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Comparison of various bioreactors on growth and artemisinin biosynthesis of Artemisia annua L. shoot cultures
Process Biochemistry 39 (2003) 45 /49 www.elsevier.com/locate/procbio
Comparison of various bioreactors on growth and artemisinin biosynthesis of Artemisia annua L. shoot cultures Chun-Zhao Liu *, Chen Guo, Yu-Chun Wang, Fan Ouyang State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, China Received 5 July 2002; received in revised form 28 August 2002; accepted 27 September 2002
Abstract Shoot cultures of Artemisia annua L. were cultivated in three different bioreactors: a modified airlift bioreactor, a multi-plate radius-flow bioreactor and an ultrasonic nutrient mist bioreactor. The shoots cultivated in the multi-plate radius-flow bioreactor and nutrient mist bioreactor showed excellent growth; however; hyperhydrated shoots were observed in the totally immersion cultivation in the modified airlift bioreactor. The dry weight increase (45 times) of shoot cultures in the mist bioreactor was higher than those (29 times and 36 times) in both the modified airlift bioreactor and the multi-plate radius-flow bioreactor. Furthermore, artemisinin production of shoot cultures in the nutrient mist bioreactor was 1.4- and 3.3-fold higher than those in both the multiplate radius-flow bioreactor and the modified airlift bioreactor, respectively. The mist bioreactor was found to be advantageous for A. annua L. shoot cultures. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Artemisia annua L.; Shoot culture; Growth; Artemisinin biosynthesis; Bioreactor; Plant-specific valuable metabolite production
1. Introduction Artemisia annua L. is known for the drug artemisinin, which has a marked activity against chloroquinineresistant and chloroquinine-sensitive strains of Plasmodium falciparum , and has also been used in treating skin and viral diseases. The commercial sources of most artemisinin are from field-grown leaves and flowering tops of A. annua L., which are subjected to seasonal and somatic variation and infestation of bacteria, fungi and insects that can affect the functional medicinal content of this plant [1,2]. Total organic synthesis is very complicated with low yields, and therefore economically unattractive [3,4]. In view of these problems, artemisinin production from in vitro plant tissue cultures has been considered as an attractive alternative. The biosynthesis of artemisinin in vitro has been detected in callus, suspension cells, shoot and hairy root cultures [5 /8]. * Corresponding author. Present address: Department of Plant Agriculture, University of Guelph, Guelph, Ont., Canada N1G 2W1. Tel.: /1-519-8244120x2727; fax: /1-519-767-0755. E-mail addresses: [email protected], [email protected] (C.-Z. Liu).
Undifferentiated callus and cell suspension cultures of A. annua L. are disappointing with respect to artemisinin production. Apparently, differentiated shoot cultures and hairy roots show promising potential for artemisinin biosynthesis [9]. Bioreactor technology is regarded as a key factor for realization of commercial production of phytochemicals from in vitro plant tissue cultures [10]. Some experimental bioreactors for shoot cultures have been developed with the aim to reduce production costs while maximizing plant growth [11 /15]. However, the use of bioreactors for large-scale cultivation has been limited because of the high costs and abnormal shoot morphogenesis associated with liquid culture [16]. Most shoot cultures are sensitive to shearing stress [17] and tend toward vitrification when in liquid culture over a prolonged period of time. Therefore, it is necessary to select a bioreactor configuration that can provide adequate biological requirements and engineering needs for shoot culture. In this work, we attempted to understand how shoot cultures of A. annua responded to different reactor microenvironments: a modified airlift bioreactor, a multi-plate radius-flow bioreactor and an ultrasonic
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 2 9 4 - 7
46
C.-Z. Liu et al. / Process Biochemistry 39 (2003) 45 /49
nutrient mist bioreactor. We were surprised to find a significant difference in the growth and artemisinin biosynthesis of A. annua L. shoot cultures that correlated with reactor type and cultivation mode.
2. Materials and methods 2.1. Plant materials and culture maintenance Shoot cultures of A. annua L. were induced and maintained on basal MS medium [18] supplemented with 0.05 mg l 1 a-naphthaleneacetic acid, 0.5 mg l 1 6-benzyladenine and 30 g l1 sucrose. The pH of the medium was adjusted to 5.8 with 1 M NaOH and agar was added at 8 g l 1 before autoclaving. The shoot cultures were subcultured at 15-day intervals and incubated at 259/1 8C under a 16-h fluorescent light of 3000 lx per day. 2.2. Bioreactor configuration and cultivation condition A schematic diagram of a modified airlift bioreactor is shown in Fig. 1A. The bioreactor was constructed of one glass column with 2.5 l working volume (100 mm i.d. /300 mm h). Three stainless steel meshes with 2 mm pore size were fixed along the height of the column. One glass concentric draught-tube with holes was fixed in the center of the bioreactor. A schematic diagram of a
multi-plate radius-flow bioreactor is shown in Fig. 1B. The bioreactor was constructed of one glass column with 2.5 l working volume (100 mm i.d. /300 mm h). Three glass plates were fixed along the height of the column. The medium supplied by peristaltic pump flowed through each plate and then returned to the medium bath. A schematic diagram of an ultrasonic nutrient mist bioreactor is shown in Fig. 1C. The bioreactor was constructed of one glass column with 2.5 l working volume (100 mm i.d. /300 mm h). Three stainless meshes with 2 mm pore size were fixed along the height of the column. One glass concentric draughttube with holes was fixed in the center of the column and its bottom was 2/3 cm over the liquid surface. A mistifier was fixed in the center under the bottom of the column. The mistifier with transducer and mist generation system was controlled by timer to produce nutrient mist periodically. Air was supplied during the periods without mist. Before starting the cultivation, the bioreactors and the whole system were sterilized by autoclaving at 121 8C for 40 min. These bioreactors were inoculated by placing shoot cultures (from flask culture) homogeneously on each mesh and plate. The cultivation conditions in various bioreactors are shown in Table 1. The loss of water due to evaporation in various bioreactors was compensated by sterile water. Triplicate bioreactors were used in all experiments.
Fig. 1. Diagrams of various bioreactors: (A) a modified airlift bioreactor; (B) a multi-plate radius-flow bioreactor; (C) an ultrasonic nutrient mist bioreactor.
C.-Z. Liu et al. / Process Biochemistry 39 (2003) 45 /49 Table 1 Cultivation conditions of A. annua L. shoot cultures in various reactors MAB MRB 1
Inoculum size (g fresh wt. l ) 3.0 Medium volume (l) 2.0 Air flow rate (vvm) 1.0 Light irradiation Liquid flow rate (ml min 1) Misting cyclea
NMB
3.0 3.0 0.4 0.4 0.4 0.4 3000 lx, 16 h per day 2.0 3/90
MAB: modified airlift bioreactor, MRB: multi-plate radius-flow bioreactor, NMB: nutrient mist bioreactor. a Minutes of misting ON/minutes of misting OFF.
2.3. Analytical methods For fresh weight determination, shoot cultures were gently pressed on filter papers to remove excess water and weighed. Then they were dried in an oven at 60 8C for 24 h and the dry weight was recorded. Artemisinin was determined by the methods described by Shen et al. [19]. Briefly, the dried powder (250 mg) of the shoot cultures was extracted with 20 ml petroleum ether (30 / 60 8C) in a supersonic bath for 30 min. The extraction mixture was filtered and the petroleum ether was evaporated. The residue was dissolved in 1 ml ethanol and centrifuged at 10 000 rpm for 10 min. After centrifugation, 100 ml ethanol sample solution was used for TLC separation using silica gel GF254 (30 /40 mm) produced by Qingdao Marine Chemical Factory, Qingdao, China, and a developing solution of petroleum ether:diethylether (5:5). The artemisinin recovered from the silica gel was treated with 0.2% NaOH at 50 8C for 30 min. After cooling down to the room temperature, the solution was determined at 292 nm by UV/visible spectrophotometer. Authentic artemisinin (Sigma, MO) was used as a standard in this experiment. Data are represented as means of three samples.
3. Results and discussion The cultivation characteristics of A. annua L. shoots in various bioreactors showed evident differences. The shoot cultures in both the nutrient mist bioreactor and the multi-plate radius-flow bioreactor showed excellent growth, but those in the modified inner-loop airlift bioreactor resulted in hyperhydration due to the poor respiration and severe physical stress of shoots submerged in liquid for a long time. The biomass results of A. annua L. shoot cultures are shown in Fig. 2A. After 25-day batch culture, the dry
47
weight increase (45 times) of shoot cultures in the nutrient mist bioreactor was higher than those (29 times and 36 times) in both the modified airlift bioreactor and the multi-plate radius-flow bioreactor. Compared with that in the multi-plate radius-flow bioreactor, where the liquid nutrient was only supplied to the bottom of the shoot cultures, the growth of shoots was improved significantly in the mist bioreactor where the liquid nutrient was made into mist form and then delivered efficiently to each part of the shoot cultures. Compared with that in the modified airlift bioreactor, the serious vitrification of shoot culture was avoided because the shoot cultures were exposed directly to gas phase in the nutrient mist bioreactor. The ratio of fresh weight to dry weight under the submerged cultivation in the modified airlift bioreactor was evidently higher than those in both the ultrasonic mist bioreactor and the multi-plate radius-flow bioreactor. The hyperhydrated shoots in the totally submerged cultivation had the highest water content. Park and Hu [20] reported that shoots of A. annua L. in a rectangular shape airlift bioreactor containing a single carbon and nitrogen source showed normal growth and root formation, but the fresh weight of A. annua shoot cultures increased eight times after 29 days under the completely submerged condition in their culture system. Fulzele et al. [21] also reported the biomass of A. annua shoots increased 4 /5-fold in 1-l capacity bioreactors under submerged conditions after 30 days. Comparing these studies with ours, we concluded that the submerged liquid cultivation in the liquid-phase modified airlift bioreactor is not suitable for A. annua L. shoot growth. In contrast, a gas-phase nutrient mist bioreactor, which we developed for growth of hairy roots [22], provided an excellent environment for shoot growth of A. annua by allowing adequate gas exchange and providing sufficient nutrients in a lower shear stress environment. In various bioreactors, the artemisinin content and productions in A. annua L. shoot cultures are shown in Fig. 2B. Artemisinin content of shoot cultures in the mist bioreactor was 2.0- and 1.1-fold higher than those in both the modified airlift bioreactor and the multiplate radius-flow bioreactor. After 25 days, artemisinin production reached 48.2 mg l 1 in the mist bioreactor. In the mist bioreactor, which provided an excellent gasphase environment and sufficient nutrient supply for both shoot growth and artemisinin biosynthesis, the artemisinin production was 3.3- and 1.4-fold higher than those in both the modified airlift bioreactor and the multi-plate radius bioreactor. Artemisinin is a sesquiterpene lactone endoperoxide found mainly in the aerial parts of A. annua plants, and contains the therapeutically active endoperoxide bridge [23]. The biosynthesis of artemisinin requires a considerable involvement of oxygen [24], and the higher content in both nutrient mist bioreactor and multi-plate radius-flow bioreactor might
48
C.-Z. Liu et al. / Process Biochemistry 39 (2003) 45 /49
Fig. 2. A. annua L. shoot growth (A) and artemisinin biosynthesis (B) in various bioreactors. Values are means of triplicate results and error bars represent standard deviations.
be partly due to the greater availability of oxygen. Kim et al. [25] reported the higher artemisinin content of A. annua L. hairy roots in mist reactors than in bubble column reactors because of oxygen availability. Our current data with A. annua shoot cultures might further support that oxygen is a key factor for artemisinin biosynthesis in A. annua tissue cultures. In addition, lesser physical stress might improve artemisinin biosynthesis of the shoot cultures in the multi-plate radius bioreactor and nutrient mist bioreactor in contrast to
the higher hydrodynamic force and physiological stress of long-term submerged liquid cultivation in the modified airlift bioreactor. In conclusion, we have demonstrated that the type of reactor environment can significantly affect growth and artemisinin biosynthesis of A. annua shoot cultures. These results not only provide a potential alternative for artemisinin production, but also permit rapid mass propagation of individual plant selection in a suitable reactor system.
C.-Z. Liu et al. / Process Biochemistry 39 (2003) 45 /49
References [1] Klayman DL. Qinghaosu (artemisinin): an antimalarial drug from China. Science 1985;228:1049 /55. [2] Luo XD, Shen CC. The chemistry, pharmacology and clinical applications of Qinghaosu (artemisinin) and its derivatives. Med Res Rev 1987;7:29 /52. [3] Avery MA, Chong WKM, Jennings-White C. Stereoselective total synthesis of (/)-artemisinin, the antimalarial constituent of Artemisia annua L. J Am Chem Soc 1992;114:974 /9. [4] Xu XX, Zhu J, Hung DZ, Zhou WS. Total synthesis of arteannuin and deoxyarteannuin. Tetrahedron 1986;42:819 /28. [5] He XC, Zeng MY, Li GF, Liang Z. Callus induction and regeneration of plantlets from Artemisia annua and changes of Qinghaosu contents. Acta Botanica Sinica 1983;25:87 /90. [6] Paniego NB, Giulietti AM. Artemisinin production by Artemisia annua L.-transformed organ cultures. Enzyme Microb Technol 1996;18:526 /30. [7] Tawfiq NK, Anderson LA, Roberts MF, Phillipson JD, Bray DH, Warhurst DC. Antiplasmodial activity of Artemisia annua plant cell cultures. Plant Cell Rep 1989;8:425 /8. [8] Weathers PJ, Cheetham RD, Follansbee E, Teoh K. Artemisinin production by transformed roots of Artemisia annua . Biotechnol Lett 1994;16:1281 /6. [9] Paniego NB, Giulietti AM. Artemisia annua L.: dedifferentiated and differentiated cultures. Plant Cell Tissue Org Cult 1994;36:163 /8. [10] Liu CZ, Wang YC, Ouyang F, Ye HC, Li GF, Guo ZC. Research advance in plant tissue culture using bioreactor technology. Eng Chem Metall 1999;20:329 /36. [11] Akita M, Takayama S. Stimulation of potato (Solanum tuberosum L.) tuberization by semicontinuous liquid medium surface level control. Plant Cell Rep 1994;13:184 /7. [12] Escalona M, Lorenzo JC, Gonzalez-Daquinta M, Gonzalez JL, Desjardins Y, Borroto CG. Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems. Plant Cell Rep 1999;18:743 /8. [13] Teisson C, Alvard D, Berthouly B, Cote F, Escalant H, Lartaud M. Simple apparatus to perform plant tissue culture by temporary immersion. Acta Hort 1996;440:521 /6.
49
[14] Weathers PJ, Giles KL. Regeneration of plants using nutrient mist culture. In Vitro Cell Dev Biol Plant 1988;24:727 /32. [15] Woo SH, Park JM. Multiple shoot culture of Dianthus caryophyllus using mist culture system. Biotechnol Tech 1993;7:697 / 702. [16] Aitken-Christie J, Kozai T, Takayama S. Automation in plant tissue culture */general introduction and overview. In: AitkenChristie J, Kozai T, Smith MAL, editors. Automation and Environmental Control in Plant Tissue Culture. Boston, MA: Kluwer Academic Publishers, 1995:1 /18. [17] Liu CZ, Moon KH, Honda H, Kobayashi T. Immobilization of rice (Oryza sativa L.) callus in polyurethane foam using a turbine blade reactor. Biochem Eng J 2000;4:169 /75. [18] Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962;15:473 / 97. [19] Shen XK, Yan KD, Luo ZY. Determination of artemisinin content by ultraviolet meter. Chin Pharm Anal 1983;3:24 /6. [20] Park JM, Hu WS. Cultuvation of Artemisia annua L. plantlets in a bioreactor containing a single carbon and a single nitrogen source. Biotechnol Bioeng 1989;34:1209 /13. [21] Fulzele DP, Heble MR, Rao PS. Production of terpenoid from Artemisia annua L. plantlet cultures in bioreactor. J Biotechnol 1995;40:139 /43. [22] Liu CZ, Wang YC, Zhao B, Guo C, Ouyang F, Ye HC, Li GF. Development of a nutrient mist bioreactor for growth of hairy roots. In Vitro Cell Dev Biol Plant 1999;35:271 /4. [23] Meshnick SR, Taylor TE, Kamchonwongpaisan S. Artemisinin and antimalarial endoperoxides: from herbal remedy to targeted chemotherapy. Microbiol Rev 1996;60:301 /15. [24] Wallaart TE, Pras N, Quax WJ. Isolation and identification of dihydroartemisinic acid hydroperoxide from Artemisia annua : a novel biosynthesis precursor of artemisinin. J Nat Prod 1999;62:1160 /2. [25] Kim Y, Wyslouzil BE, Weathers PJ. A comparative study of mist and bubble column reactors in the vitro production of artemisinin. Plant Cell Rep 2001;20:451 /5.
Comparison of various bioreactors on growth and artemisinin biosynthesis of Artemisia annua L. shoot cultures Chun-Zhao Liu *, Chen Guo, Yu-Chun Wang, Fan Ouyang State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, China Received 5 July 2002; received in revised form 28 August 2002; accepted 27 September 2002
Abstract Shoot cultures of Artemisia annua L. were cultivated in three different bioreactors: a modified airlift bioreactor, a multi-plate radius-flow bioreactor and an ultrasonic nutrient mist bioreactor. The shoots cultivated in the multi-plate radius-flow bioreactor and nutrient mist bioreactor showed excellent growth; however; hyperhydrated shoots were observed in the totally immersion cultivation in the modified airlift bioreactor. The dry weight increase (45 times) of shoot cultures in the mist bioreactor was higher than those (29 times and 36 times) in both the modified airlift bioreactor and the multi-plate radius-flow bioreactor. Furthermore, artemisinin production of shoot cultures in the nutrient mist bioreactor was 1.4- and 3.3-fold higher than those in both the multiplate radius-flow bioreactor and the modified airlift bioreactor, respectively. The mist bioreactor was found to be advantageous for A. annua L. shoot cultures. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Artemisia annua L.; Shoot culture; Growth; Artemisinin biosynthesis; Bioreactor; Plant-specific valuable metabolite production
1. Introduction Artemisia annua L. is known for the drug artemisinin, which has a marked activity against chloroquinineresistant and chloroquinine-sensitive strains of Plasmodium falciparum , and has also been used in treating skin and viral diseases. The commercial sources of most artemisinin are from field-grown leaves and flowering tops of A. annua L., which are subjected to seasonal and somatic variation and infestation of bacteria, fungi and insects that can affect the functional medicinal content of this plant [1,2]. Total organic synthesis is very complicated with low yields, and therefore economically unattractive [3,4]. In view of these problems, artemisinin production from in vitro plant tissue cultures has been considered as an attractive alternative. The biosynthesis of artemisinin in vitro has been detected in callus, suspension cells, shoot and hairy root cultures [5 /8]. * Corresponding author. Present address: Department of Plant Agriculture, University of Guelph, Guelph, Ont., Canada N1G 2W1. Tel.: /1-519-8244120x2727; fax: /1-519-767-0755. E-mail addresses: [email protected], [email protected] (C.-Z. Liu).
Undifferentiated callus and cell suspension cultures of A. annua L. are disappointing with respect to artemisinin production. Apparently, differentiated shoot cultures and hairy roots show promising potential for artemisinin biosynthesis [9]. Bioreactor technology is regarded as a key factor for realization of commercial production of phytochemicals from in vitro plant tissue cultures [10]. Some experimental bioreactors for shoot cultures have been developed with the aim to reduce production costs while maximizing plant growth [11 /15]. However, the use of bioreactors for large-scale cultivation has been limited because of the high costs and abnormal shoot morphogenesis associated with liquid culture [16]. Most shoot cultures are sensitive to shearing stress [17] and tend toward vitrification when in liquid culture over a prolonged period of time. Therefore, it is necessary to select a bioreactor configuration that can provide adequate biological requirements and engineering needs for shoot culture. In this work, we attempted to understand how shoot cultures of A. annua responded to different reactor microenvironments: a modified airlift bioreactor, a multi-plate radius-flow bioreactor and an ultrasonic
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 2 9 4 - 7
46
C.-Z. Liu et al. / Process Biochemistry 39 (2003) 45 /49
nutrient mist bioreactor. We were surprised to find a significant difference in the growth and artemisinin biosynthesis of A. annua L. shoot cultures that correlated with reactor type and cultivation mode.
2. Materials and methods 2.1. Plant materials and culture maintenance Shoot cultures of A. annua L. were induced and maintained on basal MS medium [18] supplemented with 0.05 mg l 1 a-naphthaleneacetic acid, 0.5 mg l 1 6-benzyladenine and 30 g l1 sucrose. The pH of the medium was adjusted to 5.8 with 1 M NaOH and agar was added at 8 g l 1 before autoclaving. The shoot cultures were subcultured at 15-day intervals and incubated at 259/1 8C under a 16-h fluorescent light of 3000 lx per day. 2.2. Bioreactor configuration and cultivation condition A schematic diagram of a modified airlift bioreactor is shown in Fig. 1A. The bioreactor was constructed of one glass column with 2.5 l working volume (100 mm i.d. /300 mm h). Three stainless steel meshes with 2 mm pore size were fixed along the height of the column. One glass concentric draught-tube with holes was fixed in the center of the bioreactor. A schematic diagram of a
multi-plate radius-flow bioreactor is shown in Fig. 1B. The bioreactor was constructed of one glass column with 2.5 l working volume (100 mm i.d. /300 mm h). Three glass plates were fixed along the height of the column. The medium supplied by peristaltic pump flowed through each plate and then returned to the medium bath. A schematic diagram of an ultrasonic nutrient mist bioreactor is shown in Fig. 1C. The bioreactor was constructed of one glass column with 2.5 l working volume (100 mm i.d. /300 mm h). Three stainless meshes with 2 mm pore size were fixed along the height of the column. One glass concentric draughttube with holes was fixed in the center of the column and its bottom was 2/3 cm over the liquid surface. A mistifier was fixed in the center under the bottom of the column. The mistifier with transducer and mist generation system was controlled by timer to produce nutrient mist periodically. Air was supplied during the periods without mist. Before starting the cultivation, the bioreactors and the whole system were sterilized by autoclaving at 121 8C for 40 min. These bioreactors were inoculated by placing shoot cultures (from flask culture) homogeneously on each mesh and plate. The cultivation conditions in various bioreactors are shown in Table 1. The loss of water due to evaporation in various bioreactors was compensated by sterile water. Triplicate bioreactors were used in all experiments.
Fig. 1. Diagrams of various bioreactors: (A) a modified airlift bioreactor; (B) a multi-plate radius-flow bioreactor; (C) an ultrasonic nutrient mist bioreactor.
C.-Z. Liu et al. / Process Biochemistry 39 (2003) 45 /49 Table 1 Cultivation conditions of A. annua L. shoot cultures in various reactors MAB MRB 1
Inoculum size (g fresh wt. l ) 3.0 Medium volume (l) 2.0 Air flow rate (vvm) 1.0 Light irradiation Liquid flow rate (ml min 1) Misting cyclea
NMB
3.0 3.0 0.4 0.4 0.4 0.4 3000 lx, 16 h per day 2.0 3/90
MAB: modified airlift bioreactor, MRB: multi-plate radius-flow bioreactor, NMB: nutrient mist bioreactor. a Minutes of misting ON/minutes of misting OFF.
2.3. Analytical methods For fresh weight determination, shoot cultures were gently pressed on filter papers to remove excess water and weighed. Then they were dried in an oven at 60 8C for 24 h and the dry weight was recorded. Artemisinin was determined by the methods described by Shen et al. [19]. Briefly, the dried powder (250 mg) of the shoot cultures was extracted with 20 ml petroleum ether (30 / 60 8C) in a supersonic bath for 30 min. The extraction mixture was filtered and the petroleum ether was evaporated. The residue was dissolved in 1 ml ethanol and centrifuged at 10 000 rpm for 10 min. After centrifugation, 100 ml ethanol sample solution was used for TLC separation using silica gel GF254 (30 /40 mm) produced by Qingdao Marine Chemical Factory, Qingdao, China, and a developing solution of petroleum ether:diethylether (5:5). The artemisinin recovered from the silica gel was treated with 0.2% NaOH at 50 8C for 30 min. After cooling down to the room temperature, the solution was determined at 292 nm by UV/visible spectrophotometer. Authentic artemisinin (Sigma, MO) was used as a standard in this experiment. Data are represented as means of three samples.
3. Results and discussion The cultivation characteristics of A. annua L. shoots in various bioreactors showed evident differences. The shoot cultures in both the nutrient mist bioreactor and the multi-plate radius-flow bioreactor showed excellent growth, but those in the modified inner-loop airlift bioreactor resulted in hyperhydration due to the poor respiration and severe physical stress of shoots submerged in liquid for a long time. The biomass results of A. annua L. shoot cultures are shown in Fig. 2A. After 25-day batch culture, the dry
47
weight increase (45 times) of shoot cultures in the nutrient mist bioreactor was higher than those (29 times and 36 times) in both the modified airlift bioreactor and the multi-plate radius-flow bioreactor. Compared with that in the multi-plate radius-flow bioreactor, where the liquid nutrient was only supplied to the bottom of the shoot cultures, the growth of shoots was improved significantly in the mist bioreactor where the liquid nutrient was made into mist form and then delivered efficiently to each part of the shoot cultures. Compared with that in the modified airlift bioreactor, the serious vitrification of shoot culture was avoided because the shoot cultures were exposed directly to gas phase in the nutrient mist bioreactor. The ratio of fresh weight to dry weight under the submerged cultivation in the modified airlift bioreactor was evidently higher than those in both the ultrasonic mist bioreactor and the multi-plate radius-flow bioreactor. The hyperhydrated shoots in the totally submerged cultivation had the highest water content. Park and Hu [20] reported that shoots of A. annua L. in a rectangular shape airlift bioreactor containing a single carbon and nitrogen source showed normal growth and root formation, but the fresh weight of A. annua shoot cultures increased eight times after 29 days under the completely submerged condition in their culture system. Fulzele et al. [21] also reported the biomass of A. annua shoots increased 4 /5-fold in 1-l capacity bioreactors under submerged conditions after 30 days. Comparing these studies with ours, we concluded that the submerged liquid cultivation in the liquid-phase modified airlift bioreactor is not suitable for A. annua L. shoot growth. In contrast, a gas-phase nutrient mist bioreactor, which we developed for growth of hairy roots [22], provided an excellent environment for shoot growth of A. annua by allowing adequate gas exchange and providing sufficient nutrients in a lower shear stress environment. In various bioreactors, the artemisinin content and productions in A. annua L. shoot cultures are shown in Fig. 2B. Artemisinin content of shoot cultures in the mist bioreactor was 2.0- and 1.1-fold higher than those in both the modified airlift bioreactor and the multiplate radius-flow bioreactor. After 25 days, artemisinin production reached 48.2 mg l 1 in the mist bioreactor. In the mist bioreactor, which provided an excellent gasphase environment and sufficient nutrient supply for both shoot growth and artemisinin biosynthesis, the artemisinin production was 3.3- and 1.4-fold higher than those in both the modified airlift bioreactor and the multi-plate radius bioreactor. Artemisinin is a sesquiterpene lactone endoperoxide found mainly in the aerial parts of A. annua plants, and contains the therapeutically active endoperoxide bridge [23]. The biosynthesis of artemisinin requires a considerable involvement of oxygen [24], and the higher content in both nutrient mist bioreactor and multi-plate radius-flow bioreactor might
48
C.-Z. Liu et al. / Process Biochemistry 39 (2003) 45 /49
Fig. 2. A. annua L. shoot growth (A) and artemisinin biosynthesis (B) in various bioreactors. Values are means of triplicate results and error bars represent standard deviations.
be partly due to the greater availability of oxygen. Kim et al. [25] reported the higher artemisinin content of A. annua L. hairy roots in mist reactors than in bubble column reactors because of oxygen availability. Our current data with A. annua shoot cultures might further support that oxygen is a key factor for artemisinin biosynthesis in A. annua tissue cultures. In addition, lesser physical stress might improve artemisinin biosynthesis of the shoot cultures in the multi-plate radius bioreactor and nutrient mist bioreactor in contrast to
the higher hydrodynamic force and physiological stress of long-term submerged liquid cultivation in the modified airlift bioreactor. In conclusion, we have demonstrated that the type of reactor environment can significantly affect growth and artemisinin biosynthesis of A. annua shoot cultures. These results not only provide a potential alternative for artemisinin production, but also permit rapid mass propagation of individual plant selection in a suitable reactor system.
C.-Z. Liu et al. / Process Biochemistry 39 (2003) 45 /49
References [1] Klayman DL. Qinghaosu (artemisinin): an antimalarial drug from China. Science 1985;228:1049 /55. [2] Luo XD, Shen CC. The chemistry, pharmacology and clinical applications of Qinghaosu (artemisinin) and its derivatives. Med Res Rev 1987;7:29 /52. [3] Avery MA, Chong WKM, Jennings-White C. Stereoselective total synthesis of (/)-artemisinin, the antimalarial constituent of Artemisia annua L. J Am Chem Soc 1992;114:974 /9. [4] Xu XX, Zhu J, Hung DZ, Zhou WS. Total synthesis of arteannuin and deoxyarteannuin. Tetrahedron 1986;42:819 /28. [5] He XC, Zeng MY, Li GF, Liang Z. Callus induction and regeneration of plantlets from Artemisia annua and changes of Qinghaosu contents. Acta Botanica Sinica 1983;25:87 /90. [6] Paniego NB, Giulietti AM. Artemisinin production by Artemisia annua L.-transformed organ cultures. Enzyme Microb Technol 1996;18:526 /30. [7] Tawfiq NK, Anderson LA, Roberts MF, Phillipson JD, Bray DH, Warhurst DC. Antiplasmodial activity of Artemisia annua plant cell cultures. Plant Cell Rep 1989;8:425 /8. [8] Weathers PJ, Cheetham RD, Follansbee E, Teoh K. Artemisinin production by transformed roots of Artemisia annua . Biotechnol Lett 1994;16:1281 /6. [9] Paniego NB, Giulietti AM. Artemisia annua L.: dedifferentiated and differentiated cultures. Plant Cell Tissue Org Cult 1994;36:163 /8. [10] Liu CZ, Wang YC, Ouyang F, Ye HC, Li GF, Guo ZC. Research advance in plant tissue culture using bioreactor technology. Eng Chem Metall 1999;20:329 /36. [11] Akita M, Takayama S. Stimulation of potato (Solanum tuberosum L.) tuberization by semicontinuous liquid medium surface level control. Plant Cell Rep 1994;13:184 /7. [12] Escalona M, Lorenzo JC, Gonzalez-Daquinta M, Gonzalez JL, Desjardins Y, Borroto CG. Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems. Plant Cell Rep 1999;18:743 /8. [13] Teisson C, Alvard D, Berthouly B, Cote F, Escalant H, Lartaud M. Simple apparatus to perform plant tissue culture by temporary immersion. Acta Hort 1996;440:521 /6.
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