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Journal of Plant Physiology 163 (2006) 1337—1341

0176-1617/$ - sdoi:10.1016/j.

Abbreviationhigh-performanand Skoog med�CorrespondE-mail addr

www.elsevier.de/jplph

SHORT COMMUNICATION

Effect of oxygen supply on cell growth and saponinproduction in bioreactor cultures of Panax ginseng

Nguyen Trung Thanha, Hosakatte Niranjana Murthya,b, Kee-Won Yuc,Cheol Seung Jeongc, Eun-Joo Hahna, Kee-Yoeup Paeka,�

aResearch Center for the Development of Advanced Horticultural Technology, Chungbuk National University,Cheongju 361-763, Republic of KoreabDepartment of Botany, Karnatak University, Dharwad – 580 003, IndiacCBN Biotech Co. Ltd., Songdaeri 319-9 Ochang, Cheowngwon 363-883, Republic of Korea

Received 4 February 2005; accepted 31 August 2005

KEYWORDSGinseng;Plant cell suspensionculture;Saponin;Secondarymetaboliteproduction

ee front matter & 2005jplph.2005.08.014

s: EC, electrical conducce liquid chromatograpium; SOUR, specific oxying author. Fax: +82 43 2ess: paekky@chungbuk.

SummaryThe effects of oxygen supply within the range 20.8–50% (using pure oxygen and air),on cell cultures of Panax ginseng were investigated in a balloon-type bubblebioreactor (5 L capacity, containing 4 L Murashige and Skoog medium, supplementedwith 7.0mg L�1 indolebutyric acid, 0.5mg L�1 kinetin and 30 g L�1 sucrose). A 40%oxygen supply was found to be optimal for the production of both cell mass andsaponin yielding values of 12.8 g (DW) L�1, 4.5mg (g DW)�1 on day 25, respectively.Low (20.8%, 30%) and high (50%) oxygen concentration supplies were unfavorable tocell growth and saponin accumulation. The results indicate that oxygen supple-mentation to bioreactor-based ginseng cultures was beneficial for biomassaccumulation and saponin production.& 2005 Elsevier GmbH. All rights reserved.

Introduction

Ginseng (Panax ginseng C.A. Meyer), a memberof Araliaceae family, is traditionally considered tobe one of the most potent medicinal plants.

Elsevier GmbH. All rights rese

tivity; HPLC,hy; MS, Murashigegen uptake rate75 7467.ac.kr (K.-Y. Paek).

Ginsenosides (saponins) are regarded as the mostimportant active components in ginseng roots andare attributed with cardio-protective, immunomo-dulatory, anti-fatigue, and hepato-protective phy-siological and pharmacological effects (Tang andEisenbrand, 1992).

In recent years, plant cells have been cultured inbioreactors for production of secondary metabo-lites, including pharmaceuticals, pigments andother chemicals; considerable progress has beenmade in stimulating formation and accumulation of

rved.

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Figure 1. Schematic diagram of the balloon-type bubblebioreactor culture system: a – balloon-type bubblereactor, b – air vent, c – inoculum port (5mm diameter),d – sampling port (10mm diameter), e – medium-exchange port, f – air flow meter, g – membrane filter,h – water column, i – air compressor, j – air reservoir, k –

air cooler, l – filter system, m – air dryer, n – oxygen tank,o – sparger, p – pressure gauge.

N. Trung Thanh et al.1338

secondary metabolites (Rao and Ravishankar,2002). Growth and accumulation of secondarymetabolites in bioreactors is influenced by variousphysical and chemical factors, including gaseouscomposition. In particular, oxygen supply has beenshown to significantly affect secondary metaboliteformation in cell cultures (e.g. Gao and Lee, 1992;Han and Zhong, 2003; Schlatmann et al., 1994). Gasexchange between the gas and liquid phases isespecially important in the context of scale-up ofplant cell cultures. In bioreactors, forced aerationis needed to supply oxygen and to improve fluidmixing. However, it may also lead to the removal ofsome known (e.g. CO2, ethylene) or unknowngaseous components. It has been suggested thatsuch gaseous metabolites are important for cellgrowth and/or synthesis of secondary metabolites(Gao and Lee, 1992; Schlatmann et al., 1994).

In the present study, ginseng cells were culturedin balloon-type bubble bioreactors and interactionsbetween oxygen supply, cell growth, and saponinproduction were investigated. The significance ofoxygen supply during bioreactor culture was re-vealed. This study is of relevance in the applicationof ginseng cell cultures to the large-scale produc-tion of saponin.

Materials and methods

Cell suspensions of P. ginseng were maintained inMurashige and Skoog (MS) medium, supplementedwith 2.0mg L�1 a-naphthalene acetic acid (NAA),0.1mg L�1 kinetin, and 30 g L�1 sucrose. The med-ium pH was adjusted to 5.8 before autoclaving. Cellcultures were grown in 300mL Erlenmeyer flaskswith a working volume of 100mL and weremaintained on a rotary shaker at 105 rpm in thedark at 25 1C. Cells were maintained by sub-culturing (6% inoculum) into fresh medium, every15 days.

Cell cultures were also established in a 5-Lcapacity balloon-type bubble bioreactor containing4 L of MS medium (working volume) supplementedwith 7.0mg L�1 indolebutyric acid (IBA), 0.5mg L�1

kinetin and 30 g L�1 sucrose. Sixty grams cell freshweight per liter was added as inoculum. In theballoon-type bubble bioreactor, a sintered-glasssparger was used for aeration. The airflow rate wasmaintained at 0.1 vvm using a flow meter (DwyerInc., IN, USA). To investigate the effects ofdifferent levels of oxygen on biomass and saponinproduction, the inlet air was mixed with oxygen toyield oxygen concentrations of 20.8% (control),30%, 40%, and 50%. A schematic diagram of the

experimental system is shown in Fig. 1. Thecultivation temperature in the bioreactor wascontrolled at 25 1C and the vessel was maintainedin the dark. Three identical cultivation vessels wereoperated under each set of conditions investigated,and the cultivation data shown represent averagevalues with standard deviations. The bioreactorcultures were maintained for up to 30 days.

A 30mL suspension sample was taken from eachbioreactor every 5 days. The cell suspensions werefiltered and washed several times with distilledwater for the measurement of cell (fresh and dry)weights. The culture supernatants were analyzedfor residual sugar using HPLC, following theanalytical procedures described by Zhang andZhong (1997). Specific oxygen uptake rate (SOUR)measurement was performed as described byPan et al. (2000). The supernatant conductivitywas determined using a conductivity meter (ModelLF-54, WTW GmbH, Wielhalm, Germany).

Extraction and analysis of saponin were under-taken using the method of Yu et al. (2002). Thesaponin fraction was analyzed using HPLC (Waters2690 separation module; Waters 996 photodiodearray detector; Waters millennium 2010 chromato-graphy manager) with Altec Platinum C18 column(particle size 1.5 mm, 33mm� 7mm), eluting withwater/acetonitrile at 3:1 (v/v) for 10min, then at63:37 (v/v) for 25min, with a flow rate of1.2mLmin�1. Saponin reference standards wereobtained from Chromadex Inc., USA. Total saponin

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content was calculated as the sum of the elutedsaponin fractions.

7Control

Results and discussion

The growth kinetics of P. ginseng cells cultivatedin balloon type-bubble bioreactors at four differentoxygen supply levels are presented in Fig. 2. Thecell growth and biomass accumulation increasedgradually with time and maximum biomass levelswere reached after 25 days. Similar growthpatterns have been previously reported forP. notoginseng cell cultures (Zhong et al., 1999).The maximum FW (316 g L�1) was achieved at40% oxygen; the corresponding DW was 12.8 g L�1

(Fig. 2A and B). Increase of 15% in fresh cell biomassand 10% in dry cell biomass were evident, com-pared to the control (20.8% O2 supply) bioreactorcultures. Compared to earlier reported results (e.g.Shamakov et al., 1991; Strogov et al., 1990)significantly higher fresh and dry cell weights (58%

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Figure 2. Time profiles for (A) fresh cell weight, (B) drycell weight in P. ginseng suspensions cultivated in 5 Lballoon-type bubble reactors.

and 14% more, respectively) were achieved in thepresent study with a 40% oxygen air feed to theballoon-type bubble reactor cultures. Similarlypositive effects of oxygen supply on cell growthhave been reported for tobacco suspension cultures(Gao and Lee, 1992). When the feed oxygen levelwas further increased to 50%, biomass levels fell to255 g FWL�1 and 9.0 g DWL�1. This reduction inbiomass may be due to oxygen at higher levels asreported with tobacco (Gao and Lee, 1992) andCatharanthus roseus (Tate and Payne, 1991) sus-pension cultures.

Medium conductivity reflects the uptake ofmedium salts (ions) by the cells and conductivitymeasurements have been used to indirectly assessbiomass levels, with potential for continuous, on-line monitoring of plant cell cultures (e.g. Ryu,1990). Fig. 3A shows characteristic reductions in

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Figure 5. Kinetics of ginseng saponin production bybioreactor-cultivated P. ginseng cells.

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medium conductivity over the course of the batchgrowth cycle for all conditions investigated. In thecultures supplied with 40% oxygen, the pronouncedreduction in EC values, from an initial value of5.6–1.23mS cm�1 at day 25 reflects the higherbiomass levels achieved under these conditions(Fig. 2).

Time profiles for residual sugar concentration inthe medium at different levels of O2 supply areshown in Fig. 3B. In all cases, sugar supplies werealmost exhausted when maximum biomass levelswere achieved. The biomass yield (on sucrose) at50% O2 supply [9.5 g DW (g sugar)�1] was lower thanthat of control 11.5 g DW (g sugar)�1, suggestingthat carbon flux was altered by inlet O2 concentra-tion. Similar phenomena have been reported forC. roseus (Tate and Payne, 1991) and P. notoginseng(Han and Zhong, 2003).

SOUR profiles are presented in Fig. 4 and theseprofiles are typical for plant cell suspensioncultures. Increasing SOUR values during the first10 days of cultivation reflected increased meta-bolic activity associated with inoculum response todilution and initiation of cell division. The max-imum SOUR was observed on day 10 in suspensionsupplied with 40% O2 [0.42mmol O2 (g FWh)�1].

Profiles for total saponin (ginsenoside) produc-tion are shown in Fig. 5. Highest saponin accumula-tion was recorded on days 20–25 and declinedthereafter. The maximum total saponin concentra-tions were 3.8, 4.4, 4.5 and 2.85mg (g DW)�1 at20.8%, 30%, 40%, and 50% O2 supply, respectively(Fig. 5). Highest saponin levels were achieved at40% O2 supply; the lowest levels were achieved at50% O2 supply.

Overall, supplementation of oxygen up to a valueof 40% O2 in air to bioreactor-based suspension

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Figure 4. Changes in specific oxygen uptake rate in cellcultures of P. ginseng cultivated in 5 L balloon-typebubble reactors.

cultures was found to significantly affect theaccumulation of saponin. Huang and Chou (2000)also reported that moderate oxygen levels (oxygenpartial pressure of 0.3 atm equivalent to 30%,approximately) were beneficial to cell growth andL-DOPA (3,4-dihydroxyphenylalanine) production incell cultures of Stizolobium hassjoo; higher oxygenlevels were inhibitory.

High cell density leads to increased fluid viscositywhich could significantly reduce oxygen transferefficiencies in bioreactors. The conventional ap-proach to improving oxygen transfer rate is toincrease agitation speed and/or aeration rate.However, these strategies have several limitations,including high power consumption, cell damage dueto hydrodynamic shear stress, and potential reduc-tion of productivity because of stripping of CO2 andother essential volatiles. An alternative approachwhich may partially address the problems is toimprove the quality of the inlet air via an appro-priate level of oxygen enrichment. In the presentexperiment we have shown that oxygen supplemen-tation, up to a level of 40%, improves the accumula-tion of biomass, the production of metabolites insuspension cultures of P. ginseng cultivated in apneumatically mixed laboratory-scale bioreactor.

Acknowledgements

This work is financially supported by the Ministryof Education and human Resources Development(MOE), the Ministry of Commerce, Industry andEnergy (MOCIE) and the Ministry of Labor (MOLAB)through the fostering project of the Lab ofExcellency.

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