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Kinetics of two-liquid-phase Taxus cuspidata cell culture for production of Taxol
Biochemical Engineering Journal 5 (2000) 137–142
Kinetics of two-liquid-phase Taxus cuspidata cell culture for production of Taxol Zhao-Liang Wu, Ying-Jin Yuan∗ , Zhong-Hai Ma, Zong-Ding Hu Department of Biochemical Engineering, Tianjin University, Tianjin 300072, China Received 25 August 1999; accepted 7 January 2000
Abstract The effects of different organic solvents (paraffin, organic acid, alcohol and ester) and their volumetric fractions on the cell growth and Taxol production were studied in two-liquid-phase and the two-stage culture. A kinetic model, incorporated the effects of the toxicity of organic solvents was developed for two-liquid-phase culture of Taxus cuspidata in the two-stage Taxol production. The results showed that the proposed kinetic model could fit the experimental data satisfactorily. The results also showed that Taxol production could reach the optimal value when 10−log P was in the range of 2 to 5 and the volumetric fraction of the organic solvents at the corresponding the highest Taxol production should be lower when 10−log P was high. © 2000 Elsevier Science S.A. All rights reserved. Keywords: Plant cell culture; Two-liquid-phase; Taxus cuspidata; Taxol; Kinetic model
1. Introduction
2. Materials and methods
In order to increase the supply of Taxol, an effective anticancer drug, plant cell culture is considered to be a possible source for a large scale production of Taxol [1–4]. However, due to the extremely low solubility of Taxol in the medium and its inhibiting effect on cell growth [5] and Taxol synthesis, the two-liquid-phase culture is taken as a key technology, which was confirmed to be very effective for enhancing Taxol production of plant cell cultures [6]. In situ removal of Taxol out of the aqueous phase into the hydrophobic phase leads to a shift of equilibrium towards more production and, then promoting the Taxol biosynthesis. To verify the above mentioned advantages of the two phase culture, effects of different parameters such as culture time, toxicity and volumetric fraction of organic solvents, etc. on cell growth and Taxol production were studied. In this paper, a kinetic model for the culture system was also proposed.
2.1. Materials
∗ Corresponding author. Tel.: +86-22-2740-3888. E-mail address: [email protected] (Y.-J. Yuan)
2.1.1. Plant material The cell line was initiated from young stems of Taxus cuspidata and subcultured on solid B5 medium [7] at 25◦ C in the dark. 2.1.2. Liquid medium The growth medium was B5 medium with Vitamin B1, B6, nicotinic acid and inositol doubled, sucrose 25 g/L (L represents fresh liquid medium in this paper). It was supplemented with 6-benzyladenine 0.5 mg/L and casamino acid 2 mg/L. The production medium was liquid B5 medium supplemented with sucrose 60 g/L, 6-benzyladenine 0.5 mg/L and casamino acid 1 mg/L. 2.1.3. Organic solvents The following two points were considered in the selection of organic solvents for the two-liquid-phase cultures. First, the organic solvents should be biocompatible with cultured plant cells. Second, the partition coefficient of Taxol should be high enough. It was reported [8] that organic solvents with log P>4 were biocompatible with cells on the whole (log P is logarithm of the solvent partition coefficient in a standard two-phase
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Table 1 Physical properties of organic solvents in the systems Organic solvents
log P
Hexadecane
8.7
Partition coefficient
Decanol
4.0
311
D420 = 0.773= 0.830
Oleic acid
7.7
154
D420 = 0.773= 0.894
Dibutylphthalate
5.4
236
D420 = 0.773= 1.048
5.7
the mixed extract was measured by HPLC similarly to the Taxol measurement in spent medium.
Density [10] D420 = 0.773
system i-octanol/water). The log P of organic solvents can be found in relevant literature [8,9]. Table 1 shows those organic solvents used and their physical properties. Partition coefficients of Taxol in the two-phase systems (organic solvent/liquid medium) were measured (the two-phase systems were saturated by Taxol at 25◦ C). Taxol measurement in organic solvents and in aqueous phase are given in Sections 2.3.3 and 2.3.1, respectively. The organic solvents are not soluble in water [10]. 2.2. Culture conditions Cells of T. cuspidata were cultivated in 250 ml Erlenmeyer flasks, at 25◦ C, in the dark and under continuous shaking (100 rpm) on a rotary shaker. Every flask contained initially 30 ml growth medium and 6 g fresh cells. At the end of the 10 day cell growth period (the later period of exponential growth phase), 10 ml production medium was added into the flask to enhance the production of Taxol and this process lasted for 6 days. Organic solvents were all preconditioned, i.e. mixed sufficiently with growth medium (10 ml organic solvents/90 ml growth medium) and then separated. The preconditioned organic solvents were added into the flasks on the 10th day of the cell culture. 2.3. Taxol measurements 2.3.1. Taxol measurement in spent medium The measured sample was centrifuged and separated into spent medium, cells and organic solvents. Taxol content in spent medium was measured by HPLC with a reversed-phase column (Kromasil C18 5 m 200×4.6 mm). The mobile phase consisted of acetonitrile and water (47:53 v/v). The flow rate was 1 ml/min and elution time 40 min. Taxol was detected at 227 nm with an ultraviolet detector. The injection was 10 l at a time. 2.3.2. Taxol measurement in cells The separated cells was stored in a refrigerator (−20◦ C, more than a day). The frozen cells were soaked in 5 ml cyclohexane and powdered (10 min), and then cyclohexane was abandoned. The powdered cells were mixed with 20 ml methanol, ultrasonicated for 20 min, centrifuged and separated from the mixture. This process was repeated twice with methanol. The extract (three times) was mixed and Taxol in
2.3.3. Taxol measurement in organic solvent Taxol in organic solvents was measured by HPLC with a normal-phase silica-gel column (Kromasil Sil 5 m 200×4.6 mm). The mobile phase consisted of cyclohexane and acetone (77:23 v/v). Other conditions were the same as that in Taxol measurement in spent medium. 3. Kinetic model development 3.1. Model assumption According to the experiment data, we could make the following assumptions which qualitatively represented the effects of most important parameters on cell growth and production of Taxol. Then a mathematical model was developed in order to represent the main features of the system qualitatively. 3.1.1. Fully mixed culture system The medium, cultured T. cuspidata cells and organic solvents were at the fully mixed state. 3.1.2. Two-stage culture Cell growth was partially interrelated with Taxol production in Taxus cell suspension cultures and hence two-stage culture was used. The first-stage culture aimed at enhancing cell growth, therefore, the growth medium was used. The second-stage culture aimed at enhancing Taxol production, so the production medium was used. Consequently, a model was developed for the two-phase culture in two stages. 3.2. Mathematical model According to the above assumptions, the derived mathematical model was described as follows. 3.2.1. The first-stage culture 3.2.1.1. Cell growth. Sucrose was taken as the sole limiting substrate and its concentration affected cell growth. The kinetics of cell growth could be represented by the relation proposed by Teissier [11]. dx = µmax x 1 − e−(s/K1 ) (1) dt Here x represents the concentration of dry cells (g/L), µmax represents the maximum specific cell growth rate (per day), S the concentration of sucrose (g/L) and K1 coefficient of cell growth with respect to sucrose limited (g/L). 3.2.1.2. Sucrose consumption. The rate of sucrose consumption was determined by the cell concentration and cell
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growth. The effect of Taxol production on sucrose consumption was neglected because the consumption of sucrose by Taxol production was very low.
volumetric fraction of organic solvent (g/L day), K10 and K11 represent experimental coefficients.
dx ds = −Y(s/x) − K2 x dt dt
3.2.2.2. Taxol production. Taxol production was the sum of Taxol produced in cells, those accumulated in cell-free medium and those transferred into and stored in the organic phase. Taxol production was directly proportional to the cell concentration and inversely proportional to the concentration of organic solvents and the phase equilibrium constant of Taxol in the system (ratio of mass concentration of Taxol in the organic solvent over that in the medium), and was inhibited by Taxol accumulation in cells. Taxol accumulation in the cell-free medium was at the equilibrium state with those in cells and the equilibrium constant was affected by the toxicity of an organic solvent (10−log P). Taxol accumulation in organic solvent was at the equilibrium state with those in cell-free medium.
(2)
Here Ys /x represents sucrose consumption with respect to the cell growth (g/g) and K2 the coefficient of sucrose consumption with respect to the cell concentration (g/g day). 3.2.1.3. Taxol production. Taxol production was the sum of Taxol produced in cells and accumulated in cell-free medium. The rate of Taxol production in cells was partially interrelated with the rate of cell growth. Taxol accumulation in cell-free medium was at the equilibrium state with that in cells and the equilibrium constant was unchanged. dx dM = K3 + K4 x + K5 dt dt
(3)
(7)
M = M1 + M2
dM K = K12 x K13 − K14 M1 15 + K16 (VR)K17 + K18 dt
(4)
M = M1 + M2 + M3
(8)
M = K6 M1
(5)
M2 = [K19 + K20 (10 − log P )]M1
(9)
Here M represents total Taxol (mg/L), M1 Taxol produced in cells (mg/L), M2 the Taxol accumulated in cell-free medium (mg/L), K3 the coefficient of Taxol synthesis with respect to the cell growth (g/g), K4 the coefficient of Taxol synthesis with respect to the cell concentration (g/g day), K5 the experimental coefficient (g/L day) and K6 the partition coefficient of Taxol between cell-free medium and cells. 3.2.2. The second-stage culture 3.2.2.1. Cell growth. Sucrose became a non-limiting substrate because its concentration was high enough in the second-stage culture. Cell growth was inhibited by Taxol accumulation in cells, which was more obvious for one-liquid-phase culture. Cell growth was also affected by the toxicity and the volumetric fraction of organic solvents. The log P was used to express the effects of toxicity of organic solvents on organisms in the two-liquid-phase cultures of microbes [9] and it was also used for plant cells [12] in the two-liquid-phase culture. Accordingly, the toxicity of an organic solvent was defined as 10−log P in this paper. For one-liquid-phase culture, 10−log P was zero because there were no organic solvents in the system. dx = K7 x − K8 M1 − K9 V K10 ek11 (10−log P ) dt
(6)
Here V represents volumetric fraction of organic solvents (v/v), K7 the coefficient of cell growth with respect to the cell concentration (per day), K8 the coefficient of cell growth with respect to Taxol accumulation in cells (g/g day), K9 the coefficient of cell growth with respect to toxicity and
M3 = (K21 + K22 R)VM2
(10)
Here M3 represents Taxol accumulation in organic solvent (mg/L), R the phase equilibrium constant of Taxol (for one-liquid-phase culture, R is 0). K13 represents coefficient of Taxol synthesis with respect to Taxol accumulation in cells (per day), K16 the coefficient of Taxol synthesis with respect to extracting capacity of organic solvent (VR) (g/L day), K12 the coefficient of Taxol synthesis with respect to the cell concentration g/(g day) and K13 , K15 , K17 and K18 represent experimental coefficients. K19 and K20 represent coefficients for computing partition coefficient of Taxol between cell-free medium and cells in the second-stage culture. K21 and K22 represent coefficients for computing partition coefficient of Taxol between organic solvent and cell-free medium. 3.2.3. Determination of the coefficient values of the kinetic model The model parameters were estimated on the basis of the experimental data of the two-liquid-phase culture of T. cuspidata in flasks and with the least square method of multi-parameters. The estimated model parameter values are shown in Table 2.
4. Results and discussion 4.1. Cell growth 4.1.1. Time course of cell growth Time course of cell growth is shown in Fig. 1 (the control is one-liquid-phase culture). The results showed that the
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Table 2 Model parameters correlated from experimental data K1 =212 (lag phase) K1 =11.8 (exponential growth phase) K2 =0.0746 (lag phase) K2 =0.0705 (exponential growth phase) K3 =0 (lag phase) K3 =−0.013 (exponential growth phase) K4 =0 (lag phase) K4 =0.0123 (exponential growth phase) K5 =0 (lag phase) K5 =−0.132 (exponential growth phase) K6 =0.300 K7 =0.00377 K8 =0.117 K9 =2.45
K10 =1.07 K11 =0.403 K12 =0.0485 K13 =1.60 K14 =1.60 K15 =1.60 K16 =0.0680 K17 =0.5 K18 =−3.20 K19 =0.679 K20 =0.0393 K21 =51.6 K22 =1.05 Ys/x =0.65
model curve agreeed well with the experimental data. The first-stage culture was from Day 1 to Day 10 and produced favorable conditions for the second-stage culture. Cell growth entered into the second-stage culture with the addition of production medium (the preconditioned organic solvent was also added at Day 10) and the rate of cell growth during the second-stage culture was very low. For one-liquid-phase culture, dry cell weight increased slightly, but for two-liquid-phase culture, dry cell weight decreased. As the toxicity of organic solvent increased, dry cell weight decreased markedly, especially for the two-liquid-phase culture with decanol as the organic solvent.
Fig. 2. Effects of toxicity of organic solvents on dry cell weight.
Fig. 3. Effects of volumetric fraction of organic solvents on dry cell weight.
of organic solvents increased, dry cell weight decreased gradually. 4.2. Taxol production
4.1.2. Effect of organic solvent toxicity on cell growth Although the organic solvents selected possess better biocompatibility with organisms [8], the organic solvents still affected significantly cell growth in two-liquid-phase culture of plant cells. According to the definition of the toxicity of organic solvents (10−log P) in this paper, Fig. 2 shows close relationship between dry cell weight and 10−log P in two-liquid-phase culture of T. cuspidata. As 10−log P increased, dry cell weight decreased gradually, then the effects became significant at 10−log P>5. Therefore, organic solvents with log P<5 are not suitable for the two-liquid-phase culture of T. cuspidata cells. 4.1.3. Effect of volumetric fraction of organic solvent on cell growth Fig. 3 shows the effect of volumetric fraction of organic solvents on the cell growth. As the volumetric fraction
Fig. 1. Time course of plant cell growth.
4.2.1. Time course of Taxol production Fig. 4 shows time course of Taxol production. It can be seen that there was little Taxol production during the initial four days and a little more from the fifth day to the end of the first stage, indicating that cell growth possessed partial interrelation with Taxol production in Taxus cell suspension cultures. From Day 10, Taxol production increased largely due to the two-liquid-phase and production medium in the second stage, and then the rate of Taxol production decreased gradually. For the one-liquid-phase culture or for the two-liquid-phase culture with organic solvents of lower 10−log P and low partition coefficient of Taxol such as hexadecane, Taxol production was lower at Day 16 due to high Taxol accumulation in cells, which revealed the feedback inhibition
Fig. 4. Time course of Taxol production.
Z.-L. Wu et al. / Biochemical Engineering Journal 5 (2000) 137–142
Fig. 5. Effects of organic solvent toxicity on Taxol production.
of Taxol. For the two-liquid-phase culture with organic solvents of higher 10−log P such as decanol, total Taxol production was not high since cell growth was affected by high toxicity of the organic solvent and dry cell weight at Day 16 almost was decreased to that at Day 1 (Fig. 1). However, for the two-liquid-phase culture with organic solvents of lower 10−log P and high partition coefficient of Taxol such as oleic acid or dibutylphthalate, Taxol production reached high level due to diminution of the effects of the above-mentioned unfavorable factors and might increase thereafter. Therefore, time course of Taxol production after Day 16 should be studied further. 4.2.2. Effect of organic solvent toxicity on Taxol production Fig. 5 shows the effects of the organic solvent toxicity on Taxol production. As the toxicity of organic solvents (10−log P) increased, Taxol accumulation in cells or in cell-free medium decreased gradually. Because most of Taxol was transferred into the organic solvent, there was an optimal value for Taxol production. When the toxicity of an organic solvent (10−log P) was between 2 and 5, Taxol production rearched the optimal values. Therefore, the toxicity of organic solvent was an important factor for Taxol production. 4.2.3. Effect of volumetric fraction of organic solvent on Taxol production Fig. 6 shows the relationship between Taxol production and the volumetric fractions of organic solvents. As the volumetric fractions of organic solvents increased, the toxicity of organic solvents increased [9] and the organic solvent possessed higher ability to extract Taxol, and thus Taxol ac-
Fig. 6. Effects of volumetric fraction of organic solvents on Taxol production.
141
cumulation in cells and that in cell-free medium decreased gradually. However, the relationship between Taxol accumulation in the organic solvent and the volumetric fraction of organic solvent might be more complex. As the volumetric fraction of organic solvents increased, Taxol accumulation in organic solvents increased significantly with lower toxicity of the organic solvents such as oleic acid (10−log P=2.3). However, as volumetric fraction of the organic solvents increased, Taxol accumulation in the organic solvent increased significantly initially, reached the higher level and then decreased with higher toxicity of the organic solvent such as dibutylphthalate (10−log P=4.6). Because most of Taxol was in the organic solvent in the two-liquid-phase culture, the effects of the volumetric fraction of organic solvents on Taxol production was the same as the effects of the volumetric fraction of organic solvent on Taxol accumulation in organic solvent. Therefore, for the two-liquid-phase culture with toxicity of organic solvent 10−log P<3 such as oleic acid (10−log P=2.3), suitable volumetric fraction of organic solvent was 0.08–0.10 (v/v). But for the two-liquid-phase culture with 10−log P>3, volumetric fraction of organic solvents possessed an optimal value between 0–0.1 (v/v). As the toxicity of organic solvent, 10−log P, increased, the volumetric fraction of organic solvent should be lower for the corresponding higher Taxol production. For example, the suitable volumetric fraction of organic solvent was 0.04–0.08 (v/v) for the two-liquid-phase culture with dibutylphthalate as the organic solvent.
5. Conclusions In the two-liquid-phase and two-stage cultures of T. cuspidata cells, dry cell weight decreased as organic solvent toxicity (10−log P) increased and the extent of decrease became significantly with (10−log P)>5. When the culture entered into the second stage, Taxol production increased significantly initially and the increase became less appreciable gradually, especially for the two-liquid-phase culture. As the organic solvent toxicity (10−log P) increased, Taxol production increased to the high level and then decreased. Therefore, Taxol production reached the optimal values when (10−log P) was from 2 to 5. For the two-liquid-phase with toxicity of organic solvent (10−log P)<3 such as oleic acid, the suitable volumetric fraction of organic solvents was 0.08–0.10 (v/v). For the two-liquid-phase with 10−log P>3 such as dibutylphthalate (10−log P=4.6), the suitable volumetric fraction of organic solvents was 0.04–0.08 (v/v). The kinetic model developed in the present work not only reflected the effects of toxicity of different organic solvents (paraffin, organic acid, alcohol and ester) and their volumetric fractions etc., on the cell growth and Taxol production in the two-stage and two-liquid-phase cultures, but also represented kinetics of the two-stage and one-liquid-phase cultures (10−log P=0) correctly.
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6. Nomenclature K1
K2 K3 K4 K5 K6 K7 K8 K9
K10 K11 K12 K13 K14 K15 K16
K17 K18 K19 , K20
K21 , K22
M M1 M2 M3 R S
coefficient of cell growth with respect to sucrose control (g/L) (L represents fresh liquid medium in this paper) coefficient of sucrose consumption with respect to the cell concentration (g/g day) coefficient of Taxol synthesis with respect to the cell growth (g/g) coefficient of Taxol synthesis with respect to the cell concentration (g/g day) experimental coefficient in Eq. (3) (g/L day) partition coefficient of Taxol between cell-free medium and cells in the second-stage culture coefficient of cell growth with respect to the cell concentration (per day) coefficient of cell growth with respect to Taxol accumulation in cells (g/g day) coefficient of cell growth with respect to toxicity and volumetric fraction of organic solvent (g/L day) experimental coefficient in Eq. (6) experimental coefficient in Eq. (6) coefficient of Taxol synthesis with respect to the cell concentration (g/g day) experimental coefficient in Eq. (7) coefficient of Taxol synthesis with respect to Taxol accumulation in cells (per day) experimental coefficient in Eq. (7) coefficient of Taxol synthesis with respect to extract capacity of organic solvent (VR) (g/L day) experimental coefficient in Eq. (7) experimental coefficient in Eq. (7) (g/L day) coefficient for computing partition coefficient of Taxol between cell-free medium and cells in the second-stage culture coefficient for computing partition coefficient of Taxol between organic solvent and cell-free medium in the second-stage culture Taxol production (mg/L) Taxol accumulation in cells (mg/L) Taxol accumulation in cell-free medium (mg/L) Taxol accumulation in organic solvent (mg/L) phase equilibrium constant of Taxol concentration of sucrose (g/L)
V x Ys/x µmax
volumetric fraction of organic solvent (v/v) concentration of dry cell (g/L) sucrose consumption with respect to the cell growth (g/g) maximum specific cell growth rate (per day)
Acknowledgements This work was supported by trans-century training program foundation for talents by the state education commission and key project (No. 963107211) by Science and Technology Commission of Tianjin. References [1] D.G.I. Kingston, Taxol: the chemistry and structure-activity relationships of a novel anticancer agent, Trends Biotechnol. 12 (1994) 222–227. [2] M.L. Shuler, Bioreactor engineering as an enabling technology to tap biodiversity: the case of Taxol, Ann. N. Y. Acad. Sci. 745 (1994) 455–461. [3] L.J. Pestchanker, S.C. Roberts, M.L. Shuler, Kinetics of Taxus cuspidata in shake flasks and a Wilson-type bioreactor, Enzyme Microb. Technol. 19 (1996) 256–260. [4] D.M. Gibson, R.E.B. Ketchum, T.J. Hirasuna, M.L. Shuler, Potential of plant cell culture for taxane production, in: Taxol: Science and Application, 1995, pp. 71–91. [5] J.J. Manfredi, S.B. Horuitz, in: L.A. Dethlefsen (Ed.), Taxol: An Antimitotic Agent with a New Mechnism of Action in Cell Cycle Effects of Drugs, Pregamon Press, Oxford, 1986, pp. 287–290. [6] Z.-L. Wu, Y.-J. Yuan, J.-X. Liu, H.-Y. Xuan, Z.-D. Hu, A.-C. Sun, C.-X. Hu, Study on enhanced production of Taxol from Taxus chinesis Var mairei in biphasic-liquid culture , Acta Botanica Sinica 41 (1999) 1108–1113. [7] Y.-Z. Chen, Z.-H. Chen. Cell Culture Engineering, East China University of Chemical Engineering Press, 1992, pp. 254. [8] C. Lanne, S. Boeren, K. Vos, C. Veeger, Rules for optimizaton of biocatalysis in organic solvents, Biotechnol. Bioeng. 30 (1987) 81– 87. [9] M. Vermue, J. Sikkema, A. Verheul, R. Bakker, J. Tramper, Toxicity of homologous series of organic solvents for the gram-positive bacteria Arthrobacter and Nocardia sp. and the gram-negative bacteria Acinetobacter and Pseudomonas Sp., Biotechnol. Bioeng. 42 (1993) 747–758. [10] S.-Q. Zhang, Handbook of Applied Fine Chemical Products (Volume Organic), Chemical Industry Press, Beijing, 1996. [11] Y.-Z. Qi, S.-X. Wang, Kinetics of Biochemical Reaction and Reactors, Chemical Industry Press, 1996, pp. 126. [12] L. Bassetti, J. Tramper, Organic solvent toxicity in Morinda citrifolia cell suspensions, Enzyme Microb. Technol. 16 (1994) 642–648.
Kinetics of two-liquid-phase Taxus cuspidata cell culture for production of Taxol Zhao-Liang Wu, Ying-Jin Yuan∗ , Zhong-Hai Ma, Zong-Ding Hu Department of Biochemical Engineering, Tianjin University, Tianjin 300072, China Received 25 August 1999; accepted 7 January 2000
Abstract The effects of different organic solvents (paraffin, organic acid, alcohol and ester) and their volumetric fractions on the cell growth and Taxol production were studied in two-liquid-phase and the two-stage culture. A kinetic model, incorporated the effects of the toxicity of organic solvents was developed for two-liquid-phase culture of Taxus cuspidata in the two-stage Taxol production. The results showed that the proposed kinetic model could fit the experimental data satisfactorily. The results also showed that Taxol production could reach the optimal value when 10−log P was in the range of 2 to 5 and the volumetric fraction of the organic solvents at the corresponding the highest Taxol production should be lower when 10−log P was high. © 2000 Elsevier Science S.A. All rights reserved. Keywords: Plant cell culture; Two-liquid-phase; Taxus cuspidata; Taxol; Kinetic model
1. Introduction
2. Materials and methods
In order to increase the supply of Taxol, an effective anticancer drug, plant cell culture is considered to be a possible source for a large scale production of Taxol [1–4]. However, due to the extremely low solubility of Taxol in the medium and its inhibiting effect on cell growth [5] and Taxol synthesis, the two-liquid-phase culture is taken as a key technology, which was confirmed to be very effective for enhancing Taxol production of plant cell cultures [6]. In situ removal of Taxol out of the aqueous phase into the hydrophobic phase leads to a shift of equilibrium towards more production and, then promoting the Taxol biosynthesis. To verify the above mentioned advantages of the two phase culture, effects of different parameters such as culture time, toxicity and volumetric fraction of organic solvents, etc. on cell growth and Taxol production were studied. In this paper, a kinetic model for the culture system was also proposed.
2.1. Materials
∗ Corresponding author. Tel.: +86-22-2740-3888. E-mail address: [email protected] (Y.-J. Yuan)
2.1.1. Plant material The cell line was initiated from young stems of Taxus cuspidata and subcultured on solid B5 medium [7] at 25◦ C in the dark. 2.1.2. Liquid medium The growth medium was B5 medium with Vitamin B1, B6, nicotinic acid and inositol doubled, sucrose 25 g/L (L represents fresh liquid medium in this paper). It was supplemented with 6-benzyladenine 0.5 mg/L and casamino acid 2 mg/L. The production medium was liquid B5 medium supplemented with sucrose 60 g/L, 6-benzyladenine 0.5 mg/L and casamino acid 1 mg/L. 2.1.3. Organic solvents The following two points were considered in the selection of organic solvents for the two-liquid-phase cultures. First, the organic solvents should be biocompatible with cultured plant cells. Second, the partition coefficient of Taxol should be high enough. It was reported [8] that organic solvents with log P>4 were biocompatible with cells on the whole (log P is logarithm of the solvent partition coefficient in a standard two-phase
1369-703X/00/$ – see front matter © 2000 Elsevier Science S.A. All rights reserved. PII: S 1 3 6 9 - 7 0 3 X ( 0 0 ) 0 0 0 5 2 - 8
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Z.-L. Wu et al. / Biochemical Engineering Journal 5 (2000) 137–142
Table 1 Physical properties of organic solvents in the systems Organic solvents
log P
Hexadecane
8.7
Partition coefficient
Decanol
4.0
311
D420 = 0.773= 0.830
Oleic acid
7.7
154
D420 = 0.773= 0.894
Dibutylphthalate
5.4
236
D420 = 0.773= 1.048
5.7
the mixed extract was measured by HPLC similarly to the Taxol measurement in spent medium.
Density [10] D420 = 0.773
system i-octanol/water). The log P of organic solvents can be found in relevant literature [8,9]. Table 1 shows those organic solvents used and their physical properties. Partition coefficients of Taxol in the two-phase systems (organic solvent/liquid medium) were measured (the two-phase systems were saturated by Taxol at 25◦ C). Taxol measurement in organic solvents and in aqueous phase are given in Sections 2.3.3 and 2.3.1, respectively. The organic solvents are not soluble in water [10]. 2.2. Culture conditions Cells of T. cuspidata were cultivated in 250 ml Erlenmeyer flasks, at 25◦ C, in the dark and under continuous shaking (100 rpm) on a rotary shaker. Every flask contained initially 30 ml growth medium and 6 g fresh cells. At the end of the 10 day cell growth period (the later period of exponential growth phase), 10 ml production medium was added into the flask to enhance the production of Taxol and this process lasted for 6 days. Organic solvents were all preconditioned, i.e. mixed sufficiently with growth medium (10 ml organic solvents/90 ml growth medium) and then separated. The preconditioned organic solvents were added into the flasks on the 10th day of the cell culture. 2.3. Taxol measurements 2.3.1. Taxol measurement in spent medium The measured sample was centrifuged and separated into spent medium, cells and organic solvents. Taxol content in spent medium was measured by HPLC with a reversed-phase column (Kromasil C18 5 m 200×4.6 mm). The mobile phase consisted of acetonitrile and water (47:53 v/v). The flow rate was 1 ml/min and elution time 40 min. Taxol was detected at 227 nm with an ultraviolet detector. The injection was 10 l at a time. 2.3.2. Taxol measurement in cells The separated cells was stored in a refrigerator (−20◦ C, more than a day). The frozen cells were soaked in 5 ml cyclohexane and powdered (10 min), and then cyclohexane was abandoned. The powdered cells were mixed with 20 ml methanol, ultrasonicated for 20 min, centrifuged and separated from the mixture. This process was repeated twice with methanol. The extract (three times) was mixed and Taxol in
2.3.3. Taxol measurement in organic solvent Taxol in organic solvents was measured by HPLC with a normal-phase silica-gel column (Kromasil Sil 5 m 200×4.6 mm). The mobile phase consisted of cyclohexane and acetone (77:23 v/v). Other conditions were the same as that in Taxol measurement in spent medium. 3. Kinetic model development 3.1. Model assumption According to the experiment data, we could make the following assumptions which qualitatively represented the effects of most important parameters on cell growth and production of Taxol. Then a mathematical model was developed in order to represent the main features of the system qualitatively. 3.1.1. Fully mixed culture system The medium, cultured T. cuspidata cells and organic solvents were at the fully mixed state. 3.1.2. Two-stage culture Cell growth was partially interrelated with Taxol production in Taxus cell suspension cultures and hence two-stage culture was used. The first-stage culture aimed at enhancing cell growth, therefore, the growth medium was used. The second-stage culture aimed at enhancing Taxol production, so the production medium was used. Consequently, a model was developed for the two-phase culture in two stages. 3.2. Mathematical model According to the above assumptions, the derived mathematical model was described as follows. 3.2.1. The first-stage culture 3.2.1.1. Cell growth. Sucrose was taken as the sole limiting substrate and its concentration affected cell growth. The kinetics of cell growth could be represented by the relation proposed by Teissier [11]. dx = µmax x 1 − e−(s/K1 ) (1) dt Here x represents the concentration of dry cells (g/L), µmax represents the maximum specific cell growth rate (per day), S the concentration of sucrose (g/L) and K1 coefficient of cell growth with respect to sucrose limited (g/L). 3.2.1.2. Sucrose consumption. The rate of sucrose consumption was determined by the cell concentration and cell
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growth. The effect of Taxol production on sucrose consumption was neglected because the consumption of sucrose by Taxol production was very low.
volumetric fraction of organic solvent (g/L day), K10 and K11 represent experimental coefficients.
dx ds = −Y(s/x) − K2 x dt dt
3.2.2.2. Taxol production. Taxol production was the sum of Taxol produced in cells, those accumulated in cell-free medium and those transferred into and stored in the organic phase. Taxol production was directly proportional to the cell concentration and inversely proportional to the concentration of organic solvents and the phase equilibrium constant of Taxol in the system (ratio of mass concentration of Taxol in the organic solvent over that in the medium), and was inhibited by Taxol accumulation in cells. Taxol accumulation in the cell-free medium was at the equilibrium state with those in cells and the equilibrium constant was affected by the toxicity of an organic solvent (10−log P). Taxol accumulation in organic solvent was at the equilibrium state with those in cell-free medium.
(2)
Here Ys /x represents sucrose consumption with respect to the cell growth (g/g) and K2 the coefficient of sucrose consumption with respect to the cell concentration (g/g day). 3.2.1.3. Taxol production. Taxol production was the sum of Taxol produced in cells and accumulated in cell-free medium. The rate of Taxol production in cells was partially interrelated with the rate of cell growth. Taxol accumulation in cell-free medium was at the equilibrium state with that in cells and the equilibrium constant was unchanged. dx dM = K3 + K4 x + K5 dt dt
(3)
(7)
M = M1 + M2
dM K = K12 x K13 − K14 M1 15 + K16 (VR)K17 + K18 dt
(4)
M = M1 + M2 + M3
(8)
M = K6 M1
(5)
M2 = [K19 + K20 (10 − log P )]M1
(9)
Here M represents total Taxol (mg/L), M1 Taxol produced in cells (mg/L), M2 the Taxol accumulated in cell-free medium (mg/L), K3 the coefficient of Taxol synthesis with respect to the cell growth (g/g), K4 the coefficient of Taxol synthesis with respect to the cell concentration (g/g day), K5 the experimental coefficient (g/L day) and K6 the partition coefficient of Taxol between cell-free medium and cells. 3.2.2. The second-stage culture 3.2.2.1. Cell growth. Sucrose became a non-limiting substrate because its concentration was high enough in the second-stage culture. Cell growth was inhibited by Taxol accumulation in cells, which was more obvious for one-liquid-phase culture. Cell growth was also affected by the toxicity and the volumetric fraction of organic solvents. The log P was used to express the effects of toxicity of organic solvents on organisms in the two-liquid-phase cultures of microbes [9] and it was also used for plant cells [12] in the two-liquid-phase culture. Accordingly, the toxicity of an organic solvent was defined as 10−log P in this paper. For one-liquid-phase culture, 10−log P was zero because there were no organic solvents in the system. dx = K7 x − K8 M1 − K9 V K10 ek11 (10−log P ) dt
(6)
Here V represents volumetric fraction of organic solvents (v/v), K7 the coefficient of cell growth with respect to the cell concentration (per day), K8 the coefficient of cell growth with respect to Taxol accumulation in cells (g/g day), K9 the coefficient of cell growth with respect to toxicity and
M3 = (K21 + K22 R)VM2
(10)
Here M3 represents Taxol accumulation in organic solvent (mg/L), R the phase equilibrium constant of Taxol (for one-liquid-phase culture, R is 0). K13 represents coefficient of Taxol synthesis with respect to Taxol accumulation in cells (per day), K16 the coefficient of Taxol synthesis with respect to extracting capacity of organic solvent (VR) (g/L day), K12 the coefficient of Taxol synthesis with respect to the cell concentration g/(g day) and K13 , K15 , K17 and K18 represent experimental coefficients. K19 and K20 represent coefficients for computing partition coefficient of Taxol between cell-free medium and cells in the second-stage culture. K21 and K22 represent coefficients for computing partition coefficient of Taxol between organic solvent and cell-free medium. 3.2.3. Determination of the coefficient values of the kinetic model The model parameters were estimated on the basis of the experimental data of the two-liquid-phase culture of T. cuspidata in flasks and with the least square method of multi-parameters. The estimated model parameter values are shown in Table 2.
4. Results and discussion 4.1. Cell growth 4.1.1. Time course of cell growth Time course of cell growth is shown in Fig. 1 (the control is one-liquid-phase culture). The results showed that the
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Table 2 Model parameters correlated from experimental data K1 =212 (lag phase) K1 =11.8 (exponential growth phase) K2 =0.0746 (lag phase) K2 =0.0705 (exponential growth phase) K3 =0 (lag phase) K3 =−0.013 (exponential growth phase) K4 =0 (lag phase) K4 =0.0123 (exponential growth phase) K5 =0 (lag phase) K5 =−0.132 (exponential growth phase) K6 =0.300 K7 =0.00377 K8 =0.117 K9 =2.45
K10 =1.07 K11 =0.403 K12 =0.0485 K13 =1.60 K14 =1.60 K15 =1.60 K16 =0.0680 K17 =0.5 K18 =−3.20 K19 =0.679 K20 =0.0393 K21 =51.6 K22 =1.05 Ys/x =0.65
model curve agreeed well with the experimental data. The first-stage culture was from Day 1 to Day 10 and produced favorable conditions for the second-stage culture. Cell growth entered into the second-stage culture with the addition of production medium (the preconditioned organic solvent was also added at Day 10) and the rate of cell growth during the second-stage culture was very low. For one-liquid-phase culture, dry cell weight increased slightly, but for two-liquid-phase culture, dry cell weight decreased. As the toxicity of organic solvent increased, dry cell weight decreased markedly, especially for the two-liquid-phase culture with decanol as the organic solvent.
Fig. 2. Effects of toxicity of organic solvents on dry cell weight.
Fig. 3. Effects of volumetric fraction of organic solvents on dry cell weight.
of organic solvents increased, dry cell weight decreased gradually. 4.2. Taxol production
4.1.2. Effect of organic solvent toxicity on cell growth Although the organic solvents selected possess better biocompatibility with organisms [8], the organic solvents still affected significantly cell growth in two-liquid-phase culture of plant cells. According to the definition of the toxicity of organic solvents (10−log P) in this paper, Fig. 2 shows close relationship between dry cell weight and 10−log P in two-liquid-phase culture of T. cuspidata. As 10−log P increased, dry cell weight decreased gradually, then the effects became significant at 10−log P>5. Therefore, organic solvents with log P<5 are not suitable for the two-liquid-phase culture of T. cuspidata cells. 4.1.3. Effect of volumetric fraction of organic solvent on cell growth Fig. 3 shows the effect of volumetric fraction of organic solvents on the cell growth. As the volumetric fraction
Fig. 1. Time course of plant cell growth.
4.2.1. Time course of Taxol production Fig. 4 shows time course of Taxol production. It can be seen that there was little Taxol production during the initial four days and a little more from the fifth day to the end of the first stage, indicating that cell growth possessed partial interrelation with Taxol production in Taxus cell suspension cultures. From Day 10, Taxol production increased largely due to the two-liquid-phase and production medium in the second stage, and then the rate of Taxol production decreased gradually. For the one-liquid-phase culture or for the two-liquid-phase culture with organic solvents of lower 10−log P and low partition coefficient of Taxol such as hexadecane, Taxol production was lower at Day 16 due to high Taxol accumulation in cells, which revealed the feedback inhibition
Fig. 4. Time course of Taxol production.
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Fig. 5. Effects of organic solvent toxicity on Taxol production.
of Taxol. For the two-liquid-phase culture with organic solvents of higher 10−log P such as decanol, total Taxol production was not high since cell growth was affected by high toxicity of the organic solvent and dry cell weight at Day 16 almost was decreased to that at Day 1 (Fig. 1). However, for the two-liquid-phase culture with organic solvents of lower 10−log P and high partition coefficient of Taxol such as oleic acid or dibutylphthalate, Taxol production reached high level due to diminution of the effects of the above-mentioned unfavorable factors and might increase thereafter. Therefore, time course of Taxol production after Day 16 should be studied further. 4.2.2. Effect of organic solvent toxicity on Taxol production Fig. 5 shows the effects of the organic solvent toxicity on Taxol production. As the toxicity of organic solvents (10−log P) increased, Taxol accumulation in cells or in cell-free medium decreased gradually. Because most of Taxol was transferred into the organic solvent, there was an optimal value for Taxol production. When the toxicity of an organic solvent (10−log P) was between 2 and 5, Taxol production rearched the optimal values. Therefore, the toxicity of organic solvent was an important factor for Taxol production. 4.2.3. Effect of volumetric fraction of organic solvent on Taxol production Fig. 6 shows the relationship between Taxol production and the volumetric fractions of organic solvents. As the volumetric fractions of organic solvents increased, the toxicity of organic solvents increased [9] and the organic solvent possessed higher ability to extract Taxol, and thus Taxol ac-
Fig. 6. Effects of volumetric fraction of organic solvents on Taxol production.
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cumulation in cells and that in cell-free medium decreased gradually. However, the relationship between Taxol accumulation in the organic solvent and the volumetric fraction of organic solvent might be more complex. As the volumetric fraction of organic solvents increased, Taxol accumulation in organic solvents increased significantly with lower toxicity of the organic solvents such as oleic acid (10−log P=2.3). However, as volumetric fraction of the organic solvents increased, Taxol accumulation in the organic solvent increased significantly initially, reached the higher level and then decreased with higher toxicity of the organic solvent such as dibutylphthalate (10−log P=4.6). Because most of Taxol was in the organic solvent in the two-liquid-phase culture, the effects of the volumetric fraction of organic solvents on Taxol production was the same as the effects of the volumetric fraction of organic solvent on Taxol accumulation in organic solvent. Therefore, for the two-liquid-phase culture with toxicity of organic solvent 10−log P<3 such as oleic acid (10−log P=2.3), suitable volumetric fraction of organic solvent was 0.08–0.10 (v/v). But for the two-liquid-phase culture with 10−log P>3, volumetric fraction of organic solvents possessed an optimal value between 0–0.1 (v/v). As the toxicity of organic solvent, 10−log P, increased, the volumetric fraction of organic solvent should be lower for the corresponding higher Taxol production. For example, the suitable volumetric fraction of organic solvent was 0.04–0.08 (v/v) for the two-liquid-phase culture with dibutylphthalate as the organic solvent.
5. Conclusions In the two-liquid-phase and two-stage cultures of T. cuspidata cells, dry cell weight decreased as organic solvent toxicity (10−log P) increased and the extent of decrease became significantly with (10−log P)>5. When the culture entered into the second stage, Taxol production increased significantly initially and the increase became less appreciable gradually, especially for the two-liquid-phase culture. As the organic solvent toxicity (10−log P) increased, Taxol production increased to the high level and then decreased. Therefore, Taxol production reached the optimal values when (10−log P) was from 2 to 5. For the two-liquid-phase with toxicity of organic solvent (10−log P)<3 such as oleic acid, the suitable volumetric fraction of organic solvents was 0.08–0.10 (v/v). For the two-liquid-phase with 10−log P>3 such as dibutylphthalate (10−log P=4.6), the suitable volumetric fraction of organic solvents was 0.04–0.08 (v/v). The kinetic model developed in the present work not only reflected the effects of toxicity of different organic solvents (paraffin, organic acid, alcohol and ester) and their volumetric fractions etc., on the cell growth and Taxol production in the two-stage and two-liquid-phase cultures, but also represented kinetics of the two-stage and one-liquid-phase cultures (10−log P=0) correctly.
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6. Nomenclature K1
K2 K3 K4 K5 K6 K7 K8 K9
K10 K11 K12 K13 K14 K15 K16
K17 K18 K19 , K20
K21 , K22
M M1 M2 M3 R S
coefficient of cell growth with respect to sucrose control (g/L) (L represents fresh liquid medium in this paper) coefficient of sucrose consumption with respect to the cell concentration (g/g day) coefficient of Taxol synthesis with respect to the cell growth (g/g) coefficient of Taxol synthesis with respect to the cell concentration (g/g day) experimental coefficient in Eq. (3) (g/L day) partition coefficient of Taxol between cell-free medium and cells in the second-stage culture coefficient of cell growth with respect to the cell concentration (per day) coefficient of cell growth with respect to Taxol accumulation in cells (g/g day) coefficient of cell growth with respect to toxicity and volumetric fraction of organic solvent (g/L day) experimental coefficient in Eq. (6) experimental coefficient in Eq. (6) coefficient of Taxol synthesis with respect to the cell concentration (g/g day) experimental coefficient in Eq. (7) coefficient of Taxol synthesis with respect to Taxol accumulation in cells (per day) experimental coefficient in Eq. (7) coefficient of Taxol synthesis with respect to extract capacity of organic solvent (VR) (g/L day) experimental coefficient in Eq. (7) experimental coefficient in Eq. (7) (g/L day) coefficient for computing partition coefficient of Taxol between cell-free medium and cells in the second-stage culture coefficient for computing partition coefficient of Taxol between organic solvent and cell-free medium in the second-stage culture Taxol production (mg/L) Taxol accumulation in cells (mg/L) Taxol accumulation in cell-free medium (mg/L) Taxol accumulation in organic solvent (mg/L) phase equilibrium constant of Taxol concentration of sucrose (g/L)
V x Ys/x µmax
volumetric fraction of organic solvent (v/v) concentration of dry cell (g/L) sucrose consumption with respect to the cell growth (g/g) maximum specific cell growth rate (per day)
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