The effects of oxygen supply within the range of 20.8%-50% (along with purified air), on high-density cell culture of Panax ginseng were investigated in an balloon type bioreactor (5 l capacity and containing 4 l of MS medium). A 40% oxygen supply was found optimal for the production of cell mass and ginsenoside with corresponding values of 12.8 g l-1 DW, 4.5-mg/g DW on day 25, respectively. A low (20.8%, 30%) and well as high concentration of oxygen (50%) supply was unfavorable to the cell cultures and they affect the cell growth and ginsenoside accumulation. The results indicate that oxygen is a key factor in scaling up the process of suspension cultures of Panax ginseng and supplementation of oxygen is useful for efficient large-scale production of ginsenosides by the submerged cultures.
Plants are an abundant source of a large number of useful products including pharmaceutical and food additives. Plant cell cultures are an alternative source to whole plant for the production of high-value secondary metabolites. During the past decade, a considerable progress has been made to stimulate formation and accumulation of secondary metabolites using plant cell cultures (Rao and Ravishankar, 2002). Ginseng (Panax ginseng C. A. Meyer), a member of Araliaceae, is traditionally considered one of the most potent medicinal plants. Ginsenosides have been regarded as the most important active components in ginseng roots and are attributed with cardio-protective, immunomodulatory, anti-fatigue, and hepato-protective physiological and pharmacological effects (Zhang and Zhong, 1997).
In recent years, plant cells are cultured in large-scale bioreactors for production of secondary metabolites including pharmaceuticals, pigments, and other chemicals (Rao and Ravishankar, 2002). Growth and accumulation of secondary metabolites in large-scale bioreactors is influenced by various factors such as shear stress, oxygen supply, and gas composition. A conventional stirred-tank bioreactor can produce a high shear region, while in many cases airlift and bubble column reactors are used for providing shear environment compared to turbine-agitated reactors. As reported, oxygen supply is also significant in affecting secondary metabolites formation in cell cultures (Gao and Lee, 1992; Zhong et al., 1993; Han and Zhong, 2003). Gas exchange between the gas and liquid phases is another important factor that may affect the scale-up of plant cell cultures. In bioreactors, forced aeration is needed to supply oxygen and to improve fluid mixing. However, it may also lead to the removal of some known (such as CO2 and ethylene) or unknown gaseous compounds. Such gaseous metabolites were proven or suggested to be important for cell growth and/or synthesis of secondary metabolites in plant cell cultures (Gao and Lee, 1992).
The concentration of dissolved oxygen can be easily controlled in bubble/airlift bioreactors and interaction between O2 supply, cell growth and metabolite biosynthesis can be observed. In this study, we have used balloon type bioreactors for cell cultures of ginseng and the interaction between oxygen supply, cell growth, and ginsenoside production was investigated. The significance of gas control during bioreactor culture has been established and this study is considered useful for biotechnological application ginseng cell cultures to the production of ginsenosides on a large scale.
Materials and methods
Induction and proliferation of callus
Six-year-old fresh ginseng roots (Panax ginseng C. A. Meyer) were sterilized and cultured as described by Yu, (2000).
A five-liter capacity balloon type bioreactors were used containing 4 l of MS (Murashige, Skoog, 1962) medium working volume with the culture condition as described by Thanh et al., (2004; 2005) to increase the biomass. Sixty grams cell fresh weight per liter was added as inoculum. In the bubble bioreactor, a sinter glass was used for aeration, and the airflow rate was adjusted during cultivation to homogenous mixing state. To investigate the effects of different levels of oxygen in the inlet air, air was mixed with different concentrations of oxygen i.e., 20.8% (control), 30%, 40%, and 50%. The schematic diagram of the whole experimental system is shown in Fig. 1. The cultivation temperature was controlled at 25±2oC and continuous darkness was maintained. Three identical cultivation vessels were operated under each condition, and the cultivation data shown represent average values with standard deviations. The bioreactor cultures were maintained up to 30 days.
Sampling and analyses of cell weight, medium sugar, conductivity
A sample of 30 ml of cell culture was taken once from each bioreactor at an interval of every five days. The cell suspensions were filtered and washed several times with distilled water for the measurement of cell weights (fresh and dry weights). The culture supernatants were used for analysis residual sugar, using HPLC by following analytical procedures described by Zhang and Zhong, (1997) and Woragidbum-rang et al., (2001). The electrical conductivity was from the exhausted medium using conductivity meter Wiss-teelm-werkstalten model LF-54 (WTW GmbH, Wielhalm, Germany).
Fig. 1. Schematic diagram of the balloon type bioreactor culture system used in the present study: a. body of a balloon type bioreactor, b. air vent, c. inoculum port, d. sampling port, 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.
Determination of ginsenoside content
Ginsenoside (saponin) content was determined by HPLC, and the details have been described elsewhere (Furuya and Yoshikawa, 1987; William and John, 1996).
Results and discussion
Effect of oxygen concentration on cell growth
Fig. 2 shows the growth kinetics of P. ginseng cells in 4 l balloon type bioreactors as influenced by four different levels of oxygen supply. The cell growth and biomass accumulation is gradually increased with lapse of time and optimum biomass accumulation reached after 25 days. Similar growth kinetics pattern was reported in P. notoginseng in shake flask, centrifugal impeller bioreactor and turbine reactors cultures (Zhong et al., 1999). The maximum fresh weight with the supply of 20.8% oxygen (control) was 267 g l-1 and corresponding dry weight was 11.5 g l-1 (Fig. 2A-B). It was found that optimum accumulation of fresh (316 g l-1) and dry biomass (12.8 g l-1) was with the supply 40% oxygen in the bioreactors. The biomass accumulation comparatively declined with the increase in oxygen concentration to 50% (255 g l-1 FW and 9.0 g l-1DW).
Fig. 2. Time profiles of fresh cell weight (A), dry cell weight (B) in high-density culture of Panax ginseng cells in a 5 l balloon type bioreactor.
Electrical conductivity measurements (EC) have been used as an indirect method of biomass estimation in continuous on line monitoring of plant cell cultures in bioprocess engineering studies for its accuracy and efficiency (Ryu et al., 1994). The electrical conductivity of the medium, also which reflects the uptake of medium salts (ions) by the cultured cells and linear decrease, was observed with increase in cell density during cultivation (Fig. 3A). In the cell cultures, which were supplied with 40% oxygen, showed a decrease in EC values from initial value of 5.6 mS/cm to 1.23 mS/cm (Fig. 3A). At the beginning of cultivation, the cell growth was slow and in a lag phase, and subsequently cells involved in division and multiplication and hence due to the active metabolic uptake of the medium ions by the cultured cells. Similarly, observations were recorded with P. notoginseng (Zhong et al., 1999) and rice (Wen and Zhong, 1996) suspension cultures.
Time profiles of medium sugar consumption at different levels O2 supply are shown in Fig. 3B. After inoculation, cells in all cases gradually consumed sugar and residual sugar concentration was almost exhausted when cell growth reached peak. The growth yield (on sucrose) at 50% O2 supply was lower than that of control (11.5 g l-1 versus 9.5 g l-1 on day 25) and it means that carbon flux was altered by O2 concentration. A similar phenomenon has also been reported during cell culture in Catharathus roseus (Tate and Payne, 1991) and in P. notoginseng (Han and Zhong, 2003).
Fig. 3. Time profiles of medium conductivity (A), residual sugar (B) in high-density cultures of Panax ginseng cells in a 5 l balloon type bioreactor.
Effect of oxygen concentration on metabolite production
The kinetic profile of total ginsenosides (saponin) production is shown in Fig. 4. Highest saponin accumulation was on day 20 to 25 and later it declined. Saponin content at 50% O2 supply was lower than that of control (20% O2 supply). The maximum total saponin concentrations were 3.8 mg/g DW, 4.4 mg/g DW, 4.5 mg/g DW and 2.85 mg/g DW at 20.8%, 30%, 40% and 50% O2 supply, respectively (Fig. 4). Highest saponin production was with 40% O2 supply and lowest with 50% O2 supply.
Supplementation of oxygen to the high-density suspension cultures significantly affects the accumulation of ginsenosides and these results are concurrence with earlier published reports (Gao and Lee, 1992; Zhong et al., 1993).
High cell density and fluid viscosity could significantly reduced oxygen transfer efficiencies in bioreactors and conventional way of improving oxygen transfer rate is to increase agitation speed and/or aeration rate (Huang and Chou, 2000). However, these approaches have several limitations, such as high power consumption, cell damage due to mechanical shear stress, potential reduction of productivity because of the stripping of CO2 and other essential volatiles from the system. An alternative approach is improving the quality of incoming air by with oxygen concentration. In the present experiment we have supplemented the incoming air with different ratios of pure oxygen, which facilitates oxygen transfer rates, improves the accumulation of biomass of cultured cell and in turn accumulation of metabolites.
Fig. 4. Kinetics of production of ginseng saponin of Panax ginseng cells in high-density bioreactor cultivations.
1. Gao JW, Lee JM (1992). Effect of oxygen supply on the suspension culture of genetically modified tobacco cells. Biotcehnol. Prog. 8: 285-90.
2. Furuya T, Yoshikawa T (1987). Saponin production by cultures of P. ginseng transformed with Agrobacterium rhizogens. Plant Cell Rep. 6: 449-453.
3. Han J, Zhong JJ (2003). Effects of oxygen partial pressure on cell growth and ginsenoside and polysaccharide production in high-density cell cultures of P. notoginseng. Enzyme Microb. Tech. 32: 498-503.
4. Huang SY and Chou CJ (2000). Effect of gaseous composition on cell growth and secondary metabolite production in suspension culture of Stizolobium hassjoo cell. Bioproce. Engineer., 23: 585-593.
5. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15: 473-497.
6. Rao SR, Ravishankar GA (2002). Plant cell cultures: Chemical factories of secondary metabolites. Biotech. Adv. 20: 101-153.
7. Ryu DY, Lee Schlatmann JE, Fonck E, ten Hoopen HJG, Heijnen JJ (1994). The negligible role of carbon dioxide and ethylene in ajmaliceine production by Catharanthus roseus cell suspensions. Plant Cell Rep. 14: 157-60.
8. Tate JL, Payne GF (1991). Plant cell growth under different levels of oxygen and carbon dioxide. Plant Cell Rep. 10: 22-25.
9. Thanh NT (2005). Factors affecting cell growth and ginsenoside production in P. ginseng C. A. Meyer. Ph.D. Dissertation, Chungbuk National University, Cheongju, South Korea.
10. Thanh NT., Murthy HN, Yu KW, Hahn EJ and Paek KY (2004). Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of P. ginseng in 5-l balloon type bubble bioreactors. Appl. Microb. Biotechnol., ISSN: 0175-7598 (Paper) 1432-0614 (Online).
11. Wen ZY, Zhong JJ (1996). Correlation between biomass and medium conductivity in suspension cultures of rice cells. Biotechnol. Tech. 10: 309-312.
12. William A, Jhon G, Hendel J (1996). Reversed-phase high performance liquid chromatographic determination of ginsenosides of P. quinquefolium. J. Chromatog. 77: 11-17.
13. Woragidbum-rang K, Sae-Tang P, Yao H, Han J, Chauvatchrin S, Zhong JJ (2001). Impact of conditioned medium on cell cultures of P. notoginseng in air-lift bioreactors. Process Biochem. 37: 209-313.
14. Yu KW (2000). Production of useful metabolites through bioreactor culture of Korean ginseng (P. ginseng C. A. Meyer), Ph. D. Dissertation, Chungbuk National University, Cheongju, Korea.
15. Zhang YH, Zhong JJ (1997). Hyper-production of ginseng saponin and polysaccharide by high-density cultivation of P. notoginseng cells. Enzyme Microb. Tech. 21: 59-63.
16. Zhong JJ, Chen F, Hu WW (1999). High-density cultivation of P. notoginseng cells in stirred bioreactors for the production of ginseng biomass and ginseng saponin. Process Biochem. 35: 491-496.
17. Zhong JJ, Yoshida M, Fujiyama K, Seki T, Yoshida T (1993). Enhancement of anthocyanin production by Perilla frutescens cells in stirred bioreactor with internal light irradiation. J. Ferment. Bioeng. 75: 299-303.
Thanh N. T
Department of Botany, National University Hanoi, 334 Nguyen Trai, Hanoi, Vietnam.
Yu K. W, Hahn E. J., and Paek K Y
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju, 361-763, South Korea.
Murthy H. N
Department of Botany, Karnatak University, Dharwad - 580 003, India.