of pinch points (where packing contacts or branches) and flow patterns within the reactor. Growth and maximum tissue density As a result of the severe mass-transfer limitations imposed by dense root structures, the rate of root growth within a reactor must always be evaluated in the context of the final tissue density. The objective of reactor design is not only to support high growth rates, but to sustain those growth rates at high density b y minimizing problems associated with mass-transfer limitations. Sorting out physical versus physiological limitations in growth is complicated b y difficulties in obtaining growth data. Correlation of growth with electrical conductivity is still a c o m m o n w a y of monitoring growth [2"',4,9]. However, this technique is not directly related to mass within the reactor, and can be in error if stress associated with the reactor environment changes the rates of inorganic ion uptake. For example, in the study b y Buitelaar et al. [2..], the average difference b e t w e e n actual final dry weights and those calculated with conductivity correlations indicated that errors greater than 10% were introduced for most reactor runs. While conductivity correlations are an invaluable tool in the control of production systems, they should be used with caution in evaluating reactor performance (particularly if correlations are developed from shake-flask data). Sugar usage is probably a m o r e robust measure of culture performance because it is a direct reflection of metabolism [18]. Measurement of respiration from CO 2 and 02 levels in the gas exiting the reactor is another metabolism-based m e t h o d of monitoring reactor performance; however, to date, this has not b e e n implemented for root cultures. The extent to which carbon use efficiency changes in root cultures under different environmental conditions has not b e e n adequately assessed for its impact on the accuracy of metabolic measurements of growth. Drained liquid weight provides a more direct experimental measurement of mass within the reactor [5], although roots entrain large amounts of m e d i u m which introduce considerable systematic error. Drained weights are likely to be 50-100% higher than blotted fresh weights [15"]. The m e a n residence time of an inert tracer has recently b e e n tested as a measure of tissue mass within the reactor [16"]. This technique m a y provide a more direct, non-invasive physical m e a s u r e m e n t of root mass for large-scale reactor systems. Difficulties in obtaining direct measurements of growth time courses greatly impair the ability to evaluate the
effects of reactor environments on growth. It is clear that with few exceptions, root cultures do not display sustained logarithmic growth. As a result, doubling times and specific growth rates do not have m u c h significance--and can be very misleading. The onset of internal mass transfer limitations within individual growth centers depends on the density of the inoculum and size of the root fragments. Systematic studies on the effect of inoculum density and inoculure fragmentation are needed. As inoculum densities vary b y almost an order of magnitude (Table 1) and inoculum preparation by different researchers varies considerably, comparisons of absolute rates of growth from different studies are not very meaningful. Difficulties in interpreting the effects of physical parameters on the growth of root cultures are c o m p o u n d e d b y the lack of information on the physiological requirements for growth. Information is just beginning to a p p e a r on the effects of carbon sources, inorganic substances, and gas composition. Given the tremendous adaptations exhibited by whole plants, large variations in physiological requirements can be expected. For example, the influence of carbon dioxide in largescale reactor systems is very complicated. The potential effects are beneficial (stress abatement, carbon assimilation), detrimental (reduced oxygen availability, feedb a c k repression of metabolism) and u n k n o w n (pH modulation). In nutrient-mist reactors, 1% CO 2 enhanced the tissue accumulation of beet root and safflower [14"], while sparging of 10% CO2 in shake flasks suppressed the growth of H. m u t i c u s roots [6"]. These differences in response could b e a result of the reactor environment, the plant species, or an interaction of the physiological response within a particular environment. The lack of understanding of root growth is as m u c h a reflection of the complexity of plant physiology as it is of plant root culture. Our general understanding of plant physiology lags substantially behind that of mammalian and microbial physiology, and the situation for root physiology is e v e n worse than that of plants as a whole. Fortunately, understanding is not a prerequisite for applications, and the simplistic approaches to culture used so far have b e e n very successful. Root cultures have p r o v e d to b e sufficiently amenable to culture, so that achieving industrial-scale culture should b e possible through the application of sound engineering principles. Product recovery The nature of product formation, either intracellular or secreted, will profoundly affect reactor design for a particular compound. The desire to facilitate the removal of roots has b e e n the stimulus for n u m e r o u s novel reactor designs. Keeping in mind that the ultimate scale for most secondary products will b e tens of thousands of liters, most designs are impractical. In the case of intracellular products, simple designs such as the mechanically agitated reactor or bubble column, will probably be applicable despite the slower rates of growth and/or lower volumetric productivities. For extracetlular metabolites, the ability to re-use tissue favors much higher density immobilized culture con-
Cultivation of roots in bioreactors Curtis ditions. An advantage of root structure is the ease of implementating an integrated product recovery, particularly for immobilized-culture systems that recycle media. Solid adsorbents and liquid paraffin have been used as metabolite sinks to enhance shikonin recovery from Lithospermum erythrorhizon grown in an airlift reactor [7"]. The recovery of thiophenes from Tagetes patula was enhanced in stirred tanks with biocompatible secondary phases: fluorocarbon (FC40) and hexadecane [2"']. Volatile extractants such as hexane killed the root cultures if added in situ. By contacting media with hexane in an external loop extractor, it has been shown that toxicity resulting from phase contact can be eliminated, and sesquiterpene recovery can be facilitated from root cultures into volatile solvents .to permit economical product isolation [19]. In all cases where integrated recovery has been used, productivity was enhanced, presumably as a result of a reduction in feedback repression of metabolite synthesis. Considerable progress has also been made in facilitating the release of intracellular products from root tissues. Release of intracellular nicotine from root tissue was enhanced by manipulating extracellular pH [20"]. Similarly, oxygen deprivation [9] and heat shock combined with calcium modulation [8] caused the release of pigments from beet root cultures. In these cases of stress-induced product release, it is not clear h o w much tissue death contributes to 'release'. The ability of cultures to continue growing simply indicates that the meristems are not damaged [9]. In terms of production, however, the mechanism by which release is facilitated is inconsequential, provided the reactor regenerates catalytic activity through cyclic growth and production. Such tissue turnover may be advantageous in permitting an essentially indefinite reactor operation as the age distribution within the reactor reaches a constant level. Combining release with secondary-phase trapping is a promising route to the recovery of intracellular products from root culture. In light of this progress, it is likely that high-density immobilized culture systems, such as the trickle-bed reactor, will be applicable to both intracellular and extracellular metabolites.
Problems: reproducibility and variability One of the implicit difficulties in interpreting the results of root growth in bioreactors is reproducibility. In most cases there are no replications, and in cases where runs are repeated, the reproducibility is poor, even by tissue-culture standards. Using stirred tank reactors, Buitelaar et al. [2.'] observed variabilities of 24-32% for stirred tank reactors, and 40-59% for bubble columns. McKelvey [21] observed a variability as low as 4% for shake flask culture, but it rose to as much as 24% for bubble column and trickle-bed configurations. In their work with a nutrient mist bioreactor, DiIorio et al. [13"] found that deviations of replicated final tissue weights were kept within 10%. This enhanced reproducibility could be attributed to their use of preconditioned media. As a result of the observed variability of growth in reactor systems, single-point comparisons of reactor
performance must be interpreted with extreme care. As this research field matures, more replications will have to be performed at the expense of breadth of experimentation.
Conclusion At low tissue density, roots can be cultivated in essentially any bioreactor configuration, particularly on a small scale. In cases where the intended product cannot be released to the medium, bubble columns or mechanically agitated reactors are the most promising alternative. For metabolites that are released to the medium, immobilized systems such as packed-bed or trickle-bed reactors should provide higher scale tissue loadings. Studies focusing on scale-up (with adequate replication) are needed, including the development of improved inoculation and tissue-harvest techniques.
Acknowledgements Part of this work was supported by the National Science Foundation (NSF Grant no. BCS-9110288), a n d the Pennsylvania State University College of Agriculture Inter-College Research Grant, Project Number 3211 (Root reactor design for scaled-up production of pharmaceutical proteins).
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1. •.
PAYNEG, BRINGI V, PRINCE C, SHULER M: P l a n t c e l l a n d T i s s u e c u l t u r e i n Liquid S y s t e m s . New York: Hanser; 1992:227-279. Extensive review of physiology, biochemistry, and reactor engineerhag for root cultures. BUrrELAARRM, LANGENHOFFRAM, TRAMPERJ: G r o w t h a n d Thiophene Production by Hairy Root Cultures of Tagetes p a t u l a in V a r i o u s T w o - L i q u i d - P h a s e Bioreactors. Enzyme Mtcrob Tecbnol 1991, 13:487-494, In sttu extraction for thlophene recovery has been developed. Solvents with logP greater than five were found to be biocompatible. Recovery was implemented in a variety of reactor configurations, including bubble colunm, stirred tank, a n d liquid-impelled loop. Blockage of flow w a s experienced with the liquid-impelled loop. Stirred-tank cultures grew better t h a n bubble column cultures. The greatest recovery of thiophenes was observed in bubble c o l u m n with hexadecane as an extractant. 2. ..
3.
JUNG G, TEPFER D: U s e o f G e n e t i c T r a n s f o r m a t i o n b y t h e Ri T-DNA o f Agrobacterium rhizogenes to Stimulate B i o m a s s a n d T r o p a n e A l k a l o i d P r o d u c t i o n i n Atropa belladonna and Calystegia sepium R o o t s G r o w n in vitro. Plant Sci 1987, 50:145-151.
4.
TAYA M, YOYAMA A, KONDO O, KOBAYASHI T, MATSUI C: G r o w t h C h a r a c t e r i s t i c s o f P l a n t H a i r y R o o t s a n d T h e i r C u l t u r e s i n B i o r e a c t o r s . J Chem Eng Japan 1989, 22:84-89.
5.
WILSONPDG, HILTON MG, MEEHAN PTH, WASPE CR, RHODES M: T h e c u l t i v a t i o n o f T r a n s f o r m e d Roots f r o m Labor a t o r y to Pilot Plant. In Progress in Plant Cellular and Molecular Biology. Edited by Nijkamp HJJ, v a n Der Plas LHW, Van Aartrijk J. Dordrecht, Netherlands: Kluwer Academic Publishers; 1990:700-705.
209
210
Biochemical engineering 6. •.
MCKELVEYSA, GEHRIGJ, HOLLAR KA, CURTIS WR: G r o w t h o f P l a n t Root Cultures i n Gas- a n d L i q u i d - D i s p e r s e d Reactor E n v i r o n t n e n t s . BiotechnoI Prog 1993, 9:in press. A series of small-scale reactor configurations were developed to provide quantitative information on the effects of liquid and gas dispersion within root bioreactors. Simple sparged reactors s h o w e d the poorest growth rate for H. muticus, even poorer than gas-phase reactors in which the medium was intentionally maldistributed. Reactors in which the medium was sprayed over the roots displayed growth rates comparable to gyrator*/shake-flask controls. The results suggest that roots are more capable of compensating for p o o r liquid dispersion than for p o o r gas dispersion within reactor systems. 7.
SHIMOMURAK, SUDO H, SAGA H, KAMADA H: S h i k o n i n
Production and Secretion b y Hairy Root cultures o f L i t h o s p e r m u m erythrorhixon. Plant Cell Rep 1991, 10:282-285. shikonin was recovered in an external loop for 220 days using XAD2 resin. An ammonium-free medium was required for shikonin production.
8.
of the Biotecb USA Conference, Oct 2-4, San Francisco. San KONDOO, HONDA H, TAYA M, KOBAYASHIT: C o m p a r i s o n o f G r o w t h P r o p e r t i e s o f Carrot Hairy Root i n V a r i o u s Bioreactors. Appl Microbiol Biotechnol 1989, 32:291-294.
10. •.
KINO-OKAM, HONGO Y, TAYA M, TONE S: C u l t u r e o f Red Beet H a i r y R o o t i n Bioreactor and Recovery o f Pigm e n t Released f r o m the Cells b y Repeated O x y g e n Starvation. J Chem Eng Jpn 1992, 25:490--495. Oxygen starvation for u p to 16 hours facilitated release of the pigment betanin. Repeated anoxia combined with absorption of pigments on styrene-divinybenzene copolymer resin permitted pigment recovery for 20 days. The release appeared to occur preferentially from mature tissue, permitting continued growth of meristems. 11.
Society for Agricultural Engineering; 1991. A 2-liter ebb-and-flow bioreactor was used with 1 min fill and drain cycles and 2 rain liquid and gas dwelling times. After 18 days of growth, an inoculum of 8 g FW yielded 459 g FW (23.4 g DW) which represents a higher growth rate than gyratory shake-flask controls. PRINCECL, BRINGI V, SHULERME: CoIlvorztive Mass Transfer i n Large Porous Biocatalysts: Plant O r g a n Culture. Biotechnol Prog 1991, 7:195-199.
Onion root cultures were placed inside a spherical tea ball within a defined flow field to determine the external flow rates required to induce sufficient internal flows to sustain maximal oxygen uptake. The evaluation was conducted at 320 g 1-1 FW. 13.
DIIomo AA, CHEETHAMRD, WEATHERSPJ: G r o w t h o f Tratmformed Roots i n a N u t r i e n t Mist Bioreactor: Reactor Performance and Evaluation. Appl Mtcrobiol Biotechnol
1992, 37:457-462. The authors describe the adaptation of a nutrient mist bioreactor (which was an ultrasonic transducer to produce fine mists) for application with root cultures. Culture perfusion resulted in slower
DIIORIOAA, CHEETHAMRD, WEATHERSeJ: Carbon Dioxide Improves the Growth o f Hairy Roots Culture o n Solid Mediuna and in Nutrient Mists. Appl Microbiol Biotechnol
1992, 37:463-467. Enrichment of nutrient-mist carrier gas with 1% CO 2 resulted in a 15% enhancement in the growth rate of beet (Beta vulgaris) and safflower (Carthamus tinctorius). The effects of carbon dioxide enrichment were m u c h greater in solid culture than in a bioreactor. FLORESHE, CURTIS WR: Approache~ to Understanding and Manipulating the Biosynthetic Potential o f Plant Roots. Proc N Y Acad Sci 1992, 655:188-209. H. muticus roots were grown in a 2-liter trickle-bed and a 2-liter sparged control reactor for four weeks and induced with a ffmgal elicitor. The trickle-bed reactor produces three times more solavetivone. 15.
16.
RAMAKmSHNAND, CURTIS WR: Application of Residence
Time Distribution Studies to Plant Root Culture Bioreactors. American Institute of Chemical Engineers Annual Meeting, Nov. 1-6, Miami, Florida, 1992, paper no. 164g. New York: American Institute of Chemical Engineering; 1992. A residence-time distribution technique was developed for root cultures, using blue-dextran dye as a tracer. The technique was used to correlate cell density in submerged reactor systems, and to determine liquid hold-up in trickle-bed root reactors. 17.
RODRIGUES-MENDIOLAMA, STAFFORD A, CRESSWELLR, ARIASCASTRO C: Bioreactors for Growth o f Plant Roots. Enzyme Microb Technol 1991, 13:697-702. An airlift was operated with a draft tube for TrtgoneUa foenumgraceum and a m e s h tube for Nicotiana rustica. A more uniform distribution was achieved using the mesh. 18.
TOWONEN L, OJALA M, KAUPPINEN V: I n d o l e ~ l k a l o i d
Production b y Hairy Root Cultures o f C a t h a r a n t h u s roseus: G r o w t h Kinetics and Fermentation. Biotechnol Lett 1990, 12:519-524.
CUELLOJL, WALKER PN, CURTIS WR: Ebb-and-Flow Bioreac-
tor for Hairy Root Cultures. American Society of Agricultural Engineering, International Winter Meeting, Chicago, IL, Dec. 17-20, 1991 paper no. 917528. Chicago: American
12.
14.
WEATHERSPJ, DIIORIO A, CHEETHAM RD: A Bioreactor for Differentiated P l a n t Tissues. In Biotech USA: Proceedings Francisco: Conference Management Corp; 1989:247-256.
9.
growth than media recycling, suggesting a media-conditioning effect for root cultures.
19.
CURRYJP: In situ Extraction of Secondary Metabolites from Hyoscyamus muticus in a Continuous Bioreactor. BS Honors Thesis. Pennsylvania: The Pennsylvania State University; 1991.
20.
LARSONWA, HSU JT, HUMPHREYAE: Quantification of Nico-
tine Release from a Nicotiana tabacum Hairy Root Culture. American Institute of Chemical Engineers Annual Meeting, Nov. 1-6, Miami, Florida, 1992, paper no. 154h. New York: American Institute of Chemical Engineering; 1992. Nicotine release behaved as an equilibrium ion-trapping mechanism. In response to pH perturbations, release of nicotine can be described by a two-compartment model. 21.
MCKELVEY SA: Effect of Bioreactor Design
on Growth in Agrobacterium Transformed Hairy Root Cultures of Hyoscyamus muticus. BS Honors Thesis. Pennsylvania: The Pennsylvania State University; 1992.
WR Curtis, Department of Chemical Engineering/Biotechnology Institute, The Pennyslvania State University, University Park, PA 16802, USA.