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Production and release of anthraquinone pigments by hairy roots of madder (Rubia tinctorum L.) under improved culture conditions
JOURNAL OF FERMENTATION AND BIOENGINEERING
Vol. 77, No. l, 103-106. 1994
Production and Release of Anthraquinone Pigments by Hairy Roots of Madder (Rubia tinctorum L.) under Improved Culture Conditions M A S A H I R O KINO-OKA, l KOJI MINE, 1 M A S A H I T O TAYA, 1 SETSUJI TONE, l* ~ D T A K A H I T O I C H I 2
Department of Chemical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, I and Food Color Laboratory, San-Ei Gen F.F.I. Inc., Toyonaka, Osaka 561, 2 Japan Received 25 March 1993/Accepted 14 September 1993 Carbon and nitrogen sources in the medium were selected for the culture of madder hairy roots producing anthraquinone pigments. The growth and pigment formation of the hairy roots were significantly enhanced by using modified Murashige-Skoog (MS) medium containing fructose and nitrate as the sole carbon and nitrogen sources, respectively, compared with those obtained in conventional MS medium with sucrose. Repeated-batch culture of the hairy roots was carried out, with pigment release into the medium obtained by means of O2 starvation treatment. In three pigment-release operations during 29-d culture, the total amount of released pigments was 21 m g / d m 3, representing an average production rate of 0.72 mg/(dm3.d).
Recently, the culture of hairy roots derived from higher plants has attracted much attention because hairy roots possess the outstanding properties of genetic stability and active growth as well as the capacity to synthesize secondary metabolites found in the roots of field-grown original plants (1). For the effective production of plant-derived materials from hairy root cultures, the enhancement of growth rates and product yields by modifying medium compositions is primarily required. In submerged bioreactor cultures of hairy roots, it is also desirable that metabolites of interest in the cells should be extracellularly produced, and this has been demonstrated by some workers (2-4). We previously reported that in a culture of red beet hairy roots, temporary 02 starvation treatment caused pigment release from the roots, which were capable of propagation even after this treatment (5, 6). In the present study, we investigated the culture of hairy roots derived from madder (Rubia tinctorum L.). It is known that dyestuffs which originate from root material of field-grown madder include anthraquinone derivatives (7). Sato et ai. (8) reported that in a culture of hairy roots derived from madder, the presence of 5 p m o l / d m 3 indole 3-acetic acid (IAA) or 12% sucrose in a liquid medium enhanced the root growth and anthraquinone formation. The aims of the present study were to improve the culture conditions by the selection of nitrogen and carbon sources suitable for the growth of madder hairy roots, and to apply 02 starvation treatment to a submerged culture of madder hairy roots for the release of anthraquinone pigments into the medium. Hairy roots of madder were used throughout the experiments. These were induced by the leaf-disk method with Agrobacterium rhizogenes ATCC 13332 and confirmed to be a transformant according to opine assay (9, 10). The hairy roots were maintained in Murashige-Skoog (MS) liquid medium (11) containing 30g/din 3 fructose and no phytohormone, and subcultured every month. In some experiments, the hairy root cultures were done in phytohormone-free MS basal medium with 2 0 g / d m 3 sugar
(fructose, glucose, sucrose etc.). In NH4+-free medium (denoted as NI medium), the main nitrogenous compounds in MS basal medium (1.90g/dm 3 KNO3 and 1.65 g / d m 3 NH4NO3) were altered to 6.07 g/din 3 KNO3 and 0 g / d m 3 NH4NO3 (other ingredients were the same as those of MS medium). The media were autoclaved at 121°C for 20 min after the pH was adjusted to 5.8 with 0.1 m o l / d m 3 KOH. For the experiments, madder hairy roots were cultivated in a 200-cm 3 Erlenmeyer flask (medium: 80 cm 3) or a turbine-blade fermentor (working volume: 1.0 dm 3) as described previously (5, 6). When pigment release was carried out in submerged culture in the fermentor, the air supply was first stopped and then pure N 2 gas was sparged to reduce the medium DO level to zero, keeping the pH at a given value by the addition of 0.1 m o l / d m 3 KOH. After pigment release for a given period, the culture broth containing released pigment was exchanged for fresh medium and the hairy roots were grown again by supplying air and adjusting the pH to 5.8 with 0.1 tool/din 3 HNOa. The dry weight (DW) of the hairy roots was measured gravimetrically by drying the harvested roots at 70°C for 48 h. In the culture with the fermentor, the root mass concentration on a working volume basis was determined by measuring the electrical conductivity of the medium (12). The empirical equation was predetermined as follows: X = --2.5(Ar)+Xo
(1)
where, X, X0 and Ar are root mass concentration at a given time [g-DW/dm3], initial root mass concentration [gD W / d m 3] and change of medium electrical conductivity [mS/cm], respectively. For the analysis of intracellular pigments, fresh hairy roots were homogenized in 70%(v/v) ethanol aqueous solution at 50°C using a mortar and pestle. The supernatant obtained by centrifugation (24,000 x g, 15 min, 25°C) was used as a pigment extract. The pigment concentration in the pigment extract (or in the medium) was determined by high performance liquid chromatography (HPLC) with an LC-6A System (Shimadzu Co., Kyoto) under the following conditions. Column: Nucleosil 5Cs (MachereryNagel Co., Germany); detection: absorbance at 430 nm;
* Corresponding author. 103
104
KINO-OKA ET AL.
J . FERMENT. BIOENG.,
TABLE 1. Cultures of madder hairy roots using various sugars as carbon sources Sugar None Fructose Gaiactose Glucose Lactose Maltose Sucrose
Root massa (g_DW/dm3)
Pigment content a (mg/g-DW)
0.4 (0.1) 2.8 (1.1) 0.6 (0.6) 1.0 (0.1) 0.9 (0.0) 1.2 (0.0) 1.5 (0.2)
19 (2) 19 (1) 32 (2) 29 (4) 20 (3) 27 (1) 25 (2)
(13), who reported that Catharanthus roseus hairy roots grew faster in a medium with sucrose than in one with fructose. For a detailed examination of sugar utilization, madder hairy roots were cultivated in the medium containing either sucrose, fructose, glucose or fructose and glucose, as shown in Fig. 1. At 26-d culture, the root mass concentration in the fructose-containing medium reached the maximum value of 9.7 g - D W / d m 3 (Fig. 1A), which was 2.1 times larger than that in the sucrose-containing medium (4.6 g - D W / d m 3 at 26-d culture, Fig. 1B). In suspension culture o f callus tissues it is frequently observed that sucrose is first hydrolyzed to fructose and glucose by extracellular invertase, followed by the uptake of glucose and fructose into the plant cells (14). A similar phenomenon o f sugar utilization was observed in the culture o f madder hairy roots with the sucrose-containing medium (Fig. 1B). In this case, glucose was preferentially consumed after the sucrose hydrolysis. In the culture using a fructose and glucose mixture, it was found that glucose was also consumed in preference to fructose (Fig. 1C). However, the growth o f hairy roots was very slow when glucose was added to the medium as a carbon source (Fig. 1C and D). Thus, the low growth rate of madder hairy roots in the sucrose-containing medium was due to the appearance and preferential consumption of glucose hydrolyzed from sucrose. As shown in Fig. 1, pigment contents in the roots were 20-30 m g / g - D W during the root growth phases o f the cultures, irrespective of the sugar utilized. In the culture using fructose, the increased pigment content (ca. 50 m g / g - D W ) was obtained during the stationary phase after 26 d (Fig. 1A). In a previous study (15), it was found that the growth of madder hairy roots was inhibited in a medium containing NH4 ÷. As shown in Fig. 2, in the present study, madder hairy roots were cultivated in NI medium (the medium using NO3- as a sole nitrogen source) containing 20 g / d m 3 fructose. The root mass concentration reached a plateau at 18-d culture and the growth rate was significantly enhanced in comparison with that obtained in MS medium containing fructose (see the broken line for root mass in
Cultures were carried out for 12 d in Erlenmeyer flasks containing MS medium with 20 g/din 3 sugar. a Each value is the mean of four determinations, with the S.D. in parentheses. eluent: a mixture of ethanol, water and 0.1 m o l / d m 3 KH2PO4 ( 7 : 2 : 1, by volume) at a flow rate of 0.7cm3/ min. Under these analytical conditions, the pigment mixture was eluted as two main peaks on a chromatogram with retention times o f 3.7 min (peak A) and 4.9 min (peak B). As madder roots and its hairy roots are known to form many anthraquinone derivatives (7, 8), peaks A and B may include plural pigments. In the present study, for convenience, peaks A and B were evaluated by using authentic ruberythric acid (alizarin 2-O-/%primerveroside, retention time: 3.7 min) and authentic alizarin (1,2-dihydroxyanthraquinone, retention time: 4.9 min), respectively. The amounts o f anthraquinone pigments were expressed as the sum o f the alizarin and ruberythric acid quantities. Preparations o f authentic alizarin and ruberythric acid were purchased from T o k y o Kasei Co., T o k y o and Carl Roth Co., Germany, respectively. Fructose, glucose and sucrose were analyzed by H P L C as described elsewhere (6). At first, madder hairy roots were cultivated using MS basal medium containing various sugars for the choice of carbon source suitable for the hairy root growth. As shown in Table 1, the root growth was the highest in the culture using fructose as a carbon source. This finding does not accord with the result presented by Jung et al.
A) Fructose
C) Fructose + Glucose
B) Sucrose
D)
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FIG. 1. Time courses of madder hairy root cultures using fructose, sucrose, fructose and glucose, and glucose as carbon sources. The hairy roots were cultivated in Erlenmeyer flasks containing MS medium with the indicated sugars. Each value is the mean of three determinations, with the S.D. indicated by the vertical bar. Symbols: @, root mass; A, pigment content in roots; [], fructose; ~7, glucose; ©, sucrose.
VOL. 77, 1994
NOTES 75
.
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FIG. 2. Time course of madder hairy root culture using nitrate as the sole nitrogen source. The hairy roots were cultivated in an Erlenmeyer flask containing NI medium with fructose as a carbon source. Each value is the mean of three determinations, with the S.D. indicated by the vertical bar. The symbols are the same as those in Fig. 1. The broken lines ( ...... , root mass; -.-, fructose; and .... , pigment content in roots) show the results obtained in MS medium with fructose (Fig. IA). Fig. 2). The pigment content in the roots increased with culture time and reached about 50 m g / g - D W in the stationary phase after 18 d. This pigment content was comparable to the value obtained from field-grown madder roots (4152 mg pigment per g-DW o f field-grown roots). Sato et al. (8) reported that in a culture o f madder hairy roots for 23 d, the presence o f 5/2mol/dm 3 I A A or 12% sucrose caused the three-fold enhancement o f the total pigment production ([contents o f lucidin, alizarin and purpurin] x [root mass on a fresh weight basis]), compared with a control culture in a phytohormone-free medium containing 3% sucrose. In the present study, the total pigment production ([pigment content] × [root mass on a dry weight basis]) in NI medium containing fructose at 21 d (Fig. 2) was about ten times larger than that in MS medium containing sucrose at 21 d (Fig. 1B), though a direct comparison between the results o f Sato et al. and those o f the present study is difficult. Based on the findings described above, fructose and nitrate were selected as the carbon and nitrogen sources for the culture o f madder hairy roots. The use o f a m o n o saccharide (fructose) and a single addition o f nitrogenous c o m p o u n d (nitrate) will be advantageous in hairy root cultures because it is easy to regulate the concentrations o f these nutrients in a fed-batch culture system (16). As reported in previous papers (5, 6), red beet hairy roots released betacyanin pigments into the medium, with the ability to resume cell growth, when the roots were treated by 02 starvation under appropriate conditions. In the present study, we attempted to obtain the release o f anthraquinone pigments from madder hairy roots on the basis o f the same culture strategy. The conditions for pigment release from madder hairy roots were determined in advance as follows. Period o f 02 starvation by maintaining the medium DO level at 0 p p m : 24h; p H value during O2 starvation: 7.5; culture period between pigment release by 02 starvation treatment: 8 to 10 d, cor-
¢0 •
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10 20 30 Culture time [d]
FIG. 3. Time course of repeated-batch culture of madder hairy roots with pigment release treatment. The hairy roots were cultivated in a turbine-blade fermentor containing NI medium with fructose as a carbon source. The culture broth was exchanged for fresh medium after each pigment release treatment at the times indicated by the arrows. Symbols: e , root mass; o, fructose. Lines: l , released pigment; ...... , DO; - - , pH. responding to the active root growth phase. For the successive production o f pigments, repeatedbatch culture of madder hairy roots was performed with a turbine-blade fermentor, accompanied by root growth and pigment release treatments. The time course o f the hairy root culture is presented in Fig. 3. The culture broth was exchanged for fresh medium to recover the released pigments after the 02 starvation treatment. The concentration of pigments released during each operation increased with increasing root mass concentration. In three pigment-release operations, the total amount o f released pigments was 21 rag/din 3, giving an average production rate o f 0.72 m g / (dm 3. d). At the end of the culture (29 d), the root mass concentration was 22 g - D W / d m 3 and the amount of pigment retained in the roots was 418 m g / d m 3. The ratio o f pigment recovery during culture for 29 d was 5%, where the ratio o f pigment recovery was estimated as [total amount o f released pigments]/{[total amount o f released pigm e n t s ] + [ a m o u n t o f pigments retained in roots]}. In the culture o f red beet hairy roots in a fermentor (6), the ratio of pigment recovery was 11-25% and the average production rate o f released pigments was 0.16-0.27 m g / ( d m 3. d). Thus, the average production rate o f released pigments was relatively high in the culture o f madder hairy roots, which is considered to be attributable to the enhancement o f the hairy root growth and pigment formation by modifying the medium compositions. In H P L C analysis, peak A was mainly found on a chromatogram for culture broth with the released pigments, while no significant difference was recognized between the chromatographic patterns of the pigments retained in the roots with and without 02 starvation, that is, peaks A and B were observed (data not shown). As there is a possibility that these peaks include some anthraquinone pigments, further examination will be necessary for a detailed analysis o f the pigments released into the culture broth and retained in the roots.
106
KINO-OKA ET AL.
I n conclusion, madder hairy roots showed enhanced growth and pigment f o r m a t i o n in a culture with modified MS m e d i u m using fructose a n d nitrate as the carbon and nitrogen sources, respectively. Moreover, in repeatedbatch culture of the hairy roots with root growth a n d pigment release treatment, a n average production rate of released pigments of 0.72 m g / ( d m 3. d) was achieved. The authors are grateful to Dr. M. Kawase (NGK Insulators Ltd., Nagoya) for invaluable discussions.
J. FERMENT.BIOENG.,
7. Sato, K., Goda, Y., Kawasakl, Y., Okuyama, E., Yoshihira, K., and Ozeki, Y.: Characteristic of anthraquinone production in plant roots and cell suspension cultures of Rubia tinctorum and R. akane. Plant Tissue Culture Lett., 9, 220-226 (1992). 8. Sato, K., Yamazaki, T., Okuyama, E., Yoshlhirn, K., and Shimomura, K.: Anthraquinone production by transformed root cultures of Rubia tinctrum: influence of phytohormones and sucrose concentration. Phytochem., 30, 1507-1509 (1991). 9. Tanaka, N., Hayakawa, M., Mano, Y., Ohkawa, H., and • Matsui, C.: Infection of turnip and radish storage roots with Agrobacterium rhizogenes. Plant Cell Reports, 4, 74-77 (1985).
10. Noda, T., Tanaka, N., Mann, Y., Nabeshima, S., Ohkawa, H., REFERENCES 1. Flores, H.E. and Curtis, W.R.: Approaches to understanding and manipulatingthe biosynthetic potential of plant roots. Ann. N.Y. Acad. Sd., 665, 188-209 (1992). 2. Rhodes, M. J. C., Hilton, M., Parr, A.J., Hamill, J.D., and Robins, R.J.: Nicotine production by ~hairy root" cultures of Nicotiana rustica: Fermentation and product recovery. Biotechnol. Lett., 8, 415-420 (1986). 3. Kato, Y., Uozami, N., Klmurn, T., Honda, H., and Kobayashi, T.: Enhancement of peroxidase production and excretion from horseradish hairy roots by light, NaC1 and peroxidase-adsorption in situ. Plant Tissue Culture Lett., 8, 158-165 0991). 4. Muranaka, T., Ohkawa, H., and Yamnda, Y.: Scopolamine release into media by Duboisia leichharadtii hairy root clones. Appl. Microbiol. Biotechnol., 37, 554-559 (1992).
5. Taya, M., Mine, K., Kino-oka, M., Tone, S., and lchi, T.: Production and release of pigments by culture of transformed hairy root of red beet. J. Ferment. Bioeng., 73, 31-36 (1992). 6. Kino-oka, M., Hongo, Y., Taya, M., and Tone, S.: Culture of red beet hairy root in bioreactor and recovery of pigment released from the cells by repeated treatment of oxygen starvation. J. Chem. Eng. Japan, 25, 490-495 (1992).
11. 12. 13.
14. 15. 16.
and Matsui, C.: Regeneration of horseradish hairy roots incited by Agrobacterium rhizogenes infection. Plant Cell Reports, 4, 283-286 (1987). Murashige, T. and Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15, 473--497 (1962). Taya, M., Hegglin, M., Prenosll, J.E., and Bourne, J.R.: On-line monitoring of cell growth in plant tissue cultures by conductometry. Enzyme Microb. Technol., 11, 170-176 (1989). Jung, K. H., Kwak, S, S., Kim, S. W., Lee, H., Choi, C. Y., and Liu, J.R.: Improvement of the catharanthine productivity in hairy root cultures of Catharanthus roseus by using monosaccharides as a carbon source. Biotechnol. Lett., 14, 695-700 (1992). Kato, A.: Studies on industrial biomass production of tobacco cells. Hakkokogaku, 60, 105-118 (1982). Kino-oka, M., Taya, M., and Tone, S.: Evaluation of inhibitory effect of ammonium ion on cultures of plant hairy roots. J. Chem. Eng. Japan, 26, 578-580 (1993). Uozumi, N., Kohketsu, K., Kondo, O., Honda, H., and Kobayashi, T.: Fed-batch culture of hairy root using fructose as a carbon source. J. Ferment. Bioeng., 72, 457-460 (1991).
Vol. 77, No. l, 103-106. 1994
Production and Release of Anthraquinone Pigments by Hairy Roots of Madder (Rubia tinctorum L.) under Improved Culture Conditions M A S A H I R O KINO-OKA, l KOJI MINE, 1 M A S A H I T O TAYA, 1 SETSUJI TONE, l* ~ D T A K A H I T O I C H I 2
Department of Chemical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, I and Food Color Laboratory, San-Ei Gen F.F.I. Inc., Toyonaka, Osaka 561, 2 Japan Received 25 March 1993/Accepted 14 September 1993 Carbon and nitrogen sources in the medium were selected for the culture of madder hairy roots producing anthraquinone pigments. The growth and pigment formation of the hairy roots were significantly enhanced by using modified Murashige-Skoog (MS) medium containing fructose and nitrate as the sole carbon and nitrogen sources, respectively, compared with those obtained in conventional MS medium with sucrose. Repeated-batch culture of the hairy roots was carried out, with pigment release into the medium obtained by means of O2 starvation treatment. In three pigment-release operations during 29-d culture, the total amount of released pigments was 21 m g / d m 3, representing an average production rate of 0.72 mg/(dm3.d).
Recently, the culture of hairy roots derived from higher plants has attracted much attention because hairy roots possess the outstanding properties of genetic stability and active growth as well as the capacity to synthesize secondary metabolites found in the roots of field-grown original plants (1). For the effective production of plant-derived materials from hairy root cultures, the enhancement of growth rates and product yields by modifying medium compositions is primarily required. In submerged bioreactor cultures of hairy roots, it is also desirable that metabolites of interest in the cells should be extracellularly produced, and this has been demonstrated by some workers (2-4). We previously reported that in a culture of red beet hairy roots, temporary 02 starvation treatment caused pigment release from the roots, which were capable of propagation even after this treatment (5, 6). In the present study, we investigated the culture of hairy roots derived from madder (Rubia tinctorum L.). It is known that dyestuffs which originate from root material of field-grown madder include anthraquinone derivatives (7). Sato et ai. (8) reported that in a culture of hairy roots derived from madder, the presence of 5 p m o l / d m 3 indole 3-acetic acid (IAA) or 12% sucrose in a liquid medium enhanced the root growth and anthraquinone formation. The aims of the present study were to improve the culture conditions by the selection of nitrogen and carbon sources suitable for the growth of madder hairy roots, and to apply 02 starvation treatment to a submerged culture of madder hairy roots for the release of anthraquinone pigments into the medium. Hairy roots of madder were used throughout the experiments. These were induced by the leaf-disk method with Agrobacterium rhizogenes ATCC 13332 and confirmed to be a transformant according to opine assay (9, 10). The hairy roots were maintained in Murashige-Skoog (MS) liquid medium (11) containing 30g/din 3 fructose and no phytohormone, and subcultured every month. In some experiments, the hairy root cultures were done in phytohormone-free MS basal medium with 2 0 g / d m 3 sugar
(fructose, glucose, sucrose etc.). In NH4+-free medium (denoted as NI medium), the main nitrogenous compounds in MS basal medium (1.90g/dm 3 KNO3 and 1.65 g / d m 3 NH4NO3) were altered to 6.07 g/din 3 KNO3 and 0 g / d m 3 NH4NO3 (other ingredients were the same as those of MS medium). The media were autoclaved at 121°C for 20 min after the pH was adjusted to 5.8 with 0.1 m o l / d m 3 KOH. For the experiments, madder hairy roots were cultivated in a 200-cm 3 Erlenmeyer flask (medium: 80 cm 3) or a turbine-blade fermentor (working volume: 1.0 dm 3) as described previously (5, 6). When pigment release was carried out in submerged culture in the fermentor, the air supply was first stopped and then pure N 2 gas was sparged to reduce the medium DO level to zero, keeping the pH at a given value by the addition of 0.1 m o l / d m 3 KOH. After pigment release for a given period, the culture broth containing released pigment was exchanged for fresh medium and the hairy roots were grown again by supplying air and adjusting the pH to 5.8 with 0.1 tool/din 3 HNOa. The dry weight (DW) of the hairy roots was measured gravimetrically by drying the harvested roots at 70°C for 48 h. In the culture with the fermentor, the root mass concentration on a working volume basis was determined by measuring the electrical conductivity of the medium (12). The empirical equation was predetermined as follows: X = --2.5(Ar)+Xo
(1)
where, X, X0 and Ar are root mass concentration at a given time [g-DW/dm3], initial root mass concentration [gD W / d m 3] and change of medium electrical conductivity [mS/cm], respectively. For the analysis of intracellular pigments, fresh hairy roots were homogenized in 70%(v/v) ethanol aqueous solution at 50°C using a mortar and pestle. The supernatant obtained by centrifugation (24,000 x g, 15 min, 25°C) was used as a pigment extract. The pigment concentration in the pigment extract (or in the medium) was determined by high performance liquid chromatography (HPLC) with an LC-6A System (Shimadzu Co., Kyoto) under the following conditions. Column: Nucleosil 5Cs (MachereryNagel Co., Germany); detection: absorbance at 430 nm;
* Corresponding author. 103
104
KINO-OKA ET AL.
J . FERMENT. BIOENG.,
TABLE 1. Cultures of madder hairy roots using various sugars as carbon sources Sugar None Fructose Gaiactose Glucose Lactose Maltose Sucrose
Root massa (g_DW/dm3)
Pigment content a (mg/g-DW)
0.4 (0.1) 2.8 (1.1) 0.6 (0.6) 1.0 (0.1) 0.9 (0.0) 1.2 (0.0) 1.5 (0.2)
19 (2) 19 (1) 32 (2) 29 (4) 20 (3) 27 (1) 25 (2)
(13), who reported that Catharanthus roseus hairy roots grew faster in a medium with sucrose than in one with fructose. For a detailed examination of sugar utilization, madder hairy roots were cultivated in the medium containing either sucrose, fructose, glucose or fructose and glucose, as shown in Fig. 1. At 26-d culture, the root mass concentration in the fructose-containing medium reached the maximum value of 9.7 g - D W / d m 3 (Fig. 1A), which was 2.1 times larger than that in the sucrose-containing medium (4.6 g - D W / d m 3 at 26-d culture, Fig. 1B). In suspension culture o f callus tissues it is frequently observed that sucrose is first hydrolyzed to fructose and glucose by extracellular invertase, followed by the uptake of glucose and fructose into the plant cells (14). A similar phenomenon o f sugar utilization was observed in the culture o f madder hairy roots with the sucrose-containing medium (Fig. 1B). In this case, glucose was preferentially consumed after the sucrose hydrolysis. In the culture using a fructose and glucose mixture, it was found that glucose was also consumed in preference to fructose (Fig. 1C). However, the growth o f hairy roots was very slow when glucose was added to the medium as a carbon source (Fig. 1C and D). Thus, the low growth rate of madder hairy roots in the sucrose-containing medium was due to the appearance and preferential consumption of glucose hydrolyzed from sucrose. As shown in Fig. 1, pigment contents in the roots were 20-30 m g / g - D W during the root growth phases o f the cultures, irrespective of the sugar utilized. In the culture using fructose, the increased pigment content (ca. 50 m g / g - D W ) was obtained during the stationary phase after 26 d (Fig. 1A). In a previous study (15), it was found that the growth of madder hairy roots was inhibited in a medium containing NH4 ÷. As shown in Fig. 2, in the present study, madder hairy roots were cultivated in NI medium (the medium using NO3- as a sole nitrogen source) containing 20 g / d m 3 fructose. The root mass concentration reached a plateau at 18-d culture and the growth rate was significantly enhanced in comparison with that obtained in MS medium containing fructose (see the broken line for root mass in
Cultures were carried out for 12 d in Erlenmeyer flasks containing MS medium with 20 g/din 3 sugar. a Each value is the mean of four determinations, with the S.D. in parentheses. eluent: a mixture of ethanol, water and 0.1 m o l / d m 3 KH2PO4 ( 7 : 2 : 1, by volume) at a flow rate of 0.7cm3/ min. Under these analytical conditions, the pigment mixture was eluted as two main peaks on a chromatogram with retention times o f 3.7 min (peak A) and 4.9 min (peak B). As madder roots and its hairy roots are known to form many anthraquinone derivatives (7, 8), peaks A and B may include plural pigments. In the present study, for convenience, peaks A and B were evaluated by using authentic ruberythric acid (alizarin 2-O-/%primerveroside, retention time: 3.7 min) and authentic alizarin (1,2-dihydroxyanthraquinone, retention time: 4.9 min), respectively. The amounts o f anthraquinone pigments were expressed as the sum o f the alizarin and ruberythric acid quantities. Preparations o f authentic alizarin and ruberythric acid were purchased from T o k y o Kasei Co., T o k y o and Carl Roth Co., Germany, respectively. Fructose, glucose and sucrose were analyzed by H P L C as described elsewhere (6). At first, madder hairy roots were cultivated using MS basal medium containing various sugars for the choice of carbon source suitable for the hairy root growth. As shown in Table 1, the root growth was the highest in the culture using fructose as a carbon source. This finding does not accord with the result presented by Jung et al.
A) Fructose
C) Fructose + Glucose
B) Sucrose
D)
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,
20
30 0 10 20 0 10 Culture time [ d ]
20
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FIG. 1. Time courses of madder hairy root cultures using fructose, sucrose, fructose and glucose, and glucose as carbon sources. The hairy roots were cultivated in Erlenmeyer flasks containing MS medium with the indicated sugars. Each value is the mean of three determinations, with the S.D. indicated by the vertical bar. Symbols: @, root mass; A, pigment content in roots; [], fructose; ~7, glucose; ©, sucrose.
VOL. 77, 1994
NOTES 75
.
.
.
.
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20 time
[d]
FIG. 2. Time course of madder hairy root culture using nitrate as the sole nitrogen source. The hairy roots were cultivated in an Erlenmeyer flask containing NI medium with fructose as a carbon source. Each value is the mean of three determinations, with the S.D. indicated by the vertical bar. The symbols are the same as those in Fig. 1. The broken lines ( ...... , root mass; -.-, fructose; and .... , pigment content in roots) show the results obtained in MS medium with fructose (Fig. IA). Fig. 2). The pigment content in the roots increased with culture time and reached about 50 m g / g - D W in the stationary phase after 18 d. This pigment content was comparable to the value obtained from field-grown madder roots (4152 mg pigment per g-DW o f field-grown roots). Sato et al. (8) reported that in a culture o f madder hairy roots for 23 d, the presence o f 5/2mol/dm 3 I A A or 12% sucrose caused the three-fold enhancement o f the total pigment production ([contents o f lucidin, alizarin and purpurin] x [root mass on a fresh weight basis]), compared with a control culture in a phytohormone-free medium containing 3% sucrose. In the present study, the total pigment production ([pigment content] × [root mass on a dry weight basis]) in NI medium containing fructose at 21 d (Fig. 2) was about ten times larger than that in MS medium containing sucrose at 21 d (Fig. 1B), though a direct comparison between the results o f Sato et al. and those o f the present study is difficult. Based on the findings described above, fructose and nitrate were selected as the carbon and nitrogen sources for the culture o f madder hairy roots. The use o f a m o n o saccharide (fructose) and a single addition o f nitrogenous c o m p o u n d (nitrate) will be advantageous in hairy root cultures because it is easy to regulate the concentrations o f these nutrients in a fed-batch culture system (16). As reported in previous papers (5, 6), red beet hairy roots released betacyanin pigments into the medium, with the ability to resume cell growth, when the roots were treated by 02 starvation under appropriate conditions. In the present study, we attempted to obtain the release o f anthraquinone pigments from madder hairy roots on the basis o f the same culture strategy. The conditions for pigment release from madder hairy roots were determined in advance as follows. Period o f 02 starvation by maintaining the medium DO level at 0 p p m : 24h; p H value during O2 starvation: 7.5; culture period between pigment release by 02 starvation treatment: 8 to 10 d, cor-
¢0 •
E~
5
o¢.
^ 0
0 ffl 0 0
0 0
~
10 20 30 Culture time [d]
FIG. 3. Time course of repeated-batch culture of madder hairy roots with pigment release treatment. The hairy roots were cultivated in a turbine-blade fermentor containing NI medium with fructose as a carbon source. The culture broth was exchanged for fresh medium after each pigment release treatment at the times indicated by the arrows. Symbols: e , root mass; o, fructose. Lines: l , released pigment; ...... , DO; - - , pH. responding to the active root growth phase. For the successive production o f pigments, repeatedbatch culture of madder hairy roots was performed with a turbine-blade fermentor, accompanied by root growth and pigment release treatments. The time course o f the hairy root culture is presented in Fig. 3. The culture broth was exchanged for fresh medium to recover the released pigments after the 02 starvation treatment. The concentration of pigments released during each operation increased with increasing root mass concentration. In three pigment-release operations, the total amount o f released pigments was 21 rag/din 3, giving an average production rate o f 0.72 m g / (dm 3. d). At the end of the culture (29 d), the root mass concentration was 22 g - D W / d m 3 and the amount of pigment retained in the roots was 418 m g / d m 3. The ratio o f pigment recovery during culture for 29 d was 5%, where the ratio o f pigment recovery was estimated as [total amount o f released pigments]/{[total amount o f released pigm e n t s ] + [ a m o u n t o f pigments retained in roots]}. In the culture o f red beet hairy roots in a fermentor (6), the ratio of pigment recovery was 11-25% and the average production rate o f released pigments was 0.16-0.27 m g / ( d m 3. d). Thus, the average production rate o f released pigments was relatively high in the culture o f madder hairy roots, which is considered to be attributable to the enhancement o f the hairy root growth and pigment formation by modifying the medium compositions. In H P L C analysis, peak A was mainly found on a chromatogram for culture broth with the released pigments, while no significant difference was recognized between the chromatographic patterns of the pigments retained in the roots with and without 02 starvation, that is, peaks A and B were observed (data not shown). As there is a possibility that these peaks include some anthraquinone pigments, further examination will be necessary for a detailed analysis o f the pigments released into the culture broth and retained in the roots.
106
KINO-OKA ET AL.
I n conclusion, madder hairy roots showed enhanced growth and pigment f o r m a t i o n in a culture with modified MS m e d i u m using fructose a n d nitrate as the carbon and nitrogen sources, respectively. Moreover, in repeatedbatch culture of the hairy roots with root growth a n d pigment release treatment, a n average production rate of released pigments of 0.72 m g / ( d m 3. d) was achieved. The authors are grateful to Dr. M. Kawase (NGK Insulators Ltd., Nagoya) for invaluable discussions.
J. FERMENT.BIOENG.,
7. Sato, K., Goda, Y., Kawasakl, Y., Okuyama, E., Yoshihira, K., and Ozeki, Y.: Characteristic of anthraquinone production in plant roots and cell suspension cultures of Rubia tinctorum and R. akane. Plant Tissue Culture Lett., 9, 220-226 (1992). 8. Sato, K., Yamazaki, T., Okuyama, E., Yoshlhirn, K., and Shimomura, K.: Anthraquinone production by transformed root cultures of Rubia tinctrum: influence of phytohormones and sucrose concentration. Phytochem., 30, 1507-1509 (1991). 9. Tanaka, N., Hayakawa, M., Mano, Y., Ohkawa, H., and • Matsui, C.: Infection of turnip and radish storage roots with Agrobacterium rhizogenes. Plant Cell Reports, 4, 74-77 (1985).
10. Noda, T., Tanaka, N., Mann, Y., Nabeshima, S., Ohkawa, H., REFERENCES 1. Flores, H.E. and Curtis, W.R.: Approaches to understanding and manipulatingthe biosynthetic potential of plant roots. Ann. N.Y. Acad. Sd., 665, 188-209 (1992). 2. Rhodes, M. J. C., Hilton, M., Parr, A.J., Hamill, J.D., and Robins, R.J.: Nicotine production by ~hairy root" cultures of Nicotiana rustica: Fermentation and product recovery. Biotechnol. Lett., 8, 415-420 (1986). 3. Kato, Y., Uozami, N., Klmurn, T., Honda, H., and Kobayashi, T.: Enhancement of peroxidase production and excretion from horseradish hairy roots by light, NaC1 and peroxidase-adsorption in situ. Plant Tissue Culture Lett., 8, 158-165 0991). 4. Muranaka, T., Ohkawa, H., and Yamnda, Y.: Scopolamine release into media by Duboisia leichharadtii hairy root clones. Appl. Microbiol. Biotechnol., 37, 554-559 (1992).
5. Taya, M., Mine, K., Kino-oka, M., Tone, S., and lchi, T.: Production and release of pigments by culture of transformed hairy root of red beet. J. Ferment. Bioeng., 73, 31-36 (1992). 6. Kino-oka, M., Hongo, Y., Taya, M., and Tone, S.: Culture of red beet hairy root in bioreactor and recovery of pigment released from the cells by repeated treatment of oxygen starvation. J. Chem. Eng. Japan, 25, 490-495 (1992).
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