Blotechnol. Prog. 1993, 9, 12-20
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TOPICAL PAPER Utilization of Hairy Root Cultures for Production of Secondary Metabolites Leena Toivonen Laboratory of Biochemistry and Microbiology, Department of Chemical Engineering, Helsinki University of Technology, Kemistintie 1, SF-02150 Espoo, Finland
The possibility of producing useful chemicals by plant cell cultures has been studied intensively for the past 30 years. However, problems associated with low product yields and culture instabilities have restricted wider industrial application of plant cell culture. The employment of hairy root culture technology, developed in the past 10 years, offers new opportunities for in vitro production of plant secondary metabolites. In contrast to cell suspension cultures, hairy root cultures are characterized by high biosynthetic capacity and genetic as well as biochemical stability. In this review, the establishment and cultivation of hairy root cultures as well as their properties and application for production of secondary metabolites are discussed.
Contents Introduction Root Induction by Agrobacterium rhizogenes: The Hairy Root Disease Establishment of Hairy Root Cultures Properties of Hairy Root Cultures Improvement of Secondary Metabolite Production Fermentation Systems and Scale-up F u t u r e Prospects
12 12 13 13 14 16 17
Introduction Many plant secondary products are exploited commercially as biologically active compounds in, for example, the cosmetic, food, and pharmaceutical industries. Bioactive secondary metabolites (fragrances, flavors, pharmaceuticals, pesticides) are generally of high value, but are required in relatively small quantities when compared with primary metabolites, which are obtained from plants as bulk or commodity chemicals (Balandrin and Klocke, 1988). Particularly, higher plants are still an important source of many widely used pharmaceuticals, as the rather complex structures cannot be synthesized chemically at acompetitiveprice (Berlin,1988;Fowler and Scragg, 1988). As the production of chemicals by isolation from fieldgrown plants is associated with certain problems, e.g., including susceptibility of production to environmental and geopolitical instabilities, there has been considerable interest in investigating the possibilities of exploiting plant cell cultures as an alternative source of secondary metabolite products. The potential and advances of plant cell cultures in this context have been discussed in numerous recent reviews (e.g., Rokem and Goldberg (19851, containing a table listing reviews up to 1983; Ellis, 1986; Kauppinen et al., 1986; Parr, 1988; Taticek et al., 1991). Although in vitro culture of plant cells is now a mature technology with successful applicationsin agricultural crop 875&7938/93/3009-0012$04.00/0
improvement, germ plasm storage and micropropagation (Staba, 19881, the application of in vitro culture systems in commercial production of secondary metabolites, have hitherto been most successfully achieved in the manufacture of shikoninwith Lithospermum erythrorhizon cell cultures by Mitsui Petroleum Co., Japan (Curtin, 1983). The two major limitations to wider industrial application of plant cell culture are, a t the moment, product yield and productivity. This is mainly due to the insufficient knowledge about controlling secondary metabolite production in plant cell cultures as well as the lack of stable, high-producing cell lines (Kurz, 1989). An alternative approach to the production of plant secondary metabolites in vitro is the use of organized or differentiated cultures, as their behavior has been claimed to be much more predictable when compared with that of cell suspension cultures (Parr, 1989). Secondary compound biosynthesis in organized tissue has received comparativelylittle attention until recently because such cultures tend to be rather slow growing, are difficult to handle in normal bioreactors, and provide little opportunity for alteration of product profile or yield (Charlwood et al., 1988). However, recent studies have shown that a wide range of dicotyledonous plants are susceptible to infection by Agrobacterium rhizogenes, giving rise to transformed root cultures. The employment of hairy root culture technology, developed in the past 10 years, has been claimed to revolutionize the role of plant cell culture technology in fine chemical synthesis (Flores et al., 1987a; Hamill et al., 1987; Rhodes et al., 1987; Signs and Flores, 1990). Root Induction by Agrobacterium rhizogenes: The Hairy Root Disease Hairy roots can be obtained by genetic transformation of wounded plant tissue by a pathogenic soil bacterium, Agrobacterium rhizogenes. This bacterium is capable of infecting a wide range of dicotyledonous plants, causing roots to proliferate rapidly at the infection site (hairy root disease) (White and Sinkar, 1987; Spano et al., 1985).
0 1993 Amerlcan Chemlcal Society and American Institute of Chemlcai Engineers
Biofecbnol. Rug., 1993, Vol. 9, No. 1
The infection process itself is a complex series of events, the temporal sequence of which is defined by cellular activities of the interacting partners. After bacterial colonization and attachment to plant cellsa t or near wound sites, the infection leads to insertion of one or both of two pieces of transfer DNA (TLand TR),contained in the bacterial Ri plasmid, into the plant genome (Chilton et al., 1982;White et al., 1982;Binns and Thomashow, 1988). The transformation event is triggered by activation of vir genes by wound substances synthesized in the host plant, e.g., acetosyringone (Stachel et al., 1985). The integration was shown to be random, with multiple copies of T-DNA often being inserted (Ambroset al., 1986;Petit et al., 1986). Finally, the expression of T-DNA genes coding for auxin synthesis and other rhizogenic functions results in root formation by the host plant a t the infection site. The T-DNA may also carry genes encoding the synthesis of sugar and amino acid conjugates, opines, which can be used by the invading bacteria as a source of carbon and nitrogen (Binns and Thomashow, 1988). Several Ri plasmids of different A. rhizogenes isolates have hitherto been mapped by means of restriction endonucleases (Costantinoet al., 1981; Byrne et al., 1983; Pomponi et al., 1983; Huffman et al., 1984;Jouanin et al., 1985; Combard and Baucher, 1988). The Ri plasmids, and this the bacterial isolates, were grouped into two main classes according to the opines synthesized by hairy roots and utilized by the bacterium: agropine-type strains (e.g., A4, 15834, HR1, LBA 9402), which induce roots producing agropine, mannopine, and the corresponding acids, and mannopine-type strains (e.g., 8196, TR7, TRlOl), which elicit roots containing only mannopine, mannopinic acid, and agropinic acid (Petit et al., 1983). The restriction maps of the corresponding plasmids also showed substantial differences (White and Sinkar, 1987). The agropine-type strains containing both TLand TRregions in their Ri plasmid are more often used in the establishment of hairy root cultures, as they appear to be more virulent (Rhodes et al., 1987).
Establishment of Hairy Root Cultures When establishing hairy root cultures, transformation is induced on aseptic, wounded plants or plant parts by inoculating them with a thick, viable A. rhizogenes suspension. Alternatively, cocultivation of plant protoplasts with the bacterium has been used (Wei et al., 1986). After 1-4 weeks, when roots emerge a t the site of inoculation, they are individually cut off and transferred into a hormone-free growth medium, e.g., MS (Murashige and Skoog, 1962) or B5 (Gamborg et al., 1968),containing antibiotics to kill the bacteria. The protocols for the establishment of hairy root cultures have been described in detail by Hamill et al. (1987) and Rhodes et al. (1987). The susceptibility of plant species to infection varies greatly. For successfulinfection of some species, addition of acetusyringone (Rhodes et al., 1987;Godwin et al., 1991) or auxin (Dobignyet al., 1990)may berequired. Generally, however, A. rhizogenes has a wide host range (De Cleene and De Ley, 1981). Mugnier (1988) summarized a list of successfullyestablished hairy root cultures containing over 80 dicotyledonous plant species belonging to 29 families. Subsequently, the list has been extended to include some new families such as Apocyanaceae (Parr et al., 1988; Davioud et al., 1989a), Verbenaceae (Sauerwein et al., 1991a), Campanulaceae (Yonemitsu et al., 1990), Ceraniaceae (Ishimaru and Shimomura, 1991), and Gentianaceae (Ishimaru et al., 1990),as well as several new species belonging to the families already mentioned by Mugnier (1988),especially species belonging to Asteraceae (Flores
13
LeenaToivonen received her degree of Doctor of Technology (chemical engineering) from the Department of Chemical Engineering, Helsinki University of Technology,in April 1992. Her thesis was on the utilization of Catharanthusroseushairy root and cell suspension cultures in plant biotechnology. The work was started at Plant Biotechnology Institute, Saskatoon, Saskatchewan (NRC, Canada), in 1988under the supervision of Dr. W. G. W. KUIZ,and it was continued in Finland a t Technical Research Centre of Finland under the supervision of Prof. V. Kauppinen and at Helsinki University of Technology under the supervision of Prof. S. Laakso and Dr. H. Rosenqvist during the years 1989-1992. A t the moment she is continuing the research on plant biotechnology, especially hairy root culture technology, a t Helsinki University of Technology.
et al., 1987b; Constabel and Towers, 1988; Trypsteen et al., 1991) and Solanaceae (Deno et al., 1987; Christen et al., 1989; Shimomura et al., 1991a). Members of some plant families, e.g., Papaveraceae, have been infected successfully, but the established cultures have shown disorganized morphology, being however able to grow without growth hormones (Williams et al., 1990). Interestingly, the discoveryof a hairy root culture of rye (Secale cereale), belonging to monocotyledonous plants, was also reported recently (Whitney, 1990). The synthesis of opines is a firm indication of the transformed nature of hairy root cultures (Petit et al., 1983). However, expression of the opine genes may be unstable with time (Kamadaet al., 1986). Thus, detection of the T-DNA by Southern blot hybridization is often necessary to confirm that the cultured root tissue really is transformed (Rhodes et al., 1987).
Properties of Hairy Root Cultures Hairy roots can be cultivated in a simple defined medium without addition of growth hormones. The roots are characterized by a high degree of lateral branching, a profusion of root hairs, and an absence of geotropism (Hamill et al., 1987; Rhodes et al., 1987). In contrast to cell suspension cultures, hairy roots can grow from low inocula to high final biomass densities with only a minimal lag phase being observed (Figure 1). In general,the growth rates of hairy root cultures are comparable with those of cell suspension cultures (Wilson et al., 1987). The growth rate and even the morphology of hairy root cultures may vary greatly between species (Hilton et al., 1988) and with culture conditions,such as the ionic strength of the medium (Hamill et al., 1987; Yonemitsu et al., 1990). According to Hilton et al. (1988), the shape of the batch growth curve
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BbtechrW. Prog., 1993, Vd. 9, No. 1
phase, with most of the alkaloids remaining intracellular during active growth. A key feature of hairy root cultures is that, in contrast to suspension cultures, they are genetically stable (Aird et al., 1988). Consequently, this results in biochemical stability and thus stable productivity (Flores and Filner, 1985;Kamada et al., 1986;Aird et al., 1988). The stability facilitates the optimization of secondary metabolite production and also enables the use of systematic statistical methods in studying the effects of environmental growth conditions (Toivonen et al., 1991).
Improvement of Secondary Metabolite Production Figure 1. Hairy root culture of Catharanthus roseus just after inoculation (right) and after 2 weeks of growth (left) in 1/2 B50 medium (half-diluted Gamborg’s B5 medium without growth hormones).
of transformed root cultures could be described satisfactorily by a Monod-type relationship, although the growth of hairy roots is a result of both cell division a t meristematic regions and cell expansion. By contrast, Tayaet al. (1989a) reported a kinetic expression for hairy root proliferation that was based on branching and linear extension. Table I lists hairy root cultures studied for secondary metabolite production as well as the main secondary products. The synthetic capacity of hairy roots has appeared to mirror closely that of the roots of the plant from which they are derived (e.g., Kamada et al., 1986; Constabel and Towers, 1988; Parr and Hamill, 1987), although the lack of transport mechanisms involved in product accumulation in entire plants must be taken into account (Flores et al., 1987a). The use of hairy root cultures in biotransformation processes has also been suggested (Wilson et al., 1987). Hitherto, the application of hairy root cultures for biotransformation has been reported by Japanese researchers, who used the root cultures of Panax ginseng in continuous glycosylation of 2-phenylpropionic acid (Yoshikawa and Furuya, 1990) and in the biotransformation of digitoxigenin to cardiac glycosides (Kawaguchi et al., 1990). The kineticsof secondarymetabolite production in hairy root cultures has not yet been studied very intensively. In some cases, the product synthesis was related to growth (e.g., Hamill et al., 1986; Constabel and Towers, 1988; Toivonen et al., 1990), while in others the secondary metaboliteswere accumulated only after growth had ceased (Nabeshima et al., 1986; Rhodes et al., 1989),the synthesis being related to maturing of the roots. When considering commercial production, secondary metabolite synthesis dissociated from growth would be desirable, as it would allow the use of continuous production systems (Hamill et al., 1987). The extent of secondary product release in hairy root cultures varies between species. In hairy root cultures of Nicotiana rustica, 10-5076 of the nicotine was excreted (Hamill et al., 1986), and in hairy root cultures of L. erythrorhizon, 25 76 of shikonin was excreted (Shimomura et al., 1991b), whereas in hairy root cultures of Catharanthus roseus, for example, only 3-57; of total indole alkaloids were found in the media (Toivonen e t al., 1989). Differences were obtained in product release between different root clones of the same species by Rhodes et al. (1989) for Datura stramonium. However, the excretion of the products was related to the onset of senescence in roots maintained for extended periods in the stationary
When establishing hairy root cultures, the suitable selection of starting material is of great importance. According to Rhodes et al. (1988), initiating cultures from plants with a high biosynthetic capacity is particularly beneficial. However, for example, in hairy root cultures of C. roseus the indole alkaloid spectrum was observed to be qualitatively but not quantitatively similar to that of the corresponding normal plant roots (Toivonen et al., 1989). Variations between hairy root clones of the same origin also occur both in the level and relative proportions of secondary metabolites. Thus, i t is possible to select high-producing root cultures simply by screening a large population of clones (Kamada et al., 1986; Mano et al., 1986). Disorganization of the cultured roots can also be induced by exogenous hormonal treatments to form callus or suspension cultures with consequent genetic heterogeneity, which might be further increased by mutagenesis. The resulting variant transformed cells can further be stabilized as transformed roots by returning them to hormone-free medium. This technique was applied, for example, to transformed root cultures of N. rustica (Rhodes et al., 1988). As a result, a more than 4-fold increase in alkaloid content was achieved within a population of 100regenerated variantclones. The most critical aspect of the screeningoperation is the analyticaltechnique for rapidly quantitating the compoundsof interest (Rhodes et al., 1988). The development of rapid assays for desired compounds (RIA, ELISA, fluorometric methods, for example; Fliniaux and Dubreil, 1987; Yamamoto and Yamada, 1987;Naaranlahti et al., 1989)has made extensive screening programs possible. However, to avoid the tedious screening of numerous individual clones, an alternative method has been studied. This employs selection procedures in which the variant cells are grown in medium with selection pressure allowing only the growth of the cells which produce high levels of desired compounds (Robins et al., 1987). Sincethe formation of hairy roots is based on the transfer of sections of the Ri plasmid, the system is, in addition to conventional screening and selection of clones, amenable to manipulation a t the geneticlevel. If offers the possibility to manipulate the activity of secondary product pathways in culture either by introducing structural genes that code for enzymes acting a t initial or branching points of the biosyntheticpathways or by manipulating regulatorygenes controlling the overall operation of the enzymes (Rhodes et al., 1989). While the elucidation of biosynthetic pathways has been under study (e.g., Zenk, 1990), the systems for genetic manipulations have been developed, and thus the manipulation of biochemical pathways at the genetic level is becoming a realistic possibility (e.g., Comai et al., 1985; Shahin et al., 1986). As in the case of cell suspension cultures, an increase in the productivity of hairy root cultures can be achieved
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Table I. Hairy Root Cultures Studied for Production of Secondary Metabolites family Solanaceae
Convolvulaceae Apocyanaceae Chenopodiaceae Boraginaceae Compositae Rubiaceae Asteraceae
Campanulaceae Verbenaceae Geraniaceae Gentianaceae Araliaceae Leguminosae Pabaceae Brassicaceae
genus and species Atropa belladonna
major secondary products atropine
Datura candida Datura innoxia Datura stramonium Duboisia myoporoides Hyoscyamus muticus Hyoscyamus niger Nicotiana rustica Nicotiana tabacum Nicotiana hesperis Nicotiana cavicola Scopolia japonica Scopolia tangutita Solanum laciniatum Calystegia sepium Catharanthus roseus Catharanthus trichophyllus Amsonia elliptica Beta vulgaris Lithospermum erythrorhizon Tagetes patula
scopolamine,hyoscyamine scopolamine,hyoscyamine hyoscyamine scopolamine hyoscyamine hyoscyamine nicotine, anatabine nicotine, anatabine nicotine, anabasine nicotine, nornicotine hyoscyamine hyoscyamine,scopolamine steroidal alkaloids cuscohygrine indole alkaloids 17 indole alkaloids indole alkaloids betacyanin, betaxanthin shikonin thiophenes polyacetylenes quinine alkamids thiarubrine polyactylenes and thiophenes
Cinchona ledgeriana Escinacea purpurea Chaennctis douglasii Bidens sp. Rudbeckia sp. Ambrosia sp. Lobelia inflata Lippia dulcis Geranium thunbergii Swertia japonica Panax ginseng Cassia occidentalis Cassia torosa Cassia obtusifolia Glycyrrhiza uralensis Armoracia lapathifolia
-
reference Kamada et al. (1986),Jung and Tepfer (1987), Onrej and Protiva (1987) Christen et al. (1989) Shimomura et al. (1991a) Payne et al. (1987),Jaziri et al. (1988) Den0 et al., 1987 Flores and Filner (1985) Jaziri et al. (1988) Hamill et al. (1986) Flores and Filner (1985),Parr et al. (1987) Parr et al. (1987) Parr et al. (1987) Mano et al. (1986) Shimomura et al. (1991a) Hamill et al. (1987) Jung and Tepfer (1987) Parr et al. (1988),Toivonen et al. (1989) Davioud et al. (1989a,b) Sauerwein et al. (1991b) Hamill et al. (1986) Shimomura et al. (1986 and 1991b) Flores et al. (1987b) Kyo et al. (1990) Hamill et al. (1989) Trypsteen et al. (1991) Constabel and Towers (1988) Flores et al. (1987b)
lobeline sesquiterpenes (hernandulcin) tannins amarogentin, amaroswerin, xanthons saponins germichrysone, pinselin anthraquinone
Yonemitsu et al. (1990) Sauerwein et al. (1991a) Inshimaru and Shimomura (1991) Ishimaru et al. (1990) Yoshikawa and Furuya (1987) KOet al. (1988)
glycyrrhizin peroxidase
KOet al. (1989) Taya et al. (1989~)
by manipulating environmental culture conditions. The growth rates and biomass yields have been improved by alterations in nutrient composition (Nabeshima et al., 1986; Parr et al., 1988) or pH (Mukundan and Hjortso, 1991) of the medium, as well as by addition of l-naphthaleneacetic acid (Sauerwein et al., 1991a,b), dopamine (Protacio et al., 1990), or colchicine (Westcott, 1988). In contrast to cell suspension cultures, the secondary metabolite accumulation (specific production) of hairy roots is not readily manipulated by alterations in nutrient composition or pH of the culture medium (Berlin et al., 1990;Mukundan and Hjortso, 1991;Toivonen et al., 1991). However, secondary metabolite production has been successfully enhanced by adding precursors and metabolic intermediates to the growth medium (Nabeshima et al., 1986). By feeding appropriate precursors, the quantitative balance of alkaloids produced by N. rustica could also be manipulated (Rhodes et al., 1987; Walton et al., 1988). The effect of temperature on hairy root growth is clear (e.g., Nabeshima et al., 1986), although systematic optimizations of cultivation temperature have not been published. The secondary product accumulation at different temperatures has been studied only by Hilton and Rhodes (1990),who demonstrated the effect of temperature on hyoscyamine production in transformed root cultures of D. stramonium. In these experiments, the concentration of hyoscyamine in the roots cultivated a t 35 OC was only 60 5% of that found a t 30 "C. In our laboratory we also observed that the specific production of indole alkaloids was temperaturedependent in hairy root cultures of Catharanthus roseus (Toivonen et al., 1992). The possibility of enhancing accumulation of the
secondary products in hairy root cultures by addition of biotic and abiotic elicitors (Eilert, 1987)has been studied in a few cases. The addition of a biotic (fungal) elicitor increased the accumulation of thiophene in hairy root cultures of Tagetespatula (Mukundan and Hjortao, 1990), whereas the application of nigeran and denatured RNA as abiotic elicitors had no effect on thiophene production (Westcott, 1988). In contrast, no effect of the seven different fungal culture extracts tested could be obtained in D. stramonium, whereas abiotic factors (heavy metal salts) elicited sesquiterpenoid phytoalexin production which was undetectable in unelicited cultures. No increase in tropane alkaloid accumulation could be observed, but excretion of alkaloids into the medium was increased (Furze et al., 1991). Flores and co-workers also recently reported that Bidens sulphureus hairy root cultures respond to elicitation with fungal culture filtrates by increasing the production of a specific polyacetylene (phytoalexin) (Flores et al., 1988) and that hairy roota of Hyoscyamus muticus could produce sesquiterpene phytoalexins following elicitation (Signs and Flores, 1989). Furthermore, phytoalexin (isoflavone) accumulation was obtained in transformed root cultures of Lotus corniculatus as a response to glutathione addition (Robbins et al., 1991),and sesquiterpeneproduction could be enhanced in Lippia dulcis cultures by adding chitosan as an abiotic elicitor (Sauerwein et al., 1991a). Thus, it seems likely that in organized tissue like hairy roots it is possible to enhance or induce by elicitor treatment only the accumulation of true phytoalexin compounds, which are synthesized in plants as a rapid response to, for example, fungal attack (Darvill and Albersheim, 1984).
Biot&r?o/.
16
Figure 2. Hairy root culture of Catharanthus roseus after 2 weeks of growth in air-lift fermenter (V = 3.5 L).
Although the use of elicitors did not increase the alkaloid content of hairy root cultures of D. stramonium, osmotic stress produced by addition of mannitol into the culture medium was shown to enhance alkaloid accumulation (Halperin and Flores, 1990).
Fermentation Systems and Scale-up During fermentation, the structured nature of hairy root cultures leads to formation of interconnected, nonhomogeneous material unevenly distributed throughout the fermenter (Figure 2). This results in a markedly altered rheology compared to that of cell suspension cultures and has thus made it necessary to investigate novel approaches to fermenter and process design (Wilson et al., 1987). From the technical point of view, one of the main problems in the fermentation of hairy roots is mass transfer. Mechanical agitation is not possible, since shear stress causes disorganization and callus formation with consequently lowered productivity. Furthermore, inoculation causes difficulties,because the roots cannot readily be rendered into a pumpable form. For the same reason, sampling of the roots during fermentation is not possible. Therefore, alternative methods for monitoringgrowth have to be found, e.g., measurement of nutrient consumption or conductivity (Taya et al., 1989b). On the other hand, the medium of hairy root cultures remains Newtonian and the roots are "self-immobilized", which make the continuous operation of fermenters easier (Wilson et al., 1987; Hilton et al., 1988). Despite the unusual biotechnicalproperties of hairy root cultures, they have successfullybeen grown in fermenters and maintained viable in the nongrowingstatefor extended periods in continuous fermentation modes (Table 11).The geneticand biochemical stabilities of hairy roots facilitate process design and scale-up. In contrast to cell suspension cultures, cultivation of hairy root cultures in fermenters has not affected their productivity even on a large scale (Hilton and Rhodes, 1990; Wilson et al., 1990).
Pmg., 1993, Vol. 9, No. 1
Much work on fermenter and process development has been carried out by Rhodes and co-workers for the cultivation of hairy root cultures of N. rustica and D. stramonium (Rhodeset al., 1986;Wilson etal., 1987;Hilton et al., 1988; Hilton and Rhodes, 1990;Wilson et al., 1990). A column fermenter (1.5 L)with a special stainless steel basket for the inoculum was developed to improve the contact between roots and medium. With this configuration, high final biomass densities were obtained for both D. stramonium (40 g dry wt/L in 60 days) and N. rustica (42 g dry wt/L in 64 days) in continuous operation. Furthermore, the continuous operation led to a 3.4-fold increase in the production of nicotine in N . rustica cultures (Wilson et al., 1987). Greatly improved growth characteristics were further obtained by the application of a novel reactor configuration,an impeller-mixedvessel with a mesh to distance the roots from the stirrer mechanism (Hilton and Rhodes, 1990). In batch fermentation (12 L), about 10 g dry wt/L was obtained in 40 days with a productivity of 1.9 mg of hyoscyamine/L/day. By operating the fermenter in the continuous mode, the biomass yield was improved 2-fold, with a consequent improvement in productivity to 6.4 mg of hyoscyamine/L/day, although the product remained intracellular (Hilton and Rhodes, 1990). Recently, preliminary experiments on the cultivation of D. stramonium hairy root cultures in a pilot plant fermenter (500 L) have also been reported (Wilson et al., 1990). Taya et al. (1989b) studied the growth characteristics of hairy root cultures of Armoracia rusticam in different reactor systems. In addition to free roots cultivated in a stirred-tank reactor and an air-lift column, the growth of hairy roots immobilized in reticulate polyurethane foam was tested in three different reactors without mechanical stirring. The best result (11g dry wt/L) was obtained in a 31-day culture in the air-lift column with immobilized roots (Table 11). Buitelaar e t al. (1991) tested three fermenter types, a liquid-impelled loop reactor, a stirred-tank reactor, and a bubble column, for growth and thiophene production by T.patula in a two-liquid-phasesystem. Also in this study, the use of a stirred tank reactor proved to be unsuccessful because of shear damage. The best productivity was obtained with a bubble column using hexadecane as the dispersed phase (Table 11). Shimomura e t al. (1991b) used a 2-L air-lift reactor connected with a column containing a polymeric adsorbent for continuous production of shikonin by hairy root cultures of L. erythrorhizon. The shikonin production began after 20 days and increased up to 5.9 mg/day (Table 11). In addition, air-lift-type fermenters have been successfully applied by, for example, Yoshikawa and Furuya (1990) for glycosylation of 2-phenylpropionic acid with P. ginseng hairy roots and by Sauerwein et al. (1991a) for production of hernandulcin with hairy root cultures of L. dulcis. Furthermore, Davioud et al. (1989b) used a fermenter with mechanical stirring to produce indole alkaloids with hairy root cultures of Catharanthus trichophy 1 Ius. When considering economically feasible production of secondary metabolites by employing hairy root cultures, a process based on continuous operation with release of secondary product(s) into the medium would be essential for lower priced products, although with more expensive products batch operation with standard harvesting of biomass might be sufficient (Hamill et al., 1987). Several methods have been used to achieve secondary product release in hairy root cultures. The most successful
Bbtechnol. Rug., 1993, Vol. 9, No. 1
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Table 11. Fermenter Studies Carried Out with Hairy Root Cultures species
vessel ~~
N.rustica
D.stramonium
T. patula C. sepium A. belladonna A. rusticam, free cells
vol
+
production
9
1.5 L
13 days in batch mode 16 days in cont. mode continuous mode, 64 days
1.5 L
continuous mode, 60 days
40
12 L
300 mL
batch mode, 35-40 days 8.5 continuous mode, 34 days 20.4 two-liquid-phasesystem, 7.1 (browning) batch mode, 2&30 days 5.2 batch mode, 15 days 11.8 3.3 batch mode, 25 days 4.8
300 mL 300 mL
batch mode, 31 days
2.8 11
nd nd
batch mode, 48 days
6.8
nd
batch mode, 34 days
4.7
nd
LH 500 series (air-sparged) air-sparged glass column with inoculum cage air-sparged glass column with inoculum cage STR (New Brunswick)
880 mL
STR (Applikon)
1.3 L
bubble column STR with a mesh STR with a mesh stirred tank
6L 1L
air-lift column air-lift column A. rusticam, immobilized cells medium-trickling column medium-fillin and drawing cofumn with siphon air-lift reactor C. rosew air-lift column connected L.erythrorhizon with a column containing XAD-2
biomaea yield, g/L
culture time
~
42
6-13 1 L, 3.5 L batch mode, 28 days 2L continuous mode, 220 days
techniques reported have been the ones that use continuous-flow-through of fresh medium for N. rustica hairy root cultures (Wilson et al., 1987), where 76% of total nicotine could be recovered from the medium, as well as techniques that use two-liquid-phasefermentation of hairy root cultures of T. patula (Buitelaar et al., 19911, where up to 70% of the thiophenes produced were excreted into the organic solvent phase, in contrast to 1%when the normal one-phase system was applied. Furthermore, polymeric adsorbents were demonstrated to be suitable for recovery of alkaloids from the medium of N. rustica hairy root cultures, where the stabilizing effect of the polymer led to a 60 % increase in overall yield (Robins et al., 1988). Polymeric resins were also employed to adsorb shikonin from L. erythrorhizon hairy root cultures. The production was increased 2-fold, with 85-90% of the product found in the resin (Shimomura et al., 1991b). The exploitation of secondary metabolite formation of hairy root cultures in bioreactors still requires further development. However, the production of secondary products using hairy root cultures is apparently near commercialization in Japan, where Furuya and co-workers recently reported the large-scale production of ginseng extracts using hairy root cultures of P. ginseng (20-ton cultivation tank; Scheidegger, 1990).
Future Prospects Hairy root cultures offer good prospects for the development of commercial biotechnical processes for the production of valuable secondary plant products, both because of their high potential for product formation and genetic manipulation and because of their growth characteristics and genetic stability. However, the use of hairy roots is limited to those products which are synthesized in roots. The choice of products can be extended by the use of green, photoautotrophic roots, with which it is possible to produce metabolites whose synthesis is associated with chloroplasts (Flores et al., 1988; Saito et al., 1990; Sauerwein et al., 1991a). Furthermore, a related technique, in which infection with an Agrobacterium tumefaciens mutant is used for the establishment of transformed, differentiating shoot cultures, makes it possible to extend the approach used
32.7 mg of nicotine (76%released) 94 mg (76%released)
reference Rhodes et al. (1986) Wilson et al. (1987) Wilson et al. (1987)
0.61%dw hyoscyamine 0.87 % dw hyoscyamine 1.62%dw thiophenes 1.68%dw thiophenes 0.3 % dw alkaloids 1.32%dw alkaloids nd
Hilton and Rhodes (19%)) Buitelam et al. (1991) Jung and Tepfer (1987) Taya et al. (1989b)
0.15% dw indole alkaloids Toivonen et al. (1990) max. 5.9 mgtday Shimomura et al. (1991b) (90% released)
with hairy root cultures to products synthesized in green tissues. For example, transformed shoot cultures of Mentha sp. were recently reported to produce volatile oils (terpenes) at levels comparable with those obtained in the parent plants (Spencer et al., 1990).
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