Plant Cell Reports

Plant Cell Reports (1990) 9:125-128

9 Springer-Verlag1990

Long term culture and caffeine production of immobilized coffee (Coffea arabica) L. cells in polyurethane foam T. Furuya, K. Koge, and Y. Orihara School of Pharmaceutical Sciences, Kitasato University, 9-1 Shirokane 5 Chome, Minato-ku, Tokyo 108, Japan Received December 6, 1989/Revised version received April 23, 1990 - Communicated by A. R. Gould

Abstract

Coffee (Coffea arabica L.) cells could be immobilized in p o l y u r e t h a n e foam and subcultured r e p e a t e d l y for a long time. Four phases were observed for cell growth and caffeine production, I; immobilization, II; growth, III; caffeine production, IV; regrowth. Their periods were influenced by the number of foam particles. E s p e c i a l l y in the phase III, the immobilized cells produced a relatively large amount of caffeine in the subculture numbers 5-8 (34 cubes) when the fresh weight of the immobilized cells decreased despite culture in growth medium (DK medium). Caffeine production appeared to have a negative correlation with the growth of the immobilized cells throughout the subcultures. Introduction There is a wealth of literature on p r e p a r a t i o n of immobilized plant cells in gel matrixes, such as p o l y a c r y l a m i d e derivative (Galun et al. 1983) and Ca-alginate (Brodelius et al. 1979, Furuya et al. 1984, Furusaki et al. 1988). Currently, immobilization in foam particles is r e c e i v i n g attention because this method dose not involve reagents and c o m p l i c a t e d operations which cause damage to cells and contamination with microbes. Moreover, foam particles could theoretically immobilize more cells than gel matrixes. Lindsey et al. (1983) immobilized Capsicum frutescens cells with polyurethane foam as a novel method and reported that the immobilized cells produce more capsaicin than free cells. Coffee (Coffea arabica) cells produce caffeine and t h e o b r o m i n e and release purine alkaloids in medium (Keller et al. 1972, Waller et al. 1983, Furuya et al. 1989). Therefore coffee cultured cells are thought to be suitable for a model system for continuous production of secondary This paper is Part 71 in the series of "Studies on Plant Tissue Cultures". For Part 70, see Orihara Y, Furuya T. submitted for publication.

Offprint requests to." T. Furuya

m e t a b o l i t e s using immobilized cells. A c t u a l l y it was r e p o r t e d that coffee cells immobilized in Ca-alginate beads produce purine alkaloids (Prenosil et al. 1987, Haldimann and Brodelius 1987, Furuya et al. 1989). But there is no report on immobilization of coffee cells in p o l y u r e t h a n e foam. In this paper, we report immobilization of coffee cells in p o l y u r e t h a n e foam and long term culture for s e m i - c o n t i n u o u s production of caffeine. M a t e r i a l s and methods Cell line Coffee callus was derived from seed of Coffea arabica L. from Hawaii in 1982, and subcultured every 3 weeks for about 5 years on Murashige and Skoog medium (1962) supplemented with sucrose (30 g/l), 2,4d i c h l o r o p h e n o x y a c e t i c acid (I mg/l), kinetin (0.1 mg/l), and agar (9 g/l) (DK agar medium) at 25~ in the dark (Furuya et al. 1989). Polyurethane foam particles Two types of p o l y u r e t h a n e foam [Scott Foam, Bridgestone Inc., pore density; 6 (#06), 13 (#13), 20 (#20), and 30 (#30) pores/25 mm] particle were used; the ball type (foam ball; d. ca. 2.7 cm, 3 bal~s/flask) and the cube-~ype (foam cube; Icm , 17, 34, and 68 cubes/flask). I m m o b i l i z a t i o n and cultures of coffee cells Callus (fr. wt. 15-20 g)--was transferred into 250 ml of DK liquid m e d i u m containing the p o l y u r e t h a n e foam particles in a 500 ml E r l e n m e y e r flask and cultured on a rotary shaker (149 rpm) at 25~ in the dark. After 3 weeks, the foam particles r e t a i n i n g the cells were t r a n s f e r r e d into the flask containing fresh medium and subcultured every 3 weeks under the same conditions as above. Cell growth The fresh weight of the immobilized cells and free suspended cells were measured at every subculture. When the immobilized cell cubes became too large to be drawn from the flask, the subculture was performed by the medium exchange. The fresh w e i g h ~ o f the

126 immobilized cells can be calculated by subtracting the w e i g h t of the flask, cotton plug and the m e d i u m from the weight of the whole reactor. D e t e r m i n a t i o n of caffeine in the m e d i u m At the end of each subculture, the volume of the old m e d i u m was m e a s u r e d after the i m m o b i l i z e d and the s u s p e n d e d free cells were removed. Theophylline s o l u t i o n was added to the medium as an internal standard and then the purine alkaloids were m e a s u r e d by a HPLC (Shimadzu L C - 3 A system). The H P L C c o n d i t i o n s were as follows: column; Unisil Q C18 reversed-phase column (5~m packing, 30cm x7.6mm I.D., G a s u k u r o Kogyo Inc.), eluent; methanol-water (4:6 by volume), flow rate; 1.5 ml/min., detection; UV 254nm. The calculation of peak area was performed by C h r o m a t o p a c C-R3A (Shimadzu Corporation). Results and D i s c u s s i o n Immobilization of coffee cells in ball type p o l y u r e t h a n e foam h a v i n g d i f f e r e n t pore densities was p e r f o r m e d and results at the 3rd s u b c u l t u r e shown in Fig. I A - D. The coffee cells were i m m o b i l i z e d in the foam balls in the 2nd and the 3rd s u b c u l t u r e s though i m m o b i l i z e d cells were a few in the Ist subculture. The foam balls having pore density more than 13 pores/25 mm (#13, #20, #30) s u c c e e d e d in i m m o b i l i z i n g a c o n s i d e r a b l e body of the cells (Fig. I A and C) even inside them (Fig. I B and D) at the end of the 3rd s u b c u l t u r e (fr. wt. 40-52 g/flask; dry wt. 3.2-3.6 g/flask), though the spheric surface part of the foam balls r e t a i n e d more cells than inside part. On the contrary, the immobilized cells in the foam having the least pore d e n s i t y (#06, Fig. I A and B) were the least (ft. wt. 18.4 g/flask; dry Wt. 1.1 g/flask). Free s u s p e n d e d cells r e l e a s e d from the i m m o b i l i z e d cells were I-2 g / f l a s k (fr. wt.) in #13, #20 and #30 but 26.1 g/flask (dry wt.; 2.0 g/flask) in #06. Therefore it is s u g g e s t e d that the foam balls having smaller pore size are more suitable for the i m m o b i l i z a t i o n of the coffee cells (Fig. I A and C). In foam cubes (I cm 3, ca. 80 cubes/flask), #20 and #30 i m m o b i l i z e d 95 g and 113 g (ft. wt.) of coffee cells at the end of the 3rd subculture, respectively. But #13 i m m o b i l i z e d only 70 g even at the end of the 5th. Because of the smaller body of the cubes, polyurethane foam cubes having the smaller pore size tended to i m m o b i l i z e d more cells. Thus the cubes of #30 were c o n s i d e r e d to be the most suitable of all for a f o l l o w i n g experiment. We i n v e s t i g a t e d the effect of the number of the cubes on cell growth and caffeine production for the p u r p o s e s of a long term culture. Fig. 2 suggests that the time course could be c l a s s i f i e d into 4 phases; I immobilization, II growth, III caffeine production, IV re-growth. The number of cubes i n f l u e n c e d their periods apparently. They a p p e a r e d t y p i c a l l y in 34 cubes where the largest amount of c a f f e i n e p r o d u c t i o n (24 mg /flask) was o b s e r v e d (Fig. 2 B). In the phase I, c a f f e i n e p r o d u c t i o n per

Fig. I. I m m o b i l i z e d Coffea arabica cells in ball type p o l y u r e t h a n e foam at the end of the 3rd subculture. A) Shake culture of the immobilized cells, I; pore d e n s i t y 6 pores/25 mm (#06), 2; 13 (#13). B) The whole (the upper part) and the cut face (the lower part)of I and 2. C) Shake culture, 3; 20 (#20), 4; 30 (#30). D) The whole and the cut face of 3 and 4.

fresh weight of cells was higher than in the phases II and IV (Fig. 2 A and B). It was supposed that damage to cells by grind with the cubes made some mechanical stress; namely, the stress situation might induce caffeine production. Frischknecht and Baumann (1985) reported that caffeine is stress compound, because caffeine production was stimulated by e x p o s u r e to high light intensity and high NaCI concentration. When 68 cubes were used, the phase I was not observed (Fig. 2 C). Because most of the cells were a p p e a r e d to be d e s t r o y e d by grind with so many cubes before i m m o b i l i z a t i o n and p r o d u c t i o n of caffeine.

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Subcultures Fig. 2. Growth and caffeine p r o d u c t i o n of immobilized C. arabica cells in different number of cube type p o l y u r e t h a n e foams (# 30). Immobilized C. arabica cells by p o l y u r e t h a n e foam cubes (Icm 3, 30 pores/25 mm) were s u b c u l t u r e d every 3 weeks. The number of cubes per flask were A) 17, B) 34, C) 68 which were started in duplicate. Caffeine content in medium per flask ( 0 ) , the fresh weight of the i m m o b i l i z e d ( O ) and the freely suspended cells ( X ) were m e a s u r e d when every subculture was finished. Phases are I; immobilization, II; growth, III; caffeine production, IV; re-growth. In the phase II, vigorous cell growth and lower caffeine p r o d u c t i o n were observed in all cases. The free suspended cells released from the immobilized cells hardly existed. In the least number of the cubes(17), the phase II had the longest period (Fig. 2. A and Fig. 3.); immobilized cells per a cube were the most. When the fresh weight of the whole immobilized cells exceeded about 100 g / f l a s k in 17 cubes, t~e size of cubes became larger to about 8 cm . The cubes were fixed in the flask by one another and not shaken. Only medium was shaken. They exposed partly to the air from the medium. The shaken m e d i u m was bubbled by the fixed cubes. Thus it was thought that the aeration state changed. Otherwise a clash among cubes which might give the cells the m e c h a n i c a l stress is minimized. Ishida (1988) made a device fixing foam cubes by stainless steel wire, which were supposed to create the s i t u a t i o n m e n t i o n e d above. In the phase III, the cell growth stopped, and most caffeine production was o b s e r v e d in this phase (Fig. 2 A, B and C). The immobilized cells in the cubes became soft and floated (Fig. 3). Fine free suspended things w h i c h might be almost cell walls were observed, indicating destruction of the cells. The cubes were still moving

Fig. 3. Immobilized coffee cells in cube type polyurethane foams (#30) at the end of the 7th subculture. At the end of the 7th subculture, the immobilized cells were t r a n s f e r r e d into the fresh m e d i u m and the next started. The left flask had the 17 cubes, the center the 34, and the right the 68. The immobilized cells in the left, which had not already moved, were still growing; the free suspended cells became e x t r e m e l y rare. In the 34 and the 68 cubes, the immobilized cells have become soft and floated.

128 B). Most of the i m m o b i l i z e d cells at the end of the phase III were c o n s i d e r e d to have lost capacity of purine a l k a l o i d production or viability. Corchete and Yeoman (1989) indicated that decreasing activity in immobilized cell system after the previous biotransformation could be caused by d e s t r u c t i o n of cells. Through this experiment, when the cell growth was vigorous, lower caffeine production was observed. To construct a continuous caffeine p r o d u c t i o n system using i m m o b i l i z e d cells, it is n e c e s s a r y to develop the methods p r o l o n g i n g the phase III. Foam particles, even if a ~mall particles like the cube type (I cm ) were employed, i m m o b i l i z e d much cells and they were not d e s t r o y e d by internal pressure causing by the cell growth (Fig. 4 B). Polyurethane foam is suitable for a support immobilizing some of plant cells which will be p l a n n i n g on a long term culture because of its capacity of i m m o b i l i z i n g cells and of f l e x i b i l i t y to internal pressure caused by cell growth.

Acknowledgement Fig. 4. I m m o b i l i z e d coffee cells in cube type polyurethane foams (#30) at the end of the whole subcultures. A) At the end of the whole cultivation, the 17 and the 34 cubes were not moved. B) The whole (the upper part) and the cut face (the lower part) of the immobilized cells r e t a i n e d by the 17, the 34, and the 68 cubes from the left dish. The less the number of cubes were, the larger the i m m o b i l i z e d cell cubes became.

when the fresh weight of the immobilized cells r e a c h e d even 100 g/flask. Therefore the aeration state did not change. The a e r a t i o n m i g h t be i n s u f f i c i e n t for the cell growth. O t h e r w i s e the clash one another in the flask might give the stress to the cells. A c c o r d i n g to Prenosil et al. (1987), c a f f e i n e f o r m a t i o n in coffee cells a c c e l e r a t e s nearly at the end of a c u l t i v a t i o n period, which means that most of the final amount of caffeine is s y n t h e s i z e d after the exponential period of growth, when d e p l e t i o n in sugar (glucose) was observed. Lindsey (1985) succeeded in increasing the capsaicin production of the immobilized Capsicum frutescens cells by r e s t r i c t i o n of phosphate in medium. Besides the aeration and the clash, a critical amount the cells reached might be subject to restriction of some c o m p o n e n t in the medium. In the phase IV, the cell growth began again (Fig. 2) and caffeine production leveled off a s y m p t o t i c a l l y . The change of the a e r a t i o n state as o b s e r v e d in the phase II in the 17 cubes also h a p p e n e d in the 34 (Fig. 4 A). In the 68 cubes, the cubes was still moving; the size of the cubes i m m o b i l i z i n g cells were the smallest (Fig. 4

The authors thank B r i d g e s t o n e p r o v i d i n g Scott Foam.

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for

References Brodelius P, Deus B, Mosbach K, Zenk MH (1979) FEBS Lett. 1 0 3 : 9 3 - 9 7 Corchete P, Yeoman MM (1989) Plant Cell Reports 8: 128-131 Frischknecht PM, Baumann TW (1985) P h y t o c h e m i s t r y 24: 2255-2257 Furusaki S, Nozawa T, Isohara T, Furuya T (1988) Appl. Microbiol. Biotechnol. 29: 437-441 Furuya T, Yoshikawa T, Taira M (1984) P h y t o c h e m i s t r y 23: 999-1001 Furuya T, Orihara Y, Koge K, Tsuda Y (1989) Plant Tissue Culture Letters 6: 148-151 Galun E, Aviv D, Dantes A, Freeman A (1983) Planta Med. 49: 9-13 Haldimann D, Brodelius P (1987) P h y t o c h e m i s t r y 26: 1431-1434 Ishida BK (1988) Plant Cell Reports 7: 270273 Keller H, Wanner H, Baumann TW (1972) Planta 108: 338-350 Lindsey K (1985) Planta 165: 126-133 L i n d s e y K, Yeoman MM, Black GM, Mavituna F (1983) FEBS Lett. 1 5 5 : 1 4 3 - 1 4 9 Murashige T, Skoog F (1962) Physiol. Plant 15:473-479 Prenosil JE, Hegglin M, Baumann TW, F r i s c h k n e c h t PM, K a p p e l e r AW, B r o d e l i u s P, H a l d i m a n n D (1987) Enzyme Microbiol. Technol. 9: 450-458 Waller GR, MacVean CD, Suzuki T (1983) Plant Cell Reports 2: 109-112