Biotechnol. frog. 1990, 6, 447-45 1
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Effect of Permeate Flux Rate on Alkaloid Production in a Novel Plant Cell Membrane Reactor Using Coffea arabica Cells Jeffrey A. Lang, Kwang-Hun Yoon, and Jiri E. Prenosil* Department of Chemical Engineering (TCL), Swiss Federal Institute of Technology (ETH), CH-8092 Zurich, Switzerland ~~~~~~~~~
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A novel membrane reactor was developed for phytochemical production using plant cells. The membrane reactor was a flat plate construction. An autoclavable, polypropylene membrane sheet with pore size 0.075 pm was used. The membrane was surfactant treated by the manufacturer. Medium was recirculated through medium channels under the membrane. Cells were supplied with nutrients by permeated medium and obtained oxygen from the surrounding gas phase. In this work, the effect of permeate flux rate on cell growth, glucose uptake, and alkaloid production was examined. An arbitrary pressure index scale was developed to regulate the amount of medium that permeated the membrane. It was based on differential pressures between the cell chamber and the medium recirculation loop. These pressures were measured at the medium inflow and outflow points to the membrane reactor. Three 21-day batch runs were carried out with the membrane reactor at pressure indices 38,53, and 68 and compared to parallel shake-flask runs. The pressure index 53 run was determined to be optimum for cell growth and purine alkaloid formation. Cell mass increase in the membrane reactor was 3.2-fold and in parallel suspension cultures was 3.3-fold, approximately 1.5 times that of the other two reactor runs. Glucose decreased linearly in the pressure index 53 membrane reactor run to 7.7 g/L, while glucose was completely consumed in the suspension cultures after 12 days. Final alkaloid concentration was 46.1 mg/L for the pressure index 53 membrane reactor run, approximately 2 times that of the other two membrane reactor runs and the suspension cultures.
1. Introduction Secondary product formation by plant cells has attracted a great deal of attention. This is due to the great diversity of the products as well as their value. Products produced by plant cells include medicines (codeine, diosgenin), stimulants (caffeine), insecticides (pyrethrin), perfumes (jasmine), pigments (shikonin), and flavors (quinine) (1). There are, however, very few industrial processes using plant cells (2, 3). The problems are both physical and economic. Plant cells grow slowly (doubling times greater than 20 h) and are therefore very susceptible to contamination. In addition, they are shear sensitive, making conventional stirred tanks of limited use. Work has therefore been directed toward alternate reactor types such as air lift fermentors ( 4 ) . High sparging rates in air lift reactors can, however, lead to essential gas purging and poor growth or productivity ( 5 ) . From an economic viewpoint, plant cells produce products at low levels in most cases, which makes only extremely high value products economically interesting for cell culture. Much work in the past several years has been directed toward developing immobilized cell systems (6-10). Cell densities approach in vivo levels for immobilized cells, and immobilized cells offer the possibility of maintenance of a stable, slowly dividing cell population. Product can be separated from the biomass in the reactor, reducing downstream processing and cells are protected against shear stress. Immobilized cell systems are, however, limited to cultures producing extracellular products. Immobilization techniques have included hollow fiber reactors, gel entrapment, and entrapment between stainless steel mesh (11). Hollow fiber reactors, however, suffer from nonuniform inoculation and clogging caused by cell growth.
The harsh chemical environments necessary in some systems for gel formation (acrylamide,glutaraldehyde) can result in reduced cell viability. Alginate gels, although formed under mild conditions, need a high concentration of multivalent cations and a low phosphate concentration for stability, limiting long-term use. Finally, mesh entrapment systems can be limited by the supply and removal of slightly soluble gases. In order to overcome some of the drawbacks associated with current immobilized cell systems, a novel membrane reactor for production of secondary metabolites using plant cells (12) has been developed. The cells are immobilized on a polypropylene membrane sheet and grow in a calluslike layer. Inoculation is uniform across the entire membrane surface, with the membrane giving complete cell retention. The cells rely on the surrounding gas phase for oxygen requirements and removal of waste gases. In submerged culture, cells are dependent on dissolved gases. Nutrients are supplied by medium that permeates the membrane. Medium content or wetness of the cell layer is determined by the amount of medium that permeates the membrane. The cell layer is not restricted in a confined space, so problems with cell overgrowth are eliminated. Growth in the membrane reactor also allows for simple medium changes, eliminating the manual transfer in culture on semisolid medium.
2. Materials and Methods The cell line used was Coffea arabica, obtained from Dr. T. Baumann, Institute of Plant Biology, University of Zurich, and has been maintained in our laboratory €or over 3 years. The cell line was maintained on, and all experiments were carried out on, Murashige and Skoog medium (13)supplemented with 20 g/L glucose monohydrate, 0.9
8756-7938/90/3006-0447$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1990, Vol. 6, No. 6
Harvesting Knife
Viewing window
Cover plate w/ harvesting knife Cell layer spacer plate Bottom plate
Harvested cells to collecting flask Figure 1. Membrane reactor consisting of three sections: (a) the bottom plate containing the medium channels, (b) the cell layer spacer plate onto which the membrane is glued, and (c) the cover plate with harvesting knife.
mg/L thiamine hydrochloride, 10 mg/L cysteine hydrochloride, 0.2 mg/L kinetin, and 1.0 mg/L 2,4dichlorophenoxyacetic acid (2,4-D). After adjustment to pH 5.7-5.8, the medium was autoclaved a t 121 "C for 20 min. The membrane reactor is shown in Figure 1. It consists of three sections: (a) the bottom plate containing the medium channels, (b) the cell layer spacer plate onto which the membrane is glued, and (c) the cover plate with viewing window and harvesting knife. In this study, the reactor was run batchwise, so no cells were harvested. The membrane was glued onto the bottom plate (Figure 2) with clear silicone glue (FD-plast, Karochemie AG, Zurich, Switzerland) and allowed to dry overnight. The membrane used was a Celgard K-381 polypropylene sheet (Hoechst-Celanese, Plast-Labor SA, Bulle, Switzerland) with the following characteristics: porosity 45 % , pore dimensions 0.05 X 0.125 pm, effective pore size 0.075 pm, and thickness 25 f 2.5 pm. The membrane was treated by the manufacturer with an FDA-approved surfactant to make it hydrophilic. No loss of hydrophilicity of the membrane (dry areas) was observed after any batch run. A new membrane was nevertheless used for each batch run. Figure 3 shows the batch system used in all membrane reactor experiments. Cells were filtered through 500pm mesh. The reactor was inoculated with 50 g fresh weight (gFW) of cells, and 400 mL of medium was recirculated per batch. Inoculation was done by pumping (with compressed Nz)a dense cell suspension (about 35% cells) onto the membrane. The cells were allowed to settle for several minutes, and the excess medium was then drained off. The inoculated cell layer was approximately 3 f 0.5 mm deep across the entire membrane surface (130 cm2). After inoculation, medium recirculation was started. Medium conductivity was monitored on-line by using a Phillips PW 9527 conductivity meter and PW 9513 flow conductivity probe (Phillips AG, Zurich, Switzerland). The membrane reactor was run in 3-week batches. Parallel shake flasks (3) with 200 mL of medium and 25 gFW of inoculated cells were run for each membrane reactor batch run. Medium glucose was analyzed by using a Beckman Glucose Analyzer I1 (Beckman Instruments). Purine alkaloids (caffeine and threobromine) were analyzed by using a Waters 710B autosampler, 510 pump, and M490 detector (Brechbuler AG, Zurich, Switzerland) and a Machery Nagel Nucleosil 120-5 C18 column (Machery Nagel, Duren, Germany). Medium samples were filtered (0.45 pm) and mixed 2:l with 32 mg/L theophylline as
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internal standard. The conditions were flow rate 0.7 mL/ min, solvent 45 5% methanol and 55 3 ' water, and detection a t 272 nm.
3. Experimental Section and Results The wetness of the cell layer was determined by the amount of medium that permeated the membrane. The cell layer was inaccessible to sampling, making it difficult to determine how much medium was retained per gram of cell mass. The pressure index (PI) was developed to circumvent this problem by correlating t h e crossmembrane pressures measured with the amount of medium that permeates the membrane. These pressures were measured at the in- and outflow points to the bottom plate of the reactor. The pressures were expressed in millimeters of medium head above (positive pressure) or below (negative pressure) the membrane level. By keeping PI constant, we wanted to maintain the cell layer wetness constant throughout a batch run. The pressure index is an arbitrary relative scale. It was calculated as PI = Pi, + Pout + 100 (1) Pinand Poutare the differential pressures a t the respective in- and outflow points to the membrane reactor. Pin was normally in the range of 5-30 mm of medium head. Pout was normally in the range of -50 to -80 mm of medium head (the membrane level was taken as 0). The 100 was therefore added to make PI positive. The operating pressures were dictated by the membrane characteristics, with ranges chosen to prevent flooding or drying of the cell layer. During a batch run, the medium recirculation rate was kept constant a t 42 mL/min. Moist air flow was also kept constant a t 40 mL/min throughout
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@ Temperature @ Sampleport @ Peristaltic pump Figure 3. Membrane reactor batch system. The manometers measure the pressure difference between the cell chamber and the pressure in the recirculation loop at the in- and outflow points to the reactor. Humidified air is supplied at 40 mL/min.
a batch run. The PI was therefore regulated by adjusting the height of the recirculation flask. As an example, we might want to maintain the P I constant at 65 throughout a batch run. After starting medium recirculation, Pi, is read to be 20 mm of medium head. Poutis therefore set to -55 mm of medium head (PI = 20 + (-55) + 100 = 65). Any changes can then be compensated for by raising or lowering the recirculation flask. This manual compensation method is simple and effective on a laboratory scale. In an industrial process, it would be simple to regulate PI by pressure sensors and valves in a loop control system. First, the correlation between PI and permeate flux needed to be determined for the membrane. The membrane reactor was inoculated as for batch runs (dense suspension pumping followed by removal of excess medium). Medium was recirculated at pressure indices ranging from 13 to 74 for 2 h each. The amount of medium that permeated the cell layer in 15-min time steps was measured by placing the recirculation flask on a scale and reading off the weight change. Figure 4 is a graph of permeate flux rate vs time for pressure indices ranging from 13 to 74. Permeate flux rate was calculated as the amount of medium that permeated the membrane in the previous 15-mintime interval per unit time per unit membrane area [i.e., (mL/h)/cm2]. First of all, the PI 13 curve had a negative permeate flux. This means medium was actually being removed from the cell layer. This would cause cell layer drying and eventual nutrient starvation. Second of all, as the pressure index increased, the initial permeate flux rate increased. This was evident from the increasing slopes of the curves as PI increased. Finally, all curves tended toward a flux rate of zero after about 2 h. This means that there was only an actual flux as long as the cell layer could absorb the permeated medium. A quasisteady state was reached where the amount of medium per unit cell mass stayed constant. The important fact is that although all curves tended toward zero, more medium permeated the membrane as the pressure index increased. This increasing wetness as a function of pressure index is illustrated in Figure 5. Nonuniform growth in plant cells makes "representative" samplesvery difficult to obtain. Growth has therefore been indirectly monitored by medium conductivity measurement. Taya et al. (14)determined the relationship between conductivity and cell concentration to be AX = k(AK) where
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PRESSURE INDEX Figure 5. Amount of medium that permeates the membrane in the first 2 h after starting medium recirculation for pressure indices ranging from 13 to 74. Pressure indices below 24 result in negative flux rates (medium is removed from the cell layer). AX is the change in cell concentration (grams of dry cell weight per liter), AK is the change in conductivity (millisiemans per centimeter), and k is a constant determined by Taya and co-workersto be 3.6 g cm/L.mS. The shake flasks showed a considerable decrease in medium conductivity (AK = 3.5 mS/cm) over the 21 days of cultivation. On the other hand, all three membrane reactor runsshowed very little decrease (AK = 0.4-1.0 mS/cm) in medium conductivity. The cell mass increase in the shake flasks after 21 days was 3.3-fold by dry cell weight. The best cell
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Figure 7. Alkaloid concentration vs time for the membrane reactor runs and parallel shake flasks.
mass increase in the membrane reactor was in the P I 53 run (3.2-fold). The PI 38 and 68 runs had increases of 2.4and 2.0-fold, respectively. Evaporation in the shake flasks, which amounted t o 2-3 5% , cannot explain the large differences in conductivity change. A few possible explanations for the differences in conductivity decrease between the shake flasks and the membrane reactor runs can, however, be put forward. There could have been lysis occurring in the membrane reactor replenishing the ions taken up by the growing cells. There was, however, no evidence of lysis, and the average viability of the cells in the membrane reactor after a batch run was greater than 95% (unpublished results). The second possibility is that the immobilized cells utilized less ions than the freely suspended cells. Finally, the suspended cells may have been storing ions in excess of necessary amounts. It must be noted that measuring medium conductivity is a gross measurement technique, not directed at specific ions. A more specific analysis of medium composition throughout the experiment would be needed to quantify changes in specific component concentrations. Figure 6 shows the change in glucose concentration vs time. The shake flasks completely consumed glucose after 12 days of cultivation. In the membrane reactor runs, on the other hand, glucose concentration dropped off to between 7.7 and 9.9 g/L after 21 days. The P I 53 run had the lowest final glucose concentration (7.7 g/L), followed by the PI 38 run (9.2 g/L) and the PI 68 run (9.9 g/L). The lower glucose consumption rate in the membrane reactor can be attributed to the linear growth of the immobilized cell layer. Suspension cells, on the other hand, grow exponentially. Finally, the production of extracellular alkaloids was monitored (Figure 7). Here, the greatest difference between membrane reactor runs was seen. The PI 53 run had a considerably higher level of production (46.07 mg/ L) than either of the other two membrane reactor runs (19.43 and 21.94 mg/L for t h e 38 and 68 P I runs, respectively) or the shake flasks (25.42 mg/L). Alkaloids in C. arabica are dispersed in a ratio equal to the ratio of volume of tissue to volume of nutrient medium, indicating a free exchange in the system (15). Maximum alkaloid production in C. arabica cells normally occurs after the exponential phase of growth (15). This is due to the inverse relationship between the activities of the enzymes catalyzing the methylation of theobromine to caffeine and the availability of secondary metabolic precursors. If this same inverse relationship exists in the immobilized cells, a higher production rate can be expected to occur after glucose is consumed. It is interesting to note that, in the case of the shake flasks, only a slightly higher production
rate was seen after glucose consumption. This is thought to be the result of a low-producing cell line. 4. Conclusions
The membrane reactor developed a t the E T H has already shown higher production levels of purine alkaloids than in parallel submerged culture experiments [7.2 pg/ gDW-day in suspension culture vs 63.89 pg/gDW.day in the membrane reactor (12)].What is necessary now is to improve on the process to raise production. The pressure index appears to be a good method of regulating the cell layer wetness throughout an experiment. There is a good correlation between pressure index and permeated medium. What must be considered in the future are possible changes in the membrane cbacteristics over time. An example would be a possible loss of the surfactant coating during long-term operation. This would cause a permeate flux decrease, which could be compensated for by an increase in the pressure index. The permeate flux rates measured are specific for the particular membrane used. A membrane, even with the same pore size but made out of a different substance, would exhibit different fluxes a t the same pressure index. A rigid membrane would also have different characteristics. The correlation between pressure index and permeate flux rate must therefore be determined experimentally for a given membrane. The differences in conductivity change between the three membrane reactor batch runs were only small; however, the cell growth varied from 3.2-fold (pressure index 53) to 2.0-fold (pressure index 68). The conductivity change is therefore not a very sensitive function and is perhaps not suitable for use in monitoring culture growth in the membrane reactor. Alkaloid production showed the biggest difference between membrane reactor runs. The P I 53 run had approximately a 2-fold increase over the other two membrane reactor runs. It was also approximately 2 times as good as the shake flasks even though glucose was not consumed in the membrane reactor. We can conclude that the P I 53 run is better for growth and alkaloid production than the other pressure indices. This was probably due to a favorable balance established between the medium content of the cell layer and cellular uptake of nutrients and oxygen. It must be pointed out, however, that more quantification of nutrient and oxygen gradients in the cell layer must be done to determine the limiting substrates. The membrane reactor described here could be used as the second stage in a two-step production process. Cells
Biotechnol. Prog., 1990, Vol. 6, No. 6
could be grown in another vessel and then transferred to the membrane reactor, where they would be maintained on production medium. The membrane reactor could also be used for screening cell lines for secondary metabolite production in the immobilized state. Here it would be easy to test different production media for effects on secondary pathway expression.
Acknowledgment This research was funded by Swiss National Funds Project 2000-5572. The cell line was provided by Dr. Tom Baumann, Institute of Plant Biology, University of Zurich. The membranes were supplied by Plast Labor, AG, Bulle, Switzerland.
Literature Cited (1)Fowler, M. W. Production of commercially useful compounds by plant cell culture. In Plant Biotechnology; Mantell, S . H., Smith, H., Eds.; Cambridge University Press: London, 1983; pp 3-38. (2) Rittershaus, E.; Brummer, B.; Stiller, W.; Weiss, A. Grosstechnische Fermentation von pflanzlichen Zellkulturen. Downstream Processing zur Produktgewinnung aus pflanzlichen Zellkulturen im grosstechnischen Massstab. Bioengineering 1989, 3 (4), 51-65. (3) Tabata, M.; Fujita, Y. Production of shikonin by plant cell cultures. In Biotechnology i n Plant Sciences, Relevance of Agriculture in the Eighties; Zaitlin, M., Day, P., Hollaender, A., Eds.; Academic Press: Orlando, FL, 1985; pp 207-218. (4) Bond, P. A.; Fowler, M. W.; Scragg, A. H. Growth of Catharanthus roseus cell suspensions in bioreactors: on-line analysis of oxygen and carbon dioxide levels in inlet and outlet gas streams. Biotechnol. Lett. 1988, 10, 713-718. (5) Cho, G. H. Production of purine alkaloids and bioreactor operation strategies for plant cell cultures of Coffea arabica.
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Ph.D. Thesis, Rutgers, The State University of New Jersey, New Brunswick, NJ, 1987. (6) Archambault, J.; Volesky, B.; Kurz, W. G. W. Surface immobilization of plant cells. Biotechnol. Bioeng. 1989, 33, 293-299. (7) van Gulik, W. M.; Meijer, J. J.; ten Hoopen, H. J. G.; Luyben, K. Ch. A. M.; Libbenga, K. R. Growth of a Catharanthus roseus cell suspension culture in a modified chemostat under glucose-limiting conditions. Appl. Microbiol. Biotechnol. 1989, 30, 270-275. (8) Kim, D. J.; Chang, H. N.; Liu, J. R. Plant cell immobilization in a dual hollow fiber bioreactor. Biotechnol. Tech. 1989,3, 139-144. (9) Pu, H. T.; Yang, R. Y. K.; Saus, F. L. Iontophoretic release and transport of alkaloids from Catharanthus roseus cells in a ceramic hollow fiber reactor. Biotechnol. Lett. 1989,11,8386. (10) Scragg, A. H.; Cresswell, R.; Ashton, S.; York, A.; Bond, P.; Fowler, M. W. Growth and secondary product formation of a selected Catharanthus roseus cell line. Enzyme Microb. Technol. 1988, 10, 532-536. (11) Shuler, M. L. Immobilization of cells by entrapment in membrane reactors. Methods Enzymol. 1987,135,372-387. (12) Yoon, K.-H.; Prenosil, J. E. A novel membrane reactor for plant cell culture. Swiss Biotech. 1989, 7, 13-16. (13) Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 1962, 15,473-479. (14) Taya, M.; Hegglin, M.; Prenosil, J. E.; Bourne, J. R. Online monitoring of cell growth in plant tissue cultures by conductometry. Enzyme Microb. Technol. 1989,11, 170-176. (15) Frischknecht, P. M.; Baumann, T. W. The pattern of purine alkaloid formation in suspension cultures of Coffea arabica. Plant Med. 1980,40, 245-249. Accepted September 28,1990.
