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Plant–cell bioreactors with simultaneous electropermeabilization and electrophoresis
Journal of Biotechnology 100 (2003) 13 /22 www.elsevier.com/locate/jbiotec
Plant cell bioreactors with simultaneous electropermeabilization and electrophoresis /
R.Y.K. Yang *, O. Bayraktar, H.T. Pu Bioreaction Engineering Laboratory, Department of Chemical Engineering, West Virginia University, Morgantown, WV 26506-6102, USA Received 30 October 2001; received in revised form 17 June 2002; accepted 24 June 2002
Abstract Experimental investigations on using low-level electric currents and voltages to extract, transport, and collect intracellular secondary metabolites from plant cells while maintaining their viabilities were conducted focusing on the production of: (1) ionic betalains, mainly negatively-charged betanin, from Beta vulgaris cells, and (2) ionic alkaloids, particularly positively-charged ajmalicine and yohimbine, from Catharanthus roseus cells. Three versions of tubular membrane reactors in which electropermeabilization of cell membranes and electrophoresis and diffusion of ionic products take place simultaneously, with or without convective flow, to achieve desirable extraction were developed. Concentrations of secondary metabolites produced from these plant /cell reactors under steady and oscillatory electrical forcings were recorded and the viabilities of treated cells examined. Oscillatory application of electrical field appears to produce more products while retaining higher cell viability. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Electropermeabilization; Plant /cell reactor; Membrane bioreactor; Bioseparation; Secondary-metabolite production
1. Introduction Many plant secondary metabolites of commercial interest are stored in the vacuoles within cells as in the case of Catharanthus roseus (periwinkle) and Beta vulgaris (red beet). The former contains a larger number of indole alkaloids, including ajmalicine and yohimbine; some of them are high value pharmaceuticals (Kutney, 1982). The latter is a rich source of water-soluble red pigment with its
* Corresponding author E-mail address: [email protected] (R.Y.K. Yang).
principal component being betanin (Nilsson, 1970). A variety of methods for the release of secondary metabolites from plant cells have been reported in the literature (Brodelius and Pedersen, 1993). These include electropermeabilization, chemical permeabilization, elicitation, ultrasonic technique, pressure or heat shock, oxygen or phosphate limitation, and pH gradient variation. Techniques that are applicable to most species and suitable for continuous production of secondary metabolites from plant cells without destroying their viabilities and biosynthetic capacities are of
0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 2 2 6 - 2
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significant commercial value. This paper addresses one of such techniques: electropermeabilization. Electropermeabilization as a method for the release of valuable substance from living cells is similar in mechanism to electroporation frequently used in the genetic engineering of cells (Zimmermann, 1986; Bates, 1990) and to iontophoresis technique used in transdermal or transmucosal drug delivery (Gangarosa, 1983). Both electropermeabilization and electroporation involve temporary and reversible permeabilization of cell membrane as a result of externally imposed electric field to living cells. However, in addition to the fact that the directions of movement of key substance are opposite to each other (away or into cells), the magnitudes and durations of imposed electric fields are also quite different. Electroporation normally involves extremely high intensity but only for a very short period of time (ns to ms), while electropermeabilization, as observed in our laboratory, usually involve low level electric current, e.g. 1 /5 mA, applied for several hours (Pu et al., 1989). Two types of device for electropermeabilization of plant cells, which allow for simultaneous release, transport, and collection of ionic metabolic products, have been developed in our laboratory. They are called electrophoretic tubular membrane reactor (ETMR), since inorganic membrane in tubular form is used in each. One type (two different versions) was originally constructed for batch operation, while the other was designed for continuous operation. Both types, batch ETMR and continuous ETMR, will be described in the next section.
were used. Before each experiment, 14 /17 days old cells were removed from the solid medium and washed three times with distilled-deionized water. The washed cells were then suspended in a 0.14 M citrate /phosphate buffer (pH 5.3) before loading into the ETMR. B. vulgaris callus was established from seeds (Red Ball, from Burpee Co.) following the procedure of Hunter and Kilby (1990). Briefly, seeds were germinated and grown in A3 agar medium, and segments of the seedling’s stems were placed in A1 agar medium for the development of callus. Once callus had formed, it was subcultured in A2 agar medium every 3 /4 weeks. All A1, A2 and A3 media (Hunter and Kilby, 1990) use sucrose as carbon source and Gamborg B5 (Sigma Chemical Co.) as salt base. Suspension cell culture of B. vulgaris was established and maintained using the procedure of Hunter and Kilby (1990). Approximately 5 g of callus culture of B. vulgaris was transferred into 250 ml flask having 100 ml of A2 liquid medium (pH 5.5). Each flask was placed on an orbital shaker (100 /125 rpm) and incubated at 25 8C under white fluorescent light with a 12 h light: 12 h dark cycle. The cells were subcultured every 7 /10 days by transferring half of the medium with suspended cells in each flask into 50 ml of fresh A2 liquid medium. Cell samples were taken as needed from the suspension culture in the flasks by a pipette with a hand-cut tip to allow smooth transfer of cell aggregates. Growth parameters, such as fresh and dry weights of cells and packed cell volume, were measured. 2.2. Analytical procedures
2. Materials and methods 2.1. Plant cell culture C. roseus calluses (CP-3, from Jochen Berlin, Braunschweig, Germany) were grown in an incubator at 27 /30 8C with a 12 h light: 12 h dark cycle, and subcultured every 2 weeks. A Murashige and Skoog (MS) agar medium, supplemented on per liter basis with 0.22 mg of 2,4-dichlorophenoxyacetic acid, 2 mg of kinetin, and 30 g of sucrose,
Among the indole alkaloids produced by the C. roseus cells, only the concentrations of ajmalicine and yohimbine were measured. This was carried out using a HPLC system (Waters) with a reversed-phase m-Bondapak column and a mobile phase of methanol and diammonium hydrogen phosphate buffer (pH 7.3). Same procedure as described by Pu et al. (1989) was followed. The viabilities of intact and electropermeabilized C. roseus cells were tested by the procedure of Steponkus and Lanphear (1967) using 0.6% (w/
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v) of TTC (2,3,5-triphenyltetrazolium chloride, from Sigma Chemical Co.). Cells were judged to be viable when they turned red under the TTC test. The red pigments (betacyanins) and yellow pigments (betaxanthins) are responsible for the color in red beet. The major component of the betacyanins and betaxanthins, betanin and vulgaxanthin-I, respectively, absorb at 476 /478 nm which is also the wavelengths for maximum absorbance (lmax) of vulgaxanthin-I; whereas vulgaxanthin-I does not absorb at 537 nm, the lmax of betanin. Thus, the absorbance of a mixture of red-beet pigments at 537 nm is directly proportional to the amount of betanin in the mixture (Nilsson, 1970). The charge carried by betanin and its lmax are influenced by the pH of the solution. The charge of betanin becomes positive with a charge number of one at pH less than 1.5, negative with a charge number of two at pH between 2.5 and 7.5, and a charge number of three at pH between 7.5 and 9.5. The charge of betanin becomes neutral (zero charge) and exhibits an isoelectric point at pH between 1.5 and 2.5. On the other hand, in the range of 400 /700 nm, the absorption spectra of betanin (lmax /537 nm) remain constant between pH 3.5 and 7.0. Hence phosphate buffers with pH 5.5 /6.8 were the most suitable for electrophoresis of red beet pigments, as between these pH values they do not undergo any alterations in charge (von Elbe et al., 1972). Citric/phosphate buffer (pH 5.5) was used for all runs. The concentration variations of negativelycharged betanin in this buffer solution can be continuously monitored on-line by measuring the absorbance of the solution at 537 nm spectrophotometrically. In the present study, this was carried out by using a microprocessor-controlled spectrophotometer (SPECTRONIC GENESYS 5, Milton Roy Co.). The viability of B. vulgaris cells was checked by regrowth test. In this test, approximately 3 /5 g of cells before and after electropermeabilization were transferred to pre-weighed petri dishes containing A2 solid medium. The petri dishes were then weighed again to determine the weights of cells added initially. After incubation for 21 days, petri dishes were weighed to determine the final weights. The increases in the masses of the cells which were
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not subjected to any treatment and those subjected to electropermeabilization were calculated. The ratios of the latter to the former, as well as visual inspection of cell growth, were used to judge the viability of treated B. vulgaris cells. For callus culture of B. vulgaris , re-growth tests showed that they could reproduce themselves on A2 solid medium after they had been kept in citric /phosphate buffer solution at pH 5.5 for up to 12 h. The growth of cells after this treatment was comparable to that of control, though their colors were lighter. Beyond 12 h, the viability of the cells started to decrease. 2.3. Sterilization procedures All parts of the bioreactors involved in experiments and associated tubings were sterilized by soaking in 70% (v/v) ethanol solution for 12 h. After sterilization, all components were rinsed with autoclaved deionized-distilled water and stored in a refrigerator at 4 8C if not used immediately. All steps involving transfer of cells and sterilization of parts were performed in a laminar flow hood under sterile condition. Before medium preparation all glasswares, including culture vessels (baby food jars, Sigma Co.), were washed thoroughly in soap and water, rinsed in deionized-distilled water, and then placed into an oven (160 8C) for approximately 3 /4 h. Typically, the agar solution used for solid medium was sterilized by dividing 1000 ml of medium solution into four beakers. Then the beakers, with petri dish covers placed over them, were placed into an autoclave (SM 22, Yamato Co.), together with the caps (Magenta B-Cap, Sigma Co.) for the culture vessels. The autoclave was allowed to go through a cycle whereby its interior was kept at 121 8C and 15 psig (205 kPa) for 15 min. After steam sterilization, approximately 50 ml of agar medium solution was transferred into each culture vessel inside a laminar flow hood. They were then covered with caps and allowed to cool and solidify for at least 12 h. The liquid medium used for cell suspension cultures of B. vulgaris was sterilized by transferring 100 ml of liquid medium into 250 ml flasks. Then, the flasks with their mouths plugged with cotton and wrapped with aluminum foil were
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steam sterilized at 121 8C and 15 psig (205 kPa) for 15 min in the same autoclave. 2.4. Batch electrophoretic tubular membrane reactor As mentioned earlier, two types of ETMRs were developed in our laboratory. The batch ETMR, as shown in Fig. 1, consists of a single ceramic membrane tube (3 mm ID and 5 mm OD; membrane thickness: 3 mm, and membrane pore size: 0.2 mm; from Norton) housed in an 11 cm long glass tube (16 mm ID). The ceramic tube is made of high purity alumina and has a naturally hydrophilic surface. Two platinum wires (0.4 mm diameter) were used as electrodes; one wound around spirally inside the glass tube (as anode) while the other was placed inside the ceramic tube (as cathode). A milliammeter with DC power supply of 9 V was used to provide a constant electric current at a preset value usually in the order of several milliamperes (mA). Both ajmalicine and yohimbine are positively charged under the prevailing experimental conditions. Before
Fig. 1. Batch ETMR without recirculation of aerated medium. (A) Milliammeter; (B) platinum wire; (C) ceramic membrane; (D) cell suspension; (E) syringe.
each run, 14 /17 day old C. roseus cells (from callus culture), washed three times with distilled / dionized water and suspended in citrate /phosphate buffer solution (pH 5.3), were loaded into shell region of the ETMR before electric current was applied. During the period of electric-field imposition, electropermeabilization of cell membranes and electrophoresis and diffusion of released ajamalicine and yohimbine took place simultaneously in the shell region of the ETMR. Immediately after each run, the cells were removed from the ETMR and placed in MS liquid medium (pH 5.3) for viability test. A second version of batch ETMR, which allows recirculation of aerated medium is shown in Fig. 2. The bioreactor consists of a single ceramic membrane tube positioned centrally in a 15 cm long glass tube (16 mm ID) with two openings serving as shell-side ports (one closed). The ceramic tube was the same as that described above. Studies using this ETMR included: (1) application of steady current of 1 mA for 6 h; and (2) alternatively imposing a current of 5 mA and 0 mA, i.e. on and off every minute for 72 min. For each experiment, 14-day-old C. roseus cells were loaded into the ETMR by pouring the cell suspension into one of the ports. To increase the amount of cells loading into bioreactor a piece of cheese cloth was placed under the other port to allow drainage of liquid while retaining the cells. The cells immobilized in the annular region
Fig. 2. Batch ETMR with recirculation of aerated medium (platinum electrodes not plotted). (A) Bioreactor; (B) medium reservoir; (C) extraction bottle; (D) pump; (E) air; (F) air pump; (G) air filter; (H) dissolved oxygen probe; (I) sampling valve.
R.Y.K. Yang et al. / Journal of Biotechnology 100 (2003) 13 /22
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convective flow in both radial and axial directions (mostly radial) took place in the shell region, in addition to electropermeabilization, electrophoresis, and diffusion. After electropermeabilization treatment, the immobilized cells were continuously provided with MS medium and then tested for viability after 2 weeks using the TTC test described previously. All the experiments were performed in a laminar flow hood under sterile condition.
Fig. 3. Schematic illustration of a continuous ETMR. (1) Reactor outlet (product stream); (2) gas vent; (3) polycarbonate cap (top); (4) steel brace; (5) socket for bottom end of 10; (6) socket for bottom end of 12; (7) socket for bottom end of 11; (8a) feed buffer outlet; (8b) feed buffer inlet (connected inside 9 /8a); (9) polycarbonate cap (bottom); (10) ceramic tube (Pt wire inside not plotted); (11) plastic-mesh tube (wrapping filter paper and Pt wire outside and cell mass inside not plotted); (12) PVC pipe.
between the ceramic and the glass tubes were provided with MS liquid medium (pH 5.3) for 2 weeks. Platinum electrodes were then installed into the bioreactor and the cells were exposed to the desired level of electric current. The MS medium was recirculated through the lumen at a flow rate of 0.1 ml min1 and samples were collected periodically from the outlet of the lumen for HPLC analyses. As a result of recirculation,
2.5. Continuous electrophoretic tubular membrane reactor We have also developed a continuous-flow ETMR, as shown in Fig. 3, which aims to induce as uniformly as possible convective radial-flow for transportation of secondary metabolites inside the bioreactor. The major improvement over the two batch ETMRs described above is the creation of an annular space outside the immobilized cell layer by the addition of a plastic-mesh tube (27 mm ID and 29 mm OD with 5 by 4 mm mesh size). The plastic tube with large meshes is covered by a glass-fiber filter paper (type A/C, Gelman Sciences) to retain the cells. Together with the covering paper, the plastic tube also serves as a support for the winding platinum cathode. The outer PVC pipe (3.175 cm diameter) of the ETMR and the smaller plastic-mesh tube inside are held
Fig. 4. Experimental set-up for a continuous ETMR. (A) Continuous ETMR; (B) power supply; (C) spectrophotometer; (D) buffer solution (pH 5.5); (E) collection of overflow from degasser; (F) degasser; (G) product collection; (H) pump.
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Table 1 Effect of current magnitude and time duration on the viability of C. roseus cells Time (h)
0
3
6
9
12
18
0 mA (control) 1 mA 1.5 mA 2 mA 2.5 mA 3 mA
/ / / / / /
/ / / / / /
/ / / * / /
/ / / / / /
/ * / / / /
/ / / / / /
Time (min)
0
10
20
30
40
50
60
70
80
90
100
120
150
300
5 mA 5 mA (1 min on /off)
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/, Indicates majority of cell are viable (by TTC test) in both experiments; /, indicates majority of cell are not viable (by TTC test) in both experiments; *, indicates majority of cell are viable (by TTC test) in the first experiments but not viable in the second (repeated) experiments.
together by two circular polycarbonate pieces at the top and bottom of the ETMR. The annular region between both provides the needed space for generating convective flow that is radially directed toward the center of the ceramic tube. The convective radial flow greatly increases the transfer rate of released secondary metabolites from cells toward ceramic tube for collection. B. vulgaris cells were used in all experiments related to this ETMR. For each run, the B. vulgaris cells housed in the annular space between the ceramic tube and the filter paper were bathed in citric /phosphate buffer (pH 5.5). A power supply unit (FB 154, Fisher Biotech) was used to provide a constant electrical potential between platinum electrodes. The electric field thus created caused the release of betanin from the cells as well as its electrophoretic transport toward the anode in the ceramic tube, where the betanin was pumped to the spectrophotometer for absorbance measurement. Experimental set-up for the continuous radialflow ETMR is illustrated in Fig. 4. A degasser, which was open to the atmosphere, was installed between the spectrophotometer and the bioreactor to remove the gases generated by electrolysis, so that the liquid pumped to the spectrophotometer could be as bubble free as possible. The flow rates were adjusted such that there was no considerable overflow from the degasser, and the flow rate at the outlet of ETMR was kept at 4 ml min 1. The
cells were washed with autoclaved deionized-distilled water before loading into the ETMR. Once the cells were loaded, buffer was pumped through the ETMR at a flow rate of 4 ml min 1 for about 1 h to further wash out any remaining pigments before applying a constant electrical voltage to the ETMR at t/0. A number of experiments were performed, all lasting for 6 h: (1) application of different steady electrical potential (4, 5, 10 and 20 vs. 0 V as control); and (2) application of oscillatory electrical potential in the form of rectangular waves. One of the aims of this study is to observe the dynamics of release of secondary metabolites induced by an external electrical field. Factors affecting their release may be quite complicated. The principle parameters are intensity and application pattern of electrical field, treatment time, ionic strength and pH of solution, temperature, cell concentration, cell amount, growth stage and history of the cells treated. In all of the experiments, all the parameters except electrical potential (voltage) were kept constant.
3. Results and discussion 3.1. Batch ETMR Pu et al. (1989) reported the effect of current intensity on the release of ajmalicine and yohim-
R.Y.K. Yang et al. / Journal of Biotechnology 100 (2003) 13 /22
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Fig. 5. Alkaloids produced from C. roseus cells in a batch ETMR (with medium recirculation)-constant 1 mA applied. Ajmalicine (m); Yohimbine (^).
Fig. 6. Alkaloids produced from C. roseus cells in a batch ETMR (with medium recirculation)-oscillatory 5 mA (1 min on /off) applied. Ajmalicine (m); Yohimbine (^).
bine from C. roseus cells using the batch ETMR shown in Fig. 1, but no viability data were provided. Presented in Table 1 are the results of TTC test applied to C. roseus cells after they were electropermeabilized in the same ETMR using a
current intensity of 1/5 mA for different periods of time. In Table 1, the ‘/’ sign indicates that upon visual inspection, greater than 95% of the cells stain red. Conversely, the ‘/’ sign indicates that greater than 95% of the cells show no stain.
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Note that the two different signs are distributed on the opposite sides of a diagonal line across the table, indicating that an increase in either current magnitude or time duration of its application causes a decrease in cell viability. For example, either 3 mA applied for more than 3 h, or 5 mA applied for more than 50 min, caused C. roseus cells in this ETMR to lose their viability. The results suggest that to maintain cell viability it is not advisable to use either too long a time duration (approximately greater than 12 h) or too high a current intensity (roughly greater than 2 mA). An experiment was performed to determine if cells indicated by the ‘/’ sign could recover. Cells exposed to 3 mA treatment for 3 h were chosen for this experiment and were provided with solid MS medium for 2 weeks of culturing. A TTC test performed on those cells was found to be negative, indicating irreparable damage to the cells. The same ETMR was also used to investigate the effect of extracellular pH on the release of ajmalicine and yohimbine under 2 mA of current. It was found that for viable cells the amount of each alkaloid found in the lumen increased ap-
proximately 25% as the value of extracellular pH decreased from 7.3 to 5.3. Production of ajmalicine and yohimbine from C. roseus cells using the batch ETMR with recirculation of medium (see Fig. 2) was also studied. This included: (1) application of 1 mA for 6 h; and (2) application of 5 mA (on/off every minute) for 72 min. The results are presented in Figs. 5 and 6. The data indicate that both alkaloids continuously migrate into the lumen over the experimental period, and the amount of yohimbine collected is higher than that of ajmalicine. While the oscillatory input of 0 and 5 mA appears to have no significant effect on the release of ajmalicine, it does noticeably enhance the release of yohimbine.
3.2. Continuous ETMR All experimental runs on continuous ETMR were conducted with B. vulgaris cells totally submerged in citric /phosphate buffer for a period of 6 h, well below the 12 h threshold period when the viability of the cells may start to decline even
Fig. 7. Effect of constant electrical potentials (15 and 20 V) on the release of betanin from 10 g fresh weight of B. vulgaris cells (from different batches of culture). Control run for 20 V ( */); treatment with 20 V (. . .); treatment with 15 V ( / ×/ /); control run for 15 V ( ).
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without imposing any electric potential, as pointed out earlier in this paper. Furthermore, a slight but steady release of pigments from viable cells submerged in citric /phosphate buffer was observed. The amount of released pigments appeared to be approximately proportional to the cell mass. Hence, cell washes before and after the cells were loaded into the ETMR, as described earlier, were essential to ensure that the absorbance reading reflected the amount of betanin released only as a result of the imposed electric field. For all control runs (no voltage applied), no significant changes in absorbance with respect to time were observed; while for runs with non-zero electrical potentials, absorbances increased with an increase in the voltage imposed. Some representative results for continuous ETMR showing absorbance at 537
21
nm, which is directly proportional to the concentration of betanin, are presented in Figs. 7 and 8. In both figures, second cell washes took place during the 1 h period (t//1 to 0) before the electric field was applied at t/0. The absorbance for the 20-V control run remained more or less constant around 0.08, as shown in Fig. 7. However, with the application of 20 V, the absorbance became slightly higher (0.085) initially, and then increased suddenly from 0.085 to 0.135 after about 30 min. Fifteen minutes after the abrupt increase in absorbance, foaming, which indicates cell death, was observed. A sudden decrease in the absorbance was observed at t/5 h. Also shown in Fig. 7 is the result of a 15V run, together with its control, with cells from a different batch of culture. The absorbance for the
Fig. 8. Effect of oscillatory electrical potential (hourly alteration between 5 and 20 V) on the release of betanin from 10 g fresh weight of B. vulgaris cells. Oscillatory treatment (. . .); control run for this oscillatory-treatment case ( */).
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control run remained almost constant at 0.045; application of 15 V increased the absorbance from 0.046 to 0.050 initially and then to 0.055 along with foaming after t/1 h. The degree of foaming was less compared with that of the 20-V run. Re-growth tests showed that B. vulgaris cells treated in a continuous ETMR with electrical potential up to 10 V did not lose their viabilities and their abilities to reproduce. Their growth rates were comparable to that of cells treated in a continuous ETMR without being imposed to an electric field. For cells subject to such a control run for 6 h, their viabilities were lost to a certain degree even though no electric potential was applied. As an example, 5 g of cells were taken from cell suspension culture and placed on A2 solid medium for subculturing. Another 5 g of cells, also taken from the same suspension culture, but were first treated with citric /phosphate buffer solution (pH 5.5) for 6 h in a continuous electric-field free ETMR before subculturing on solid A2 medium. After 21 days, cell masses of both batches were found to increase, respectively, to 8.10 and 7.44 g, indicating approximately 80% less in weight gain for the cells subject to ETMR run. Shown in Fig. 8 is the result of imposing two cycles of oscillatory variation in voltages (between 5 and 20 V) for 6 h to cells from a different batch. The curve reveals a totally different pattern from that of steady 20-V run, with gradual increase in absorbance over time and the observation of some foaming at the end of the run. Similar result was obtained for three cycles of oscillatory voltage variation between 5 and 20 V for cells from the same batch of culture as the two-cycle run and over the same period of time. Although further work is being conducted to investigate and optimize the effects of oscillations in voltage or current on the productivity of continuous ETMR, it is pertinent at this point in time to say that oscillatory forcing in electric field can improve the performance of a plant /cell ETMR.
Acknowledgements Support, in part, for this work from the National Science Foundation and the International Program, West Virginia University, are gratefully acknowledged. We thank James Beach for establishing the B. vulgaris callus culture used in this work, Jochen Berlin for providing the original batch of C. roseus callus, and Frank Saus for assistance with HPLC. Support to Oguz Bayraktar from Turkish Ministry of National Education during the period of this work is also acknowledged.
References Bates, G.W., 1990. Transfer of tobacco mosaic virus resistance by asymmetric protoplast fusion. In: Nijkamp, H.J.J., van der Plas, L.H.W., van Aartrijk, J. (Eds.), Progress in Plant Cellular and Molecular Biology. Kluwer Academic Publisher, Dordrecht, The Netherlands, pp. 293 /298. Brodelius, P., Pedersen, H., 1993. Increasing secondary metabolite production in plant /cell culture by redirecting transport. TIBTECH 11, 30 /36. Gangarosa, L.P., 1983. Iontophoresis in Dental Practice. Quintessence Publishing Company, Chicago. Hunter, S.C., Kilby, N.J., 1990. Betanin production and release in vitro from suspension cultures of Beta vulgaris . In: Methods in Molecular Biology, vol. 6. Humana Press, Clifton, pp. 545 /552. Kutney, J.P., 1982. Studies in plant tissue culture: potential sources of clinically important antitumor agents. Pure Appl. Chem. 54, 2523 /2536. Nilsson, T., 1970. Studies into the pigments in beetroot. Lantbrukshogskolans annuler 36, 179 /219. Pu, H.T., Yang, R.Y.K., Saus, F.L., 1989. Iontophoretic release and transport of alkaloids from Catharanthus roseus cells in a ceramic hollow fibre reactor. Biotechnol. Lett. 11, 83 /86. Steponkus, P.L., Lanphear, F.O., 1967. Refinement of the triphenyl tetrazolium chloride method of determining cold injury. Plant Physiol. 42, 1423 /1426. von Elbe, J.H., Siok, H., Il-Young, M., Gabelman, W.H., 1972. Quantitative analysis of betacyanins in red table beets (Beta vulgaris ). J. Food Sci. 37, 932 /934. Zimmermann, U., 1986. Electrical breakdown, electropermeabilization and electrofusion. Rev. Physiol. Biochem. Pharmacol. 105, 176 /250.
Plant cell bioreactors with simultaneous electropermeabilization and electrophoresis /
R.Y.K. Yang *, O. Bayraktar, H.T. Pu Bioreaction Engineering Laboratory, Department of Chemical Engineering, West Virginia University, Morgantown, WV 26506-6102, USA Received 30 October 2001; received in revised form 17 June 2002; accepted 24 June 2002
Abstract Experimental investigations on using low-level electric currents and voltages to extract, transport, and collect intracellular secondary metabolites from plant cells while maintaining their viabilities were conducted focusing on the production of: (1) ionic betalains, mainly negatively-charged betanin, from Beta vulgaris cells, and (2) ionic alkaloids, particularly positively-charged ajmalicine and yohimbine, from Catharanthus roseus cells. Three versions of tubular membrane reactors in which electropermeabilization of cell membranes and electrophoresis and diffusion of ionic products take place simultaneously, with or without convective flow, to achieve desirable extraction were developed. Concentrations of secondary metabolites produced from these plant /cell reactors under steady and oscillatory electrical forcings were recorded and the viabilities of treated cells examined. Oscillatory application of electrical field appears to produce more products while retaining higher cell viability. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Electropermeabilization; Plant /cell reactor; Membrane bioreactor; Bioseparation; Secondary-metabolite production
1. Introduction Many plant secondary metabolites of commercial interest are stored in the vacuoles within cells as in the case of Catharanthus roseus (periwinkle) and Beta vulgaris (red beet). The former contains a larger number of indole alkaloids, including ajmalicine and yohimbine; some of them are high value pharmaceuticals (Kutney, 1982). The latter is a rich source of water-soluble red pigment with its
* Corresponding author E-mail address: [email protected] (R.Y.K. Yang).
principal component being betanin (Nilsson, 1970). A variety of methods for the release of secondary metabolites from plant cells have been reported in the literature (Brodelius and Pedersen, 1993). These include electropermeabilization, chemical permeabilization, elicitation, ultrasonic technique, pressure or heat shock, oxygen or phosphate limitation, and pH gradient variation. Techniques that are applicable to most species and suitable for continuous production of secondary metabolites from plant cells without destroying their viabilities and biosynthetic capacities are of
0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 2 2 6 - 2
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significant commercial value. This paper addresses one of such techniques: electropermeabilization. Electropermeabilization as a method for the release of valuable substance from living cells is similar in mechanism to electroporation frequently used in the genetic engineering of cells (Zimmermann, 1986; Bates, 1990) and to iontophoresis technique used in transdermal or transmucosal drug delivery (Gangarosa, 1983). Both electropermeabilization and electroporation involve temporary and reversible permeabilization of cell membrane as a result of externally imposed electric field to living cells. However, in addition to the fact that the directions of movement of key substance are opposite to each other (away or into cells), the magnitudes and durations of imposed electric fields are also quite different. Electroporation normally involves extremely high intensity but only for a very short period of time (ns to ms), while electropermeabilization, as observed in our laboratory, usually involve low level electric current, e.g. 1 /5 mA, applied for several hours (Pu et al., 1989). Two types of device for electropermeabilization of plant cells, which allow for simultaneous release, transport, and collection of ionic metabolic products, have been developed in our laboratory. They are called electrophoretic tubular membrane reactor (ETMR), since inorganic membrane in tubular form is used in each. One type (two different versions) was originally constructed for batch operation, while the other was designed for continuous operation. Both types, batch ETMR and continuous ETMR, will be described in the next section.
were used. Before each experiment, 14 /17 days old cells were removed from the solid medium and washed three times with distilled-deionized water. The washed cells were then suspended in a 0.14 M citrate /phosphate buffer (pH 5.3) before loading into the ETMR. B. vulgaris callus was established from seeds (Red Ball, from Burpee Co.) following the procedure of Hunter and Kilby (1990). Briefly, seeds were germinated and grown in A3 agar medium, and segments of the seedling’s stems were placed in A1 agar medium for the development of callus. Once callus had formed, it was subcultured in A2 agar medium every 3 /4 weeks. All A1, A2 and A3 media (Hunter and Kilby, 1990) use sucrose as carbon source and Gamborg B5 (Sigma Chemical Co.) as salt base. Suspension cell culture of B. vulgaris was established and maintained using the procedure of Hunter and Kilby (1990). Approximately 5 g of callus culture of B. vulgaris was transferred into 250 ml flask having 100 ml of A2 liquid medium (pH 5.5). Each flask was placed on an orbital shaker (100 /125 rpm) and incubated at 25 8C under white fluorescent light with a 12 h light: 12 h dark cycle. The cells were subcultured every 7 /10 days by transferring half of the medium with suspended cells in each flask into 50 ml of fresh A2 liquid medium. Cell samples were taken as needed from the suspension culture in the flasks by a pipette with a hand-cut tip to allow smooth transfer of cell aggregates. Growth parameters, such as fresh and dry weights of cells and packed cell volume, were measured. 2.2. Analytical procedures
2. Materials and methods 2.1. Plant cell culture C. roseus calluses (CP-3, from Jochen Berlin, Braunschweig, Germany) were grown in an incubator at 27 /30 8C with a 12 h light: 12 h dark cycle, and subcultured every 2 weeks. A Murashige and Skoog (MS) agar medium, supplemented on per liter basis with 0.22 mg of 2,4-dichlorophenoxyacetic acid, 2 mg of kinetin, and 30 g of sucrose,
Among the indole alkaloids produced by the C. roseus cells, only the concentrations of ajmalicine and yohimbine were measured. This was carried out using a HPLC system (Waters) with a reversed-phase m-Bondapak column and a mobile phase of methanol and diammonium hydrogen phosphate buffer (pH 7.3). Same procedure as described by Pu et al. (1989) was followed. The viabilities of intact and electropermeabilized C. roseus cells were tested by the procedure of Steponkus and Lanphear (1967) using 0.6% (w/
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v) of TTC (2,3,5-triphenyltetrazolium chloride, from Sigma Chemical Co.). Cells were judged to be viable when they turned red under the TTC test. The red pigments (betacyanins) and yellow pigments (betaxanthins) are responsible for the color in red beet. The major component of the betacyanins and betaxanthins, betanin and vulgaxanthin-I, respectively, absorb at 476 /478 nm which is also the wavelengths for maximum absorbance (lmax) of vulgaxanthin-I; whereas vulgaxanthin-I does not absorb at 537 nm, the lmax of betanin. Thus, the absorbance of a mixture of red-beet pigments at 537 nm is directly proportional to the amount of betanin in the mixture (Nilsson, 1970). The charge carried by betanin and its lmax are influenced by the pH of the solution. The charge of betanin becomes positive with a charge number of one at pH less than 1.5, negative with a charge number of two at pH between 2.5 and 7.5, and a charge number of three at pH between 7.5 and 9.5. The charge of betanin becomes neutral (zero charge) and exhibits an isoelectric point at pH between 1.5 and 2.5. On the other hand, in the range of 400 /700 nm, the absorption spectra of betanin (lmax /537 nm) remain constant between pH 3.5 and 7.0. Hence phosphate buffers with pH 5.5 /6.8 were the most suitable for electrophoresis of red beet pigments, as between these pH values they do not undergo any alterations in charge (von Elbe et al., 1972). Citric/phosphate buffer (pH 5.5) was used for all runs. The concentration variations of negativelycharged betanin in this buffer solution can be continuously monitored on-line by measuring the absorbance of the solution at 537 nm spectrophotometrically. In the present study, this was carried out by using a microprocessor-controlled spectrophotometer (SPECTRONIC GENESYS 5, Milton Roy Co.). The viability of B. vulgaris cells was checked by regrowth test. In this test, approximately 3 /5 g of cells before and after electropermeabilization were transferred to pre-weighed petri dishes containing A2 solid medium. The petri dishes were then weighed again to determine the weights of cells added initially. After incubation for 21 days, petri dishes were weighed to determine the final weights. The increases in the masses of the cells which were
15
not subjected to any treatment and those subjected to electropermeabilization were calculated. The ratios of the latter to the former, as well as visual inspection of cell growth, were used to judge the viability of treated B. vulgaris cells. For callus culture of B. vulgaris , re-growth tests showed that they could reproduce themselves on A2 solid medium after they had been kept in citric /phosphate buffer solution at pH 5.5 for up to 12 h. The growth of cells after this treatment was comparable to that of control, though their colors were lighter. Beyond 12 h, the viability of the cells started to decrease. 2.3. Sterilization procedures All parts of the bioreactors involved in experiments and associated tubings were sterilized by soaking in 70% (v/v) ethanol solution for 12 h. After sterilization, all components were rinsed with autoclaved deionized-distilled water and stored in a refrigerator at 4 8C if not used immediately. All steps involving transfer of cells and sterilization of parts were performed in a laminar flow hood under sterile condition. Before medium preparation all glasswares, including culture vessels (baby food jars, Sigma Co.), were washed thoroughly in soap and water, rinsed in deionized-distilled water, and then placed into an oven (160 8C) for approximately 3 /4 h. Typically, the agar solution used for solid medium was sterilized by dividing 1000 ml of medium solution into four beakers. Then the beakers, with petri dish covers placed over them, were placed into an autoclave (SM 22, Yamato Co.), together with the caps (Magenta B-Cap, Sigma Co.) for the culture vessels. The autoclave was allowed to go through a cycle whereby its interior was kept at 121 8C and 15 psig (205 kPa) for 15 min. After steam sterilization, approximately 50 ml of agar medium solution was transferred into each culture vessel inside a laminar flow hood. They were then covered with caps and allowed to cool and solidify for at least 12 h. The liquid medium used for cell suspension cultures of B. vulgaris was sterilized by transferring 100 ml of liquid medium into 250 ml flasks. Then, the flasks with their mouths plugged with cotton and wrapped with aluminum foil were
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steam sterilized at 121 8C and 15 psig (205 kPa) for 15 min in the same autoclave. 2.4. Batch electrophoretic tubular membrane reactor As mentioned earlier, two types of ETMRs were developed in our laboratory. The batch ETMR, as shown in Fig. 1, consists of a single ceramic membrane tube (3 mm ID and 5 mm OD; membrane thickness: 3 mm, and membrane pore size: 0.2 mm; from Norton) housed in an 11 cm long glass tube (16 mm ID). The ceramic tube is made of high purity alumina and has a naturally hydrophilic surface. Two platinum wires (0.4 mm diameter) were used as electrodes; one wound around spirally inside the glass tube (as anode) while the other was placed inside the ceramic tube (as cathode). A milliammeter with DC power supply of 9 V was used to provide a constant electric current at a preset value usually in the order of several milliamperes (mA). Both ajmalicine and yohimbine are positively charged under the prevailing experimental conditions. Before
Fig. 1. Batch ETMR without recirculation of aerated medium. (A) Milliammeter; (B) platinum wire; (C) ceramic membrane; (D) cell suspension; (E) syringe.
each run, 14 /17 day old C. roseus cells (from callus culture), washed three times with distilled / dionized water and suspended in citrate /phosphate buffer solution (pH 5.3), were loaded into shell region of the ETMR before electric current was applied. During the period of electric-field imposition, electropermeabilization of cell membranes and electrophoresis and diffusion of released ajamalicine and yohimbine took place simultaneously in the shell region of the ETMR. Immediately after each run, the cells were removed from the ETMR and placed in MS liquid medium (pH 5.3) for viability test. A second version of batch ETMR, which allows recirculation of aerated medium is shown in Fig. 2. The bioreactor consists of a single ceramic membrane tube positioned centrally in a 15 cm long glass tube (16 mm ID) with two openings serving as shell-side ports (one closed). The ceramic tube was the same as that described above. Studies using this ETMR included: (1) application of steady current of 1 mA for 6 h; and (2) alternatively imposing a current of 5 mA and 0 mA, i.e. on and off every minute for 72 min. For each experiment, 14-day-old C. roseus cells were loaded into the ETMR by pouring the cell suspension into one of the ports. To increase the amount of cells loading into bioreactor a piece of cheese cloth was placed under the other port to allow drainage of liquid while retaining the cells. The cells immobilized in the annular region
Fig. 2. Batch ETMR with recirculation of aerated medium (platinum electrodes not plotted). (A) Bioreactor; (B) medium reservoir; (C) extraction bottle; (D) pump; (E) air; (F) air pump; (G) air filter; (H) dissolved oxygen probe; (I) sampling valve.
R.Y.K. Yang et al. / Journal of Biotechnology 100 (2003) 13 /22
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convective flow in both radial and axial directions (mostly radial) took place in the shell region, in addition to electropermeabilization, electrophoresis, and diffusion. After electropermeabilization treatment, the immobilized cells were continuously provided with MS medium and then tested for viability after 2 weeks using the TTC test described previously. All the experiments were performed in a laminar flow hood under sterile condition.
Fig. 3. Schematic illustration of a continuous ETMR. (1) Reactor outlet (product stream); (2) gas vent; (3) polycarbonate cap (top); (4) steel brace; (5) socket for bottom end of 10; (6) socket for bottom end of 12; (7) socket for bottom end of 11; (8a) feed buffer outlet; (8b) feed buffer inlet (connected inside 9 /8a); (9) polycarbonate cap (bottom); (10) ceramic tube (Pt wire inside not plotted); (11) plastic-mesh tube (wrapping filter paper and Pt wire outside and cell mass inside not plotted); (12) PVC pipe.
between the ceramic and the glass tubes were provided with MS liquid medium (pH 5.3) for 2 weeks. Platinum electrodes were then installed into the bioreactor and the cells were exposed to the desired level of electric current. The MS medium was recirculated through the lumen at a flow rate of 0.1 ml min1 and samples were collected periodically from the outlet of the lumen for HPLC analyses. As a result of recirculation,
2.5. Continuous electrophoretic tubular membrane reactor We have also developed a continuous-flow ETMR, as shown in Fig. 3, which aims to induce as uniformly as possible convective radial-flow for transportation of secondary metabolites inside the bioreactor. The major improvement over the two batch ETMRs described above is the creation of an annular space outside the immobilized cell layer by the addition of a plastic-mesh tube (27 mm ID and 29 mm OD with 5 by 4 mm mesh size). The plastic tube with large meshes is covered by a glass-fiber filter paper (type A/C, Gelman Sciences) to retain the cells. Together with the covering paper, the plastic tube also serves as a support for the winding platinum cathode. The outer PVC pipe (3.175 cm diameter) of the ETMR and the smaller plastic-mesh tube inside are held
Fig. 4. Experimental set-up for a continuous ETMR. (A) Continuous ETMR; (B) power supply; (C) spectrophotometer; (D) buffer solution (pH 5.5); (E) collection of overflow from degasser; (F) degasser; (G) product collection; (H) pump.
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Table 1 Effect of current magnitude and time duration on the viability of C. roseus cells Time (h)
0
3
6
9
12
18
0 mA (control) 1 mA 1.5 mA 2 mA 2.5 mA 3 mA
/ / / / / /
/ / / / / /
/ / / * / /
/ / / / / /
/ * / / / /
/ / / / / /
Time (min)
0
10
20
30
40
50
60
70
80
90
100
120
150
300
5 mA 5 mA (1 min on /off)
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/, Indicates majority of cell are viable (by TTC test) in both experiments; /, indicates majority of cell are not viable (by TTC test) in both experiments; *, indicates majority of cell are viable (by TTC test) in the first experiments but not viable in the second (repeated) experiments.
together by two circular polycarbonate pieces at the top and bottom of the ETMR. The annular region between both provides the needed space for generating convective flow that is radially directed toward the center of the ceramic tube. The convective radial flow greatly increases the transfer rate of released secondary metabolites from cells toward ceramic tube for collection. B. vulgaris cells were used in all experiments related to this ETMR. For each run, the B. vulgaris cells housed in the annular space between the ceramic tube and the filter paper were bathed in citric /phosphate buffer (pH 5.5). A power supply unit (FB 154, Fisher Biotech) was used to provide a constant electrical potential between platinum electrodes. The electric field thus created caused the release of betanin from the cells as well as its electrophoretic transport toward the anode in the ceramic tube, where the betanin was pumped to the spectrophotometer for absorbance measurement. Experimental set-up for the continuous radialflow ETMR is illustrated in Fig. 4. A degasser, which was open to the atmosphere, was installed between the spectrophotometer and the bioreactor to remove the gases generated by electrolysis, so that the liquid pumped to the spectrophotometer could be as bubble free as possible. The flow rates were adjusted such that there was no considerable overflow from the degasser, and the flow rate at the outlet of ETMR was kept at 4 ml min 1. The
cells were washed with autoclaved deionized-distilled water before loading into the ETMR. Once the cells were loaded, buffer was pumped through the ETMR at a flow rate of 4 ml min 1 for about 1 h to further wash out any remaining pigments before applying a constant electrical voltage to the ETMR at t/0. A number of experiments were performed, all lasting for 6 h: (1) application of different steady electrical potential (4, 5, 10 and 20 vs. 0 V as control); and (2) application of oscillatory electrical potential in the form of rectangular waves. One of the aims of this study is to observe the dynamics of release of secondary metabolites induced by an external electrical field. Factors affecting their release may be quite complicated. The principle parameters are intensity and application pattern of electrical field, treatment time, ionic strength and pH of solution, temperature, cell concentration, cell amount, growth stage and history of the cells treated. In all of the experiments, all the parameters except electrical potential (voltage) were kept constant.
3. Results and discussion 3.1. Batch ETMR Pu et al. (1989) reported the effect of current intensity on the release of ajmalicine and yohim-
R.Y.K. Yang et al. / Journal of Biotechnology 100 (2003) 13 /22
19
Fig. 5. Alkaloids produced from C. roseus cells in a batch ETMR (with medium recirculation)-constant 1 mA applied. Ajmalicine (m); Yohimbine (^).
Fig. 6. Alkaloids produced from C. roseus cells in a batch ETMR (with medium recirculation)-oscillatory 5 mA (1 min on /off) applied. Ajmalicine (m); Yohimbine (^).
bine from C. roseus cells using the batch ETMR shown in Fig. 1, but no viability data were provided. Presented in Table 1 are the results of TTC test applied to C. roseus cells after they were electropermeabilized in the same ETMR using a
current intensity of 1/5 mA for different periods of time. In Table 1, the ‘/’ sign indicates that upon visual inspection, greater than 95% of the cells stain red. Conversely, the ‘/’ sign indicates that greater than 95% of the cells show no stain.
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Note that the two different signs are distributed on the opposite sides of a diagonal line across the table, indicating that an increase in either current magnitude or time duration of its application causes a decrease in cell viability. For example, either 3 mA applied for more than 3 h, or 5 mA applied for more than 50 min, caused C. roseus cells in this ETMR to lose their viability. The results suggest that to maintain cell viability it is not advisable to use either too long a time duration (approximately greater than 12 h) or too high a current intensity (roughly greater than 2 mA). An experiment was performed to determine if cells indicated by the ‘/’ sign could recover. Cells exposed to 3 mA treatment for 3 h were chosen for this experiment and were provided with solid MS medium for 2 weeks of culturing. A TTC test performed on those cells was found to be negative, indicating irreparable damage to the cells. The same ETMR was also used to investigate the effect of extracellular pH on the release of ajmalicine and yohimbine under 2 mA of current. It was found that for viable cells the amount of each alkaloid found in the lumen increased ap-
proximately 25% as the value of extracellular pH decreased from 7.3 to 5.3. Production of ajmalicine and yohimbine from C. roseus cells using the batch ETMR with recirculation of medium (see Fig. 2) was also studied. This included: (1) application of 1 mA for 6 h; and (2) application of 5 mA (on/off every minute) for 72 min. The results are presented in Figs. 5 and 6. The data indicate that both alkaloids continuously migrate into the lumen over the experimental period, and the amount of yohimbine collected is higher than that of ajmalicine. While the oscillatory input of 0 and 5 mA appears to have no significant effect on the release of ajmalicine, it does noticeably enhance the release of yohimbine.
3.2. Continuous ETMR All experimental runs on continuous ETMR were conducted with B. vulgaris cells totally submerged in citric /phosphate buffer for a period of 6 h, well below the 12 h threshold period when the viability of the cells may start to decline even
Fig. 7. Effect of constant electrical potentials (15 and 20 V) on the release of betanin from 10 g fresh weight of B. vulgaris cells (from different batches of culture). Control run for 20 V ( */); treatment with 20 V (. . .); treatment with 15 V ( / ×/ /); control run for 15 V ( ).
R.Y.K. Yang et al. / Journal of Biotechnology 100 (2003) 13 /22
without imposing any electric potential, as pointed out earlier in this paper. Furthermore, a slight but steady release of pigments from viable cells submerged in citric /phosphate buffer was observed. The amount of released pigments appeared to be approximately proportional to the cell mass. Hence, cell washes before and after the cells were loaded into the ETMR, as described earlier, were essential to ensure that the absorbance reading reflected the amount of betanin released only as a result of the imposed electric field. For all control runs (no voltage applied), no significant changes in absorbance with respect to time were observed; while for runs with non-zero electrical potentials, absorbances increased with an increase in the voltage imposed. Some representative results for continuous ETMR showing absorbance at 537
21
nm, which is directly proportional to the concentration of betanin, are presented in Figs. 7 and 8. In both figures, second cell washes took place during the 1 h period (t//1 to 0) before the electric field was applied at t/0. The absorbance for the 20-V control run remained more or less constant around 0.08, as shown in Fig. 7. However, with the application of 20 V, the absorbance became slightly higher (0.085) initially, and then increased suddenly from 0.085 to 0.135 after about 30 min. Fifteen minutes after the abrupt increase in absorbance, foaming, which indicates cell death, was observed. A sudden decrease in the absorbance was observed at t/5 h. Also shown in Fig. 7 is the result of a 15V run, together with its control, with cells from a different batch of culture. The absorbance for the
Fig. 8. Effect of oscillatory electrical potential (hourly alteration between 5 and 20 V) on the release of betanin from 10 g fresh weight of B. vulgaris cells. Oscillatory treatment (. . .); control run for this oscillatory-treatment case ( */).
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control run remained almost constant at 0.045; application of 15 V increased the absorbance from 0.046 to 0.050 initially and then to 0.055 along with foaming after t/1 h. The degree of foaming was less compared with that of the 20-V run. Re-growth tests showed that B. vulgaris cells treated in a continuous ETMR with electrical potential up to 10 V did not lose their viabilities and their abilities to reproduce. Their growth rates were comparable to that of cells treated in a continuous ETMR without being imposed to an electric field. For cells subject to such a control run for 6 h, their viabilities were lost to a certain degree even though no electric potential was applied. As an example, 5 g of cells were taken from cell suspension culture and placed on A2 solid medium for subculturing. Another 5 g of cells, also taken from the same suspension culture, but were first treated with citric /phosphate buffer solution (pH 5.5) for 6 h in a continuous electric-field free ETMR before subculturing on solid A2 medium. After 21 days, cell masses of both batches were found to increase, respectively, to 8.10 and 7.44 g, indicating approximately 80% less in weight gain for the cells subject to ETMR run. Shown in Fig. 8 is the result of imposing two cycles of oscillatory variation in voltages (between 5 and 20 V) for 6 h to cells from a different batch. The curve reveals a totally different pattern from that of steady 20-V run, with gradual increase in absorbance over time and the observation of some foaming at the end of the run. Similar result was obtained for three cycles of oscillatory voltage variation between 5 and 20 V for cells from the same batch of culture as the two-cycle run and over the same period of time. Although further work is being conducted to investigate and optimize the effects of oscillations in voltage or current on the productivity of continuous ETMR, it is pertinent at this point in time to say that oscillatory forcing in electric field can improve the performance of a plant /cell ETMR.
Acknowledgements Support, in part, for this work from the National Science Foundation and the International Program, West Virginia University, are gratefully acknowledged. We thank James Beach for establishing the B. vulgaris callus culture used in this work, Jochen Berlin for providing the original batch of C. roseus callus, and Frank Saus for assistance with HPLC. Support to Oguz Bayraktar from Turkish Ministry of National Education during the period of this work is also acknowledged.
References Bates, G.W., 1990. Transfer of tobacco mosaic virus resistance by asymmetric protoplast fusion. In: Nijkamp, H.J.J., van der Plas, L.H.W., van Aartrijk, J. (Eds.), Progress in Plant Cellular and Molecular Biology. Kluwer Academic Publisher, Dordrecht, The Netherlands, pp. 293 /298. Brodelius, P., Pedersen, H., 1993. Increasing secondary metabolite production in plant /cell culture by redirecting transport. TIBTECH 11, 30 /36. Gangarosa, L.P., 1983. Iontophoresis in Dental Practice. Quintessence Publishing Company, Chicago. Hunter, S.C., Kilby, N.J., 1990. Betanin production and release in vitro from suspension cultures of Beta vulgaris . In: Methods in Molecular Biology, vol. 6. Humana Press, Clifton, pp. 545 /552. Kutney, J.P., 1982. Studies in plant tissue culture: potential sources of clinically important antitumor agents. Pure Appl. Chem. 54, 2523 /2536. Nilsson, T., 1970. Studies into the pigments in beetroot. Lantbrukshogskolans annuler 36, 179 /219. Pu, H.T., Yang, R.Y.K., Saus, F.L., 1989. Iontophoretic release and transport of alkaloids from Catharanthus roseus cells in a ceramic hollow fibre reactor. Biotechnol. Lett. 11, 83 /86. Steponkus, P.L., Lanphear, F.O., 1967. Refinement of the triphenyl tetrazolium chloride method of determining cold injury. Plant Physiol. 42, 1423 /1426. von Elbe, J.H., Siok, H., Il-Young, M., Gabelman, W.H., 1972. Quantitative analysis of betacyanins in red table beets (Beta vulgaris ). J. Food Sci. 37, 932 /934. Zimmermann, U., 1986. Electrical breakdown, electropermeabilization and electrofusion. Rev. Physiol. Biochem. Pharmacol. 105, 176 /250.