fermentation pilot plant 41 3.0 BIOREACTORS FOR PLANT CELL TISSUE AND ORGAN CULTURES (by Shinsaku Takayama) 3.1 Background of the Technique--Historical Overview Haberlandt! first reported plant cell, tissue, and organ cultures in 1902. He separated plant tissues and attempted to grow them in a simple nutrient medium. He was able to maintain these cells in a culture medium for 20 to 27 days. Although these cells increased eleven-fold in the best case,no cell division was observed. Gautheret 2 was the first to succeed in multiply ing the cells from the culture in 1934. He used the cambial tissues of Acer pseudoplatanus, Salix capraea, Sambucus nigra. After 15 to 18 months in subculture, cell activity ceased. He reasoned that this inactiveness was due to the lack of essential substances for cell division. He suspected that auxin nay have been one of the deficient substances. This compound was first eported in 1928 and was isolated by Kogel in the 1930s. Addition of auxin to the medium prompted plant cell growth. This finding was reported almost simultaneously by gautheret! 3I and White!l in 1939. Plant cell tissue and organ culture techniques rapidly developed, and in the mid-1950s another important phytohormone, cytokinins, had been discovered(Miller, Skoog, Okumura, Von Saltza and Strong 1955). 15) By 1962 Murashige and Skoogl6) had reported a completely defined medium which allowed the culture of most plant cells. Their medium has now become the mostly widely used medium in laboratories around the world After these initial discoveries and some significant improvements in media, scientific research on the cultivation of plant cell, tissue, and organs shifted to the area of basic physiological research. Industrial applications were also sought in the production of secondary metabolites, clonal plants and the improvement of various plant tissues Plant cell, tissue, and organ culture can be performed by either solid or liquid culture methods, however, in order to scale up the culture to the level of industrial processes, the liquid culture method must be employed Recently, pilot bioreactors as large as 20 kl have been constructed in the research laboratories of japan tobacco and Salt Co. and in those ofNitto Denko Co. Solid culture methods were used in large scale pilot experiments for the production of tobacco cells, and liquid culture methods were used in the production of Panax ginseng cells. An outstanding example of cell suspension culture in a pilot scale bioreactor(750 D)was the production of shikonins by Mitui Petrochemical Industries. In all these examples, various technologies have been used to improve the productivity of the metabolites
Fermentation Pilot Plant 41 3.0 BIOREACTORS FOR PLANT CELL TISSUE AND ORGAN CULTURES fly Shinsaku Takayama) 3.1 Background of the Technique-Historical Overview HaberlandtL'] first reported plant cell, tissue, and organ cultures in 1902. He separated plant tissues and attempted to grow them in a simple nutrient medium. He was able to maintain these cells in a culture medium for 20 to 27 days. Although these cells increased eleven-fold in the best case, no cell division was observed. GautheretL2] was the first to succeed in multiplying the cells from the culture in 1934. He used the cambial tissues of her pseudoplatanus, Salix capraea, Sambucus nigra. After 15 to 18 months in subculture, cell activity ceased. He reasoned that this inactiveness was due to the lack of essential substances for cell division. He suspected that auxin may have been one of the deficient substances. This compound was first reported in 1928 and was isolated by Kogel in the 1930's. Addition of auxin to the medium prompted plant cell growth. This finding was reported almost simultaneously by Gauthered3] and in 1939. Plant cell tissue and organ culture techniques rapidly developed, and in the mid-1950's another important phytohormone, cytokinins, had been discovered (Miller, Skoog, Okumura, Von Saltza and Strong 1955).15] By 1962 Murashigeand Skoog[6] had reported a completely defined medium which allowed the culture of most plant cells. Their medium has now become the mostly widely used medium in laboratories around the world. After these initial discoveries and some significant improvements in media, scientific research on the cultivation of plant cell, tissue, and organs shifted to the area of basic physiological research. Industrial applications were also sought in the production of secondary metabolites, clonal plants, and the improvement of various plant tissues. Plant cell, tissue, and organ culture can be performed by either solid or liquid culture methods, however, in order to scale up the culture to the level of industrial processes, the liquid culture method must be employed. Recently, pilot bioreactors as large as 20 kl have been constructed in the research laboratories of Japan Tobacco and Salt Co. and in those ofNitto Denko Co. Solid culture methods were used in large scale pilot experiments for the production of tobacco cells, and liquid culture methods were used in the production of Panax ginseng cells. An outstanding example of cell suspension culture in a pilot scale bioreactor (750 1) was the production of shikonins by Mitui Petrochemical Industries. In all these examples, various technologies have been used to improve the productivity of the metabolites
42 Fermentation and Biochemical Engineering Handbook The technologies include: (i) selection of a high yielding cell strain, (ii) screening of the optimum culture condition for metabolite production, (iii) addition of precursor metabolites, (iv) immobilized cell culture, and (v) differentiated tissue and/or organ culture. The productivity of various metabolites such as ginsenoside, anthraquinones, rosmarinic acid, shikonins ubiquinones, glutathione, tripdiolide, etc., reached or exceeded the amount produced by intact plants. To date, the production costs remain very high which is why most of the metabolites are still not produced on an industrial orpilot plant scale. Development oflarge scaleindustrial culture systems and techniques for plant cell, tissue, and organs, and the selection of the target metabolites are the chief prerequisites for the establishment of the industrial production of plant metabolites Culture Collection Intact Plant Breeding Genetic Engineering Cell Culture Organ Culture Clonal Plant mobilized Isolation and Purification of the Cell Culture Metabolites Useful Plant Metabolites Figure 17. The area of plant cell, tissue and organ cultures
42 Fermentation and Biochemical Engineering Handbook The technologies include: (i) selection of a high yielding cell strain, (ii) screening of the optimum culture condition for metabolite production, (iii) addition of precursor metabolites, (iv) immobilized cell culture, and (v) differentiated tissue andor organ culture. The productivity of various metabolites such as ginsenoside, anthraquinones, rosmalinic acid, shikonins, ubiquinones, glutathione, tripdiolide, etc., reached or exceeded the amount produced by intact plants. To date, the production costs remain very high which is why most of the metabolites are still not produced on an industrial or pilot plant scale. Development oflarge scale industrial culture systems and techniques for plant cell, tissue, and organs, and the selection of the target metabolites are the chief prerequisites for the establishment of the industrial production of plant metabolites. I Culture Collection I I I Cell Culture Figure 17. The area of plant cell, tissue and organ cultures
Fermentation Pilot plant 3.2 Media Formulations The formulation of the medium for plant cell, tissue, and organ culture depend primarily on nutritional requirements. Intact plants grow photoau totrophically in the soil, (i.e, they use CO2 as the principal carbon source and synthesize sugars by photosynthesis). In the case of aseptic cultures however, establishment of an autotrophic culture is not achieved so that heterotrophic or mixotrophic growth becomes the distinguishing characteristic. Therefore such cultures require the addition of carbon as an energy source. Given this fact, the culture medium must be formulated as a chemically defined mixture of mineral salts(macro-and microelements)in combination with a carbon source(usually sucrose). In addition to these constituents, organic constitu- ents such as vitamins, amino acids, sugar alcohols, and plant growth regulators are usually added to the medium. Media commonly used are listed in Table 11 Table 11. Formulations of most frequently used plant tissue culture media Ingredients(mg (l) MS Heller NH4小2SO4 134 (NHAN 1650 NaNO KNO3 1900 2500 Ca(NO3)2 CaCl3 2H2O 440 150 MgSO4 7H,O 200 KH2PO4 170 125 NaH_PO4 H2O l50 16.5 KCI 750 FeSO, 7H,O 27.8 27.8 EDTA 37.3 37.3 Cont'd next page)
Fermentation Pilot Plant 43 3.2 Media Formulations The formulation ofthe medium for plant cell, tissue, and organ culture depend primarily on nutritional requirements. Intact plants grow photoautotrophically in the soil, (i.e., they use CO, as the principal carbon source and synthesize sugars by photosynthesis). In the case ofaseptic cultures however, establishment of an autotrophic culture is not achieved so that heterotrophic or mixotrophic growth becomes the distinguishing characteristic. Therefore, such cultures require the addition of carbon as an energy source. Given this fact, the culture medium must be formulated as a chemically defined mixture of mineral salts (macro- and microelements) in combination with a carbon source (usually sucrose). In addition to these constituents, organic constituents such as vitamins, amino acids, sugar alcohols, and plant growth regulators are usually added to the medium. Media commonly used are listed in Table 11. Table 11. Formulations of most frequently used plant tissue culture media Ingredients (mg C1) MS B5 White Heller (NH4)2s04 W4W3 KNo3 NaNO, Ca(N03)2 CaCI,*2H20 MgS04*7H20 Na2SO4 =32po4 NaH,P O4.H2O KCI FeS O4.7H2O Na,,EDTA 134 1650 1900 2500 80 300 440 150 370 250 720 200 170 150 16.5 65 27.8 27.8 37.3 37.3 600 75 250 125 75 0 (Cont’d next page)
44 Fermentation and Biochemical Engineering Handbook Table 11.( Cont'd Formulations of most frequently used plant tissue culture media Ingredients(mg (.) MS White Heiler FeCl3 6H2O 1.0 Fe2(sO4) 3 MnSO4 4H2O 22.3 0.0l MnSO4 H2O ZnSO47H2O HaBO 6.2 0.83 075 Na,MoO 2H.O 0.25 CuSo→5H20 0.025 0.025 0.03 CoCl2 6H2O 0.025 NiCl2 6H,O AlCl3 0.03 100 Nicotinic acid 0.5 0.5 Pyridoxine HCI 0.5 0. Thiamine HCl Glycine 2.0 3.0 1.0 30,00020,00020,00020,000 Kinetin 0.04-100l 2.4D 0l-1.0 6.0 IAA pH 5.7-5.8 5.5 5.5
44 Fermentation and Biochemical Engineering Handbook Table 11. (Cont'd.) Formulations of most frequently used plant tissue culture media. Ingredients (mg 6-l) MS B5 White Heiler FeCl3*6H2O Mnso4-4H20 MnS04.H20 ZnS04.7H,0 K1 N%Mo04.2H20 CuS04.5H20 CoC12.6H20 NiC12*6H20 AlCI, Myo-inositol Nicotinic acid P yridoxine.HC1 Thiamine.HC1 Glycine Ca D-pantothenic acid Sucrose Kinetin Fe2(S04)3 H3BO3 2,4-D IAA PH 22.3 8.6 6.2 0.83 0.25 0,025 0.025 100 0.5 0.5 0.1 2.0 30,000 0.04- 10 1.0-30 5.7-5.8 10 2 3 0.75 0.25 0.025 0.025 100 1 .o 1 .o 10.0 20,000 0.1 0.1-1 .o 5.5 2.5 7 3 1.5 0.75 0.5 0.1 0.1 3 .O 1 .o 20,000 6.0 5.5 1 .o 0.01 1 1 0.01 0.03 0.03 0.03 1.0 20,000
Fermentation Pilot Plant 45 3.3 General applications The most important fields of research for industrial applications, plant cell tissue and organ cultures are clonal propagation and secondary metabo- lite production. Plants cultivated in vitro have great changes in their morphological features, from cell tissue to differentiated embryo, roots, shoots or plantlets Applications to Secondary Metabolite Production. Plant tissue culture is a potential method for producing secondary metabolites. Both shikonins(Fujita and Tabata 1987)]and ginseng saponins(Ushiyama etal 1986)[] have now been produced on a large scale by this method. However, the important secondary metabolites are usually produced by callus or cell suspension culture techniques. the amounts of some metabolites in the cell have exceeded the amounts of metabolites in the cells of the original plants grown in the soil. So it is expected that cell culturing may be applicable to industrial processes for the production of useful secondary metabolites. It is common knowledge that when a cell culture is initiated and then transferred, the productivity of the metabolite decreases(Kurz and Constabel, 1979).19) Once productivity decreases, it becomes very difficult to arrest or reverse the decrease. In order to avoid this phenomenon, many cell strains were screened to select those which would maintain metabolite productivity. Some metabo- lites such as anthocyanins, shikonins, vinca alkaloids, and ubiquinones have been reported to have increased their productivity significantly. Deus Neumann and Zenk(1984) ol have checked the stability of the productivity of the selected cell strains reported in the literature and noted that the production of some metabolites such as anthraquinone Morinda citrifolia), rosmalinic acid (Colius blumei), visnagin (Ammi visnaga), diosgenin (Dioscorea deltoidea, etc were stable after several subcultures, but some metabolites such as nicotine(Nicotiana rustica), shikonin(Lithospermum erythrorhizon), ajmalicine(Catharanthus roseus), rotenoids( Derris elliptica) anthocyan(Daucus carota), etc, were shown to be unstable after several subcultures Clonal Plant Propagation. Plants are propagated clonally from vegetative tissue or organs via bypass sex. Conventional clonal propagation can be performed by leafor stem cutting and layering or dividing ofthe plants however the efficiency is very low. Recently, many plants were propagated efficiently through tissue culture. This technique was first reported in 1960 by G. Morell] for the propagation of orchids and since then, many plants have been propagated by tissue culture. Today there are many commercial
Fermentation Pilot Plant 45 3.3 General Applications The most important fields of research for industrial applications, plant cell tissue and organ cultures are clonal propagation and secondary metabolite production. Plants cultivated in vitro have great changes in their morphological features, from cell tissue to differentiated embryo, roots, shoots or plantlets. Applications to Secondary Metabolite Production. Plant tissue culture is a potential method for producing secondary metabolites. Both shikonins (Fujita and Tabata 1987)r'I and ginseng saponins (Ushiyama et al., 1986)[*] have now been produced on a large scale by this method. However, the important secondary metabolites are usually produced by callus or cell suspension culture techniques. The amounts of some metabolites in the cell have exceeded the amounts of metabolites in the cells of the original plants grown in the soil. So it is expected that cell culturing may be applicable to industrial processes for the production of useful secondary metabolites. It is common knowledge that when a cell culture is initiated and then transferred, the productivity of the metabolite decreases (Kurz and Constabel, 1979).['1 Once productivity decreases, it becomes very difficult to arrest or reverse the decrease. In order to avoid this phenomenon, many cell strains were screened to select those which would maintain metabolite productivity. Some metabolites such as anthocyanins, shikonins, vinca alkaloids, and ubiquinones have been reported to have increased their productivity significantly. DeusNeumann and Zenk ( 1984)[1°1 have checked the stability of the productivity of the selected cell strains reported in the literature and noted that the production of some metabolites such as anthraquinone (Uorinda citrofoliu), rosmalinic acid (Colius blumei), visnagin (Ammi visnaga), diosgenin (Dioscoreu deltoidea), etc., were stable after several subcultures, but some metabolites such as nicotine (Nicotiana rusticu), shikonin (Lithospermum erythrorhizon), ajmalicine (Catharanthus roseus), rotenoids (Derris eliptica), anthocyan (Duucus carota), etc., were shown to be unstable after several subcultures. Clonal Plant Propagation. Plants are propagated clonally from vegetative tissue or organs via bypass sex. Conventional clonal propagation can be performed by leaf or stem cutting and layering or dividing ofthe plants, however the efficiency is very low. Recently, many plants were propagated efficiently through tissue culture. This technique was first reported in 1960 by G. Morel["] for the propagation of orchids and since then, many plants have been propagated by tissue culture. Today there are many commercial
46 Fermentation and Biochemical Engineering Handbook tissue culture nurseries throughout the world. Most of these tissue culture nurseries are using flasks or bottles containing agar medium for commercial propagation, but the efficiency is also low. In order to improve the efficiency, use of a bioreactor is desirable. Using a small bioreactor (4 to 10 liters), the author has produced over 4, 000 to 10, 000 plantlets within I to 2 months. The bioreactor system allows the induction of somatic embryos from vegetative cells which then leads to the production of artificial seeds(Rodenbaugh et al 1987).2 3. 4 Bioreactors-Hardware Configuration The configuration of bioreactors most frequently used for plant cell tissue, and organ cultures is fundamentally the sameas that used for microbial or animal cell cultures. However, in plants, the cells, tissues, and organs are all susceptible to mechanical stresses by medium aeration and agitation. At times, the production of both cells mass and metabolites is repressed severely and the bioreactor must therefore have the characteristics of low shear stresses and efficient oxygen supply. For these reasons, different bioreactors (Fig. 18)have been investigated in order to select the most suitable design Wagner and Vogelmann( 1977)3 have studied the comparison of different types of bioreactors for the yield and productivity of cell mass and anthraqui- none(Fig. 19). Among different types of bioreactors, the yield of anthraqui- nones in the air-lift bioreactor was about double that found in those bioreactors with flat blade turbine impellers, perforated disk impellers, or draft tube bioreactors with Kaplan turbine impellers. It was also about 30% higher than that of a shake flask culture. Thus, the configuration of the bioreactor is very important and development efforts are underway for both bench scale and pilot scale bioreactors Aeration-Agitation Bioreactor. This type of bioreactor(Fig. 20)is popular and is fundamentally the same as that used with microbial cultures For small scale experiments, the aeration-agitation type bioreactors is widely used. However, when the culture volume is increased, many problems arise The following are some of the scale-up problems in large aeration-agitation bioreactors: (i)increasing mechanical stresses by impeller agitation and (ii) increasing foaming and adhesion of cells on the inner surface of the bioreactor. Despite these problems, a large scale pilot bioreactor(volume 20 kl)was constructed. It successfully produced both cell mass and metabolites This bioreactor is therefore the most important type for bioreactor systems
46 Fermentation and Biochemical Engineering Handbook tissue culture nurseries throughout the world. Most of these tissue culture nurseries are using flasks or bottles containing agar medium for commercial propagation, but the efficiency is also low. In order to improve the efficiency, use of a bioreactor is desirable. Using a small bioreactor (4 to 10 liters), the author has produced over 4,000 to 10,000 plantlets within 1 to 2 months. The bioreactor system allows the induction of somatic embryos from vegetative cells which then leads to the production of artificial seeds (Redenbaugh et al., 1987).['*] 3.4 Bioreactors-Hardware Configuration The configuration of bioreactors most frequently used for plant cell, tissue, and organ cultures is hndamentally the same as that used for microbial or animal cell cultures. However, in plants, the cells, tissues, and organs are all susceptible to mechanical stresses by medium aeration and agitation. At times, the production of both cells mass and metabolites is repressed severely and the bioreactor must therefore have the characteristics of low shear stresses and efficient oxygen supply. For these reasons, different bioreactors (Fig. 18) have been investigated in order to select the most suitable design. Wagner and Vogelmann ( 1977)[131 have studied the comparison of different types of bioreactors for the yield and productivity ofcell mass and anthraquinone (Fig. 19). Among different types of bioreactors, the yield of anthraquinones in the air-lift bioreactor was about double that found in those bioreactors with flat blade turbine impellers, perforated disk impellers, or draft tube bioreactors with Kaplan turbine impellers. It was also about 30% higher than that of a shake flask culture. Thus, the configuration of the bioreactor is very important and development efforts are underway for both bench scale and pilot scale bioreactors. Aeration-Agitation Bioreactor. This type of bioreactor (Fig. 20) is popular and is fundamentally the same as that used with microbial cultures. For small scale experiments, the aeration-agitation type bioreactors is widely used. However, when the culture volume is increased, many problems arise. The following are some of the scale-up problems in large aeration-agitation bioreactors: (i) increasing mechanical stresses by impeller agitation and (ii) increasing foaming and adhesion of cells on the inner surface of the bioreactor. Despite these problems, a large scale pilot bioreactor (volume 20 kl) was constructed. It successfully produced both cell mass and metabolites. This bioreactor is therefore the most important type for bioreactor systems
tt B D E (12 Rs H Figure 18. Different types of bioreactors for plant cells, tissues and organs. (A)Shake Flask.(B) Aeration-Agitation. (C) Percolated Impeller.(D) Draught Tube Air-lift.(E Draft Tube with Kaplan Turbine. (F) Air-lift loop.(G)Rotating Drum. (B) Light Emittir Draught Tube. ( )Spin Filter. () Bubble Column. (K) Aeration.()Gaseous Phase
Fermentation Pilot Plant 47
48 Fermentation and Biochemical Engineering Handbook 目 dry weight四 metabolite口 dry weight■ metabolite productivity ity for cell mass and anthraquinones in systems.(1)Shake Fla Flat Blade Turbine. ()Perfolated Disk (4 Draft Tube Bioreactor with Turbine, (5)Air-lift Bioreactor. Air Driven Bioreactors. The simplest design is the air-driven bioreactor equipped with sparger at the bottom of the vessel. It is widely used for plant cell, tissue, and organ cultures. In cases wherethe cells grow rapidly and the cell mass occupies 40-60% of the reactor volume, the flow charac teristics become non Newtonian and the culture medium can no longer be agitated by simple aeration Rotating Drum bioreactor The rotating drum bioreactor(Fig. 21) turns on rollers and the oxygen supply mechanism is entirely different from either the mechanically agitated or the air-lift bioreactor. Tanaka et al (1983), 4) reported that the oxygen transfer coefficient is affected by a change of airflow rate under all rotational speeds(Fig. 22). This character istic is suitable not only for the growth of plant cell, tissue, and organs but also for the production of metabolites under high viscosity and high density cultures. It is superior to the cultures using either mechanically agitated air-lift bioreactors since the cultures are supplied ample oxygen and are only used for a pilot scale experiment (Tanaka 1987). ye was constructed and weakly stressed. Recently a I kl bioreactor of this typ
48 Fermentation and Biochemical Engineering Handbook 10 I 0.5 wrn 100 rprn I 101 0.5 wm 100 rprn I u4 75 I 0.33 vvm 350 rprn dry weight metabolite JJ dry weight metabolite yield productivity Figure 19. Comparison of yield and productivity for cell mass and anthraquinones in various bioreactor systems. (1) Shake Flask. (2) Flat Blade Turbine. (3) Perfolated Disk Impeller. (4) Draft Tube Bioreactor with Kaplan Turbine. (5) Air-lift Bioreactor. Air Driven Bioreactors. The simplest design is the air-driven bioreactor equipped with sparger at the bottom ofthe vessel. It is widely used for plant cell, tissue, and organ cultures. In cases where the cells grow rapidly and the cell mass occupies 40-60% of the reactor volume, the flow characteristics become non-Newtonian and the culture medium can no longer be agitated by simple aeration, Rotating Drum Bioreactor. The rotating drum bioreactor (Fig. 21) turns on rollers and the oxygen supply mechanism is entirely different from either the mechanically agitated or the air-lift bioreactor. Tanaka et al., ( 1983),[14] reported that the oxygen transfer coefficient is affected by a change of airflow rate under all rotational speeds (Fig. 22). This characteristic is suitable not only for thegrowth ofplant cell, tissue, and organs but also for the production of metabolites under high viscosity and high density cultures. It is superior to the cultures using either mechanically agitated or air-lift bioreactors since the cultures are supplied ample oxygen and are only weakly stressed. Recently a 1 kl bioreactor of this type was constructed and used for a pilot scale experiment (Tanaka 1987).[15]
fermentation pilot plant 49 99 Figure 20. Ninety-five liter automated bioreactor for plant cell, tissue and organ cultures Photo courtesy ofK. F. Engineering Co, Ltd, Tokyo) BAFFLE PLATE SENSOR AIR一 Figure 21. Schematic diagram of the rotating drum bioreactor(Tanaka, H, et al., 1983)
Fermentation Pilot Plant 49 Figure 20. Ninety-five liter automated bioreactor for plant cell, tissue and organ cultures. (photo courtesy ofK. F. Engineering Co., Ltd., Tokyo). BAFFLE OXYGEN PLATE SENSOR ¥ ~8 AIR-~ (!) Figure 21. Schematic diagram of the rotating drum bioreactor (Tanaka, H., et al., 1983)
o Fermentation and Biochemical Engineering Handbook AIR FLOW RATE (vm Figure 22. Effect of the airflow rate on ku a in rotating drum fermenter. (Tanaka, H, et al Spin Filter Bioreactor. This type of bioreactor(Styer, 1985)16 is equipped with a filter driven by a magnetic coupling in the stir plate( Fig. 26) tic coup This spinning filter operates as a medium agitator without generating shear stress and also serves as an excellent filter for the removal ofthe medium from filter bie will be most suitable for the continuous culture of plant cells. When a conventional bioreactor was used and the feeding rate ofthe medium was increased, the cell density was decreased because of washout. However, when a spin filter bioreactor was used, the cell density was maintained constant and half of the spent medium was effectively removed through the spin filter Gaseous Phase Bioreactor, As shown in Fig. 24, this type of bioreactor is equipped with filters on which the culture is supported and with a shower nozzle for spraying on the medium(Ushiyama et al., 1984; 7J Ushiyama, 1988). 18 Seed cultures are inoculated on the filters and the medium is supplied to the culture by spraying from a shower nozzle. The drained medium is collected on the bottom of the bioreactor. This type of bioreactor is excellent for plant cell, tissue, and organ cultures because there is no mechanical agitation(.g, driven impeller, aerator) and, therefore, the growth rate and the secondary metabolite production are enha Light Introducing Bioreactor. Plants are susceptible to light irradiation and as a consequence various metabolic and/or physiological changes are generated. Some important reactions are: (i) photosynthesis (ii) activation of specific enzymes such as phenylalanine ammonia lyase (PAL) and to induce the production of flavonoids or anthodyanins, (iii) photomorphogenesis such as development of leaves. For these reactions, the
Ro!ation speed 5:: 0 AIR FLOW RATE (wm) 5 Figure 22. Effect ofthe airflow rate on k,a in rotating drum fermenter. (Tanaka, H., et al., 1983) Spin Filter Bioreactor. This type of bioreactor (Styer, 1985)[161 is equipped with a filter driven by amagnetic coupling in the stir plate (Fig. 26). This spinning filter operates as a medium agitator without generating shear stress and also serves as an excellent filter for the removal ofthe medium from the bioreactor without the cells plugging it. The spin filter bioreactor will be most suitable for the continuous culture of plant cells. When a conventional bioreactor was used and the feeding rate ofthe medium was increased, the cell density was decreased because of washout. However, when a spin filter bioreactor was used, the cell density was maintained constant and half of the spent medium was effectively removed through the spin filter. Gaseous Phase Bioreactor. As shown in Fig. 24, this type of bioreactor is equipped with filters on which the culture is supported and with a shower nozzle for spraying on the medium (Ushiyama et al., 1984;[”] Ushiyama, 1988).[’*] Seed cultures are inoculated on the filters and the medium is supplied to the culture by spraying from a shower nozzle. The drained medium is collected on the bottom of the bioreactor. This type of bioreactor is excellent for plant cell, tissue, and organ cultures because there is no mechanical agitation (e.g., driven impeller, aerator) and, therefore, the growth rate and the secondary metabolite production are enhanced. Light Introducing Bioreactor. Plants are susceptible to light irradiation and as a consequence various metabolic and/or physiological changes are generated. Some important reactions are: (i) photosynthesis, (ii) activation of specific enzymes such as phenylalanine ammonia lyase (PAL) and to induce the production of flavonoids or anthodyanins, (iii) photomorphogenesis such as development of leaves. For these reactions, the