上海交通大学：《细胞工程》精品课程教学资源（学习文献）第7讲 文献-immobilization

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol.70,N0.5,317-321.1990 Enhanced Production of Shikonin by Lithospermum erythrorhizon Cells Immobilized in Polyurethane Foam Matrices YOUNG HOON PARK,1*WEON TAEK SEO,'AND JANG RYOL LIU2 Biochemical Process Laboratory and Plant Cell Biology Laboratory,?Genetic Engineering Center,The Korea Institute of Science and Technology,P.O.Box 17,Daeduk Science Town,Taejon 305-333,Korea Received 18 April 1990/Accepted 11 September 1990 Production of shikonin by Lithospermum erythrorhizon cells immobilized in polyurethane foam matrices was investigated.The cells were effectively entrapped inside the polymer matrices prepared in a cubic form with a lateral length of 0.5 cm.They proliferated further by incubation and resulted in immobilized polymer foam cubes which were fully packed with cell aggregates.Shikonin production was observed to increase significantly when the immobilized cells were used.It was excreted by the cells and directly adsorbed,by hydrophobic inter- action,to the polymer site.The product could be recovered,after the cells were separated,by direct solvent extraction from the shikonin-adsorbed polyurethane matrices.It is more interesting to note that a single-stage culture with immobilized L.erythrorhizon cells yielded higher shikonin productivity than the conventional two-stage culture system,which is considered to be of practical importance for industrial applications. Plant cell culture technology has been developing rap- tion.With the cell line used in this study,SH medium was idly since the 1970s,and successfully applied to the produc- found to be superior for cell growth to other media like tion of many high value-added chemicals.Among them, MG-5 described by Fujita et al.(6).Modifications of the shikonin has been reported as the most successful example SH medium were made;SH medium containing 2 mg//of for the mass production of plant secondary metabolites. p-chlorophenoxyacetic acid and 0.1 mg//of kinetin was Large scale production of shikonin has been carried out by designated as SHCK medium,and that with 1.75 mg//of a two-staged culture method using the improved media indole-3-acetic acid and 0.1 mg//of kinetin as SHIK me- MG-5 and M-9 for cell growth and for product forma- dium.The cell line was maintained by regular subcultures tion,respectively (1). in SHCK liquid and agar media every eight days. Immobilization techniques have also been successfully Support material A sheet of polyurethane foam (Sam applied to plant cell culture systems;entrapment of grow- Kwang Chemical Co.,Seoul,Korea)was cut into cubic ing plant cells within inert polysaccharides(2)and synthe- forms with a lateral length of 0.5 cm.The foam matrices tic resin polymers (3),and their adsorption to porous poly- had a porosity of around 0.97 and pore size of 40 pores per mer materials(4)are typical examples.By immobilization. linear inch.Polyurethane foam cubes prepared in such a plant cells are protected from external environmental fac- manner were washed several times with ethanol and distilled tors such as shear stress,and the cellular activity itself can water and then dried before they were used. be further enhanced by facilitated cell-to-cell contact.The Cell cultivation in shake flasks Cells of L.ery- possibility of ease of scale-up and of improvement in proc. throrhizon were cultivated in 250 ml Erlenmeyer flasks ess productivity resulting from employing immobilized containing 40 ml liquid medium in a gyratory shaking in- cells should therefore be carefully considered in any plant cubator at 25C and 100 rpm under dark condition.After cell culture system in order to achieve proper economic 9d of cultivation,cells were inoculated to a specified cul- feasibility. ture medium.10%(v/v)inoculum was used for a single- In the present work,polyurethane foam was selected as stage culture,or for the first stage of a two-stage culture a support material for immobilization of Lithospermum system.In the two-stage culture,the cells precultured in erythrorhizon cells to produce shikonin derivatives.Cell the first stage were harvested on a stainless steel mesh growth and shikonin production were examined by using (mesh size,ca.30 um)and transferred to the second-stage the immobilized plant cells.Considerable improvements flask containing a specified medium. were observed in the process productivity of the plant cell Immobilization of the cells Polyurethane foam culture system,and the results are reported in this paper. cubes prepared as above were added to the culture flask such that their total weight constituted 1%(w/v)of the MATERIALS AND METHODS culture broth and then sterilized at 120C for 15 min. Since each foam cube weighed approximately 0.0025- Cell line and culture media All experiments were per- 0.0030 g,the content was roughly estimated to be about formed using L.erythrorhizon KCTC PCL 52001.which 4,000 cubes per liter of culture broth.10%(v/v)inoculum was obtained from Korean Collections for Type Cultures was then made with free cells of L.erythrorhizon Genetic Engineering Center,The Korea Institute of cultivated as above such that the flask contained a cell con- Science and Technology,Taejon,Korea. centration of ca.40 g of fresh cell weight//.When the flask The basal media used were Shenk and Hildebrandt (SH) was incubated on a gyratory shaker,the cells became en- medium(5)for cell growth and M-9(6)for product forma- trapped in the pores of the polyurethane foam.It was ob- served that the cells were almost completely entrapped in Corresponding author. the pores within 2-3 d of incubation.Since free cells of L. 317

JOURNAL OF FERMENTATION AND BIOENGINEERING VOI. 70, No. 5, 317-321. 1990 Enhanced Production of Shikonin by Lithospermum erythrorhizon Cells Immobilized in Polyurethane Foam Matrices YOUNG HOON PARK, 1. WEON TAEK SEO, 1 AND JANG RYOL LIU 2 Biochemical Process Laboratory ~ and Plant Cell Biology Laboratory, 2 Genetic Engineering Center, The Korea Institute of Science and Technology, P.O. Box 17, Daeduk Science Town, Taejon 305-333, Korea Received 18 April 1990/Accepted 11 September 1990 Production of shikonin by Lithospermum erythrorhizon cells immobilized in polyurethane foam matrices was investigated. The cells were effectively entrapped inside the polymer matrices prepared in a cubic form with a lateral length of 0.5 cm. They proliferated further by incubation and resulted in immobilized polymer foam cubes which were fully packed with cell aggregates. Shikonin production was observed to increase significantly when the immobilized cells were used. It was excreted by the cells and directly adsorbed, by hydrophobic inter￾action, to the polymer site. The product could be recovered, after the cells were separated, by direct solvent extraction from the shikonin-adsorbed polyurethane matrices. It is more interesting to note that a single-stage culture with immobilized L. erythrorhizon cells yielded higher shikonin productivity than the conventional two-stage culture system, which is considered to be of practical importance for industrial applications. Plant cell culture technology has been developing rap￾idly since the 1970s, and successfully applied to the produc￾tion of many high value-added chemicals. Among them, shikonin has been reported as the most successful example for the mass production of plant secondary metabolites. Large scale production of shikonin has been carried out by a two-staged culture method using the improved media MG-5 and M-9 for cell growth and for product forma￾tion, respectively (1). Immobilization techniques have also been successfully applied to plant cell culture systems; entrapment of grow￾ing plant cells within inert polysaccharides (2) and synthe￾tic resin polymers (3), and their adsorption to porous poly￾mer materials (4) are typical examples. By immobilization, plant cells are protected from external environmental fac￾tors such as shear stress, and the cellular activity itself can be further enhanced by facilitated cell-to-cell contact. The possibility of ease of scale-up and of improvement in proc￾ess productivity resulting from employing immobilized cells should therefore be carefully considered in any plant cell culture system in order to achieve proper economic feasibility. In the present work, polyurethane foam was selected as a support material for immobilization of Lithospermum erythrorhizon cells to produce shikonin derivatives. Cell growth and shikonin production were examined by using the immobilized plant cells. Considerable improvements were observed in the process productivity of the plant cell culture system, and the results are reported in this paper. MATERIALS AND METHODS Cell line and culture media All experiments were per￾formed using L. erythrorhizon KCTC PCL 52001, which was obtained from Korean Collections for Type Cultures, Genetic Engineering Center, The Korea Institute of Science and Technology, Taejon, Korea. The basal media used were Shenk and Hildebrandt (SH) medium (5) for cell growth and M-9 (6) for product forma- * Corresponding author. 317 tion. With the cell line used in this study, SH medium was found to be superior for cell growth to other media like MG-5 described by Fujita et al. (6). Modifications of the SH medium were made; SH medium containing 2 mg/l of p-chlorophenoxyacetic acid and 0.1 mg/l of kinetin was designated as SHCK medium, and that with 1.75 mg/l of indole-3-acetic acid and 0.1 mg/l of kinetin as SHIK me￾dium. The cell line was maintained by regular subcultures in SHCK liquid and agar media every eight days. Support material A sheet of polyurethane foam (Sam Kwang Chemical Co., Seoul, Korea) was cut into cubic forms with a lateral length of 0.5 cm. The foam matrices had a porosity of around 0.97 and pore size of 40 pores per linear inch. Polyurethane foam cubes prepared in such a manner were washed several times with ethanol and distilled water and then dried before they were used. Cell cultivation in shake flasks Cells of L. ery￾throrhizon were cultivated in 250 ml Erlenmeyer flasks containing 40 ml liquid medium in a gyratory shaking in￾cubator at 25°C and 100 rpm under dark condition. After 9 d of cultivation, cells were inoculated to a specified cul￾ture medium. 10~ ~ (v/v) inoculum was used for a single￾stage culture, or for the first stage of a two-stage culture system. In the two-stage culture, the cells precultured in the first stage were harvested on a stainless steel mesh (mesh size, ca. 30/zm) and transferred to the second-stage flask containing a specified medium. Immobilization of the cells Polyurethane foam cubes prepared as above were added to the culture flask such that their total weight constituted 1% (w/v) of the culture broth and then sterilized at 120°C for 15min. Since each foam cube weighed approximately 0.0025- 0.0030 g, the content was roughly estimated to be about 4,000 cubes per liter of culture broth. 10% (v/v) inoculum was then made with free cells of L. erythrorhizon cultivated as above such that the flask contained a cell con￾centration of ca. 40 g of fresh cell weight/l. When the flask was incubated on a gyratory shaker, the cells became en￾trapped in the pores of the polyurethane foam. It was ob￾served that the cells were almost completely entrapped in the pores within 2-3 d of incubation. Since free ceils of L

318 PARK ET AL J.FERMENT.BIOENG., erythrorhizon range in size from 50-300 um,it was believed mobilized cells,each sample of 20 cell-packed foam cubes that the amount of polyurethane foam added provided was randomly taken from the culture broth.The cells were enough space for the entrapment.Further incubation re- then easily separated from the polyurethane foam matrices sulted in active proliferation of the entrapped cells,and by simple squeezing and washing.Cells collected in such a the polyurethane foam cubes became fully packed with cell manner were then filtered,and the FCW and DCW of the aggregates. sample were determined as above.Since shikonin is highly Operation of bioreactor A packed-bed type bioreac- insoluble to water,and thus adsorbs well onto poly- tor(Fig.1)was employed for production of shikonin by urethane matrices,loss of the product during cell separa- using the immobilized plant cells.The internal diameter tion is considered negligible. and height of the reactor were 80 and 300 mm,respectively. Shikonin was then extracted from the polyurethane The column was made of a Pyrex glass tube with a foam cubes with chloroform and its content was deter- sintered glass filter at the bottom,through which pre- mined by using the method described by Mizukami et al. humidified air was supplied at a fixed rate of 0.5 vvm. (8).Its concentration was estimated as mg of acetyl About 40g of fresh cells (equivalent to 2g dry cell shikonin per liter of culture broth.An authentic sample of weight)precultivated in SHIK medium were harvested and acetyl shikonin was obtained from the Plant Cell Biology transferred asceptically to the reactor containing 1 of the Laboratory,Genetic Engineering Center,KIST,Taejon, same medium and 10 g of polyurethane cubes prepared as Korea. above. The temperature was maintained at 25C by circulating constant-temperature water through the jacket.The work- RESULTS AND DISCUSSION ing liquid volume was kept at 1.0/and the column was Shake flask culture with free cells Figure 2 shows the covered with aluminum foil to ensure darkness during the time courses for sucrose consumption and accompanying reactor operation. cell growth with a free suspension culture of L Analytical methods Sugar concentration was deter- erythrorhizon in SHCK and SHIK media.In SHCK me- mined by the DNS method (7). dium,cell growth reached up to 19g DCW//(450g To determine the cell mass with a free suspension culture FCW/after 10 d and sucrose was almost completely con- of the plant cells,the cells were first separated by using sumed at this point.Decrease of cell mass thereafter was stainless steel mesh (mesh size ca.30 um)and the fresh considered due to cell autolysis.Similar observations have cell weight(FCW)was measured from the filtered sample. been reported in other plant cell culture systems (9,10).It The dry cell weight(DCW)was then determined by drying was noted that cell growth was slightly retarded in SHIK the sample in an oven at 95C to a constant weight.For im- medium and reached up to 14g DCW//(330 g FCW/) after 14 d.With the cell line used in the present study,the shikonin produced was observed to be retained inside the cells during the culture period(Fig.3A),but its concentra- tion was negligibly small,as shown in Fig.2 Cell growth was seen to be closely related to the con- 40 20 30 (501X) 30 75 0 Q 云20 ● FIG.1.Schematic diagram of the packed-bed bioreactor system Time (d> with immobilized L.ervthrorhizon cells.I.Air flow meter,2.air FIG.2.Time courses of sucrose consumption,cell growth and filter,3.air humidifying vessel,4.sintered glass filter,5.sampling shikonin production with free cells of L.erythrorhizon in SHCK and port,6.water jacket,7.packed-bed of immobilized cells,8.inoculum SHIK media.Data points marked by circles and squares indicate port,9.antifoam inlet,10.condensor. those obtained from SHCK and SHIK media,respectively

318 PARK ET AL. J. FERMENT. BIOENG., erythrorhizon range in size from 50-300 pm, it was believed that the amount of polyurethane foam added provided enough space for the entrapment. Further incubation re￾sulted in active proliferation of the entrapped cells, and the polyurethane foam cubes became fully packed with cell aggregates. Operation of bioreactor A packed-bed type bioreac￾tor (Fig. 1) was employed for production of shikonin by using the immobilized plant cells. The internal diameter and height of the reactor were 80 and 300 mm, respectively. The column was made of a Pyrex glass tube with a sintered glass filter at the bottom, through which pre￾humidified air was supplied at a fixed rate of 0.5 vvm. About 40g of fresh cells (equivalent to 2g dry cell weight) precultivated in SHIK medium were harvested and transferred asceptically to the reactor containing 1 l of the same medium and 10 g of polyurethane cubes prepared as above. The temperature was maintained at 25°C by circulating constant-temperature water through the jacket. The work￾ing liquid volume was kept at 1.01 and the column was covered with aluminum foil to ensure darkness during the reactor operation. Analytical methods Sugar concentration was deter￾mined by the DNS method (7). To determine the cell mass with a free suspension culture of the plant cells, the cells were first separated by using stainless steel mesh (mesh size ca. 30/zm) and the fresh cell weight (FCW) was measured from the filtered sample. The dry cell weight (DCW) was then determined by drying the sample in an oven at 95°C to a constant weight. For ira- 11 :_.,. 10 8 dl = AIR 1 611 7 --'l 4 FIG. 1. Schematic diagram of the packed-bed bioreactor system with immobilized L. erythrorhizon cells. 1. Air flow meter, 2. air filter, 3. air humidifying vessel, 4. sintered glass filter, 5. sampling port, 6. water jacket, 7. packed-bed of immobilized cells, 8. inoculum port, 9. antifoam inlet, 10. condensor. mobilized cells, each sample of 20 cell-packed foam cubes was randomly taken from the culture broth. The cells were then easily separated from the polyurethane foam matrices by simple squeezing and washing. Cells collected in such a manner were then filtered, and the FCW and DCW of the sample were determined as above. Since shikonin is highly insoluble to water, and thus adsorbs well onto poly￾urethane matrices, loss of the product during cell separa￾tion is considered negligible. Shikonin was then extracted from the polyurethane foam cubes with chloroform and its content was deter￾mined by using the method described by Mizukami et al. (8). Its concentration was estimated as mg of acetyl shikonin per liter of culture broth. An authentic sample of acetyl shikonin was obtained from the Plant Cell Biology Laboratory, Genetic Engineering Center, KIST, Taejon, Korea. RESULTS AND DISCUSSION Shake flask culture with free cells Figure 2 shows the time courses for sucrose consumption and accompanying cell growth with a free suspension culture of L. erythrorhizon in SHCK and SHIK media. In SHCK me￾dium, cell growth reached up to 19g DCW/I (450g FCW//) after 10 d and sucrose was almost completely con￾sumed at this point. Decrease of cell mass thereafter was considered due to cell autolysis. Similar observations have been reported in other plant cell culture systems (9, 10). It was noted that cell growth was slightly retarded in SHIK medium and reached up to 14g DCW/I (330g FCW//) after 14 d. With the cell line used in the present study, the shikonin produced was observed to be retained inside the cells during the culture period (Fig. 3A), but its concentra￾tion was negligibly small, as shown in Fig. 2. Cell growth was seen to be closely related to the con- 40 ; ~ I00 ,3{ b~ .20+ I~ ° ° 20 ~ 10 25 0 - 0 4 8 12 16 0 Time (d) FIG. 2. Time courses of sucrose consumption, cell growth and shikonin production with free cells of L. erythrorhizon in SHCK and SHIK media. Data points marked by circles and squares indicate those obtained from SHCK and SHIK media, respectively

VoL.70,1990 SHIKONIN PRODUCTION BY IMMOBILIZED PLANT CELL 319 A 100丛 白 B 20 1004 FIG.3.Microphotographs of L.erythrorhizon cells.(A)Cell ag- gregates from free suspension culture containing shikonin (Arrows indicate shikonin granules inside cells);(B)cells immobilized in poly- urethane pore matrices. FIG.4.Cell growth and shikonin production in SHCK (A)and SHIK (B)media with immobilized L.erythrorhizon cells. sumption of sucrose in the medium.Growth yields based on sucrose consumption were calculated to be 0.54 g cells being confined in the polyurethane pores.Most of DCW/g of sucrose and 0.47 g DCW/g of sucrose in SHCK shikonin produced was excreted by the cells and practically and SHIK medium,respectively.Maximum specific adsorbed onto the polyurethane matrices.The adsorption growth rates were 0.291 d-and 0.286 d in SHCK and mechanism is believed to be hydrophobic interaction,since SHIK medium,respectively. both shikonin and polyurethane are highly hydrophobic. Production of shikonin by immobilized plant cells Shikonin could be adsorbed on the polymer material up to Increased production of plant secondary metabolites has 180 mg/g of polyurethane (data not shown)in an ethanol- often been observed with immobilized growing plant cells water (1:10)mixture. (11,12).In the present study,we also observed that by Intracellular localization and secretion of shikonin by employing immobilized cells the production of shikonin in- Lithospermumn cells have been well studied (1),and a simi- creased significantly.When the cells of L.erythrorhizon lar secretory activity should be applicable to the present im- were inoculated to flasks with SHCK and SHIK media, mobilized cell system.In the free cell culture shikonin accu- and cultivated in the presence of polyurethane foam cubes, mulated on the cell walls as numerous red granules the cells became entrapped in the polyurethane foam pores whereas in the immobilized cell system it directly adsorbed and proliferated,with the active induction of shikonin onto the polymer matrices.Intracellular concentration of (Fig.3B).Within 2-3 d of shaking incubation most of the shikonin would therefore be reduced by its being trans free cells were entrapped in the foam matrices,resulting ported out to polymer matrix sites,and the consequent in a practically clear broth. reduction in intracellular product concentration would Figure 4 shows the time courses of cell growth and further enhance its biosynthetic activity.In addition, shikonin production in SHCK(A)and SHIK (B)media.It there is also a possibility of lowering the chances of prod- was noted that although cell growth was slightly reduced uct decomposition,which should result in an improved re- compared to the free suspension culture,shikonin produc- covery yield. tion was much more effective with immobilized cells than Comparison of single and two-stage cultures Tabata with the suspension culture of free cells (see Fig.2).The et al.(1)found that the ammonium ion,essential to increase of shikonin production was considered probably achieve sufficient cell growth,inhibited shikonin synthesis, due to enhanced cell-to-cell contact resulting from the and accordingly they developed an ammonium-free pro-

VoL. 70, 1990 SHIKONIN PRODUCTION BY IMMOBILIZED PLANT CELL 319 FIG. 3. Microphotographs of L. erythrorhizon cells. (A) Cell ag￾gregates from free suspension culture containing shikonin (Arrows indicate shikonin granules inside cells); (B) cells immobilized in poly￾urethane pore matrices. sumption of sucrose in the medium. Growth yields based on sucrose consumption were calculated to be 0.54g DCW/g of sucrose and 0.47 g DCW/g of sucrose in SHCK and SHIK medium, respectively. Maximum specific growth rates were 0.291 d ' and 0.286 d ~ in SHCK and SHIK medium, respectively. Production of shikonin by immobilized plant cells Increased production of plant secondary metabolites has often been observed with immobilized growing plant cells (11, 12). In the present study, we also observed that by employing immobilized cells the production of shikonin in￾creased significantly. When the cells of L. erythrorhizon were inoculated to flasks with SHCK and SHIK media, and cultivated in the presence of polyurethane foam cubes, the cells became entrapped in the polyurethane foam pores and proliferated, with the active induction of shikonin (Fig. 3B). Within 2-3 d of shaking incubation most of the free cells were entrapped in the foam matrices, resulting in a practically clear broth. Figure 4 shows the time courses of cell growth and shikonin production in SHCK (A) and SHIK (B) media. It was noted that although cell growth was slightly reduced compared to the free suspension culture, shikonin produc￾tion was much more effective with immobilized cells than with the suspension culture of free cells (see Fig. 2). The increase of shikonin production was considered probably due to enhanced cell-to-cell contact resulting from the A "~. "~, -. ~ i ~0 "" ,O 15 15 "'"" 10 ~l "o. I~me i d FIG. 4. Cell growth and shikonin production in SHCK (A) and SHIK (B) media with immobilized L. erythrorhizon cells. cells being confined in the polyurethane pores. Most of shikonin produced was excreted by the cells and practically adsorbed onto the polyurethane matrices. The adsorption mechanism is believed to be hydrophobic interaction, since both shikonin and polyurethane are highly hydrophobic. Shikonin could be adsorbed on the polymer material up to 180 mg/g of polyurethane (data not shown) in an ethanol￾water (1 : 10) mixture. Intracellular localization and secretion of shikonin by Lithospermum cells have been well studied (1), and a simi￾lar secretory activity should be applicable to the present im￾mobilized cell system. In the free cell culture shikonin accu￾mulated on the cell walls as numerous red granules, whereas in the immobilized cell system it directly adsorbed onto the polymer matrices. Intracellular concentration of shikonin would therefore be reduced by its being trans￾ported out to polymer matrix sites, and the consequent reduction in intracellular product concentration would further enhance its biosynthetic activity. In addition, there is also a possibility of lowering the chances of prod￾uct decomposition, which should result in an improved re￾covery yield. Comparison of single and two-stage cultures Tabata et al. (1) found that the ammonium ion, essential to achieve sufficient cell growth, inhibited shikonin synthesis, and accordingly they developed an ammonium-free pro-

320 PARK ET AL. J.FERMENT.BIOENG., TABLE 1.Culture data from single and two-stage cultures of L.erythrorhizon immobilized in polyurethane foam Single-stage Two-stage SHCK SHIK SHCKSHCK SHIK /SHIK /M-9 /M-9 Final cell mass 17.45 12.48 12.63 17.25 7.5 (g DCW/ Final shikonin conc. 25 112 56 12 78 100 (mg/0 Culture time 18 20 7/11 7/9 7/9 (d) Volumetric productivity 1.4 5.6 3.1 0.8 4.9 (mg/I/d) Culture times for two-stage culture indicate the durations in the first stage/second stage;the cultivation period in the first stage was fixed at 7 d. duction medium,M-9.They reported that,with a selected cell line of high yield,shikonin production was maximized with a two-stage culture system;cells were propagated in a large quantity in the first stage containing MG-5 medium, which lasted for 9 d,and then 1.5 g//of shikonin titre was induced during the subsequent 14-d second stage with M-9 Time (d) medium. In the present study,single and two-stage culture sys- FIG.5.Typical time course of shikonin production in a packed- bed bioreactor with immobilized L.erythrorhizon cells. tems were compared by using the immobilized plant cells; the media used were SHCK and SHIK for the single-stage and SHCK/SHIK,SHCK/M-9 and SHIK/M-9 combina- Fig.4). tions for the two-stage cultures.Culture data of final cell Direct comparison of the productivity in the present growth and shikonin titre are summarized in Table 1.It bioreactor system with that in other studies,such as the was noted that the single-stage culture with SHIK medium one by Tabata et al.(1),is very difficult because the activ- was much more effective for shikonin production than the ities of the cell lines used are quite different;the shikonin two-stage culture with SHCK/M-9.The two-stage culture synthetic activity of the cell line used in the present study is with SHIK/M-9 gave higher shikonin production than lower by an order of magnitude than those used by Tabata SHCK/M-9 but lower cell growth.In terms of volumetric er al.However,it is still considered that the present im- productivity,there was about a 15%increase with the mobilization technique can offer similar improvements to single-stage culture (SHIK)compared to the two-stage, systems with cell lines of higher activity,since it can con- SHIK/M-9 system.This is probably due to the fact that siderably reduce the culture steps and process time.We effective induction of shikonin is supported by enhanced believe that room still exists for further optimization of the productivity in SHIK medium.It has been reported that bioreactor system,and that it should be able to give higher natural auxin IAA (indole-3-acetic acid)is more favorable productivity than the present result. for shikonin production than the synthetic auxins such as 2,4-D(2,4-dichlorophenoxyacetic acid)(1).This agreed well with the result obtained from the present work REFERENCES employing immobilized cells that shikonin production was 1.Tabata,M.and Fujita,Y.:Production of shikonin by plant cell higher in SHIK than in SHCK medium (Fig.4).Noting cultures,p.207-218.Biotechnology in plant science.Academic that SH medium does contain ammonium (ca.2.6 mM), Press,New York,N.Y.(1985). which was reported to be inhibitory for shikonin biosyn- 2.Hulst,A.C.,Tramper,J.,Brodelius,P.,Leon,J.C.,and thesis (1),the present results indicate that the synthetic Luyben,K.A.M.:Immobilized plant cells;respiration and oxy activity can be controlled by using appropriate growth gen transfer.J.Chem.Tech.Biotech.,35B,198-204 (1985). regulators like IAA.Immobilization in the polymer 3.Nakajima,H..Sonomoto,K.,Morikawa,H.,Sato,F. matrices should also provide the plant cells with more Ichimura,K.,Yamada,Y.,and Tanaka,A.: Entrapment of favorable conditions for shikonin production as described Lavandula vera cells with synthetic resin prepolymers and its ap- plication to pigment production.Appl.Microbiol.Biotechnol. above. 24,266-270(1986). Operation of packed-bed bioreactor A typical time 4.Mavituna,F.and Park,J.M.:Growth of immobilized plant cells course of shikonin production in packed-bed bioreactor is in reticulate polyurethane foam matrices.Biotech.Lett.,7 (9), shown in Fig.5.The packed-bed reactor system was a 637-640(1985). single-stage batch culture using SHIK medium for both 5.Shenk,R.U.and Hildebrandt,A.C.:Medium and techniques cell growth and shikonin production.As in the flask cul- for induction and growth of monocotyledonous and ture,the cells grew to yield fully-packed immobilized cell dicotyledonous plant cell cultures.Can J.Bot.,50.199-204 aggregates and the shikonin produced was directly ad- (1972). sorbed to the polyurethane matrices,resulting in a practi- 6.Fujita,Y.,Hara,Y.,Suga,C.,and Morimoto,T.:Production of shikonin derivatives by cell suspension cultures of Lithospermum cally clear culture broth.It was noted that the product titre erythrorshizon.Plant Cell Reports,1,61-63 (1981). was higher than in flask culture,although slow cell growth 7.Chaplin,M.F.and Kennedy,J.F.(eds.):Carbohydrate analysis; and the initial lag in shikonin production were similar(see a practical approach,p.3.IRL Press,Washington,D.C.(1986)

320 PARK ET AL. TABLE 1. Culture data from single and two-stage cultures of L. erythrorhizon immobilized in polyurethane foam Single-stage Two-stage SHCK SHIK SHCK SHCK SHIK /SHIK /M-9 /M-9 Final cell mass 17.45 12.48 12.63 17.25 7.5 (g DCW//) Final shikonin conc. 25 112 56 12 78 (mg//) Culture time ~ 18 20 7/11 7/9 7/9 (d) Volumetric productivity 1.4 5.6 3.1 0.8 4.9 (mg/l/d) Culture times for two-stage culture indicate the durations in the first stage/second stage; the cultivation period in the first stage was fixed at 7 d. duction medium, M-9. They reported that, with a selected cell line of high yield, shikonin production was maximized with a two-stage culture system; cells were propagated in a large quantity in the first stage containing MG-5 medium, which lasted for 9 d, and then 1.5 g/l of shikonin titre was induced during the subsequent 14-d second stage with M-9 medium. In the present study, single and two-stage culture sys￾tems were compared by using the immobilized plant cells; the media used were SHCK and SHIK for the single-stage, and SHCK/SHIK, SHCK/M-9 and SHIK/M-9 combina￾tions for the two-stage cultures. Culture data of final cell growth and shikonin titre are summarized in Table 1. It was noted that the single-stage culture with SHIK medium was much more effective for shikonin production than the two-stage culture with SHCK/M-9. The two-stage culture with SHIK/M-9 gave higher shikonin production than SHCK/M-9 but lower cell growth. In terms of volumetric productivity, there was about a 15%0 increase with the single-stage culture (SHIK) compared to the two-stage, SHIK/M-9 system. This is probably due to the fact that effective induction of shikonin is supported by enhanced productivity in SHIK medium. It has been reported that natural auxin IAA (indole-3-acetic acid) is more favorable for shikonin production than the synthetic auxins such as 2, 4-D (2, 4-dichlorophenoxyacetic acid) (1). This agreed well with the result obtained from the present work employing immobilized cells that shikonin production was higher in SHIK than in SHCK medium (Fig. 4). Noting that SH medium does contain ammonium (ca. 2.6 raM), which was reported to be inhibitory for shikonin biosyn￾thesis (I), the present results indicate that the synthetic activity can be controlled by using appropriate growth regulators like IAA. Immobilization in the polymer matrices should also provide the plant cells with more favorable conditions for shikonin production as described above. Operation of packed-bed bioreactor A typical time course of shikonin production in packed-bed bioreactor is shown in Fig. 5. The packed-bed reactor system was a single-stage batch culture using SHIK medium for both cell growth and shikonin production. As in the flask cul￾ture, the cells grew to yield fully-packed immobilized cell aggregates and the shikonin produced was directly ad￾sorbed to the polyurethane matrices, resulting in a practi￾cally clear culture broth. It was noted that the product titre was higher than in flask culture, although slow cell growth and the initial lag in shikonin production were similar (see J. FERMENT. BIOENG., 3( x. = ~"-o. Q A \ ,9 --+ r5 x \ Time ( d ) FIG. 5. Typical time course of shikonin production in a packed￾bed bioreactor with immobilized L. erythrorhizon cells. Fig. 4). Direct comparison of the productivity in the present bioreactor system with that in other studies, such as the one by Tabata et al. (1), is very difficult because the activ￾ities of the cell lines used are quite different; the shikonin synthetic activity of the cell line used in the present study is lower by an order of magnitude than those used by Tabata et al. However, it is still considered that the present im￾mobilization technique can offer similar improvements to systems with cell lines of higher activity, since it can con￾siderably reduce the culture steps and process time. We believe that room still exists for further optimization of the bioreactor system, and that it should be able to give higher productivity than the present result. REFERENCES 1. Tabata, M. and Fujita, Y.: Production of shikonin by plant cell cultures, p. 207-218. Biotechnology in plant science. Academic Press, New York, N.Y. (1985). 2. Hulst, A.C., Tramper, J., Brodelius, P., Leon, J.C., and Luyhen, K. A. M.: Immobilized plant cells; respiration and oxy￾gen transfer. J. Chem. Tech. Biotech., 35B, 198-204 (1985). 3. Nakajima, H., Sonomoto, K., Morikawa, H., Sato, F., Iehimura, K., ¥amada, Y., and Tanaka, A.: Entrapment of Lavandula vera cells with synthetic resin prepolymers and its ap￾plication to pigment production. Appl. Microbiol. Biotechnol., 24, 266-270 (1986). 4. Mavituna, F. and Park, J. M.: Growth of immobilized plant cells in reticulate polyurethane foam matrices. Biotech. Lett., 7 (9), 637-640 (1985). 5. Shenk, R. U. and Hiidebrandt, A.C.: Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J. Bot., 50, 199-204 (1972). 6. Fujita, Y., Hara, Y., Suga, C., and Morimoto, T.: Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorshizon. Plant Cell Reports, 1, 61-63 (1981). 7. Chaplin, M. F. and Kennedy, J. F. (eds.): Carbohydrate analysis; a practical approach, p. 3. IRL Press, Washington, D.C. (1986)

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VOL. 70, 1990 SHIKONIN PRODUCTION BY IMMOBILIZED PLANT CELL 321 8. Mizukami, H., Konoshima, M., and Tabata, M.: Effect of nutri￾tional factors on shikonin derivative formation in LithoSpermum callus cultures. Phytochemistry, 16, 1183-1186 (1977). 9. Sahai, O.P. and Shuler, M.L.: Environmental parameters in￾fluencing phenolic production by batch cultures'of Nicotiana tabacum. Biotechnol. Bioeng., 26, 111-120 (1984). 10. Maurel, B. and Pareilleux, A.: Effect of carbon dioxide on the growth of cell suspension of Catharanthus roseus. Biotechnol. Lett., 7(5), 313-318 (1985). 11. Brodelius, P. and Mosbaeh, K.: Immobilized plant cells. Adv. Appl. Microbiol., 28, 1-25 (1982). 12. Kargi, F.: Alkaloid formation by Catharanthus roseus cells in a packed bed column biofilm reactor. Biotechnol. Lett., 181-186 (1988)