Plant cell suspension cultures_ some engineering considerations

Journal of Biotechnology 59 (1997) 39–52

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Plant cell suspension cultures: some engineering considerations

P.M. Kieran a,*, P.F. MacLoughlin b, D.M. Malone b

a Biochemical Engineering Research Group, School of Biological Sciences, Dublin City Uni6ersity, Dublin 9, Irelandb Department of Chemical Engineering, Uni6ersity College Dublin, Belfield, Dublin 4, Ireland

Received 1 October 1996; received in revised form 3 February 1997; accepted 25 July 1997

Abstract

Higher plants are the source of a vast array of biochemicals which are used as drugs, pesticides, flavourings andfragrances. For some of these compounds, plant cell culture can provide a potential production alternative totraditional cultivation methods or chemical synthesis routes. Many systems have been patented and the last 20 yearshave seen considerable industrial and academic interest in the development of large scale cultures to producepharmaceutically active, high value substances. However, the industrial application of plant cell suspension cultureshas, to date, been limited. Commercialisation has essentially been impeded by economic feasibility, arising from bothbiological and engineering considerations. This paper reviews the commercial development of the technology to dateand focuses on the impact of specific engineering-related factors, in particular, the shear sensitivity of plant cellsuspension cultures. Evidence of sensitivity to hydrodynamic shear in bioreactors has generally been attributed to thephysical characteristics of the suspended cells. Recent studies indicate that shear sensitivity may not be as important,in some cases, as initially anticipated. © 1997 Elsevier Science B.V.

Keywords: Plant cells; Shear sensitivity; Bioreactor; Scale-up

1. Introduction

Higher plants are recognised as importantsources of a wide range of biochemicals, used asdrugs, pesticides, flavourings and fragrances. Tra-

ditionally, these substances have been extractedfrom naturally grown whole plants. On a com-mercial basis, this approach involves large-scalecrop cultivation (e.g. alkaloids from Catharanthusroseus). Many plant products can now be pro-duced by chemical synthesis, which can be a morereliable, consistent and cost-effective method.Plant cell culture provides an alternative ap-

* Corresponding author. Tel.: +353 1 7045584; fax: +3531 7045412; e-mail: [email protected]

0168-1656/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved.

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P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 39–5240

proach, which may be attractive under certaincircumstances: if, for example, the source plant isdifficult to cultivate, has a long cultivation periodor has a low metabolite yield; if chemical synthe-sis has not been achieved or if it is technicallyproblematic. Metabolite yield by the cell culturemay significantly exceed that observed in the par-ent plant. Thus, using this technology, themetabolite can be produced under controlled andreproducible conditions, independent of geo-graphical and climatic factors.

The anti-cancer drug Taxol® (a registered trade-mark of Bristol-Myers Squibb) is a very impor-tant example of a potential candidate forproduction by cell culture methods. Taxol® wasoriginally isolated from the bark of the PacificYew tree, Taxus bre6ifolia. This slow growing treeis principally found in the Pacific North-West. Toobtain 1 kg of Taxol® requires the bark of morethan 1000 trees, each up to 100 years old. Analternative approach to production is essential ifTaxol® supplies are to be assured and if thePacific Yew population is not to be destroyed.Total synthesis of Taxol® has recently been re-ported (Holton et al., 1994, Nicolaou et al., 1994)but is not economically sustainable on a largescale and Bristol-Myers Squibb currently employsa semi-synthetic process for the production ofTaxol®, involving taxane precursors extractedfrom various yew species. There is also prelimi-nary evidence to suggest that fungi growing onthe Pacific Yew can produce both Taxol® andother taxanes (Stierle et al., 1995). If the biosyn-thetic pathway for Taxol® can be fully elucidated,genetically engineered synthesis may be possible.Plant cell culture may yet provide another pro-duction route (Fett-Neto and DiCosmo, 1996;Seki et al., 1997).

From an engineering perspective, cell suspen-sion culture has more immediate potential forindustrial application than plant tissue or organcultures, due to the extensive body of expertisewhich has been amassed for the treatment ofsubmerged microbial cultures. While tissue androot cultures (Flores and Curtis, 1992) offer ge-netic stability as well as, in some instances, supe-rior metabolic performance over suspensioncultures of the same lines, the development of

appropriate reactors and processing techniquesfor these systems will involve enormous de novoinvestment. Accordingly, most research effort hasbeen directed towards the commercialisation ofplant cell suspension culture. The purpose of thispaper is to provide an overview of the commercialdevelopment of plant cell suspension culture tech-nology to date and to focus on a limited numberof engineering issues, specifically hydrodynamicshear sensitivity, which have hampered its ex-ploitation on an industrial scale.

2. Commercial development

That plant cell culture offers the tantalising, butas yet largely unattained prospect of long-termcommercial success, is evidenced in many recentreviews concerning the technology and strategiesfor its optimisation (Zenk, 1991; Verpoorte et al.,1991; Scragg, 1992, 1995; Buitelaar and Tramper,1992; Kreis, 1993; Shuler, 1993; Su, 1995; Di-Cosmo and Misawa, 1995; Dornenburg andKnorr, 1995; Zhong et al., 1995; Schlatmann etal., 1996). Despite its enormous potential, indus-trial processes involving plant cell cultures havebeen limited to a handful of applications, includ-ing the production of shikonin from Lithosper-mum erythrorhizon (Fujita, 1988), berberine fromCoptis japonica (Fujita and Tabata, 1987) andginsenosides from Panax ginseng (Ushiyama,1991). Many other processes have been investi-gated and patented but, to date, few have provento be economically viable. Plant cell cultures havepredominantly been valued as a source of natu-rally occurring secondary metabolites. However,they can also be used for biotransformations suchas the glucosylation of hydroquinone to arbutinby Rau6olfia serpentina (Lutterbach and Stockigt,1992). In Japan, workers at Shiseido have alterna-tively employed C. roseus for the production ofarbutin by a similar process (Yokoyama andYanagi, 1991; Inomata et al., 1991). As a sourceof enzymes for the genetically engineered synthe-sis of natural products (Scott, 1994), they offergreat promise. And large-scale processes for theuse of cell cultures as a source of biomass (Hashi-moto and Azechi, 1988) have been investigated.

P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 39–52 41

Reviews on the potential of plant systems for theproduction of valuable products by these routesare presented by Parr (1989), Pras (1992), Alfer-mann and Petersen (1995) and Stockigt et al.(1995).

The recent literature and patent libraries (Ver-poorte et al., 1991; Su, 1995) reveal the predomi-nance of Japanese companies in the search forcommercial applications of plant culture technol-ogy. Spearheaded by the company Plant Cell Cul-ture Technology, research in Japan has receivedstrong support from both government and indus-try (Misawa, 1991; Hara, 1996) and has producedalmost all of the commercialised processes. In theUSA and Europe, however, there have been fewercommercial applications and most research efforthas been concentrated on a limited number ofhigh value products, including Taxol® and theindole alkaloids. Phyton Catalytic recently filed apatent for the production of Taxol® from high-yielding cell cultures of Taxus chinensis (Bringi etal., 1995). It has been reported (Anonymous,1994) that ESCAgenetics, which filed a patent forthe production of vanillin using tissue culture(Knuth and Sahai, 1991), was also involved inTaxol® production scale-up trials.

Limited commercialisation of the technology todate can essentially be attributed to matters ofeconomic feasibility which, in turn, derive largelyfrom a combination of biological and engineeringfactors. Plant cell culture, on an industrial scale, isan inherently capital intensive process. Productconcentrations and productivities are typicallylow. On this basis, its use can only be justified ifit offers an economic advantage over chemicalsynthesis or traditional extraction processes, or ifno other alternative production route exists.Shuler (1993) predicted that pharmaceuticals pro-duced naturally by slow-growing, woody plants(e.g. Taxol®) are the most likely candidates fordevelopment. In comprehensive reviews on theproduction of alkaloids, Verpoorte et al. (1991,1993) compared the results of a number of eco-nomic evaluation studies for the production ofalkaloids by cell culture. Their analysis confirmedthat while product price would be high and pro-cess optimisation essential, cell culture processescould be feasible for speciality chemicals. How-

ever, the calculated product prices vary substan-tially with the choice of production strategy (e.g.batch culture with product retained intracellu-larly; spontaneous or induced product release,etc.). However, economic feasibility studies areunanimous in acknowledging productivity as alimiting factor. For example, Drapeau et al.(1987) estimated that a 40-fold increase in theajmalicine productivity of C. roseus would berequired to justify the production of this com-pound by cell culture methods.

With regard to secondary metabolites, the pro-ductivity of plant cell suspensions is of the orderof 10−2–10−1 g l−1 per day (Scragg, 1995). Dueto the fact that productivity depends on productyield, the organism growth rate and prevailingbiomass levels, it is clear that there is a role forboth biologists and engineers in improving systemperformance. The question of economic feasibilitycould be largely resolved by the development ofstable, high-yielding strains (Berlin, 1988) and theidentification of optimal cultivation conditions.While the high biomass levels required for eco-nomic viability necessarily limit the volume of freemedium available, in an ideal system the cellswould actively secrete the product into the sus-pending process fluid (Buitelaar and Tramper,1992), thereby simplifying downstream process-ing. However, to achieve these objectives, a fullerunderstanding of the biosynthetic pathways is es-sential, in addition to a clearer picture of theinteractions between growth kinetics, system mor-phology, cell–cell interaction and product synthe-sis.

3. Engineering considerations

From an engineering perspective, the primarychallenges lie in the area of process scale-up. Plantcell suspensions can now be almost routinely cul-tivated in small-scale configurations. Moreover, inaddition to the commercial processes mentionedearlier, there are a number of examples of large-scale, albeit non-commercial systems. Nicotianatabacum cultures have been successfully cultivatedin a 20 m3 stirred tank reactor (Noguchi et al.,1977). Westphal (1990) reported on long-term cul-


Table 1Summary of biocatalyst characteristics

Qoa (mmol l−1 h−1)Doubling time (h)Biocatalyst Shape Size (mm) Cell wall Aggregates

Yes 20–100Plant cells Spherical l: 100–500 Yes 10−1

Cylindrical d: 20–50

No No 20Animal cells 10−1Spherical d: 10–20

0.5–10Yes/No 103Bacteria YesSpherical d: B1Cylindrical l: B5Spiral

10Yes 102Yeasts Yes/NoSpherical d: 5–10

Yes/No 10–20Moulds Mycelial d: 5–10 102Yesl: B100

a Characteristic oxygen consumption rate for biocatalyst in liquid culture.

tivation of a number of lines in 50 m3 vessels.However, in many cases, scale-up has been ac-companied by a reduction in system productivity(Schiel and Berlin, 1987; Ikeda, 1991; Taticek etal., 1991), variously attributed to mixing and masstransfer problems in the characteristically non-Newtonian broths, which exhibit varying degreesof aggregation and which, ideally, have highbiomass concentrations. Product recovery is com-plicated, both technically and economically, bythe fact that most systems retain the product ofinterest intracellularly, in the vacuole, thereby ne-cessitating either cell lysis or non-destructive per-meabilisation.

3.1. System characteristics and bioreactor design

A number of reviews have dealt with the choiceof bioreactors for plant cell suspension culture(Kargi and Rosenberg, 1987; Payne et al., 1987;Panda et al., 1989; Doran, 1993) and most em-phasis has been placed on modifications of theconventional stirred tank reactor (STR), withbubble aeration, employing a variety of impellerdesigns. However, equipment normally employedfor microbial cultivation may not be immediatelyapplicable to plant cells, due to differences be-tween the characteristics of both individual cellsand suspensions of the respective systems. By wayof illustration, a summary of some of the physicalproperties of biological systems is presented inTable 1.

3.1.1. AggregationPlant cells are significantly larger and slower

growing than most microbial organisms. Individ-ual plant cells have a typically characteristiclength of the order of 101–102 mm and may bespherical to cylindrical in shape. Aggregation iscommon, largely due to the failure of cells toseparate after division, although the secretion ofextracellular polysaccharides (ECP), particularlyin the later stages of batch growth, may con-tribute to increased aggregation (Taticek et al.,1991). The aggregation phenomenon has beenused in the development of self-immobilisationmethods (Prenosil et al., 1987; Hegglin et al.,1990). Aggregates, comprising up to 102 cells, maybe many millimetres in diameter (Tanaka, 1982)and typically exhibit a tendency to settle. Aggre-gation patterns, variously studied using imageanalysis (Kieran et al., 1995) and sieving (Mavi-tuna and Park, 1987) techniques, vary signifi-cantly between cell lines and also as aconsequence of culture age and cultivation condi-tions. For example, N. tabacum, which is fre-quently described as highly aggregated(Hashimoto and Azechi, 1988; Hooker et al.,1989; 1990) exists, under certain conditions asunbranched chains of up to 50 cells (DeJong etal., 1967). Zenk et al. (1975) reported that 60% ofa Morinda citrifolia culture existed as single cellsor cells of two chains, whereas, in a study byKieran et al. (1993) the corresponding figure laybetween approximately 10% (lag and stationary


phases) and 50% (exponential phase). Wagner andVogelmann (1977) observed a change in the mor-phology of suspensions of C. roseus from pelletedto single cells on scale-up from shake flasks to anair-lift reactor. These examples emphasise the im-portance of identifying morphological trends un-der actual growth conditions and with duereference to culture age. Deviations from expectedaggregate distributions may be indicative of cul-ture variations in response to environmental fac-tors. Although the role of cell–cell interactions inundifferentiated systems has yet to be conclusivelyestablished, Shuler (1993) discusses evidence tosuggest that metabolite productivity may be sig-nificantly influenced by the degree of cellular asso-ciation and may, therefore, be affected byvariations in aggregation patterns arising onscale-up.

3.1.2. RheologyAggregation, aggregate interactions, high

biomass concentrations (e.g. up to 70 g l−1 on adry weight basis (Matsubara and Fujita, 1991))and, in some cases, ECP secretions, result in highwhole-broth viscosities for plant cell suspensions.There have been no comprehensive on-line rheo-logical studies. Data have been collected for avariety of plant cell systems using conventionalviscometers, although, as with other microbialsuspensions, care must be taken to avoid sedimen-tation effects (Scragg et al., 1986) and/or aggre-gate disruption (Rosenberg, 1987). Using theconcept of the apparent viscosity of a fluid (Met-zner and Otto, 1957) rotational devices fitted withpurpose-built impellers have also been used forthe rheological characterisation of plant cell sus-pensions: for example, helical ribbon impellers,designed to satisfy the need for good suspensionmixing at the widest possible range of laminarflow conditions (Jolicouer et al., 1992; Kieran,1993). A summary of relevant studies is presentedin Table 2 and it is apparent that the majority ofsuspension cultures investigated exhibit non-New-tonian, shear-thinning characteristics. Many sys-tems also show evidence of a yield stress.Thixotropic behaviour has been observed, al-though only in isolated samples (Wagner and

Vogelmann, 1977). In common with many micro-bial suspensions, the apparent viscosity is foundto be strongly dependent on biomass concentra-tion (Tanaka, 1982; Zhong et al., 1992a; Kieran,1993), although the data reported in the literaturedo not, in general, refer to the high density sus-pensions required for economically viable large-scale processing. The influence of the morphologyof the suspended cells and/or aggregates on theapparent viscosity of the suspension requires fur-ther investigation (Zhong et al., 1992a; Curtis andEmery, 1993); it should also be noted that the useof biomass concentration (on a dry weight basis)as a correlating factor for viscosity can maskeffects attributable to variations in individual cellsize and water content over the course of thegrowth cycle.

3.1.3. Oxygen and aeration effectsThe oxygen requirements of plant cells are com-

paratively modest. Specific oxygen consumptionrates, on a dry weight basis, are of the order of10−6 g g−1 per second, (Bond et al., 1988;Dubuis et al., 1995; Ho et al., 1995). However,high cell densities and high fluid viscosities canreduce oxygen mass transfer efficiencies in biore-actor systems. Although critical dissolved oxygenconcentrations of approximately 15–20% air satu-ration are commonly quoted for plant cell suspen-sion cultures (Payne et al., 1992), the critical valuefor cell growth may be significantly lower thanthat for metabolite synthesis and recent studies(Schlatmann et al., 1995) point to the importanceof dissolved oxygen concentration for metaboliteproductivity.

Studies of the effects of aeration on plant sus-pension cultures have focused largely on the influ-ence of kLa, the system mass transfer coefficient,in which the combined effects of aeration andagitation are inextricably linked. Kato et al.(1975), Tanaka (1981), Smart and Fowler (1981)and Leckie et al. (1991) all investigated the effectof initial mass transfer coefficients on system per-formance in a variety of bioreactor configura-tions. Although the results are system specific, itwas generally concluded that for each system, alower limiting value of kLa exists, below which the


Table 2Rheological characterisation of plant cell suspension cultures

Characterisationb ReferenceViscometric deviceSystem Biomass concentra-tiona (g l−1)

B14.5 NewtonianPapa6er som- Brookfield; modified Stormer concentric Curtis and Emeryniferum (1993)cylinder

Curtis and EmeryBrookfield; modified Stormer concentricNicotianatabacum (1993)cylinder

Pseudoplastic,Batch cultiva- =9.0tion n=0.6

=7.9 NewtonianSemi-continu-ous

Kato et al. (1978)513 Pseudoplastic, Brookfield-typeNicotianatabacum n:0.7

Bingham plastic Brookfield-typePerilla frutescens B20 Zhong et al.(1992a)

5450 (fresh weight) Modified Weissenberg rheogonimeter Ballica et al.Datura stramo- Casson plastic(1992)nium

Contraves rheomat; Brookfield Ballica and Ryu(1993)

Pseudoplastic, Kieran (1993)Morinda citrifolia 5450 (fresh weight) Double-helical ribbon impellern:0.8Pseudoplastic, Helical ribbon impeller527 Jolicouer et al.Catharanthus

roseus 0.1BnB0.9 (1992)Pseudoplastic Tanaka (1982)Cudriana tricuspi- 515

datan:0.53Catharantus

roseusNicotiana

tabacum

a Dry weight basis unless otherwise indicated.b n, flow behaviour index.

culture is inhibited; reduced productivity ob-served at higher gassing rates is variously at-tributed to the stripping of CO2 and essentialvolatiles from the system (Kato et al., 1975;Ducos and Pareilleux, 1986) or shear-related ef-fects (Ballica and Ryu, 1993). Using a gas re-circulation bioreactor for scale-up studiesinvolving C. roseus, Schlatmann et al. (1993)confirmed the importance of dissolved gaseouscomponents for system performance and con-cluded (Schlatmann et al., 1994) that loss of anunidentified essential volatile factor was respon-sible for the reduced ajmalicine synthesis ob-served on scale-up from a shake flask to anaerated bioreactor.

Aeration of bioreactors can lead to foamingand in plant cell suspension cultures this prob-

lem can be particularly severe (Zhong et al.,1992b). Although there have been few compre-hensive studies of this phenomenon and allhave been limited to laboratory or pilot scalesystems, foaming has typically been correlatedwith aeration rates and extracellular proteinconcentrations (Wongasmuth and Doran, 1994).However, the contribution of extracellularpolysaccharides and other medium componentsto foaming potential or foam stability has yetto be established. A number of antifoams havebeen used to control foaming in plant cell sus-pensions (Smart and Fowler, 1981; Zhong etal., 1992b; Wongasmuth and Doran, 1994; Liet al., 1995) resulting, in some cases (Smart andFowler, 1981; Wongasmuth and Doran, 1994)in reduced system productivities.


3.1.4. Bioreactor designAs with microbial systems, approaches to over-

coming suspension, mass transfer and mixing-re-lated problems typically include improved reactordesign and, more commonly, increasing agitationand aeration intensity. However, solutions mustbe achieved without concomitant negative shear-related effects. In STRs, large, slow-moving im-pellers often provide good mixing at relatively lowrotational speeds. Oxygen transfer may be limit-ing, due to poor bubble dispersion. However,Jolicouer et al. (1992) reported on the successfuluse of a double helical ribbon impeller, in an 11 lsurface-baffled vessel used for the cultivation ofC. roseus cultures. Bubble columns and air-liftloop reactors offer the promise of a low-shearenvironment (Moo-Young and Chisti, 1988) andthey have been used by a number of workers forplant cell suspension systems (Smart and Fowler,1981; Hegarty et al., 1986; Fulzele and Heble,1994; Matsushita et al., 1994). Here again, perfor-mance is limited by mixing efficiency (Doran,1993) which is reduced at the broth viscositiesassociated with the high biomass levels necessaryfor an economically viable process.

There have been many reports on the develop-ment of novel bioreactors for plant cell suspen-sion culture. For example, rotating drum reactors(RDR) have been used for the cultivation of C.roseus (Tanaka et al., 1983), N. tabacum(Shibasaki et al., 1992) and L. erythrorhizon(Takahashi and Fujita, 1991). In the latter case,the choice of an RDR in preference to either anair-lift reactor or a paddle-agitated STR wasbased on its superior performance in terms ofsuspension homogeneity, low-shear environmentand reduced wall growth. Fluidised-bed reactorshave been proposed for industrial scale use(Dubuis et al., 1993, 1995; Khlebnikov et al.,1995), offering the possibility of perfusion opera-tion with a facility for cell harvesting, a low-shearenvironment and increased cell–cell interaction.Light irradiation has been shown to have a stimu-lating effect on the secondary metabolism of somesystems (Zhong et al., 1991; Furusaki et al., 1993),but its integration into the operation of large-scalebioreactors is problematic.

3.1.5. Shear sensiti6ity in plant cell suspensioncultures

The hydrodynamic shear sensitivity of biologi-cal systems, including prokaryotic and eukaryoticsuspensions and enzyme solutions, has attractedconsiderable research attention in recent yearsand has been comprehensively reviewed by anumber of authors (Thomas, 1990; Markl et al.,1991; Merchuk, 1992; Hua et al., 1993; Joshi etal., 1996). These systems encompass a range ofparticle sizes, as well as varying degrees of struc-tural and metabolic complexity, all of which im-pact on their sensitivity to a given shearenvironment and on the extent of their responses.While few submerged systems are adversely af-fected by the hydrodynamic environment associ-ated with laboratory maintenance conditions,process scale-up and the cultivation of high-den-sity suspensions increase the mass transfer re-quirements of the system and accordingly, withconventional processing equipment, the intensityof the aeration and agitation conditions.

The response of a biological system to anyhydrodynamic environment depends on the dura-tion and intensity of the applied conditions as wellas on the physiological characteristics of the sys-tem itself. The response may be positive (e.g. anincrease in biomass yield or secondary metabolitesynthesis). Investigations of shear sensitive sys-tems, however, generally focus on the negative ordamaging effects associated with the hydrody-namic conditions encountered in process equip-ment. System response to shear may be measuredand assessed in a number of ways. Techniquesemployed for plant cell systems are summarised inTable 3. With a view to system scale-up, it isinteresting to note how few of the measurementtechniques listed lend themselves to on-line appli-cations. In a number of studies (Dunlop et al.,1994; Takeda et al., 1994), it is emphasised thatthe quantitative evaluation of shear-related ef-fects, in particular of sub-lytic effects, dependscrucially on the choice of damage indicator, whichfrequently precludes the direct comparison of re-sults collected from different studies.

Shear studies on biological systems, in general,can be broadly divided into two categories,classified in terms of the prevailing shear environ-


Table 3Methods used for the assessment of shear-related effects in plant cell suspension cultures

Parameter measured ReferenceSystem response

Growth rate Rosenberg (1987), Meijer et al. (1993)Reduction in viabilityRegrowth potential Rosenberg (1987), Scragg et al. (1988)Membrane integrity

Dye exclusion Takeda et al. (1994), Kieran et al. (1995)Dual isotope labelling Parr et al. (1984); Kieran (1993)Dielectric permitivity Markx et al. (1991)

Release of intracellular components pH variation Wagner and Vogelmann (1977)Meijer et al. (1993)Protein releaseMeijer et al. (1993)Total organic carbon

Secondary metabolite release Hooker et al. (1989)

Change in metabolism Oxygen uptake rate Ho et al. (1995)Rosenberg (1987), Takeda et al. (1994), Zhong et al.Respiration activity (TTC

reduction) (1994)ATP concentration Takeda et al. (1994)Metabolite productivity Hooker et al. (1989); Zhong et al. (1994)

Tanaka et al. (1988)Cell wall composition

Aggregate size/shape Takeda et al. (1994), Kieran et al. (1995)Changes in morphology and/oraggregation patterns

Expansion index Zhong et al. (1994)

ment and the duration of cell exposure. In the firstcategory, the biological suspension is exposed toshear forces under growth conditions, for theduration of cultivation or a significant portionthereof (Scragg et al., 1988; Meijer et al., 1994;Takeda et al., 1994; Zhong et al., 1994; Kieran etal., 1995). Although these studies tend to behighly system-specific, due to the variety of biore-actors employed, it is arguable that analysis underactual growth conditions, or in a scaled downversion of a production bioreactor, offers thegreatest potential for successful scale-up of results(Meijer et al., 1994; Ho et al., 1995). The hydro-dynamic environment is generally regulated bychanging the rate or method of agitation and/oraeration. Due to the difficulties involved in quan-tifying the levels of shear to which an organism isexposed in the turbulent field of an agitated biore-actor, the intensity of the environment has beengenerally related to impeller speed or power input.

The benefits of examining the shear sensitivityof a system under actual or proposed operatingconditions are not to be underestimated. As theapparatus is normally designed for sterile opera-

tion, the experimental exposure time is limitedonly by the kinetics of the system itself and by theability of the organism to survive under the pre-vailing conditions. Use of chemostat culture (Mei-jer et al., 1993) facilitates the investigation oflong-term effects, but does not, in general, reflectactual production conditions, where batch pro-cessing is most commonly employed and, more-over, may be less appropriate for systems yieldingnon-growth associated metabolites.

In the second type of study, cells are exposed towell-defined, laminar or turbulent flow conditions(e.g. in couette, capillary and submerged jetdevices), for short periods of time, generally undernon-growth conditions. Studies involving plantcell suspension cultures are summarised in Table4. An exponential decay model is commonly em-ployed to describe viability loss with increasingexposure time to the imposed shearing conditions;the resultant death or decay rate is used to quan-tify system response to a given hydrodynamicenvironment (Kieran et al., 1995). Frequently, theconcept of a critical shear stress is employed. Forexample, on the basis of system response to lami-


Table 4Defined flow-field shear experiments using plant cell suspension cultures

Flow regime Damage indicatorCell suspension Apparatus Reference

Laminar/turbulent Dye exclusionMorinda citrifo- Kieran et al. (1995)Recirculating flow capillaryMorphologylia

Turbulent Dye exclusion MacLoughlin et al. (1997)Submerged jetMorphology

Morphology Rosenberg (1987)Daucus carota Couette viscometer LaminarRegrowthCell lysis

Hooker et al. (1989)ViabilityCouette-typeNicotiana Transitional/turbulenttabacum Cell lysis

Metabolite production

Zhong et al. (1995)Couette-type Respiration activityPerilla frutescens

nar flow conditions in a viscometric device, Vogel-mann et al. (1978) suggested a critical shear stressof between 80 and 200 N m−2 for M. citrifolia ;using regrowth potential as an indicator of systemresponse, a critical shear stress of 50 N m−2 wasidentified for suspensions of Daucus carota(Rosenberg, 1987). Recent studies (Dunlop andNamdev, 1993; Kieran et al., 1995; MacLoughlinet al., 1997) have pointed to the use of energydissipation as a correlating factor for shear-re-lated damage. However, given the variety of ag-gregate morphologies exhibited by plant cellsystems, no single mechanism for cell damage hasbeen conclusively identified. Because these devicesare generally operated under non-sterile condi-tions, system response is most frequently moni-tored in terms of loss of viability and accordingly,more subtle, non-lytic effects may be overlooked(Namdev and Dunlop, 1995).

There are a number of important conclusions tobe drawn from these studies which have signifi-cant consequences for the success of commercialsystems in conventional bioreactors. Interestingly,it appears that plant cell suspensions are morerobust than initially anticipated and that signifi-cant losses of viability are not, in general, to beexpected under normal operating conditions in aconventional STR. However, other less dramaticshear-related effects may be observed, including

reductions in metabolite yield (Hooker et al.,1990) and biomass productivity (Ho et al., 1995).Metabolic responses may be related to changes incell–cell interactions effected by shear-induced ag-gregate disruption. System response may vary sig-nificantly between cell lines, and may also dependon culture age and cultivation history. For exam-ple, Wagner and Vogelmann (1977) reported evi-dence of shear sensitivity in cultures of C. roseus.In subsequent studies by Meijer et al. (1993), C.roseus was found to be considerably more robustthan cultures of Cinchona robusta and Tabernae-montana di6aricata. Namdev and Dunlop (1995)recently highlighted limitations of shear-relatedstudies, as conducted to date and proposed amodel for a more systematic and holistic evalua-tion of the response of plant cell systems tohydrodynamic environments, focusing on calciumtransport, stress protein expression, osmo-regula-tion and aggregation. Overall, however, it appearsas if research effort might be more profitablydevoted to the development of shear-resistant celllines, rather than low-shear bioreactors.

4. Conclusions

This paper has considered only a limited num-ber of engineering-related issues which impinge


upon the commercialisation of plant cell suspen-sion technology. The overall focus of related re-search is the achievement of large-scale cultivationof high-yielding cell lines and efficient productrecovery, through the integration of biologically-and engineering-based approaches. Process mod-elling has been successfully used as a tool for thedesign, analysis and optimisation of many biolog-ical systems, including plant cell suspension cul-tures (Hooker and Lee, 1992; van Gulik et al.,1993), but there is scope for development of mod-els which take account of the fact that suspen-sions may not be homogeneous with respect tometabolite synthesis. In the area of process con-trol, a large-scale operation of plant systems ishampered by difficulties associated with the on-line analysis of parameters such as cell or aggre-gate size distributions which may play animportant role in metabolite productivity. Differ-ent operating strategies (e.g. batch, two-stage,continuous, etc.) may be required for cell linesexhibiting different metabolite synthesis kinetics(e.g. growth associated and non-growth associ-ated) and which either store the product of inter-est intracellularly or, preferably, secrete it into thesuspending fluid. Non-lethal methods for inducingproduct release and the use of two-phase systems(Buitelaar and Tramper, 1992) can facilitate con-tinuous processing without loss of biomass. Theproduct can be concentrated and by removingfeed-back inhibition effects, productivity may beimproved. Immobilisation (Hulst and Tramper,1989) offers the possibility of a low-shear environ-ment, enhanced cell–cell contact, biomass re-useand, if product secretion can be achieved, a facil-ity for product removal. Some of these advan-tages have already been demonstrated usingself-immobilised plant cell systems in fluidised-bedreactors (Dubuis et al., 1993; Khlebnikov et al.,1995). Despite the obvious attractions of theseintegrated processing approaches, problems ofboth economic and technical feasibility must beresolved before their application on an industrialscale can be considered. Through concerted re-search efforts in the areas discussed in this paperand in those highlighted above, wide-scale com-mercialisation of plant cell suspension technologymay soon be feasible.

References

Alfermann, A.W., Petersen, M., 1995. Natural product forma-tion by plant cell biotechnology. Plant Cell Tissue OrganCult. 43, 199–205.

Anonymous, 1994. ESCA scales up taxol. Chemical MarketingReporter. May 24, p. 7.

Ballica, R., Ryu, D.D.Y., 1993. Effects of rheological proper-ties and mass transfer on plant cell bioreactor perfor-mance: production of tropane alkaloids. Biotechnol.Bioeng. 42, 1181–1189.

Ballica, R., Ryu, D.D.Y., Powell, R., Owen, D., 1992. Rheo-logical properties of plant cell suspensions. Biotechnol.Prog. 8, 413–420.

Berlin, J., 1988. Formation of secondary metabolite in cul-tured plant cells and its impact on pharmacy. In: Bajaj,Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry:Medicinal and Aromatic Plants I, vol. 4. Springer-Verlag,Berlin, pp. 37–59.

Bond, P.A., Fowler, M.W., Scragg, A.H., 1988. Growth ofCatharanthus roseus cell suspensions in bioreactors: on-lineanalysis of oxygen and carbon dioxide levels in inlet andoutlet gas streams. Biotechnol. Lett. 10, 713–718.

Bringi, V., Kadkade, P.G., Kane, E.J., Prince, C.L., Roach,B., Schubmehl, B.F., 1995. High yield production of taxoland taxane production from Taxus species cell cultures: bycultivation in a nutrient medium cells derived from callusand/or suspension cultures of Taxus species useful forcancer treatment, US Patent 5 407 816.

Buitelaar, R.M., Tramper, J., 1992. Strategies to improve theproduction of secondary metabolites with plant cell cul-tures: a literature review. J. Biotechnol. 23, 111–141.

Curtis, W.R., Emery, A.H., 1993. Plant cell suspension culturerheology. Biotechnol. Bioeng. 42, 520–526.

DeJong, D.W., Jansen, E.F., Olson, A.C., 1967. Oxidoreduc-tive hydrolytic enzyme patterns in plant suspension culturecells. Exp. Cell Res. 47, 139–156.

DiCosmo, F., Misawa, M., 1995. Plant cell and tissue culture:alternatives for metabolite production. Biotechnol. Adv.13, 425–453.

Doran, P.M., 1993. Design of reactors for plant cells andorgans. Adv. Biochem. Eng. Biotechnol. 48, 117–168.

Dornenburg, H., Knorr, D., 1995. Strategies for the improve-ment of secondary metabolite production in plant cellcultures. Enzym. Microb. Technol. 17, 674–684.

Drapeau, D., Blanch, H.W., Wilke, C.R., 1987. Economicassessment of plant cell culture for the production ofajmalicine. Biotechnol. Bioeng. 30, 946–953.

Dubuis, B., Pluss, R., Romette, J.L., Kut, O.M., Prenosil,J.E., Bourne, J.R., 1993. Physical factors affecting thedesign and scale-up of fluidized-bed bioreactors for plantcell culture. In: Nienow, A.W. (Ed.), Bioreactor and Bio-process Fluid Dynamics, BHR Group Conference Series,vol. 5. MEP, London, pp. 89–100.

Dubuis, B., Kut, A.M., Prenosil, J.E., 1995. Pilot-scale cul-tures of Coffea arabica in a novel loop fluidised-bed reac-tor. Plant Cell Tissue Organ Cult. 43, 171–183.


Ducos, J.-P., Pareilleux, A., 1986. Effect of aeration rate andinfluence of pCO2 in large-scale cultures of Catharanthusroseus cells. Appl. Microbiol. Biotechnol. 25, 101–105.

Dunlop, E.H., Namdev, P.K., 1993. Effect of fluid forces onplant cell suspensions. In: Nienow, A.W. (Ed.), Bioreactorand Bioprocess Fluid Dynamics. MEP, London, pp. 447–455.

Dunlop, E.H., Namdev, P.K., Rosenberg, M.Z., 1994. Effectof fluid shear forces on plant cell suspensions. Chem. Eng.Sci. 49, 2263–2276.

Fett-Neto, A.G., DiCosmo, F., 1996. Production of Paclitaxeland related Taxoids in cell cultures of Taxus cuspidata :perspectives for industrial applications. In: DiCosmo, F.,Misawa, M. (Eds.), Plant Cell Culture SecondaryMetabolism–Toward Industrial Application. CRC Press,Boca Raton, FL, pp. 139–166.

Flores, H.E., Curtis, W.R., 1992. Approaches to understand-ing and manipulating the biosynthetic potential of plantroots. Ann. New York Acad. Sci. 665, 188–209.

Fujita, Y., 1988. Shikonin: production by plant (Lithospermumerythrorhizon) cell cultures. In: Bajaj, Y.P.S. (Ed.), Bio-technology in Agriculture and Forestry: Medicinal andAromatic Plants I, vol. 4. Springer-Verlag, Berlin, pp.225–236.

Fujita, Y., Tabata, M., 1987. Secondary metabolites fromplant cells: pharmaceutical applications and progress incommercial production. In: Green, C.E., Somers, D.A.,Hackett, W.P., Biesboer, D.D. (Eds.), Plant Tissue andCell Culture. Alan R. Liss, New York, pp. 169–185.

Fulzele, D.P., Heble, M.R., 1994. Large-scale cultivation ofCatharanthus roseus cells: production of ajmalicine in a 20-lairlift bioreactor. J. Biotechnol. 35, 1–7.

Furusaki, S., Seki, M., Kurata, H., Furuya, T., 1993. Theeffect of light irradiation on secondary metabolite produc-tion by Coffea arabica cells. In: Yoshida, T., Tanner, R.D.(Eds.), Bioproducts and Bioprocesses, vol. 2. Springer-Ver-lag, Berlin, pp. 271–282.

Hara, Y., 1996. Research on the production of useful com-pounds by plant cell cultures in Japan. In: DiCosmo, F.,Misawa, M. (Eds.), Plant Cell Culture SecondaryMetabolism–Toward Industrial Application. CRC Press,Boca Raton, FL, pp. 187–202.

Hashimoto, T., Azechi, S., 1988. Bioreactors for large-scaleculture of plant cells. In: Bajaj, Y.P.S. (Ed.), Biotechnologyin Agriculture and Forestry: Medicinal and AromaticPlants I, vol. 4. Springer-Verlag, Berlin, pp. 104–122.

Hegarty, P.K., Smart, N.J., Scragg, A.H., Fowler, M.W.,1986. The aeration of Catharanthus roseus L.G. Don sus-pension cultures in airlift bioreactors: the inhibitory effectat high aeration rates on culture growth. J. Exp. Bot. 37,1911–1920.

Hegglin, M., Prenosil, J.E., Bourne, J.R., 1990. Reaktorsystemzur Massenkultivation von pflanzlichen Zellkulturen beiniedrigem hydrodynamischen Stress. Chimia 44, 26–32.

Ho, C.-H., Henderson, K.A., Rorrer, G.L., 1995. Cell damageand oxygen mass transfer during cultivation of Nicotianatabacum in a stirred-tank bioreactor. Biotechnol. Prog. 11,140–145.

Holton, R.A., Somoza, C., Kim, H.-B., Liang, F., Biediger,R.J., Boatman, P.D., Shindo, M., Smith, C.C., Kim, S.,Nadizadeh, H., Suzuki, Y., Tao, C., Vu, P., Tang, S.,Zhang, P., Murthi, K.K., Gentile, L.N., Liu, J.H., 1994.First total synthesis of taxol 1. Functionalization of the Bring. J. Am. Chem. Soc. 116, 1597–1598.

Hooker, B.S., Lee, J.M., 1992. Application of a new structuredmodel to tobacco cell cultures. Biotechnol. Bioeng. 39,765–774.

Hooker, B.S., Lee, J.M., An, G., 1989. Response of planttissue culture to a high-shear environment. Enzym. Mi-crob. Technol. 11, 484–490.

Hooker, B.S., Lee, J.M., An, G., 1990. Cultivation of plantcells in a stirred vessel: effect of impeller design. Biotech-nol. Bioeng. 35, 296–304.

Hua, J., Erickson, L.E., Yiin, T.-Y., Glasgow, L.A., 1993.Review of the effects of shear and interfacial phenomenaon cell viability. Crit. Rev. Biotechnol. 13, 305–328.

Hulst, A.C., Tramper, J., 1989. Immobilized plant cells: aliterature survey. Enzym. Microb. Technol. 11, 546–558.

Ikeda, T., 1991. Production of anti-plant-viral protein byMirabilis jalapa L. cells in suspension culture. In: Ko-mamine, A., Misawa, M., DiCosmo, F. (Eds.), Plant CellCulture in Japan. CMC, Tokyo, pp. 45–57.

Inomata, S., Yokoyama, M., Seto, S., Yanagi, M., 1991.High-level production of arbutin from hydroquinone insuspension cultures of Catharanthus roseus plant cells.Appl. Microbiol. Biotechnol. 36, 315–319.

Jolicouer, M., Chavarie, C., Carreau, P.J., Archambault, J.,1992. Development of a helical-ribbon impeller bioreactorfor high-density plant cell suspension culture. Biotechnol.Bioeng. 39, 511–521.

Joshi, J.B., Elias, C.B., Patole, M.S., 1996. Role of hydrody-namic shear in the cultivation of animal, plant and micro-bial cells. Chem. Eng. J. 62, 121–141.

Kargi, F., Rosenberg, M.Z., 1987. Plant cell bioreactors:present status and future trends. Biotechnol. Prog. 3, 1–8.

Kato, A., Shimizu, Y., Nagai, S., 1975. Effect of initial kLa onthe growth of tobacco cells in batch culture. J. Ferment.Technol. 53, 744–751.

Kato, A., Kawazoe, S., Soh, Y., 1978. Viscosity of the brothof tobacco cells in suspension culture. J. Ferment. Technol.56, 224–228.

Khlebnikov, A., Dubuis, B., Kut, O.M., Prenosil, J.E., 1995.Growth and productivity of Beta 6ulgaris cell culture influidized-bed reactors. Bioproc. Eng. 14, 51–56.

Kieran, P.M., 1993. An investigation of the hydrodynamicshear susceptibility of suspension cultures of Morinda cit-rofolia. Ph.D. Thesis, University College Dublin, Ireland.

Kieran, P.M., Malone, D.M., MacLoughlin, P.F., 1993. Varia-tion of aggregate size in plant cell suspension batch andsemi-continuous cultures. Food and bioproducts process-ing. Trans. I. Chem. E. (C) 71, 40–46.

Kieran, P.M., O’Donnell, H.J., Malone, D.M., MacLoughlin,P.F., 1995. Fluid shear effects on suspension cultures ofMorinda citrifolia. Biotechnol. Bioeng. 45, 415–425.


Knuth, M.E., Sahai, O.P., 1991. Flavor composition andmethod, US Patent 5 057 424.

Kreis, W., 1993. Arzneistoffe aus Pflanzlichen Zell- und Gewe-bekulturen: Moglichkeiten und Grenzen. Dtsch. Apoth.Ztg. 133, 17–42.

Leckie, F., Scragg, A.H., Cliffe, K.C., 1991. An investigationinto the role of initial kLa on the growth and alkaloidaccumulation by cultures of Catharanthus roseus. Biotech-nol. Bioeng. 37, 364–370.

Li, G.Q., Shin, J.H., Lee, J.M., 1995. Mineral oil addition asa means of foam control for plant cell cultures in stirredtank fermenters. Biotechnol. Tech. 9, 713–718.

Lutterbach, R., Stockigt, J., 1992. High yield formation ofarbutin from hydroquinone by cell-suspension cultures ofRauwolfia serpentina. Helv. Chim. Acta. 75, 2009–2011.

MacLoughlin, P.F., Malone, D.M., Murtagh, J.T., Kieran,P.M., 1997. The effects of turbulent jet flows on plant cellsuspension cultures. Biotechnol. Bioeng. (in press).

Markl, H., Bronnenmeier, R., Wittek, B., 1991. The resistanceof micro-organisms to hydrodynamic stress. Int. Chem.Eng. 31, 185–197.

Markx, G.H., ten Hoopen, H.J.G., Meijer, J.J., Vinke, K.L.,1991. Dielectric spectroscopy as a novel and convenienttool for the study of the shear sensitivity of plant cells insuspension culture. J. Biotechnol. 19, 145–158.

Matsubara, K., Fujita, Y., 1991. Production of berberine. In:Komamine, A., Misawa, M., DiCosmo, F. (Eds.), PlantCell Culture in Japan. CMC, Tokyo, pp. 39–44.

Matsushita, T., Koga, N., Ogawa, K., Fujino, K., Funatsu,K., 1994. High density culture of plant cells using an air liftcolumn for production of valuable metabolites. In: Ryu,D.D.Y., Furusaki, S. (Eds.), Studies in Plant Science 4:Advances in Plant Biotechnology. Elsevier, Amsterdam,pp. 339–353.

Mavituna, F., Park, J.M., 1987. Size distribution of plant cellaggregates in batch culture. Chem. Eng. J. 35, B9–B14.

Meijer, J.J., ten Hoopen, H.J.G., Luyben, K.Ch.A.M.,Libbenga, K.R., 1993. Effects of hydrodynamic stress oncultured plant cells: a literature survey. Enzym. Microb.Technol. 15, 234–238.

Meijer, J.J., ten Hoopen, H.J.G., van Gameren, Y.M., Luy-ben, K.Ch.A.M., Libbenga, K.R., 1994. Effects of hydro-dynamic stress on the growth of plant cells in batch andcontinuous culture. Enzym. Microb. Technol. 16, 467–477.

Merchuk, J.C., 1992. Shear effects on suspended cells. Adv.Biochem. Eng. Biotechnol. 44, 65–122.

Metzner, A.B., Otto, R.E., 1957. Agitation of non-Newtonianfluids. A.I.Ch.E. J. 3, 3–10.

Misawa, M., 1991. Research activities in Japan. In: Ko-mamine, A., Misawa, M., DiCosmo, F. (Eds.), Plant CellCulture in Japan. CMC, Tokyo, pp. 3–7.

Moo-Young, M., Chisti, Y., 1988. Considerations for design-ing bioreactors for shear-sensitive culture. Bio/Technol. 6,1291–1296.

Namdev, P.K., Dunlop, E.H., 1995. Shear sensitivity of plantcells in suspension. Appl. Biochem. Biotechnol. 54, 109–131.

Nicolaou, K.C., Yang, Z., Liu, J.J., Ueno, H., Nantermet,P.G., Guy, R.K., Claiborne, C.F., Renaud, J., Cou-ladouros, E.A., Paulvannan, K., Sorensen, E.J., 1994. To-tal synthesis of taxol. Nature 367, 630–634.

Noguchi, M., Matsumoto, T., Hirata, Y., Yamamoto, K.,Katsuyama, A., Kato, A., Azechi, S., Kato, K., 1977.Improvement of growth rate of plant cell cultures. In:Barz, W., Reinhard, E., Zenk, M.H. (Eds.), Plant TissueCulture and its Biotechnological Application. Springer-Verlag, Berlin, pp. 85–94.

Panda, A.K., Saroj, M., Bisaria, V.S., Bhojwani, S.S., 1989.Plant cell reactors: a perspective. Enzym. Microb. Technol.11, 386–397.

Parr, A.J., 1989. The production of secondary metabolites byplant cell cultures. J. Biotechnol. 10, 1–26.

Parr, A.J., Smith, J.I., Robins, R.J., Rhodes, M.J.C., 1984.Apparent free space and cell volume estimation: a non-de-structive method for assessing the growth and membraneintegrity/viability of immobilised plant cells. Plant CellRep. 3, 161–164.

Payne, G.F., Shuler, M.L., Brodelius, P., 1987. Large scaleplant cell culture. In: Lydersen, B.K. (Ed.), Large ScaleCell Culture Technology. Hanser, Munich, pp. 193–229.

Payne, G.F., Bringi, V., Prince, C.L., Shuler, M.L., 1992.Plant Cell and Tissue Culture in Liquid Systems, Hanser,Munich.

Pras, N., 1992. Bioconversion of naturally occurring precur-sors and related synthetic compounds using plant cellcultures. J. Biotechnol. 26, 29–62.

Prenosil, J.E., Hegglin, M., Bourne, J.R., Hamilton, R., 1987.Purine alkaloid production by free and immobilized Coffeaarabica cells. Ann. New York Acad. Sci. 501, 390–394.

Rosenberg, M.Z., 1987. The hydrodynamic shear sensitivity ofsuspension cultured plant cells. Ph.D. Thesis. WashingtonUniversity, St. Louis, MO.

Schiel, O., Berlin, J., 1987. Large scale fermentation andalkaloid production of cell suspension cultures of Catha-ranthus roseus. Plant Cell Tiss. Org. Cult. 8, 153–161.

Schlatmann, J.E., Nuutila, A.M., van Gulik, W.M., tenHoopen, H.J.G., Verpoorte, R., Heijnen, J.J., 1993. Scale-up of ajmalicine production by plant cell cultures ofCatharanthus roseus. Biotechnol. Bioeng. 41, 253–262.

Schlatmann, J.E., Fonck, E., ten Hoopen, H.J.G., Heijnen,J.J., 1994. The negligible role of carbon dioxide andethylene in ajmalicine production by Catharanthus roseuscell suspensions. Plant Cell Rep. 14, 157–160.

Schlatmann, J.E., Vinke, J.L., ten Hoopen, H.J.G., Heijnen,J.J., 1995. Relation between dissolved oxygen concentra-tion and ajmalicine production rate in high-density culturesof Catharanthus roseus. Biotechnol. Bioeng. 45, 435–439.

Schlatmann, J.E., ten Hoopen, H.J.G., Heijnen, J.J., 1996.Large-scale production of secondary metabolites by plantcell cultures. In: DiCosmo, F., Misawa, M. (Eds.), PlantCell Culture Secondary Metabolism–Toward IndustrialApplication. CRC Press, Boca Raton, FL, pp. 11–52.

Scott, A., 1994. Genetically engineered synthesis of naturalproducts. J. Nat. Prod. 57, 557–573.


Scragg, A.H., 1992. Large scale plant cell culture: methods,applications and products. Curr. Opin. Biotechnol. 3,105–109.

Scragg, A.H., 1995. The problems associated with highbiomass levels in plant cell suspension cultures. Plant CellTissue Organ Cult. 43, 163–170.

Scragg, A.H., Allan, E.J., Bond, P.A., Smart, N.J., 1986.Rheological properties of plant cell suspension cultures.In: Morris, P., Scragg, A.H., Stafford, A., Fowler, M.W.(Eds.), Secondary Metabolism in Plant Cell Cultures.Cambridge University Press, Cambridge, pp. 178–194.

Scragg, A.H., Allan, E.J., Leckie, F., 1988. Effect of shearon the viability of plant cell suspensions. Enzym. Microb.Technol. 10, 361–367.

Seki, M., Ohzora, C., Takeda, M., Furusaki, S., 1997. Taxol(Paclitaxel) production using free and immobilized cellsof Taxus cuspidata. Biotechnol. Bioeng. 53, 214–219.

Shibasaki, N., Hirose, K., Yonemoto, T., Takadi, T., 1992.Suspension culture of Nicotiana tabacum cells in a rotary-drum bioreactor. J. Chem. Technol. Biotechnol. 53, 359–363.

Shuler, M.L., 1993. Strategies for improving productivity inplant cell, tissue and organ culture in bioreactors. In:Yoshida, T., Tanner, R.D. (Eds.), Bioproducts and Bio-processes, vol. 2. Springer-Verlag, Berlin, pp. 235–245.

Smart, N.J., Fowler, M.W., 1981. Effect of aeration onlarge-scale cultures of plant cells. Biotechnol. Lett. 3,171–176.

Stierle, A., Strobel, G., Stierle, D., Grothaus, P., Bignami,G., 1995. The search for a taxol-producing micro-organ-ism among the endophytic fungi of the Pacific Yew,Taxus bre6ifolia. J. Nat. Prod. 58, 1315–1324.

Stockigt, J., Obitz, P., Falkenhagen, H., Lutterbach, R., En-dress, S., 1995. Natural products and enzymes from plantcell cultures. Plant Cell Tissue Organ Cult. 43, 97–109.

Su, W.W., 1995. Bioprocessing technology for plant cell sus-pension cultures. Appl. Biochem. Biotechnol. 50, 189–230.

Takahashi, S., Fujita, Y., 1991. Production of Shikonin. In:Komamine, A., Misawa, M., DiCosmo, F. (Eds.), PlantCell Culture in Japan. CMC, Tokyo, pp. 72–78.

Takeda, T., Seki, M., Furusaki, S., 1994. Hydrodynamicdamage of cultured cells of Catharanthus tinctorius in astirred tank reactor. J. Chem. Eng. Jpn. 27, 466–471.

Tanaka, H., 1981. Technological problems in cultivation ofplant cells at high density. Biotechnol. Bioeng. 23, 1203–1218.

Tanaka, H., 1982. Oxygen transfer in broths of plant cells athigh density. Biotechnol. Bioeng. 24, 425–442.

Tanaka, H., Nishijima, F., Suwa, M., Iwamoto, T., 1983.Rotating drum fermenter for plant cell suspension cul-tures. Biotechnol. Bioeng. 25, 2359–2370.

Tanaka, H., Semba, H., Jitsufuchi, T., Harada, H., 1988.The effect of physical stress on plant cells in suspensioncultures. Biotechnol. Lett. 10, 485–490.

Taticek, R.A., Moo-Young, M., Legge, R.L., 1991. Thescale-up of plant cell culture: engineering considerations.Plant Cell Tissue Organ Cult. 24, 139–159.

Thomas, C.R., 1990. Problems of shear in biotechnology. In:Winkler, M.A. (Ed.), Critical Reports on Applied Chem-istry: Chemical Engineering Problems in Biotechnology,vol. 29. Elsevier Applied Science, London, pp. 23–94.

Ushiyama, K., 1991. Large scale culture of ginseng. In: Ko-mamine, A., Misawa, M., DiCosmo, F. (Eds.), Plant CellCulture in Japan. CMC, Tokyo, pp. 92–98.

van Gulik, W.M., ten Hoopen, H.J.G., Heijnen, J.J., 1993.A structured model describing carbon and phosphate lim-ited growth of Catharanthus roseus plant cell suspensionsin batch and chemostat cultures. Biotechnol. Bioeng. 41,771–780.

Verpoorte, R., van der Heijden, R., van Gulik, W., tenHoopen, H.J.G., 1991. Plant biotechnology for the pro-duction of alkaloids: present status and prospects. In:Brossi, A. (Ed.), The Alkaloids, vol. 40. Academic Press,London, pp. 2–187.

Verpoorte, R., van der Heijden, R., Schripsema, J., Hoge,J.H.C., ten Hoopen, H.J.G., 1993. Plant cell biotechnol-ogy for the production of alkaloids: present status andprospects. J. Nat. Prod. 56, 186–207.

Vogelmann, H., Bischof, A., Pape, D., Wagner, F., 1978.Some aspects on mass cultivation. In: Alfermann, A.W.,Reinhard, E. (Eds.), Production of Natural Compoundsby Cell Culture Methods. GSF, Munich, pp. 130–146.

Wagner, F., Vogelmann, H., 1977. Cultivation of plant tissuecultures in bioreactors and formation of secondarymetabolites. In: Barz, W., Reinhard, E., Zenk, M.H.(Eds.), Plant Tissue Culture and Its Biotechnological Ap-plication. Springer-Verlag, Berlin, pp. 245–252.

Westphal, P.R., 1990. Large scale production of new biologi-cally active compounds in plant cell cultures. In: Ni-jkamp, H.J.J., vander Plan, L.H.W., van Aartrijk, J.(Eds.), Progress in plant cellular and molecular biology.Kluwer, Dordrecht, pp. 601–608.

Wongasmuth, M., Doran, P.M., 1994. Foaming and cellflotation in suspended plant cell cultures and the effect ofchemical antifoams. Biotechnol. Bioeng. 44, 481–488.

Yokoyama, M., Yanagi, M., 1991. High-level production ofarbutin by biotransformation. In: Komamine, A., Mis-awa, M., DiCosmo, F. (Eds.), Plant Cell Culture inJapan. CMC, Tokyo, pp. 79–91.

Zenk, M.H., 1991. Chasing the enzymes of secondarymetabolism: plant cell cultures as a pot of gold. Phyto-chemistry 30, 3861–3863.

Zenk, M.H., El-Shagi, H., Schulte, U., 1975. Anthraquinoneproduction by cell suspension cultures of Morinda citrifo-lia, Planta Med. Suppl. 79–101.

Zhong, J.-J., Seki, T., Kinoshita, S., Yoshida, T., 1991. Ef-fect of light irradiation on anthocyanin production bysuspended culture of Perilla frutescens. Biotechnol. Bio-eng. 38, 653–658.


Zhong, J.-J., Seki, T., Kinoshita, S., Yoshida, T., 1992a.Rheological characterisation of cell suspension and cellculture of Perilla frutescens. Biotechnol. Bioeng. 40, 1256–1262.

Zhong, J.-J., Seki, T., Kinoshita, S., Yoshida, T., 1992b.Effects of surfactants on cell growth and pigment produc-tion in suspension cultures of Perilla frutescens. World J.Microbiol. Biotechnol. 8, 106–109.

Zhong, J.-J., Fujiyama, K., Seki, T., Yoshida, T., 1994. Aquantitative analysis of shear effects on cell suspension andcell cultures of Perilla frutescens in bioreactors. Biotechnol.Bioeng. 44, 649–654.

Zhong, J.-J, Yu, J.-T., Yoshida, T., 1995. Recent advances inplant cell cultures in bioreactors. World J. Microbiol.Biotechnol. 11, 481–487.

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