Critical Reviews in Biotechnology, 27:29–43, 2007Copyright ©c Informa HealthcareISSN: 0738-8551 print / 1549-7801 onlineDOI: 10.1080/07388550601173918

Hairy Root Culture for Mass-Productionof High-Value Secondary Metabolites

Smita Srivastavaand Ashok K. SrivastavaDepartment of BiochemicalEngineering and Biotechnology,Indian Institute of Technology,New Delhi, India

ABSTRACT Plant cell cultivations are being considered as an alternative toagricultural processes for producing valuable phytochemicals. Since many ofthese products (secondary metabolites) are obtained by direct extraction fromplants grown in natural habitat, several factors can alter their yield. The use ofplant cell cultures has overcome several inconveniences for the production ofthese secondary metabolites. Organized cultures, and especially root cultures,can make a significant contribution in the production of secondary metabo-lites. Most of the research efforts that use differentiated cultures instead of cellsuspension cultures have focused on transformed (hairy) roots. Agrobacteriumrhizogenes causes hairy root disease in plants. The neoplastic (cancerous) rootsproduced by A. rhizogenes infection are characterized by high growth rate, ge-netic stability and growth in hormone free media. These genetically transformedroot cultures can produce levels of secondary metabolites comparable to thatof intact plants. Hairy root cultures offer promise for high production and pro-ductivity of valuable secondary metabolites (used as pharmaceuticals, pigmentsand flavors) in many plants. The main constraint for commercial exploitationof hairy root cultivations is the development and scaling up of appropriatereactor vessels (bioreactors) that permit the growth of interconnected tissuesnormally unevenly distributed throughout the vessel. Emphasis has focused ondesigning appropriate bioreactors suitable to culture the delicate and sensitiveplant hairy roots. Recent reactors used for mass production of hairy roots canroughly be divided as liquid-phase, gas-phase, or hybrid reactors. The presentreview highlights the nature, applications, perspectives and scale up of hairyroot cultures for the production of valuable secondary metabolites.

KEYWORDS hairy roots, secondary metabolites, bioreactors

INTRODUCTIONThe large and diverse group of chemicals produced by plants, which include

alkaloids, anthraquinones, anthocyanins, flavanoids, saponins and terpenes, hasplayed an important role in the pharmaceuticals, cosmetics, perfumeries, dye-ing and flavor industry. Plants produce many of these compounds throughsecondary metabolism. Secondary metabolites are not essential to plant growthand hence are produced in small amounts (Kim et al., 2002b). These often ac-cumulate in specialized tissues, e.g. trichomes at distinct developmental stages,

Address correspondence to Ashok K.Srivastava, Department of BiochemicalEngineering and Biotechnology,Indian Institute of Technology, HauzKhas, New Delhi 110016, India. E-mail:ashokks@dbeb.iitd.ernet.in

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making their extraction, isolation and purification dif-ficult (Kim et al., 2002b). These compounds usuallyhave very complicated structures and/or exhibit chi-rality (Mukundan et al., 1997). Consequently, in manycases organic synthesis is not cost effective, and ex-traction from field-grown plants has been the majormethod used to economically obtain these importantsecondary metabolites (Balandrin et al., 1985; Dicosmoand Misawa, 1995). Depending on the plant species,traditional agricultural methods often require monthsto years to obtain a crop. Furthermore, the levels of sec-ondary metabolites from plants are affected by manyfactors, including pathogens and climate changes. Inaddition to factors mentioned above, decreased plantresources and high labor cost involved in the extrac-tion of plant secondary metabolites have acceleratedthe use of plant cell cultivation for their effective pro-duction. Plant cell culture is not affected by changesin environmental conditions, so improved productionmay be available in any place or season. Studies on theproduction of useful metabolites by plant cell cultiva-tion have been carried out on an increasing scale sincethe 1950s. Despite considerable efforts, only a few com-mercial processes have been achieved using plant cellcultures (Kieran et al., 1997). The biggest challenge ofproducing secondary metabolites from plant cell sus-pension cultures is that secondary metabolites are usu-ally produced by specialized cells and/or at distinct de-velopmental stages (Balandrin et al., 1985; Mukundanet al., 1997). Some compounds are not synthesized if thecells remain undifferentiated (Berlin et al., 1985). Theundifferentiated plant cell cultures being genetically un-stable often lose, partially or totally, their biosyntheticability to accumulate secondary products (Rokem andGoldberg, 1985; Charlwood and Charlwood, 1991).

A new route for enhancing secondary metabolite pro-duction in tissue culture system is by transformationof desirable plant species using the natural vector sys-tem Agrobacterium rhizogenes. It is the causative agent ofhairy root disease in plants (Giri and Narasu, 2000;Bourgaud et al., 2001). In nature, the gram-negative soilbacterium A. rhizogenes genetically engineers dicotyle-donous plant species into chemical producers of anAgrobacterium food source (opines). This transforma-tion process leads to the emergence of “hairy roots” atthe site of infection of the plant (Shanks and Morgan,1999). These genetically transformed (hairy) roots arecapable of unlimited growth in culture media free ofgrowth hormones.

Hairy root cultures of a number of dicotyle-donous/monocotyledonous plants have been estab-lished and found to produce the same secondarymetabolites as natural roots and hence offer apromising system for secondary metabolite production(Mukundan et al., 1997; Doran, 2002; Rudrappa et al.,2005). Tepfer (1990) has listed some of the plant speciesin which fast-growing productive hairy root lines havebeen established.

The greatest advantage of hairy roots is that theyoften exhibit about the same or greater biosyntheticcapacity for secondary metabolite production as com-pared to their mother plants (Kim et al., 2002a, b). Manyvaluable secondary metabolites are synthesized in rootsin vivo, and often the synthesis is linked to root dif-ferentiation (Flores et al., 1999). Members of a num-ber of families including Balsaminaceae, Chenopodiaceae,Compositae, Juglandaceae, Labiatae, Moraceae, Ranuncu-laceae, Solanaceae, Asteraceae, Cucurbitaceae, Plumbagi-naceae, Apocynaceae, Asclepiadaceae and Umbelliferae havebeen reported to induce hairy root disease symptomson getting wounded and inoculated with A. rhizogenesin a green house (De Cleene and De Ley, 1981; Dawdaet al., 1997). Even in cases where a particular secondarymetabolite accumulates only in the aerial part of anintact plant, hairy root cultures have been shown toaccumulate the same metabolite (Wallaart et al., 1999).Moreover, transformed roots are often able to regener-ate whole viable plants and maintain their genetic sta-bility during continuous subculturing and plant regen-eration. Hairy root cultures are also known to produce aspectrum of secondary metabolites that are not presentin the parent plant (Veeresham, 2004). Furthermore, atransgenic root system offers tremendous potential forintroducing additional genes along with the Ri plas-mid, especially with modified genes, into plant cellswith A. rhizogenes vector systems. Hairy root cultureshave turned out to be a valuable tool to study the bio-chemical properties and the gene expression profile ofmetabolic pathways. Moreover, hairy root culture canbe used to elucidate the intermediates and key enzymesinvolved in the biosynthesis of secondary metabolites(Hu and Du, 2006). Various advantages of hairy root cul-ture highlighted above have led to its promising role inthe mass-production of high-value secondary metabo-lites. Although scale up of hairy roots is not an easy taskdue to its complex features, various processes are cur-rently under investigation for making its commercialapplication feasible. Despite the few attempts reported

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in the literature (Wysokinska and Chmiel, 1997; Shanksand Morgan, 1999; Giri and Narasu, 2000; Kim et al.,2002b), a greater insight is required to deal with the chal-lenges of commercial application of hairy root culturetechnology for production of plant secondary metabo-lites. To comprehend the same, the present review dealsin depth with new perspectives on the prospects andchallenges in the effective production of secondarymetabolites from hairy root culture technology. It dis-cusses the importance of the use of various cultivationmethods and the different bioreactors configurationssuitable for the scale-up of hairy root culture.

HAIRY ROOT INDUCTIONDiscovery

The name “hairy root” was first introduced in theliterature by Steward et al. (1900). Riker et al. (1930)later described and named the hairy-root-causing mi-croorganism as Phytomonas rhizogenes, which was laterrenamed Agrobacterium rhizogenes. This conclusion wasaccepted and given wide recognition by many otherresearchers (White, 1972). The first directed transfor-mation of higher plants using A. rhizogenes was madeby Ackermann in 1973 (Ackermann, 1977). A largenumber of small, fine, hairy roots covered with roothairs originate directly from the explant in response toA. rhizogenes infection (Tepfer and Tempe, 1981) andhence the term “hairy root”. Porter (1991) reported thatmore than 450 species of many different genera andfamilies are known to be susceptible to the infectionby A. rhizogenes (Hamill and Lidgett, 1997); since thenmany more additions have been made to the list.

MechanismInvasion of dicotyledonous plant tissues by A. rhizo-

genes soil bacteria usually occur at a wounded site, pos-sibly caused by insect or mechanical damage. Woundedsite produces phenolic compounds that attract A. rhi-zogenes by chemotaxis, which subsequently infects theplant cell at the wounded site. This activity causeshairy root disease (a number of small roots protrudeas fine hairs at the infection site and proliferate rapidly)(Balandrin et al., 1985). This phenotypic response (Hairyroot) results from the insertion of T-DNA (transferDNA) into the plant genome carried by the bacterialRi-plasmid (root inducing plasmid), which codes forauxin synthesis (Petit et al., 1983; Ambros et al., 1986).

The integrated segment, T-DNA, also contains genesfor opine biosynthesis, which are used by A. rhizogenesas the sole carbon and nitrogen sources for furthergrowth. Products of virulence (vir) genes located onnon-transferred segment of the Ri plasmid (root induc-ing plasmid) are responsible for excision of the T-DNAfor transfer into the plant cell, and possibly for chro-mosomal integration in the nucleus of the recipient cell(Giri and Narasu, 2000).

ConfirmationThe transformation of a plant cell with A. rhizogenes

can be confirmed by typical transformed root morphol-ogy exhibited by hairy roots obtained after infectionand their transformed regenerants. As described earlier,transformed roots have an altered phenotype, profusionof laterals, and show lack of geotropism (Tepfer, 1984).Also, the transformed regenerants of hairy roots inheritan aberrant phenotype in having wrinkled leaves andshortened internode compared to their normal coun-terparts (Chilton et al., 1982; Ooms et al., 1985; Guercheet al., 1987).

Since the opine synthesis in A. rhizogenes infectedplant cells is encoded by T-DNA of Ri plasmid (Whiteet al., 1982, 1985), its detection serves as an effectivebiochemical marker in elucidating the transformed na-ture of the cultured root tissue (Petit et al., 1983; Tepfer,1984). There are literature reports in which the trans-formation of hairy root culture has been confirmed bythe detection of opines through paper electrophoresis(Bakkali et al., 1997; Sasaki et al., 1998; Bais et al., 2001).Although synthesis of opines is a firm indication thathairy roots are indeed transformed, the expression ofopine genes in hairy root tissue may become unstablewith time (Kamada et al., 1986).

T-DNA localization in the host plant genome actsas a reliable genetic marker to confirm transforma-tion (Mukundan et al., 1997). There are a number oftechniques available to demonstrate and locate T-DNAincorporation in the host plant chromosomal DNA.These include localization of T-DNA by southern hy-bridization (White et al., 1982). It remains one of theearliest used methods and is also still widely employedtoday. The transformation of hairy root cultures ofDacus carrota (David et al., 1988), Cinchona ledgeriana(Hamill et al., 1989), Nicotiana rustica (Rhodes et al.,1994), Artemisia annua (Chen et al., 1999), Cucurbita pepoL. (Leljak-Levani et al., 2004) are some of the examples of

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confirmation by southern hybridization. Other meth-ods include verification of the transformed nature of atissue by screening for the presence of a foreign genesequence by DNA “blot dotting” (Draper and Scott,1988), localization of T-DNA in plant chromosome tis-sue by in situ hybridization (Ambros et al., 1986; Donget al., 1992), and verification of transgenes as well asdetermination of changes in a particular gene sequenceresulting from tissue culture by polymerase chain reac-tion (PCR) (Jaziri et al., 1994; Dong et al., 1992). Trans-formed root cultures of (hairy root) Brugmansia candidaand Ginkgo biloba L have been confirmed using PCR(Giulietti et al., 1993; Ayadi and Tremouillaux-Guiller,2003); however, it should be noted that it is importantto verify that the plants are cured of the agrobacteriumto avoid false positives if PCR is used.

CharacteristicsHairy roots are characterized by a high degree of lat-

eral branching, profusion of root hairs and absence ofgeotropism (Tepfer, 1984). They often grow as fast as orfaster than normal roots (untransformed) due to theirextensive branching, resulting in the presence of manymeristems (Charlwood and Charlwood, 1991; Floreset al., 1999) and they do not require phytohormones inthe medium (Rao and Ravishankar, 2002). The increasein the number of branches is approximately logarithmicduring the early stages of growth and thus the overallpattern of growth is similar to cell suspension cultures(Flores and Filner, 1985; Flores, 1986; Flores and Curtis,1992). Owing to the highly organized and small-celledregion of the meristem in each lateral, cell cycle timesfor hairy roots average less than 10 h (Gould, 1982).Hairy roots do not necessarily require conditioning ofthe medium but physical and chemical conditioningis known to result in improved growth and metabo-lite production (Rhodes et al., 1987; Veeresham, 2004).Stable integration of Ri T-DNA (root inducing transferDNA) into host plant genome accounts for the geneticstability of transformed root cultures. The most impor-tant characteristic of transformed roots is their capa-bility of synthesizing secondary metabolites specific tothat plant species from which they have been devel-oped (Doran, 1989; Flores, 1986, 1992). They exhibitbiochemical stability that leads to a high growth ratewith a stable and high level of production of secondarymetabolites (Kamada et al., 1986; Aird et al., 1988a, b).

SECONDARY METABOLITEPRODUCTION FROM HAIRY ROOTSVarious advantages of hairy root culture over cell sus-

pension culture include genotypic and biochemical sta-bility, cytodifferentiation and growth in hormone freemedium. These factors play a vital role during secondarymetabolite production. Fast growth, low doubling time,ease of maintenance of hairy roots and their ability tosynthesize a large range of chemical compounds offer anadditional advantage as a continuous source for the pro-duction of valuable secondary metabolites (Bourgaudet al., 2001). A number of secondary metabolites havebeen reported to be produced from hairy root cultures(Giri and Narasu, 2000). Hairy root cultures often ex-hibit about the same or greater biosynthetic capacityfor secondary metabolite production as compared totheir normal roots (Table 1). Hairy roots have also beenobserved to synthesize novel secondary metabolites,which are not present in the untransformed (control)tissue (Banerjee et al., 1995). Even in cases where sec-ondary metabolites accumulate only in the aerial part ofan intact plant, hairy root cultures have been shown toaccumulate the metabolites. For example, lawsone nor-mally accumulates only in the aerial part of the plant,but hairy roots of Lawsonia inermis grown in half- or full-strength MS medium (Murashige and Skoog, 1962) canproduce lawsone under dark conditions (Bakkali et al.,1997). Similarly, artemisinin was thought to accumu-late only in the aerial part of the Artemisia annua plant(Wallaart et al., 1999) but several reports have shown thathairy roots can also produce artemisinin (Weathers et al.,1994; Jaziri et al., 1995; Liu et al., 1999; Giri et al., 2000).

Hairy root culture follows a definite growth pattern;however, secondary metabolite production may or maynot be growth related. Secondary metabolite biosynthe-sis in transformed roots is genetically controlled (Hamilland Rhodes, 1988) but it is strongly influenced by nutri-tional and environmental factors (Hilton and Rhodes,1993). Other factors like elicitors (Pitta-Alvarez et al.,2000), biotransformations of precursors and genetic ma-nipulations through the Ri plasmid of A. rhizogenes alsoinfluence the yield of secondary metabolites from hairyroots (Rao and Ravishankar, 2002). To obtain a high-density culture of roots, the culture conditions shouldbe maintained at the optimum level. The compositionof the culture medium affects secondary metabolite pro-duction (De-Eknamkul and Ellis, 1984). Variables ex-amined for their influence on growth and secondary

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TABLE 1 Hairy Root in which Specific Metabolites are Synthesized at Levels Higher than in Untransformed Tissue

Plant species Secondary metabolite Reference

Atropa belladonna Scopolamine Bonhomme et al., 2000A. belladonna Atropine and hysocyamine Jung and Tepfer, 1987Cinchona ledgeriana Quinine, quinidine, and cinchonidine Hamill et al., 1989Datura innoxia Hysocyamine and scopolamine Shimomura et al., 1991aD. quercifolia Scopolamine and hysocyamine Dupraz et al., 1994D. candida Scopolamine and hysocyamine Christen et al., 1991Duboisia leichhardtii Scopolamine Mano et al., 1989Fagopyrun esculentum (+) Catechin Trotin et al., 1993

(−) epicatechin-3-O-gallateprocyanidin B2-3’-O-gallate

Hyoscyamus niger Hysocyamine and scopolamine Shimomura et al., 1991aHyoscyamus niger Scopolamine Zhang et al., 2004Rubia tinctoria Anthraquinone Saito et al., 1991Solanum khasianum Solasodine Jacob and Malpathak, 2004Tagetes patula Thiophene Croes et al., 1989Valeriana officinalis Valpotriates Granicher et al., 1992

metabolite production from hairy roots include differ-ent basal media (Christen et al., 1992), sucrose level(Uozumi et al., 1993), exogenous supply of growth hor-mone (Bais et al., 2001), nature of the nitrogen sourceand their relative amounts (Norton and Towers, 1986),and phosphate concentration (Taya et al., 1994). Physi-cal factors including light (Hirata et al., 1991; Yu et al.,2005), temperature (Hilton and Rhodes, 1994; Yu et al.,2005), presence of chemicals inducing physical stress(Sim et al., 1994), and magnetic field induction (Katoet al., 1989) have also been reported to affect secondarymetabolite production from hairy roots. Betacyanin re-lease from hairy roots of Beta vulgaris was achieved byoxygen starvation (Giri and Narasu, 2000). Addition ofXAD-2, liquid paraffin stimulated the production ofshikonin (Shimomura et al., 1991b). Permeabilizationtreatment using Tween-80 (Polyoxy ethylene sorbilanemonolaurate) released a high yield of hyoscyaminefrom roots of Datura innoxia without any detrimentaleffects (Boitel et al., 1995). Treatment with 5 mM hy-drogen peroxide induced a transient release of tropanealkaloids from transformed roots without affecting vi-ability (Lee et al., 1998). The fact that individual hairyroots may have different requirements for nutrient con-ditions suggests that the culture conditions should beoptimized separately for each species and for individualclones. Despite such attempts sometimes the efficiencyof secondary metabolite production is not as desired.Metabolic engineering offers new perspectives for im-proving the production of secondary metabolites by

the over-expression of single genes in hairy root cul-ture. This approach may lead to an increase of someenzymes involved in metabolism and consequently re-sults in the accumulation of the target products (Hu andDu, 2006). The hairy roots of A. belladonna transformedwith the rabbit P450 2E1 gene displayed increased lev-els of the metabolites (Banerjee et al., 2002). Catharan-thus roseus hairy roots harboring hamster 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase (HMGR)cDNA without the membrane-binding domain werefound to produce more ajmalicine and cantharanthineor serpentine and campesterol than the control (Ayora-Talavera et al., 2002). Secondary metabolite productionhas also been improved by over-expression of enzymesthat are already located in a plant (Hu and Du, 2006).The tobacco putrescine N -methyltransfersase (PMT)was transformed into Datura metel L. and Hyoscyamusmuticus L. (Moyano et al., 2003). The enzyme catalyzedthe first committed step in the tropane alkaloid path-way and stimulated the growth of transgenic roots andthe accumulation of tropane alkaloid. Oxygen defi-ciency is a usual problem in hairy root culture caused bypoor mixing and mass transfer conditions. To improvethe low oxygen conditions that affect growth duringfermentation, two enzymes, namely (ADH) and pyru-vate decarboxylase, were transferred into the hairy rootsof Arabidopsis thaliana L. The transformant root linesmaintained a similar growth rate under conditions oflow oxygen to the rate achieved with full aeration (Shiaoet al., 2003).

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SCALE-UP OF SECONDARYMETABOLITE PRODUCTION FROM

HAIRY ROOTSThe vast potential of hairy root cultures as a sta-

ble source of biologically active chemicals has focusedthe attention of the scientific community for its ex-ploitation. Scaling up of hairy roots in novel biore-actors can provide the best conditions for optimumgrowth and secondary metabolite production, compa-rable to or higher than that in native roots. Thoughthe need for developing bioreactors suitable for thehairy root cultivation has long been recognized, rootcultures present unique challenges (Wilson et al., 1987).The complex fibrous structure of the roots makes thegrowth analysis and development of a large-scale cul-ture system difficult. Hairy root growth is not homo-geneous, which affects the reactor performance. Fur-thermore, the hairy root morphology is quite plastic asthe roots respond to the changes in the local environ-ment. Changes in morphology, including changes inthe density and length of the root hairs, directly affectthe secondary metabolite production from hairy roots(Carvalho et al., 1997). Thus, bioreactor design for rootcultures is a balancing act between the biological needsof the tissues, without inducing an additional, unde-sirable biological response (Wyslouzil et al., 2000). Re-views on hairy roots by Shanks and Morgan (1999) andGiri and Narasu (2000) briefly discuss the importanceof the use of bioreactors for hairy root cultures. Me-chanical agitation causes wounding of hairy roots andleads to callus formation. Due to branching, the rootsform an interlocked matrix that exhibits resistance tonutrient flow. Hairy roots are hetrotrophic, respiratoryorganisms that rely on oxygen for energy generationand other metabolic functions. Substantial progress hasbeen made in understanding the mechanisms of oxygenlimitation, one of the principle challenges for large-scalegrowth of hairy root cultures (Curtis et al., 2001). Be-cause of the solid phase nature of the roots and thedevelopment of oxygen gradients within root tissues,relatively small reductions in the dissolved oxygen con-centration in the medium can lead to a significant de-crease in growth rate and may also affect the synthesisof certain secondary metabolites. In fact, hairy roots canbe oxygen limited even in shake flask cultures (Kanok-waree and Doran, 1997). Restriction of nutrient oxygendelivery to the central mass of tissue gives rise to a pocketof senescent tissues. Mass transfer resistances near the

liquid and solid boundary affect the oxygen delivery tothe growing hairy roots (Asplund and Curtis, 2001).

Thus, exploitation of hairy root culture as a sourceof bioactive chemicals depends on the development ofsuitable bioreactor system where several physical andchemical parameters (nutrient availability, nutrient up-take, oxygen, and hydrogen depletion in the medium,mixing and shear sensitivity) must be taken into ac-count. The design of bioreactors for hairy root cul-tures should also take into consideration factors suchas the requirement for a support matrix and the pos-sibility of flow restriction by the root mass in certainparts of the bioreactor. Several bioreactor designs havebeen reported for hairy root culture taking into con-sideration the above factors that permit the growth ofinterconnected tissue unevenly distributed throughoutthe culture vessel. Reactors used to culture hairy rootscan roughly be divided into three types: liquid-phase,gas-phase, or hybrid reactors that are a combination ofboth (Kim et al., 2002b).

Liquid-Phase ReactorsIn liquid-phase reactors, roots are submerged in the

medium, and therefore the term “submerged reactors”is commonly applied. Some of the reactor configura-tions belonging to this category in literature are stirredtank (Wilson et al., 1987; Taya et al., 1989; Buitelaar et al.,1991; Kim et al., 2002b; Jeong et al., 2002), bubble col-umn (Buitelaar et al., 1991; Tescione et al., 1997; Kimet al., 2001, 2002a; Suresh et al., 2004), airlift (Taya et al.,1989), liquid-impelled loop (Buitelaar et al., 1991), andsubmerged convective flow (Carvalho and Curtis, 1998;Kim et al., 2002b) reactors, etc. Those successfully usedfor hairy root cultivation are explained as follows.

Stirred Tank ReactorThis type of bioreactor used to culture hairy roots

includes impeller or turbine blades, which facilitatemass transfer to the roots supported on a wire mesh(Figure 1) (Kim et al., 2002b). Though the stirred tankreactor is used for high-density cultures, it is usuallynot found suitable for hairy root cultivation becauseof the wound response and callus formation that re-sults from the shear stress caused by the impeller rota-tion (Taya et al., 1989; Hilton and Rhodes, 1990). Theenvironment inside the reactor vessel is characterizedby high shear, which damages the roots. Drawbacks of

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FIGURE 1 Stirred tank reactor (Kim et al., 2002b).

the stirred tank reactor for hairy root cultivation alsoinclude poor control of critical gas concentrations, in-sufficient nourishment of the biomass at high concen-trations due to poor liquid circulation and chemicalgradients in the medium when the reactor is denselypacked. Secondary metabolites are generally producedat low levels, so a high ratio of medium to metabolitevolume can complicate the separation of the products.However, recently some modified stirred tank reactorshave been developed that have large impellers and baf-fles that are agitated at a very low speed to provideproper mixing and a low shear stress environment (Giriand Narasu, 2000). Modification also includes additionof a steel cage or mesh inside a stirred tank reactor toisolate the roots from the impeller (Kondo et al., 1989;Hilton and Rhodes, 1990).

Airlift BioreactorsThese are similar to stirred tank reactors but lack an

impeller. It is a gas liquid bioreactor, where compressedair is used for aeration and agitation and is based ondraught tube principle. Hairy roots require a compara-tively low oxygen sparge rate of about 0.05–0.4 vol ofair/vol of liquid/min. Humidified air is passed througha glass grid that functions as aerator (Giri and Narasu,2000). This is useful for mixing and oxygenation. Asshown in Figure 2, a reverse teardrop-type glass vesselhas been used as an airlift reactor, in which humidi-fied air was introduced into the bottom of the reac-tor through a sintered glass sparger to culture hairyroots of carrot (Kondo et al., 1989). Shimomura et al.(1991b) used an airlift reactor connected to a columncontaining a polymorphic adsorbent for the continu-ous production of shikonin by hairy root cultures of

FIGURE 2 Airlift reactors (Kondo et al., 1989).

Lithospermum erythrorhizon. Despite the fact that airliftreactors have been successfully used for culturing differ-ent hairy roots (Taya et al., 1989; Tescione et al., 1997),there are some demerits associated with them. These in-clude development of dead zones inside the vessels dueto insufficient mixing and non-uniform nutrient sup-ply caused by high biomass density. These limitationscan have a negative impact on hairy root growth andsecondary metabolite production, and therefore modi-fications are required in the design. Novel airlift reactorfittings have been designed and used to more easily ac-complish tissue inoculation, distribution, and harvestduring the kinetic study of Solanum chrysotrichum hairyroots (Caspeta et al., 2005).

Bubble Column ReactorLike an airlift bioreactor, in a bubble column the

bubbles create less shear stress, so that it is useful forthe growth of organized structures such as hairy roots(Figure 3) (Kim et al., 2002a). In this case, the bubblingrate needs to be gradually increased with the growthof hairy roots. Moreover, the division of a bubble col-umn into segments, and installation of multiple sparg-ers increases the overall mass transfer (Buitelaar et al.,1991). Buitelaar et al. (1991) tested growth and thio-phene production by Tagetes patula hairy roots in threedifferent types of reactors and the best productivity wasfound with a bubble column bioreactor. Sim and Chang(1993) proposed a two-phase bubble column reactorfor increased shikonin production from hairy roots ofLithospermum erythrorhizon. Hairy root cultures of redbeet (Beta vulgaris) were grown in a 3-liter bubble col-umn reactor for studying enhancement in betalaine pro-duction by elicitor addition (Suresh et al., 2004).

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FIGURE 3 Bubble column reactor (Kim et al., 2003).

Convective Flow ReactorThis reactor consists mainly of a stirred tank and a

tubular culture chamber (Figure 4) (Kim et al., 2002). Al-though the convective flow reactor showed improvedperformance compared to a bubble column reactor, itmay not be a realistic large-scale system due to the pres-sure required to circulate the culture medium at a veloc-ity high enough to overcome the flow resistance of theroot bed. Hyoscyamus muticus hairy roots cultivated in aconvective flow reactor gave a 79% increase in biomassas compared to that obtained in a bubble column re-actor (Carvalho and Curtis, 1998). Reactors similar tothe convective flow reactor have been successfully usedto culture hairy roots. Kino-Oka et al. (1999) culturedred beet hairy roots in a single column reactor with anoptimum superficial velocity of 15 m h−1. Similarly,a packed bed recirculation reactor has been used toculture hairy roots of Atropa belladonna, which facili-tated minimization of the liquid–solid hydrodynamicboundary layer at the root surface (William and Doran,1999).

FIGURE 4 Convective flow reactor (Kim et al., 2002b).

FIGURE 5 Turbine blade reactor (Kondo et al., 1989).

Turbine Blade ReactorThis is a combination of an airlift and a stirred tank

reactor. Here, cultivation space is separated from agita-tion space by a stainless steel mesh, so that hairy rootsdo not come in contact with the impeller and humid-ified air is introduced from the bottom and dispersedby an impeller that agitates the medium. Growth prop-erties of carrot hairy root cells in various bioreactorswere investigated and the most efficient growth was ob-served in turbine-blade reactor (Figure 5). Maximumgrowth rate of 0.63 g/l per day and 10 g/l dry cell masswas obtained after 30 days, while only 4 g/l was ob-tained in shake flask cultures (Kondo et al., 1989). It isan efficient reactor configuration for the developmentof hairy roots (Uozumi et al., 1993).

Rotating Drum BioreactorA reactor configuration that operates on the princi-

ples similar to a fill and drain reactor is the rotatingdrum (Figure 6) (Kondo et al., 1989). This consists of adrum-shaped container mounted on rollers for supportand rotation. The drum is rotated at only 2–6 rpm tominimize the shear pressure on the hairy roots. Hairyroots adhere to the walls of the reactor and as the drumrotates, the roots tend to break up. To overcome this

FIGURE 6 Rotating drum bioreactor (Kondo et al., 1989).

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problem, a polyurethane foam sheet is fixed onto thesurface of the drum, to which the hairy roots get at-tached. This results in higher growth without any de-tachment. The rotating motion of the drum reactorfacilitates proper mixing of gas-liquid in the reactors,thereby promoting efficient oxygen transfer to biomassat high densities. It imparts less hydrodynamic stress.Kondo et al. (1989) used this system to culture hairyroots of carrot. A cylindrical glass vessel was used as areactor. The drum reactor was rotated at 5 rpm on a cellrotator and humidified air was supplied to the mediumthrough a submerged nozzle. In order to use the im-mobilized system, a polyurethane foam sheet was fixedon the inner wall by stainless steel wires to be used as asupport. After 30 days of culture, 10 g/1 of hairy rootbiomass was obtained and the maximum growth ratewas found to be 0.61 g/1 per day for the culture. Themajor disadvantage of this type of reactor is its depen-dence on the high-energy requirement for large-scaleoperation (Ravishankar and Venkataraman, 1990).

LimitationsLiquid-phase reactors are associated with limitations

and this has resulted in gas-phase and hybrid reac-tors being favored more recently. In liquid-phase reac-tors, the increased root hair density increases the pres-sure drop across the reactor and limits mass transport(Carvalho et al., 1997). A proliferation of root tips com-pared with elongated cells can increase oxygen demandtenfold. This extra aeration required to meet the oxygendemand of the growing roots in a liquid phase reactorbecomes more difficult because the pressure drop acrossthe reactor also increases with increased branching. Theproblem is compounded if the extra oxygen stimulatesroot hair formation and further increases fluid drag.Finally, the mucilage present on roots grown in liq-uid can supply additional resistance to oxygen transfer(Williams and Doran, 1999).

Gas Phase ReactorsReactors in which liquid is the dispersed phase and

gas is the continuous phase (aeroponic culture system)appear to offer ideal conditions for growth and pro-ductivity of root cultures because of the high gas con-tent available to growing roots (Kondo et al., 1989;Dilorio et al., 1992a, b; Whitney, 1992; McKelvey et al.,1993). In gas-phase reactors, roots are exposed to air

and liquid nutrients are either sprayed onto the roots(Williams and Doran, 2000) or delivered as a mistof micron-sized droplets (Weathers et al., 1999). Useof aeroponics in gas-phase reactors minimizes manyof the limitations associated with liquid phase reactorsdescribed earlier in the text. One of the major advan-tages of aeroponics is the complete control of gases inthe culture environment. There are four primary en-vironmental factors that affect aeroponic root culture:moisture, temperature, mineral nutrition, and the gasphase composition (mainly carbon dioxide, ethyleneand oxygen) (Weathers and Zobel, 1992). Moisture isrequired by all plant tissues and optimum timing (du-ration and cycle) and intensity of moisture applicationsvaries with species, temperature and configuration ofthe spray apparatus (Weathers and Zobel, 1992). Differ-ent types of gas-phase reactors being used in research ac-tivities include: gas sparged, trickle bed (Taya et al., 1989;Flores and Curtis, 1992; McKelvey et al., 1993), dropletphase (Wilson, 1997), liquid-dispersed (Williams andDoran, 2000), radial flow reactors (Kino-Oka et al.,1999) and nutrient mist reactors (Dilorio et al., 1992a,b; Whitney, 1992; Buer et al., 1996; Woo et al., 1996;Liu et al., 1999; Weathers et al., 1999; Wyslouzil et al.,2000; Kim et al., 2001, 2002a, b). Some of these reactorsare described below for a deeper insight.

Trickle Bed ReactorIn this type of bioreactor, the medium trickles over

the support on the top of which roots are inoculated(Figure 7). The spent medium is drained from the bot-tom of the bioreactor to a reservoir and is recirculatedat a specific rate. The degree of distribution of liquidvaries according to the mechanism of liquid delivery atthe top of the reactor chamber. For better dispersion,spraying is done by mixing humidified air with medium(Dilorio et al., 1992; Whitney, 1992). Analogous to gas

FIGURE 7 Trickle bed reactor.

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phase channeling encountered in bubble column, thetrickle bed reactor is subjected to channeling of theliquid phase as it flows down over the root matrix. Outof the different gas phase reactor configurations, thetrickle/spray mode has been identified as a potentiallyscaleable configuration (Flores and Curtis, 1992) sincethe flow patterns are strongly influenced by gravity,which acts uniformly over the bed in contrast to local-ized power input from mechanical agitation. The reac-tor exhibits excellent growth (McKelvey et al., 1993) andproduction (Singh, 1995) characteristics for root cul-tures. Trickle-bed root culture reactors have been shownto achieve tissue concentrations as high as 36 g DW/L(752 g FW/L) at a scale of 14 L. Trickle-bed reactor sys-tems can sustain tissue concentrations, growth rates andvolumetric biomass productivities substantially higherthan other reported bioreactor configurations (Ramakr-ishnan and Curtis, 2004).

Radial Flow ReactorsThe liquid flow conditions in the bioreactors are

important for hairy root growth because the changein liquid flow reflects on both the factors of oxygentransfer in the intertwined root clumps (Yu and Doran,1994) and hydraulic stress against root cells (Ramakr-ishnan and Curtis, 1994). Knowledge of these factorscontributed in the development of radial flow reactor(Figure 8) (Kim et al., 2002), a more preferable reactorsystem for the cultivation of hairy roots. It improvesthe oxygen supply and promotes high-density cultureof the hairy roots. A radial flow reactor based on spe-cific cross-sectional area (a ratio of cross-sectional areato volume in the growth chamber) had been construct-ed in which the air-saturated medium entered through

FIGURE 8 Radial flow reactors (Kim et al., 2002b).

the ports on the sidewall of the reactor and exitedthrough the ports at the center of the top and bottomplates (Kino-Oka et al., 1999). A substantial increase inbiomass of Hyoscyamus muticus hairy roots was achievedas compared to that obtained in the convective flowreactor used by Carvalho and Curtis (1998).

Nutrient Mist BioreactorThe concept of nutrition supply as a mist originated

from Weathers and Giles (1988). The development ofnutrient mist bioreactors was conducted by Weathersand Giles (1988) and Whitney (1992). Mists can be pro-duced by using either nozzles and compressed air orultrasonics (Figure 9) (Weathers et al., 1997; Kim et al.,2002b). In addition to avoiding damage from the liq-uid shear associated with sprays, mists are more wa-ter efficient, thus eliminating the need for extensiverecirculation equipment. Use of mists also decreases thethickness of the liquid film deposition on the surface ofthe roots, thereby preventing nutrient and gas transferlimitation. The nutrient mist reactor is unique amongall other bioreactors used to culture hairy roots due tothe following benefits: gas composition in the reactorenvironment can be easily controlled; oxygen is nota limiting factor (Weathers et al., 1999); pressure dropsand shear rates are both low; high nutrient transfer ratesdue to a large mass transfer area; rapid replenishment of

FIGURE 9 Nutrient mist reactors (Weathers et al., 1997; Kimet al., 2002b).

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nutrients and removal of toxic metabolites; and easy op-eration and scale up (Huang et al., 2004). Low cost mistbioreactors were designed to grow Artemisia annua trans-formed roots. The ratio of the final to initial fresh weightwas found to be 7.4 after 24 days (Chatterjee et al., 1997).Liu et al. (1998) developed an inter-loop ultrasonic nu-trient mist bioreactor for cultivating Artemisia annua L.The growth index (final dry weight/initial dry weight)reached 68. Under an appropriate mist cycle (e.g., 3 minon, 30 min off) for 25 days, dry weight of Artemisia an-nua L hairy roots reached 13.6 g/l (Liu et al., 1999).Cichorium intybus L. hairy roots grown in an acousticmist bioreactor showed the best performance in termsof increased specific growth rate (0.072 d−1) and esculincontent (18.5 g/l). Esculin content was almost doubleas compared to that present in the roots grown in bub-ble column and nutrient sprinkle bioreactors (Bais et al.,2002). An acoustic mist bioreactor, which enables effi-cient mass transfer and higher productivity, has beendesigned by Suresh et al. (2005) using Tagetes patula L.hairy roots as a model system.

Hybrid ReactorThe disadvantage of gas-phase reactors is that there is

no way to uniformly distribute the roots in the growthchamber without manual loading. In order to make useof the respective advantages and at the same time toovercome limitations associated with both kinds of re-actor systems, a combination of liquid-phase and gas-phase reactor system was proposed for effective hairyroot cultivation. Flores and Curtis (1992) suggested thata combination of a bubble column/trickle bed reactoroffers the best compromise between the two extremes.By inoculating the reactor under bubble column condi-tions the roots were free to circulate, anchor to packingrings, and distribute evenly in the reactor. When thetissue concentration became high enough that the bub-ble column mode was no longer effective, the reactorwas switched to trickle bed operation, and the advan-tages of a gas-phase reactor were realized. This strategywas efficiently used to inoculate, distribute, and culturehairy roots in pilot-scale reactors (Ramakrishnan et al.,1994; Wilson, 1997). A hybrid reactor system made upof bubble column and nutrient mist bioreactor was usedto study the transient growth characteristics and nutri-ent utilization rates of Artemisia annua hairy roots (Kimet al., 2003).

Measurement of Growthin Bioreactors

One of the major challenges during scale-up ofhairy root cultures has been biomass measurement.Since the growth is uneven, non-homogeneous andin the form of entangled mass, hence the intermittentbiomass estimation becomes difficult during bioreac-tor studies. Taking into consideration the limitationsmentioned above, biomass estimation can be dividedinto direct and indirect methods being reported in theliterature.

Hairy root growth can be directly determined on thebasis of fresh weight and dry cell weight. Fresh weightcan be measured after removing culture medium by fil-tration through a preweighed Whatman #1 filter paperunder vacuum. Dry cell weight can be determined bydrying the sample at 60◦C for 48 h (Nguyen et al., 1992).Growth indices for hairy roots can be expressed as theratio of fresh weight of roots at harvest to fresh weightof inoculum (Hook, 1994).

Indirect measurement of growth in terms of biomasscan be done by correlating it with the conductivity andosmolarity of the medium (Taya et al., 1989; Inomataet al., 1993). Conductivity (microsiemen) measurementscan be done using a conductivity meter and osmo-larity (osmol/kg) can be measured by using an auto-matic cryoscopic osmometer. Studies relating to theon-line estimation of conductivity and osmolarity topredict the growth of hairy root cultures have been dis-cussed by Bais et al. (2002). A model of liquid nutri-ent uptake and osmolality to account for changing spe-cific water content of root has been used for biomasscorrelation to estimate fresh weight time course dataof hairy root cultivation in a shake flask and biore-actor (Ramakrishnan et al., 1999). The mass balancetechnique permits the accurate aseptic online estima-tion of dry cell weight, fresh weight and liquid vol-ume in root culture, utilizing either refractive indexor electrical conductivity of the medium along withliquid medium osmolality. A new technique has beendeveloped for estimation of biomass considering thechanges in biomass volume during a high- density cul-ture of hairy roots (Jung et al., 1998). In this method,ratio of the volume of liquid medium to the volumeof biomass decreased continuously with the growthof hairy roots, thus the change in biomass volumehas been introduced as a new parameter for biomassestimation.

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CONCLUSIONThe employment of hairy root culture technology

has offered new opportunities for in vitro productionof valuable plant secondary metabolites. Hairy rootcultivation offers good prospects for feasible commer-cial production of high value secondary metabolitesof plants. The biotechnological application of hairyroot cultures is promising for a number of reasons:(1) stable, high level secondary metabolite production;(2) fast auxin-independent growth; and (3) suitabilityfor adaptation to reactor systems. At the same timeit provides many challenges during large-scale cultiva-tion. The unusual rheological properties of hairy rootcultures have made it necessary to investigate novel ap-proaches to fermentor and process design. Good growthcharacteristics, high oxygen transfer and ability to fol-low the progress of growth non-invasively are some ofthe important factors in the development of a suit-able bioreactor configuration for hairy root cultivation.A key aspect of future use of hairy roots in a com-mercial bioprocess depends on the low capital and/oroperating cost. At the same time, the selected designshould meet Good Manufacturing Practice (GMP) re-quirements. To scale up hairy root cultivation, vari-ous operational factors such as medium, inoculum size,measurement of growth, interpretation of effects of var-ious physical parameters on growth, product recovery(intracellular/extracellular) and reproducibility of re-sults have to be assessed before the technology is readyfor commercialization.

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