Enzyme and Microbial Technology 32 (2003) 498–503

Rapid communication

Effects of oxygen partial pressure on cell growth and ginsenosideand polysaccharide production in high density cell cultures

of Panax notoginseng

Jin Han, Jian-Jiang Zhong∗State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road,

Shanghai 200237, China

Accepted 8 December 2002

Abstract

The effects of oxygen partial pressure (pO2), within the range 10.6–36.5 kPa, on high density cell cultures ofPanax notoginsengwereinvestigated in a bioreactor. ApO2 of 21.3–29.3 kPa was found optimal for the production of cell mass, ginseng saponin and polysaccharidewith corresponding values of 24, 1.75 and 3.0 g l−1 on Day 15, respectively. A lowpO2 was unfavorable to the cell cultures due to oxygenlimitation. HighpO2 (36.5 kPa) inhibited cell growth and reduced the production of ginseng saponin and polysaccharide, which was due tothe detrimental effect of oxidative burst. The effect ofpO2 on heterogeneity of ginsenosides produced in the cell cultures was also studied.A high pO2 (at 36.5 kPa) stimulated Rb1 biosynthesis in the initial stage of cultivation, while a lowpO2 (at 10.6 kPa) inhibited Rg1 andRb1 formation during cultivation.© 2002 Elsevier Science Inc. All rights reserved.

Keywords:Oxygen partial pressure; Plant cell suspension culture;Panax notoginseng; Ginsenoside; Secondary metabolite production; Traditional Chinesemedicinal herb

1. Introduction

Panax notoginsengis a famous traditional Chinesemedicinal herb. Ginseng saponins (ginsenosides) and gin-seng polysaccharides are known as its major bioactivemetabolites. Ginsenosides, for example, are attributedwith cardio-protective, immunomodulatory, anti-fatigue,and hepato-protective physiological and pharmacologicaleffects.

Although the oxygen requirement of plant cells is rela-tively modest compared to microbial cells, high cell den-sity and fluid viscosity could significantly reduce oxygentransfer efficiencies in bioreactors. A conventional wayof improving oxygen transfer rate is to increase agitationspeed and/or aeration rate. However, these approaches haveseveral limitations, such as high power consumption, celldamage due to the mechanical shear stress, and potentialreduction of productivity because of the stripping of CO2and other essential volatiles from the system. An alternativeapproach to avoid oxygen limitation in bioreactors is viamanipulation of oxygen partial pressure (pO2).

∗ Corresponding author. Tel.:+86-21-64252091; fax:+86-21-64253904.E-mail address:jjzhong@ecust.edu.cn (J.-J. Zhong).

pO2 is known to be important in aerobic microbial fer-mentations[1,2]. In plant cell and tissue cultures,pO2has been shown to affect cell proliferation and differentia-tion of somatic embryos[3–5]. For secondary metaboliteproduction by plant cells, Huang et al. reported that apO2 of 0.3 atm was beneficial to cell growth andl-DOPA(3,4-dihydroxyphenylalanine) production in cell culturesof Stizolobium hassjoo; higherpO2 values were inhibitory[6,7]. However, there are no published studies of the effectsof pO2 on cell growth and metabolite production by ginsengspecies.

In this article, the effects ofpO2 on the cell growth andaccumulation of ginseng saponin and ginseng polysaccha-ride by suspension cultures ofP. notoginsengwere studiedin a small-scale airlift bioreactor. DifferentpO2 levels wereobtained by mixing air with different ratios of pure oxygenor nitrogen while the total aeration rate was maintainedconstant. At a highpO2, as oxygen toxicity on the cellcultures was observed, H2O2 production was also mon-itored for evidence of oxidative burst. Furthermore, theeffect ofpO2 on the heterogeneity of ginsenosides was alsoinvestigated. The information obtained in this work is con-sidered useful for efficient large-scale culture of the plantcells.

0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.doi:10.1016/S0141-0229(02)00337-X

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2. Materials and methods

2.1. Cell subculture

Suspension cells ofP. notoginsengwere grown inMurashige and Skoog (MS) medium and subculturedevery 2 weeks. The subculture conditions were as reportedpreviously[8,9].

2.2. Bioreactor and variation of pO2

A 1 l (working volume) airlift bioreactor was used in thiswork to investigate the effect ofpO2 on cell cultures. Thebioreactor structure was as described elsewhere[9]. ThepO2 of the inlet gas stream was varied by mixing air withpure oxygen or nitrogen at different ratios at total pressureof 102 kPa and total aeration rate of 0.8 vvm.pO2 setpointsof 10.6, 17.3, 21.3, 29.3 and 36.5 kPa were investigated. Inaddition, considering the dilution of CO2 when air mixedwith pure oxygen or nitrogen, CO2 at flowrates of 0.12 and0.045 ml min−1 was also added to the inlet gas stream atpO2 of 10.6 and 36.5 kPa to make the CO2 level the sameas control (air alone).

2.3. Culture conditions

All cultures in this work were high-density batch cul-tures. A modified MS medium containing 1.0 mM copper,3.75 mM phosphate and 50 g l−1 sucrose was used, and theinoculum size was 50 g l−1 fresh cells[8]. Total aeration ratewas adjusted to 0.8 vvm during cultivation to obtain goodmixing in the bioreactor. Two or three identical cultivationvessels were operated under each condition, and the culti-vation data shown represent average values with standarddeviations.

2.4. Sampling and analyses of cell weight, medium sugar,and specific oxygen uptake rate (SOUR)

For sampling in 1 l airlift bioreactors, about 20–30 mlof cell culture was taken once from each reactor. The cellsuspensions were filtered and washed several times withdistilled water for the measurement of dry cell weights(DWs). The culture supernatants were used for analysisof residual medium sugar. The analytical procedures andcalculation of growth yield on sugar were as reported previ-ously [8,9]. The measurement of SOUR was also describedelsewhere[10].

2.5. Analyses of ginseng saponin and polysaccharide

TLC colorimetry and the carbazole-sulfuric acid methodwere used to determine total saponin and polysaccharidecontent in the cultured cells, respectively[8,9]. Individualginsenosides were assayed by HPLC, and the details havebeen described elsewhere[11].

2.6. Assay of oxidative burst

H2O2 originates from superoxide generated by a plasmamembrane-associated NADH oxidase in challenged plantcells. H2O2 generation is associated with the so-called ox-idative burst[12]. H2O2 production was assayed by thescopoletin fluorescence oxidative quenching method (ex-citation wavelength: 350 nm; emission: 460 nm)[13]. Tomeasure H2O2 production, 0.2 ml 50 mM stock solutionof scopoletin (Fluka) in DMSO (Sigma Chemical Co., St.Louis, MO) and 5 ml 2 mg ml−1 stock solution of peroxi-dase (Sino-American Biotechnology Co., Shanghai) wereadded to cell culture in 1 l airlift bioreactor. Scopoletinwas progressively oxidized, and the production of reactiveoxygen species (ROS) was calculated from the fluores-cence decrease using a calibration curve established in thepresence of H2O2. Aliquots of medium were collected atvarious intervals over the 60-min period following inocu-lation and monitored by a spectrofluorimeter (Varian CaryEclipse, CA). A standard curve by adding scopoletin to thesolutions at different H2O2 concentrations was prepared byusing cell-free medium. In order to detect whether aerationaffects scopoletin oxidation, scopoletin was added to thecell-free medium under aerated and unaerated conditions.It was confirmed that aeration had no obvious effect onscopoletin oxidation. In addition, pH value did not varywith pO2 levels during the first hour after inoculation.

3. Results and discussion

3.1. Effect of pO2 on cell growth

Fig. 1A shows the kinetics of cell growth at three dif-ferent pO2s. At a pO2 level of 10.6 kPa, cell growth waslimited, and the maximum DW was 17.5 ± 0.9 g l−1 onDay 17, which was only 70% of that at apO2 of 21.3 kPa(control). The SOUR level at 10.6 kPa was lower than thatof control during the cultivation (Fig. 1C), which coincidedwell with the cell growth profile. According to the massbalance of oxygen, the dissolved oxygen (DO) during cul-tivation was estimated, and oxygen limitation atpO2 of10.6 kPa was found. For example on Day 8,kLa was 24 h−1

at pO2 of 10.6 kPa,C∗ (saturated DO) is 0.133 mmol l−1,the SOUR was 0.36 mmol O2 g−1 DW h−1), the dry cellmass was 10.6 g l−1, thus, DO was calculated to be below0%. This theoretical estimation was also confirmed in othershort-term cultivation experiments by measuring DO with aDO sensor.

By comparing cell growth under air mixed with pureoxygen (atpO2 of 36.5 kPa) with that under control con-ditions, it was found that the maximum DW in the formercase was lower than that in the latter, i.e. 20.5 ± 0.4 g l−1

versus 24.6 ± 0.2 g l−1 on Day 15. Similar SOUR patternswere observed under all conditions. In order to investigatewhether the dilution of CO2 due to the incorporation of

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Fig. 1. Time course of DW (A), medium sugar (B) and SOUR (C) incultivations of P. notoginsengcells in 1-l airlift bioreactors at differentpO2s: 10.6 kPa (�); 21.3 kPa (�); 36.5 kPa (�).

pure oxygen or nitrogen into air caused the different cellgrowth, the inlet air stream was supplemented with CO2 toyield a CO2 partial pressure the same as that of control (airalone). It was observed that CO2 dilution had no effect oncell growth, because the growth pattern (data not shown)and DW (Table 1) were almost the same for the cases withor without CO2 supplementation.

In another aspect, an increase inpO2 may have toxiceffects on plant cells if oxygen concentration is too high[14]. Oxidative burst usually occurs within a few minutesof stress imposition with the production of ROS[15]. Inorder to investigate the oxidative burst at highpO2, H2O2production was assayed.Fig. 2 shows H2O2 productionprofiles atpO2 of 36.5 and 21.3 kPa (control) during a short

Fig. 2. H2O2 production in cell suspensions ofP. notoginsengat pO2 of21.3 kPa (�) and 36.5 kPa (�).

period of cultivation. After inoculation, significant H2O2production was observed atpO2 of 36.5 kPa, which reached1.02 ± 0.03 mmol g−1 DW after 50 min. In contrast, cellsin the control showed lower H2O2 production levels. ROSplay an important role in endonuclease activation and con-sequent DNA damage[15], which may be responsible forcell growth inhibition observed atpO2 of 36.5 kPa. Theinhibition of cell growth at a highpO2 was also reportedin flask cultures ofCatharanthus roseuscells [14], but theoxidative burst was not considered in that work.

Cell growth atpO2 of 17.3 and 29.3 kPa was similar tothat under control conditions (data not shown), and the max-imum DW was 22.1 ± 0.1 and 22.5 ± 0.6 g l−1 on Day 15,respectively (Table 1).

Time profiles of medium sugar consumption atpO2 of10.6, 21.3, and 36.5 kPa are shown inFig. 1B. After inoc-ulation, sugar was gradually consumed by cells in cases.Oxygen supply limitation occurring atpO2 of 10.6 kPa re-duced sugar consumption rate after 5 days cultivation. Inthis case, the residual sugar concentration was still high(18.6 ± 0.4 g l−1) when the maximum DW was obtained(Fig. 1A). In contrast, the residual sugar was almost ex-hausted when the cell growth reached a peak atpO2 of 21.3and 36.5 kPa. It is interesting that although the maximumDW at pO2 of 36.5 kPa was 17% lower than that of control,the consumption of residual sugar was very close in the twocases. The growth yield (on sucrose) atpO2 of 36.5 kPawas lower than that of control (0.324 g/g versus 0.406 g/g).It means that carbon flux was altered bypO2. A similarphenomenon has also been reported for flask cultures ofC. roseus[14].

Air mixed with pure oxygen or nitrogen caused the di-lution of CO2. The above results indicate that CO2 dilutionhad no significant effects on the cell growth, sugar consump-tion and SOUR during the cultivation. The work suggeststhat pO2 is an important factor for plant cell suspensioncultures.

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Table 1Effects ofpO2 on production and productivity of dry cells, ginseng saponin and polysaccharide in high-density cell cultures ofP. notoginsengin bioreactors

pO2 (kPa) Maximum concentration (g l−1) Productivity (mg l−1 per day)

Dry cells Saponin Polysaccharide Dry cells Saponin Polysaccharide

10.6 17.5± 0.9 (17)a 1.04 ± 0.10 1.62± 0.12 702± 52 37.6± 5.8 65.9± 7.310.6 + CO2

b 18.1 ± 0.7 (17) 1.00± 0.06 1.70± 0.10 735± 39 35.3± 3.6 70.6± 5.617.3 22.1± 0.1 (15) 1.57± 0.10 2.38± 0.15 1168± 6 83.5± 6.7 127.1± 10.121.3 24.6± 0.2 (15) 1.76± 0.06 3.01± 0.17 1267± 13 90.7± 4.2 167.3± 11.629.3 22.5± 0.6 (15) 1.74± 0.10 2.99± 0.23 1199± 40 89.3± 6.7 166.0± 15.336.5 20.5± 0.4 (15) 1.50± 0.08 2.64± 0.08 993± 26 73.3± 5.3 142.7± 5.536.5 + CO2

b 20.6 ± 0.4 (15) 1.46± 0.07 2.49± 0.18 1000± 29 70.7± 4.7 132.7± 11.9Shake flask 19.2± 0.6 (17) 1.31± 0.05 2.00± 0.18 860± 34 58.4± 3.0 89.6± 10.6

a The number in the parentheses refers to the time (day) when the maximum DW was obtained.b CO2 (0.12 and 0.045 ml min−1) was added to the inlet gas stream atpO2 of 10.6 and 36.5 kPa to yield the same CO2 partial pressure as under

control conditions.

3.2. Effects of pO2 on metabolite production

Ginseng polysaccharide content atpO2 of 10.6 kPa waslower than that atpO2 of 21.3 and 36.5 kPa (Fig. 3A). CO2supplementation had no obvious effect on polysaccharidecontent (data not shown).Fig. 3Bshows the time profiles oftotal polysaccharide concentration at differentpO2. In the

Fig. 3. Kinetics of polysaccharide content (A), total polysaccharide (B), saponin content (C) and total saponin (D) in cell cultures ofP. notoginsenginbioreactors at differentpO2 levels. Symbols as inFig. 1.

initial 5 days, polysaccharide accumulated slowly becauseof slow cell growth. Thereafter, it increased quickly, achiev-ing maximum value of 1.62± 0.12 (Day 17), 3.01± 0.17(Day 15) and 2.64±0.08 g l−1 (Day 15) atpO2 of 10.6, 21.3and 36.5 kPa, respectively. AtpO2 of 17.3 and 29.3 kPa,total polysaccharide concentration reached maximum val-ues of 2.38± 0.15 and 2.99± 0.23 on Day 15, respectively

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(Table 1). The results suggest that the low polysaccharideproduction at a lowpO2 may be due to the oxygen limita-tion. CO2 supplementation again had no significant effect ontotal polysaccharide concentration (Table 1). Polysaccharideproductivity was 65.9±7.3 and 127.1±10.1 mg l−1 per dayat pO2 of 10.6 and 17.3 kPa, respectively, i.e. about 40 and75% of the control values (Table 1). At high pO2 (36.5 kPa),although the level of polysaccharide content was similar tothe control value, the inhibition of cell growth caused lowerproduction and productivity. The optimal range ofpO2 forthe polysaccharide production was from 21.3 to 29.3 kPa(Table 1).

Ginseng saponin production profiles (total and specificlevels) atpO2 of 10.6, 21.3 and 36.5 kPa are shown inFig. 3Cand D. The observed fluctuations in saponin content are sim-ilar to those previously reported[8,11]. Saponin content atpO2 of 10.6 kPa was lower than that at 21.3 or 36.5 kPa.At pO2 of 17.3 and 29.3 kPa, the saponin content was sim-ilar to that of control (data not shown). When the inlet gaswas supplemented with CO2 at pO2 of 36.5 and 10.6 kPa,the pattern and range of saponin content were very similarto those without CO2 addition (data not shown). The maxi-mum total ginseng saponin concentrations were 1.04±0.10,1.57± 0.10, 1.76± 0.06, 1.74± 0.10 and 1.50± 0.08 g l−1

at pO2 of 10.6, 17.3, 21.3, 29.3 and 36.5 kPa, respectively(Table 1). Dilution of CO2 again had no obvious effect onsaponin production (Table 1). For saponin production, it isclear that the range ofpO2 from 21.3 to 29.3 kPa was op-timal; a highpO2 of 36.5 kPa was harmful because of itsinhibition of cell growth.

3.3. Effect of pO2 on ginsenoside heterogeneity

It has been reported that different ginsenosides have dif-ferent biological activities[11]. For example, Rg1 has theeffect of stimulating the central nervous system, whereasRb1 has tranquilizing effects on the central nervous system.Because the physico-chemical characteristics of differentginsenosides are very similar, separation and purificationof individual ginsenosides is an expensive and complicatedprocess. From the viewpoint of biotechnological applica-tion, it would be very advantageous to be able to intention-ally manipulate the heterogeneity of ginsenosides in ginsengcell cultures[11]. Here, profiles of ginsenoside Rg1, Re andRb1 content atpO2 of 10.6, 21.3 and 36.5 kPa are shownin Fig. 4. The fluctuations during cultivation are similarto those previously reported[11]. Rg1 content atpO2 of10.6 kPa was lower than that at higherpO2 values (21.3 and36.5 kPa). Re content fluctuated between 0.12 and 0.15 mgper 100 mg DW atpO2 of 10.6 kPa; a higherpO2 was alsobeneficial to Re accumulation in the later stages of culture.Rb1 content at apO2 of 10.6 kPa decreased from inoculationuntil Day 15, and was also lower than at a higherpO2. In theinitial 5 days, there was a significant increase in Rb1 contentatpO2 of 36.5 kPa compared to the control. The Rb1 contentwas 0.46 mg per 100 mg DW on Day 5, which was about

Fig. 4. Dynamic changes of the content of ginsenoside Rg1 (A), Re (B),Rb1 (C) in cell cultures ofP. notoginsengcells in bioreactors at differentpO2 levels. Symbols as inFig. 1.

twice that of control, but it decreased thereafter, fallingto approximately control levels by Day 15. Supplementa-tion of CO2 at pO2 of 10.6 and 36.5 kPa had no obviouseffect on ginsenoside formation (data not shown). The re-sults imply thatpO2 plays a significant role in ginsenosidebiosynthesis. At lowpO2 (10.6 kPa), oxygen limitationmight have led to limited ginsenoside formation. At highpO2 (36.5 kPa), Rb1 formation was greatly stimulated in theinitial period of cultivation, although it decreased at the laterstage.

ROS, which were produced at a higherpO2, are secondmessengers that activate downstream defense reactions, such

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as synthesis of pathogenesis-related proteins, glutathioneS-transferase, glutathione peroxidase, and ubiquitin, as wellas phytoalexin accumulation[13]. Yuan et al. reported thatpaclitaxel formation induced by fungal elicitation was pro-portional to the intensity of oxidative burst in suspensioncultures ofTaxus chinensis[16]. Zhao et al. also demon-strated the positive relationship between oxidative burst andbiosynthesis of indole alkaloids in cell cultures ofC. roseus[17]. In this work, Rb1 formation was stimulated signifi-cantly in the initial 5 days at a highpO2 (Fig. 4), whichmay be associated with the oxidative burst (Fig. 2). How-ever, the reason for the decrease of Rb1 content at thelater stage of cultivation is still unclear and requires furtherstudy.

4. Conclusions

pO2 was found to impact on cell growth and metaboliteproduction byP. notoginsengcells in 1-l airlift bioreactors.Cell growth was obviously limited at a lowpO2 (10.6 kPa)due to oxygen limitation. Inhibition of cell growth occurredat a highpO2 (36.5 kPa) because of the oxygen toxicity,which was associated with H2O2 production. Productionof ginseng saponin and polysaccharide was also limited ata pO2 of 10.6 kPa.pO2 levels in the range 21.3–29.3 kPawere found to be optimal for the production of cell mass,ginseng saponin and polysaccharide yielding total produc-tion levels of about 24, 1.75 and 3.0 g l−1, respectively. Inthis work, CO2 dilution or supplementation had no obviouseffects on cell growth and metabolite production, which isdifferent from other reports[6,7,18] but similar to the caseof ajmalicine production byC. roseuscell suspensions[19].The effect ofpO2 on heterogeneity of secondary metabo-lite formation was demonstrated here for the first time. Alow pO2 was found to be detrimental to ginsenoside (Rg1,Re and Rb1) formation. A high pO2 (at 36.5 kPa) couldsignificantly stimulate the Rb1 accumulation at the initialstage, where oxidative burst might have played a criticalrole.

Acknowledgments

Financial support from the National Natural ScienceFoundation of China (NSFC project nos. 20076011,30270038 and 20236040) and the Key Discipline Devel-opment Project of Shanghai Municipality (Shanghai-shiZhongdian Xueke Jianshe Xiangmu) is gratefully acknowl-edged. We also thank the support from the Cheung KongScholars Program administered by the Ministry of Educa-tion of China and the National Science Fund for Distin-guished Young Scholars administered by NSFC (project no.20225619).

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