Biotechnology Letters 24: 1927–1930, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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High density cell culture of Panax notoginseng for production of ginseng saponin and polysaccharide in an airlift bioreactor Jin Han & Jian-Jiang Zhong∗ State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China ∗ Author for correspondence (Fax: +86-21-64253904; E-mail:
[email protected]) Received 29 July 2002; Revisions requested 23 August 2002; Revisions received 17 September 2002; Accepted 17 September 2002
Key words: airlift bioreactor, ginseng saponin and polysaccharide, high density cell culture, Panax notoginseng, plant cell culture
Abstract High cell density of Panax notoginseng in a 17 l airlift bioreactor was achieved in batch cultivation using a modified MS medium. The dry cell weight, ginseng saponin and polysaccharide reached 24, 1.7 and 2.8 g l−1 , respectively, after 15 d. A strategy of sucrose feeding based on changes in the specific O2 uptake rate was applied to the cell cultures, which increased these respective yields to 30, 2.3 and 3.2 g l−1 .
Introduction Plant cell culture is a promising technology to obtain plant-specific valuable metabolites (Verpoorte et al. 1999) including paclitaxel, artemisinin, and ginseng saponin (Choi et al. 2000, Linden et al. 2001, Verpoorte et al. 1999, Zhong 2001). A commercial process using plant cell culture for production of fine chemicals requires large-scale cultivation and a high product yield (Scragg 1990). During the scale up of plant cell cultures, a decreased productivity has often been reported (Scragg et al. 1987, Zhong 2001). Panax notoginseng (Sanchi-ginseng), which is one of the most valuable oriental herbs, has been used as a healing drug and health tonic in China since ancient times. Ginseng saponin and ginseng polysaccharide are its major bioactive metabolites (Zhang & Zhong 1997). In our previous work, a relatively stable and high saponin producing cell culture of P. notoginseng was established, and high density cell culture for production of ginseng saponin and polysaccharide was reached in a 500 ml shake flask and a 1 l airlift bioreactor (Zhang & Zhong 1997, Hu et al. 2001). In this work, the kinetics of high density cultivations of P. notoginseng cells in a 17 l airlift bioreactor were investigated. A feeding strategy based on changes of
specific O2 uptake rate, which was proposed in 1 l bioreactor cultivations (Hu et al. 2001), was also examined in this study.
Materials and methods Cell cultivation Suspension cells of Panax notoginseng were grown in MS medium and subcultured every 2 weeks (Zhang & Zhong 1997). They were grown in a 17 l airlift bioreactor with a working volume of 10 l. For the bioreactor, its column diameter, column height, draft tube diameter, and draft tube height were 12.8 cm, 102 cm, 8.5 cm, and 53 cm, respectively. The draft tube was placed 5 cm above the bottom. The cultivation temperature and aeration rate was controlled at 25 ◦ C and 7 l min−1 during cultivation. For high density batch cultivation, a modified MS medium containing 1 µM Cu2+ , 3.75 mM phosphate and 50 g sucrose l−1 was used, and the inoculum size was 50 g fresh cells l−1 (Zhang & Zhong 1997). A fed-batch cultivation in the bioreactor was operated by feeding sucrose at the later stage of cultivation (Hu et al. 2001).
1928 Sampling and analyses of cell weight, residual sugar and specific O2 uptake rate About 100–150 ml of cell culture was taken for analyses from the bioreactor. The cell suspensions were filtered and washed several times with sufficient amount of distilled water for the measurements of dry cell weights (DW). The culture supernatants were used for analyses of residual medium sugar. The analytical procedures were the same as reported previously (Hu et al. 2001). Calculation of growth yield on sugar and measurement of specific O2 uptake rate were described elsewhere (Hu et al. 2001). Assay of ginseng saponin and polysaccharide TLC colorimetry and carbazole-sulfuric acid method were used to determine the content of ginseng saponin and polysaccharide, respectively (Zhang & Zhong 1997).
Results and discussion High density batch cultivation As shown in Figure 1A, dry cell weight increased slowly in the initial 5 d in batch cultivation in a 17-l airlift bioreactor. From day 5, it increased quickly and reached a peak of 24 g l−1 on day 15. After day 15, a sharp decrease of dry cell weight was also observed, which might be related to the cell autophagy. Cell autophagy occurs when major medium components were almost consumed (Moriyasu & Ohsumi 1996). A similar phenomenon was also reported in a shake flask and a 1 l airlift bioreactor (Hu et al. 2001). Sucrose in the medium was consumed linearly and was nearly exhausted by day 15. The final growth yield on sucrose was 0.42 g g−1 , which was similar to that achieved in a 1 l airlift bioreactor (Hu et al. 2001). Dynamic changes of the content and total production of ginseng polysaccharide and saponin are shown in Figures 2A and 2B, respectively. For the formation of ginseng polysaccharide, its content changed within a range of 7.3–11.5 mg/100 mg DW. The total polysaccharide concentration increased slowly in the initial 5 d because of the slow cell growth. Thereafter it increased quickly with rapid cell growth and reached a maximal value of 2.8 g l−1 on day 15 with a corresponding productivity of 158 mg l−1 d−1 . Ginseng saponin content fluctuated between 6.7 and 7.9 mg/100 mg DW. A similar phenomenon of
Fig. 1. Time course of dry cell weight, residual medium sugar and specific O2 uptake rate in batch (A) and fed-batch (B) cultivations of Panax notoginseng cells in a 17-l airlift bioreactor. Dry cells (); medium sugar (); specific O2 uptake rate (SOUR) (). All cultivations were repeated and the data shown represent average values of two fermentations with maximum errors from two independent samples.
fluctuation in its content was also reported (Zhang & Zhong 1997, Hu et al. 2001). The total saponin concentration increased slowly at the beginning of cultivation and reached a maximal value of 1.7 g l−1 on day 15 with a productivity of 91 mg l−1 d−1 . Compared to that in a 1-l bioreactor (Hu et al. 2001), the above results indicate that the high density batch cultivation was successfully achieved in the 17-l airlift bioreactor without loss of the metabolites production and productivity.
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Fig. 2. Dynamic profiles of the content and total production of ginseng polysaccharide and saponin in the batch (A, B) and fed-batch (C, D) cultures of Panax notoginseng cells in a 17-l airlift bioreactor. P: Polysaccharide content (), total polysaccharide (); saponin content (), total saponin (). All cultivations were repeated and the data shown represent average values of two fermentations with maximum errors from two independent samples.
High-density fed-batch cultivation As reported previously in 1-l bioreactors (Hu et al. 2001), sucrose feeding before a sharp decrease of specific O2 uptake rate was important to the efficient fed-batch cultivation of P. notoginseng cells. To obtain a high-density cell culture process in a large bioreactor, this study examined whether the above feeding strategy could be applied to 17-l bioreactor cultures. As shown in Figure 1B, after inoculation, the specific O2 uptake rate value increased and reached its maximum value on day 8; thereafter, it decreased slowly and had a sharp decrease on day 13, when sucrose was fed into the bioreactor. It indicates that the su-
crose feeding did promote the final cell density, and a high dry cell density of 30 g l−1 was obtained on day 17 (Figure 1B) with a productivity of 1453 mg l−1 d−1 . The dry cell weight and its productivity increased by about 25 and 15% compared to those in batch cultivations, respectively. Figures 2C and 2D show the changes of the content and total production of ginseng polysaccharide and saponin in fed-batch cultures in a 17-l bioreactor. The polysaccharide content changed ranging from 6.9 to 11 mg/100 mg DW during the cultivation (Figure 2C). Generally, the total polysaccharide concentration increased with an increase of dry cell
1930 weight, and it reached a maximum value of 3.2 g l−1 on day 17, which was about 15% higher than that in batch cultivation. The volumetric productivity of ginseng polysaccharide was 164 mg l−1 d−1 , which was also higher than that in batch cultivation. The total production and productivity of polysaccharide in fedbatch cultivation were similar to those in a 1-l airlift bioreactor (Hu et al. 2001). The content of ginseng saponin fluctuated from 6.9 to 7.9 mg/100 mg DW during the fed-batch cultivation (Figure 2D). The total saponin production was improved by feeding sucrose at the later stage of cultivation (on day 13), and it reached a peak of 2.3 g l−1 on day 17 with a corresponding productivity of 113 mg l−1 d−1 . Its concentration and productivity was about 35 and 25% higher than that in batch cultivation, respectively. In the 1-l airlift bioreactor, the maximum saponin concentration and productivity in fed-batch cultures was 2.1 g l−1 and 106 mg l−1 d−1 , respectively (Hu et al. 2001). The above results confirmed the usefulness of specific O2 uptake rate-based feeding strategy in a 17-l airlift bioreactor. The high-density culture process of P. notoginseng cells was achieved for the first time in a 17-l airlift bioreactor without loss in the production titer and productivity of dry cell weight, ginseng saponin and ginseng polysaccharide compared to those in a 1-l bioreactor. The success provides technical feasibility for the cell culture process in bioreactors and it is another step of advance towards large-scale production of the proposed metabolites by P. notoginseng cells. Further work to scale up the cell culture process is under way in our laboratory.
Acknowledgements Financial support from the National Natural Science Foundation of China (NSFC project no. 20076011),
the Science & Technology Commission of Shanghai Municipality (Qimingxing Jihua), and the Key Discipline Development Project of Shanghai Municipality (Shanghai-shi Zhongdian Xueke Jianshe Xiangmu) is gratefully acknowledged. J.J.Z. also thanks the support from the Cheung Kong Scholars Program administered by the Ministry of Education of China and the National Science Fund for Distinguished Young Scholars administered by NSFC.
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