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On-line monitoring of cell growth in plant tissue cultures by conductometry
On-line monitoring of cell growth in plant tissue cultures by conductometry M. Taya*, M. Hegglin, J. E. Prenosilt and J. R. Bourne D e p a r t m e n t o f Chemical Engineering (TCL), Swiss Federal Institute o f Technology (ETH), CH-8092 Zurich, Switzerland
(Received 18 May 1987; revised 2 March 1988)
Use of conductivity change in the medium for the on-line monitoring of cell mass growth was investigated in cultures of Coffea arabica, Nicotiana tabacum, Withania somnifera, and Catharanthus roseus. It was found that the effect of p H (4.5-7.0) and sugar concentration (0-1%) changes on medium conductivity can be neglected. Linear relationships between the dry cell mass and conductivity change were observed for all the four cell lines: AX = k(Ax), where the coefficient k = 3.6, 2.8, 3.2, and 4.1 g . cm • l -~ • ms -I for C. arabica, N. tabacum, W. somnifera, and C. roseus, respectively. By this method it was possible to estimate the cell mass concentrations in situ during the growth phase for various plant cell cultures.
Keywords: Plant cell culture; monitoring of cell growth; medium conductivity measurement
Introduction Plant tissue cultures are becoming increasingly important for the production of economically valuable biochemicals, including enzymes, flavonoids, pigments, vitamins, and cell mass itself. 1-3 In the cultures, success in achieving high yield of the product is largely dependent on the availability of a reliable and fast method for the estimation of the cell growth during the cultivation period. Thus it is indispensable to understand the growth kinetics of plant cells, which offers the most important and fundamental information for the design, optimization, and control of largescale plant tissue cultures. Usually, plant cells are much bigger than microbes and grow in aggregates or as a callus. 4 These facts make it difficult to obtain a homogeneous sample of cell suspension or to employ official methods, routinely used for determination of cell mass concentration in microbial cultures. The methods generally adopted for cell mass determination in plant tissue cultures involve gravimetric and volumetric measurement on a wet or dry basis and microscopic counting of the cells after protoplast-forming treatment. 5 However, these methods include time- and labor-
* Present address: Faculty of Engineering Science, Osaka University, Toyonaka 560, Osaka, Japan t To whom all correspondence should be addressed
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consuming procedures and are often lacking the desired accuracy. Moreover, withdrawing aliquots of plant cell suspension from a culture system may increase the risks of microbial contamination. In the course of investigating plant cell reactors, it was found necessary to develop a suitable strategy for plant cell mass determination during the cultivations. The present work reports on the utility of the conductivity change of medium caused by the cell uptake of minerals, as a tool for in situ estimation of plant cell mass.
Materials and methods Cell lines a n d c u l t u r e s Coffea arabica and Catharanthus roseus cells were obtained from the Institute of Plant Biology, University of Zurich. Nicotiana tabacum and Withania somnifera cells were obtained from the Institute of Biotechnology, ETH Zurich. These cell lines were maintained in shake flasks on an orbital shaker (120 rev min -I) at 25°C in darkness with subculturing weekly. The basic medium used for the plant cells was Murashige and Skoog's medium 6 (Flow Laboratories, Scotland) modified by the following supplementary components: 1.0 mg 1-1 thiamine • HCI, 10 mg 1-1 cysteine • HC1, 1.0 mg 1-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 mg 1-1 kinetin, and 20 g 1-I glucose (unless otherwise noted) for C. arabica, W. somnifera, and C. roseus, and 2.0 mg 1-1 indole-3-acetic acid, 0.2 © 1989 Butterworth Publishers
Conductometric monitoring of plant cell growth: 114. Taya et al. mg 1-1 kinetin, and 20 g 1-1 sucrose for N. tabacum. After the pH adjustments to 5.7 (C. arabica, W. somnifera, and C. roseus) and 6.0 (N. tabacum), the media were autoclaved for 20 rain at 121°C. For suspension culture, 200-ml Erlenmeyer flasks with 50-ml cultures of the four cell lines were shaken on an orbital shaker with 120 rev min -1 at 25°C. The triplet of whole culture broth obtained from the flasks was used as a sample at a given time during the cultivations. The cultivation using a membrane-bottom boat was done as described previously. 7'a The membrane boat consisted of a Plexiglas ring (ca. 5.5 cm in diameter and 2 cm in height) with a semipermeable membrane (Celgard 3500, microporous polypropylene engineering film, Celanese Corp., USA). The boat loaded with cells was floated on 50 ml medium in a plastic flask. After a batch cultivation, the boat with cells was transferred to fresh medium. Data were obtained from the average of 5 to 10 runs.
Conductivity measurement Specific conductivity of medium was determined using a digital conductivity meter, PW9527, and a dip and flow-type conductivity cell, PW9513/00 (Philips Co., England). After removal of cell material by vacuum filtration, culture broth was submitted to the conductivity measurement at 25°C, unless otherwise noted.
Analyses For cell mass determination, the plant cell mass was harvested by filtration and rinsed with a large amount of water on a glass filter. Dry cell weight was measured after drying the mass at 80°C for 3-5 days. In the expanded bed reactor system, the cell growth was estimated from cell packed volume, and the volume was converted into dry cell weight by an empirical relationship (1000 cm 3 settled cells = 37 g dry cells). The elemental composition (C, H, N) of the cells was determined by an elemental analyzer (Elemental Analyzer 240, Perkin Elmer, USA). The following analyses of medium were carried out using cell-free culture broth: Glucose concentration was determined with a glucose analyzer (Beckman Instruments Inc., USA). Sucrose and fructose concentrations were determined by an HPLC system (Waters Associates, Inc., USA) with an interference refractometer (Multiref 902, Optilab, Inc., Sweden) as a detector and with a Waters Sugar-PAK I column equipped with a precolumn (Micro-Guard TM, Bio-Rad Laboratories, USA). The running conditions were as follows: eluent 0.1 mM calcium acetate, flow rate 0.5 ml min -1, column temperature 95°C, and detector temperature 82°C. The concentrations of CI-, NO3 and SO4z- were determined by the Waters HPLC equipped with a conductivity detector (Model 213-505, Wescan Instruments Inc., USA) and with a Wescan 269-001 Anion Column equipped by a precolumn. The running conditions were as follows: eluent 4 mM potassium hydrogen phthalate (pH 4.5), flow rate 2.0
ml min -1, and column and detector temperature 25°C. The concentrations of NH~- and PO43+ were measured colorimetrically using phenol 9 and molybdate, 1° respectively. The amounts of K-, Ca z+, and Mg 2+ were determined by atomic absorption spectrometry (Model 751, Instrumentation Laboratory, USA).
Results
Preliminary experiments The purpose of the initial experiments was to check the relationship between conductivity and concentration of the medium. The medium contains many ion species, and thus deviations from the linear relationship may be expected in a relatively concentrated medium. The measurements have shown clearly linear relationship up to 1.5-fold concentration of the usual medium. This observation was important, supporting the idea of use of conductivity for the measurement of plant tissue biomass, because the degree of conductivity change with salt concentrations in the culture is relatively small. If necessary, moreover, it will be possible to cultivate plant cells in fed-batch and continuous culture systems, where the nutrient concentrations can be kept at a lower level.
Effects of environmental changes on medium conductivity In practice, the progress of plant cell culture is accompanied by other environmental changes besides a decrease in medium conductivity. Therefore, in the present work we took into account the main environmental factors that could possibly influence the medium conductivity. Figure 1 shows the effects of glucose and sucrose (typical carbon sources for plant tissue cultures) concentrations, cell fresh weight (FW), and pH on specific conductivity of the medium. Nearly constant values of specific conductivity were observed for both the sugars up to 10 g 1-1 of sugar concentration. In the range of high sugar concentrations (or high ratio of nonelectrolytes to electrolytes), the sugar molecules may cause a lowering of conductivity by decreasing the ionic mobility in the medium. The effect of pH on specific conductivity of the medium was investigated by pH adjustment of the medium. In this experiment, graded amounts of 0.01 N HC1, KCI, and KOH were added to the medium with a constant dilution ratio (additive : medium = 1 : 4). The changes of K + and C1concentrations were regarded as being negligible because of the very small total amounts added. As shown in Figure 1, the conductivity was little affected by pH in the range investigated. This observation was important for the successful application of conductometry to plant tissue cultures because the pH changes are usually not too large (pH 4.5-7.0). The cell suspension conductivity was relatively sensitive to the cell concentration, as shown in Figure 1. Therefore, all conductivity measurements were made in a cell-free medium. Temperature change alters the conductivity of Enzyme Microb. Technol., 1989, vol. 11, March
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sion cultures of C. arabica, N. tabacum, W. somnifera, and C. roseus. For each culture, a linear relationship was observed and the straight lines were obtained:
the salt solution strongly. However, this effect was eliminated satisfactorily by a temperature compensation function built into the conductivity meter. In the present study, the reference temperature was set at 25°C.
AX = k(AK) where X = dry cell mass (g 1-1), k = empirical coefficient (g cm 1-l mS-l), and K = specific conductivity (mS cm-1). Here, the values of k obtained by linear regression forced through zero were 3.6 (C. arabica), 2.8 (N. tabacum), 3.2 (W. somnifera), and 4.1 (C. roseus) g cm 1-1 mS -l (Table 1). Thus, it was possible to estimate the cell mass concentration from the conductivity measurement, using the above empirical equation.
Relationship between cell mass grown and conductivity decrease Plant cells require various inorganic nutrients as well as a carbon source for their propagation. Therefore, the uptake of ionic materials by plant cells is expected to cause the lowering of medium conductivity. Figure 2 shows the relationship between cell mass grown and conductivity decrease during batch suspen-
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Conductometric monitoring of plant cell growth: 114. Taya et al. Table 1 Coefficients (k) and elemental compositions of plant cells
Discussion Plant cell cultures potentially provide an alternative route to numerous plant-derived products that are too expensive to be synthesized chemically. Though largescale cultivation of plant cells has undergone a vigorous development in the past decade, some problems still remain to be solved. One of them is the development of a simple and reliable method to monitor plant cell growth. In general, plant cells very often form the desired products only for a brief period or in a limited growth phase. In the present communication, we were able to present the measurement of culture broth conductivity as a useful method for the estimation of plant cell mass concentration. The application of conductometry to plant cell cultures has the following advantages:
Elemental composition
(wt%)
C. roseus C. arabica W. somnifera N. tabacum
k value (g cm 1-1 mS -1)
C
N
H
4.1 3.6 3.2 2.8
45.4 41.5 39.6 43.1
4.12 4.56 6.45 6.17
6.61 5.46 5.24 5.65
Estimation o f cell growth in various cultures based on conductivity measurement Figures 3-6 show the results using the suspension cultures of C. arabiea, N. tabacum, W. somnifera, and C. roseus, respectively. In these figures, the lines indicating the cell mass were calculated using the corresponding equations as mentioned above. For all the four cultures, the lines agreed very well with the experimental data. Towards the end of the culture (or in the declining phase), however, the calculated values tended to be higher than those obtained gravimetrically. This may be due to the failure to follow cellular changes such as lysis and vacuolation by the conductivity measurements. Nevertheless, these deviations do not present any problems for the cell mass growth measurements during the main culture period, especially in the fed-batch culture system as described below. Figure 7 shows the cultivation results of C. arabica in the membrane-bottom boat system. The calculated values of the cell mass concentration are expressed as the solid lines, based on the specific conductivity measured. It was possible to estimate the cell growth by conductometry in good agreement with the gravimetric determinations.
1. possibility of in situ or on-line measurements; 2. the absence of cell mass sampling reduces greatly the risk of contamination; 3. simple and reproducible procedure without complicated and time-consuming steps. 4. negligible temperature influence due to the compensation function built in conductivity instruments; plant tissue cultures can be, and often are, carried out under coarsely thermostated conditions or at ambient temperature; 5. it is especially useful for immobilized cell systems such as membrane containment (Figure 7) and gel entrapment 2.~1-14. 6. high sensitivity of the changes of conductivity with the cell mass concentration changes (Figure 2). On the other hand, the limitation is that the cell mass evaluation is based on an indirect method. The decrease in cell mass concentration was not monitored exactly by the conductometric determination at the ends of the batch cultures as shown in Figures 3--6. Furthermore, this method could not be applied
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E n z y m e M i c r o b . T e c h n o l . , 1989, v o l . 11, M a r c h
C o n d u c t o m e t r i c m o n i t o r i n g o f p l a n t c e l l g r o w t h : M. Taya e t al.
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without modifications to the cultivation system followed by the large change of sugar concentration (Figure 1). However, these problems do not arise in fedbatch, semi-continuous, and continuous cultures, as illustrated in Figure 7. Hahlbrock et al. iSJ6 suggested the possible use of conductometry for determining specific growth stages and concomitant peaks in some enzyme activities of cells during plant cell suspension cultures. In the present work, we propose the quantitative monitoring of the C. arabica, N. tabacum, W. somnifera, and C. roseus cultures on the basis of the medium conductivity measurement. For each cell line, the empirical coefficient k was determined between cell mass grown and conductivity decrease (Figure 2). The coefficient values appeared to reflect the amount of inorganic
Table 2 Consumption of main inorganic nutrients and cell yields based on overall mineral uptake during suspension cultures of C. arabica and IV. tabacum
Cultivation time (h) Minerals consumed (mmol 1-1) NH~ K+ Ca2+ Mg 2÷ NO; CISO42-
C. arabica
N. tabacum
358
305
P043-
16 7 1.1 0.2 24 0 0.7 1.2
17 12 1.4 0.7 30 4.2 1.6 1.4
Total
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68.3
Cell mass grown (g 1-1) Cell yield based on overall mineral uptake (g mmo1-1)
8.36
9.99
0.167
0.146
nutrients consumed by the cells. Hahlbrock et al. 16 reported that the main contribution to medium conductivity decrease was nitrate uptake in soybean cell culture. Thus, the elemental compositions of the plant cells were determined and are given together with the k values in Table 1. As a result, it was found that the cells with higher nitrogen content did not necessarily correspond to those with lower values of k. This fact suggested that the overall uptake of ionic material by the cell was responsible for the decrease of medium conductivity. Table 2 shows the consumption of the main inorganic nutrients in the media during the cell growth, corresponding to the results presented in Figures 3 and 4. The cell yields based on overall mineral uptake and cell mass grown are shown as well.
Conclusions The present study demonstrated the feasibility of the on-line monitoring of cell mass growth in plant cell tissue cultures by a conductometric method. For C. arabica, N. tabacum, W. somnifera, and C. roseus cells it showed that linear relationships exist between dry cell mass and medium conductivity change. Furthermore, it was shown that this method could be used for accurate estimation of the cell mass concentration in suspension and membrane-bottom boat cultures. On the basis of our results with four different cell lines, it can be assumed that other plant cell cultures will behave similarly. Conductometry could be tentatively considered as a general method for cell mass determination in plant tissue culture, provided the values of the empirical factor k are known. Further work may be required using more cell lines to establish a wide experimental data base and to determine the inherent cell parameters, such as overall mineral uptake yield, governing the proportionality factor k, so that its value can be eventually predicted. E n z y m e M i c r o b . T e c h n o l . , 1989, v o l . 11, M a r c h
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Papers Theoretically, this method should be applicable also to animal cell cultures. Preliminary investigations in our laboratory are in progress.
Acknowledgements The authors wish to express their thanks to Drs. T. W. Baumann and P. M. Frischknecht (Institute of Plant Biology, University of Zurich) and Dr. P. Brodelius (Institute of Biotechnology, Honggerberg, ETH Zurich) for kindly providing the plant cell lines. The authors also wish to thank Mrs. V. Kaspar for her assistance with the experiments. This work was supported in part by research grants of the Swiss National Science Foundation (NF 2.902 and NF 2.815).
4 5 6 7 8 9 10
11 12 13
References 1 2 3
176
Staba, E. J., ed. in Plant Tissue Culture as a Source o f Biochemicals CRC Press, Boca Raton, 1980 Prenosil, J. E. and Pedersen, H. Enzyme Microb. Technol. 1983, 5, 323-331 Lee, J. M. and An, G. Enzyme Microb. Technol. 1986, 8, 260-265
Enzyme Microb. Technol., 1989, vol. 11, March
14 15 16
Tanaka, H. Biotechnol. Bioeng. 1982, 24, 425-442 Dixon, R. A., ed. in Plant Tissue Culture-A Practical Approach IRL Press, Oxford and Washington DC, 1985, pp. 1-20 Murashige, T. and Skoog, F. Physiol. Plant. 1962, 15, 473-479 Prenosil, J. E. et al. Enzyme Microb. Technol. 1987, 9, 450-457 Prenosil, J. E., Hegglin, M., Bourne, J. R. and Hamilton, R. M. in Enzyme Engineering (Laskin, A. I., et al., eds.) Ann. N.Y. Acad. Sci. 1987, 8, 390-394 Merck, E. Klinisches Labor 12th Ed., Darmstadt, 1974 (in German) LePage, G. A. in Manometric Techniques--A Manual Describing Methods Applicable to the Study o f Tissue Metabolism (Umbreit, W.W., Burris, R. M. and Stauffer, J. F., eds.) Burgess Publishing, Minneapolis, 1957, pp. 268-281 Brodelius, P. and Mosbach, K. Adv. Appl. Microbiol. 1982, 28, 1-26 Brodelius, P. and Nilsson, K. FEBS Lett. 1980, 122, 312-316 Brodelius, P. in Enzymes and Immobilized Cells in Biotechnology (Laskin, A. I., ed.) Benjamin/Cummings Publishing, Menlo Park, California, 1985, pp. 109-148 Nakajima, H. et al. Appl. Microbiol. Biotechnol. 1986, 24, 266-270 Hahlbrock, K. and Kuhlen, E. Planta (Berl.) 1972, 108, 271-278 Hahlbrock, K., Ebel, J. and Oaks, A. Planta (Berl.) 1974, 118, 75-84
(Received 18 May 1987; revised 2 March 1988)
Use of conductivity change in the medium for the on-line monitoring of cell mass growth was investigated in cultures of Coffea arabica, Nicotiana tabacum, Withania somnifera, and Catharanthus roseus. It was found that the effect of p H (4.5-7.0) and sugar concentration (0-1%) changes on medium conductivity can be neglected. Linear relationships between the dry cell mass and conductivity change were observed for all the four cell lines: AX = k(Ax), where the coefficient k = 3.6, 2.8, 3.2, and 4.1 g . cm • l -~ • ms -I for C. arabica, N. tabacum, W. somnifera, and C. roseus, respectively. By this method it was possible to estimate the cell mass concentrations in situ during the growth phase for various plant cell cultures.
Keywords: Plant cell culture; monitoring of cell growth; medium conductivity measurement
Introduction Plant tissue cultures are becoming increasingly important for the production of economically valuable biochemicals, including enzymes, flavonoids, pigments, vitamins, and cell mass itself. 1-3 In the cultures, success in achieving high yield of the product is largely dependent on the availability of a reliable and fast method for the estimation of the cell growth during the cultivation period. Thus it is indispensable to understand the growth kinetics of plant cells, which offers the most important and fundamental information for the design, optimization, and control of largescale plant tissue cultures. Usually, plant cells are much bigger than microbes and grow in aggregates or as a callus. 4 These facts make it difficult to obtain a homogeneous sample of cell suspension or to employ official methods, routinely used for determination of cell mass concentration in microbial cultures. The methods generally adopted for cell mass determination in plant tissue cultures involve gravimetric and volumetric measurement on a wet or dry basis and microscopic counting of the cells after protoplast-forming treatment. 5 However, these methods include time- and labor-
* Present address: Faculty of Engineering Science, Osaka University, Toyonaka 560, Osaka, Japan t To whom all correspondence should be addressed
170
Enzyme Microb. Technol., 1989, vol. 11, March
consuming procedures and are often lacking the desired accuracy. Moreover, withdrawing aliquots of plant cell suspension from a culture system may increase the risks of microbial contamination. In the course of investigating plant cell reactors, it was found necessary to develop a suitable strategy for plant cell mass determination during the cultivations. The present work reports on the utility of the conductivity change of medium caused by the cell uptake of minerals, as a tool for in situ estimation of plant cell mass.
Materials and methods Cell lines a n d c u l t u r e s Coffea arabica and Catharanthus roseus cells were obtained from the Institute of Plant Biology, University of Zurich. Nicotiana tabacum and Withania somnifera cells were obtained from the Institute of Biotechnology, ETH Zurich. These cell lines were maintained in shake flasks on an orbital shaker (120 rev min -I) at 25°C in darkness with subculturing weekly. The basic medium used for the plant cells was Murashige and Skoog's medium 6 (Flow Laboratories, Scotland) modified by the following supplementary components: 1.0 mg 1-1 thiamine • HCI, 10 mg 1-1 cysteine • HC1, 1.0 mg 1-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 mg 1-1 kinetin, and 20 g 1-I glucose (unless otherwise noted) for C. arabica, W. somnifera, and C. roseus, and 2.0 mg 1-1 indole-3-acetic acid, 0.2 © 1989 Butterworth Publishers
Conductometric monitoring of plant cell growth: 114. Taya et al. mg 1-1 kinetin, and 20 g 1-1 sucrose for N. tabacum. After the pH adjustments to 5.7 (C. arabica, W. somnifera, and C. roseus) and 6.0 (N. tabacum), the media were autoclaved for 20 rain at 121°C. For suspension culture, 200-ml Erlenmeyer flasks with 50-ml cultures of the four cell lines were shaken on an orbital shaker with 120 rev min -1 at 25°C. The triplet of whole culture broth obtained from the flasks was used as a sample at a given time during the cultivations. The cultivation using a membrane-bottom boat was done as described previously. 7'a The membrane boat consisted of a Plexiglas ring (ca. 5.5 cm in diameter and 2 cm in height) with a semipermeable membrane (Celgard 3500, microporous polypropylene engineering film, Celanese Corp., USA). The boat loaded with cells was floated on 50 ml medium in a plastic flask. After a batch cultivation, the boat with cells was transferred to fresh medium. Data were obtained from the average of 5 to 10 runs.
Conductivity measurement Specific conductivity of medium was determined using a digital conductivity meter, PW9527, and a dip and flow-type conductivity cell, PW9513/00 (Philips Co., England). After removal of cell material by vacuum filtration, culture broth was submitted to the conductivity measurement at 25°C, unless otherwise noted.
Analyses For cell mass determination, the plant cell mass was harvested by filtration and rinsed with a large amount of water on a glass filter. Dry cell weight was measured after drying the mass at 80°C for 3-5 days. In the expanded bed reactor system, the cell growth was estimated from cell packed volume, and the volume was converted into dry cell weight by an empirical relationship (1000 cm 3 settled cells = 37 g dry cells). The elemental composition (C, H, N) of the cells was determined by an elemental analyzer (Elemental Analyzer 240, Perkin Elmer, USA). The following analyses of medium were carried out using cell-free culture broth: Glucose concentration was determined with a glucose analyzer (Beckman Instruments Inc., USA). Sucrose and fructose concentrations were determined by an HPLC system (Waters Associates, Inc., USA) with an interference refractometer (Multiref 902, Optilab, Inc., Sweden) as a detector and with a Waters Sugar-PAK I column equipped with a precolumn (Micro-Guard TM, Bio-Rad Laboratories, USA). The running conditions were as follows: eluent 0.1 mM calcium acetate, flow rate 0.5 ml min -1, column temperature 95°C, and detector temperature 82°C. The concentrations of CI-, NO3 and SO4z- were determined by the Waters HPLC equipped with a conductivity detector (Model 213-505, Wescan Instruments Inc., USA) and with a Wescan 269-001 Anion Column equipped by a precolumn. The running conditions were as follows: eluent 4 mM potassium hydrogen phthalate (pH 4.5), flow rate 2.0
ml min -1, and column and detector temperature 25°C. The concentrations of NH~- and PO43+ were measured colorimetrically using phenol 9 and molybdate, 1° respectively. The amounts of K-, Ca z+, and Mg 2+ were determined by atomic absorption spectrometry (Model 751, Instrumentation Laboratory, USA).
Results
Preliminary experiments The purpose of the initial experiments was to check the relationship between conductivity and concentration of the medium. The medium contains many ion species, and thus deviations from the linear relationship may be expected in a relatively concentrated medium. The measurements have shown clearly linear relationship up to 1.5-fold concentration of the usual medium. This observation was important, supporting the idea of use of conductivity for the measurement of plant tissue biomass, because the degree of conductivity change with salt concentrations in the culture is relatively small. If necessary, moreover, it will be possible to cultivate plant cells in fed-batch and continuous culture systems, where the nutrient concentrations can be kept at a lower level.
Effects of environmental changes on medium conductivity In practice, the progress of plant cell culture is accompanied by other environmental changes besides a decrease in medium conductivity. Therefore, in the present work we took into account the main environmental factors that could possibly influence the medium conductivity. Figure 1 shows the effects of glucose and sucrose (typical carbon sources for plant tissue cultures) concentrations, cell fresh weight (FW), and pH on specific conductivity of the medium. Nearly constant values of specific conductivity were observed for both the sugars up to 10 g 1-1 of sugar concentration. In the range of high sugar concentrations (or high ratio of nonelectrolytes to electrolytes), the sugar molecules may cause a lowering of conductivity by decreasing the ionic mobility in the medium. The effect of pH on specific conductivity of the medium was investigated by pH adjustment of the medium. In this experiment, graded amounts of 0.01 N HC1, KCI, and KOH were added to the medium with a constant dilution ratio (additive : medium = 1 : 4). The changes of K + and C1concentrations were regarded as being negligible because of the very small total amounts added. As shown in Figure 1, the conductivity was little affected by pH in the range investigated. This observation was important for the successful application of conductometry to plant tissue cultures because the pH changes are usually not too large (pH 4.5-7.0). The cell suspension conductivity was relatively sensitive to the cell concentration, as shown in Figure 1. Therefore, all conductivity measurements were made in a cell-free medium. Temperature change alters the conductivity of Enzyme Microb. Technol., 1989, vol. 11, March
171
Papers Z
02 I
>,
100
g
98
O
96
94 0
I
I
I
2
4
6
glucose, sucrose
[%];
cells
8
[%FW/V];
pH
Figure 1 Effects of glucose, sucrose, cell fresh weight, and pH on medium conductivity. Relative influence for a medium with spec. conductivity of 5.8 mS cm -1. The value for pH 5.5 was set to 100%. Symbols: pH (A), cell fresh weight (I), glucose (o), and sucrose (x)
sion cultures of C. arabica, N. tabacum, W. somnifera, and C. roseus. For each culture, a linear relationship was observed and the straight lines were obtained:
the salt solution strongly. However, this effect was eliminated satisfactorily by a temperature compensation function built into the conductivity meter. In the present study, the reference temperature was set at 25°C.
AX = k(AK) where X = dry cell mass (g 1-1), k = empirical coefficient (g cm 1-l mS-l), and K = specific conductivity (mS cm-1). Here, the values of k obtained by linear regression forced through zero were 3.6 (C. arabica), 2.8 (N. tabacum), 3.2 (W. somnifera), and 4.1 (C. roseus) g cm 1-1 mS -l (Table 1). Thus, it was possible to estimate the cell mass concentration from the conductivity measurement, using the above empirical equation.
Relationship between cell mass grown and conductivity decrease Plant cells require various inorganic nutrients as well as a carbon source for their propagation. Therefore, the uptake of ionic materials by plant cells is expected to cause the lowering of medium conductivity. Figure 2 shows the relationship between cell mass grown and conductivity decrease during batch suspen-
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172
Enzyme Microb. Technol., 1989, vol. 11, March
Conductometric monitoring of plant cell growth: 114. Taya et al. Table 1 Coefficients (k) and elemental compositions of plant cells
Discussion Plant cell cultures potentially provide an alternative route to numerous plant-derived products that are too expensive to be synthesized chemically. Though largescale cultivation of plant cells has undergone a vigorous development in the past decade, some problems still remain to be solved. One of them is the development of a simple and reliable method to monitor plant cell growth. In general, plant cells very often form the desired products only for a brief period or in a limited growth phase. In the present communication, we were able to present the measurement of culture broth conductivity as a useful method for the estimation of plant cell mass concentration. The application of conductometry to plant cell cultures has the following advantages:
Elemental composition
(wt%)
C. roseus C. arabica W. somnifera N. tabacum
k value (g cm 1-1 mS -1)
C
N
H
4.1 3.6 3.2 2.8
45.4 41.5 39.6 43.1
4.12 4.56 6.45 6.17
6.61 5.46 5.24 5.65
Estimation o f cell growth in various cultures based on conductivity measurement Figures 3-6 show the results using the suspension cultures of C. arabiea, N. tabacum, W. somnifera, and C. roseus, respectively. In these figures, the lines indicating the cell mass were calculated using the corresponding equations as mentioned above. For all the four cultures, the lines agreed very well with the experimental data. Towards the end of the culture (or in the declining phase), however, the calculated values tended to be higher than those obtained gravimetrically. This may be due to the failure to follow cellular changes such as lysis and vacuolation by the conductivity measurements. Nevertheless, these deviations do not present any problems for the cell mass growth measurements during the main culture period, especially in the fed-batch culture system as described below. Figure 7 shows the cultivation results of C. arabica in the membrane-bottom boat system. The calculated values of the cell mass concentration are expressed as the solid lines, based on the specific conductivity measured. It was possible to estimate the cell growth by conductometry in good agreement with the gravimetric determinations.
1. possibility of in situ or on-line measurements; 2. the absence of cell mass sampling reduces greatly the risk of contamination; 3. simple and reproducible procedure without complicated and time-consuming steps. 4. negligible temperature influence due to the compensation function built in conductivity instruments; plant tissue cultures can be, and often are, carried out under coarsely thermostated conditions or at ambient temperature; 5. it is especially useful for immobilized cell systems such as membrane containment (Figure 7) and gel entrapment 2.~1-14. 6. high sensitivity of the changes of conductivity with the cell mass concentration changes (Figure 2). On the other hand, the limitation is that the cell mass evaluation is based on an indirect method. The decrease in cell mass concentration was not monitored exactly by the conductometric determination at the ends of the batch cultures as shown in Figures 3--6. Furthermore, this method could not be applied
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Figure 3 Cell growth monitoring during suspension culture of C. arabica. The solid line is the cell mass concentration calculated from the conductivity measurements. Symbols: pH (&), specific conductivity (R), dry cell mass (Ig), and glucose (0)
Enzyme Microb. Technol., 1989, vol. 11, March
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Figure 4 Cell growth monitoring during suspension culture of N. tabacum. The solid line is the cell mass concentration calculated from the conductivity measurements. Symbols: pH (&), specific conductivity ([El), dry cell mass (i), sucrose (x), glucose (0), and fructose (+)
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E n z y m e M i c r o b . T e c h n o l . , 1989, v o l . 11, M a r c h
C o n d u c t o m e t r i c m o n i t o r i n g o f p l a n t c e l l g r o w t h : M. Taya e t al.
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Figure 7 Cell growth monitoring during membrane-bottom boat culture of C. arabica. The boats with cells were transferred to 60 ml of fresh medium with 20 g 1-1 of glucose at the time indicated by broken lines. The other caption and symbols are the same as those shown in Figure 3
without modifications to the cultivation system followed by the large change of sugar concentration (Figure 1). However, these problems do not arise in fedbatch, semi-continuous, and continuous cultures, as illustrated in Figure 7. Hahlbrock et al. iSJ6 suggested the possible use of conductometry for determining specific growth stages and concomitant peaks in some enzyme activities of cells during plant cell suspension cultures. In the present work, we propose the quantitative monitoring of the C. arabica, N. tabacum, W. somnifera, and C. roseus cultures on the basis of the medium conductivity measurement. For each cell line, the empirical coefficient k was determined between cell mass grown and conductivity decrease (Figure 2). The coefficient values appeared to reflect the amount of inorganic
Table 2 Consumption of main inorganic nutrients and cell yields based on overall mineral uptake during suspension cultures of C. arabica and IV. tabacum
Cultivation time (h) Minerals consumed (mmol 1-1) NH~ K+ Ca2+ Mg 2÷ NO; CISO42-
C. arabica
N. tabacum
358
305
P043-
16 7 1.1 0.2 24 0 0.7 1.2
17 12 1.4 0.7 30 4.2 1.6 1.4
Total
50.2
68.3
Cell mass grown (g 1-1) Cell yield based on overall mineral uptake (g mmo1-1)
8.36
9.99
0.167
0.146
nutrients consumed by the cells. Hahlbrock et al. 16 reported that the main contribution to medium conductivity decrease was nitrate uptake in soybean cell culture. Thus, the elemental compositions of the plant cells were determined and are given together with the k values in Table 1. As a result, it was found that the cells with higher nitrogen content did not necessarily correspond to those with lower values of k. This fact suggested that the overall uptake of ionic material by the cell was responsible for the decrease of medium conductivity. Table 2 shows the consumption of the main inorganic nutrients in the media during the cell growth, corresponding to the results presented in Figures 3 and 4. The cell yields based on overall mineral uptake and cell mass grown are shown as well.
Conclusions The present study demonstrated the feasibility of the on-line monitoring of cell mass growth in plant cell tissue cultures by a conductometric method. For C. arabica, N. tabacum, W. somnifera, and C. roseus cells it showed that linear relationships exist between dry cell mass and medium conductivity change. Furthermore, it was shown that this method could be used for accurate estimation of the cell mass concentration in suspension and membrane-bottom boat cultures. On the basis of our results with four different cell lines, it can be assumed that other plant cell cultures will behave similarly. Conductometry could be tentatively considered as a general method for cell mass determination in plant tissue culture, provided the values of the empirical factor k are known. Further work may be required using more cell lines to establish a wide experimental data base and to determine the inherent cell parameters, such as overall mineral uptake yield, governing the proportionality factor k, so that its value can be eventually predicted. E n z y m e M i c r o b . T e c h n o l . , 1989, v o l . 11, M a r c h
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Papers Theoretically, this method should be applicable also to animal cell cultures. Preliminary investigations in our laboratory are in progress.
Acknowledgements The authors wish to express their thanks to Drs. T. W. Baumann and P. M. Frischknecht (Institute of Plant Biology, University of Zurich) and Dr. P. Brodelius (Institute of Biotechnology, Honggerberg, ETH Zurich) for kindly providing the plant cell lines. The authors also wish to thank Mrs. V. Kaspar for her assistance with the experiments. This work was supported in part by research grants of the Swiss National Science Foundation (NF 2.902 and NF 2.815).
4 5 6 7 8 9 10
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References 1 2 3
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Staba, E. J., ed. in Plant Tissue Culture as a Source o f Biochemicals CRC Press, Boca Raton, 1980 Prenosil, J. E. and Pedersen, H. Enzyme Microb. Technol. 1983, 5, 323-331 Lee, J. M. and An, G. Enzyme Microb. Technol. 1986, 8, 260-265
Enzyme Microb. Technol., 1989, vol. 11, March
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Tanaka, H. Biotechnol. Bioeng. 1982, 24, 425-442 Dixon, R. A., ed. in Plant Tissue Culture-A Practical Approach IRL Press, Oxford and Washington DC, 1985, pp. 1-20 Murashige, T. and Skoog, F. Physiol. Plant. 1962, 15, 473-479 Prenosil, J. E. et al. Enzyme Microb. Technol. 1987, 9, 450-457 Prenosil, J. E., Hegglin, M., Bourne, J. R. and Hamilton, R. M. in Enzyme Engineering (Laskin, A. I., et al., eds.) Ann. N.Y. Acad. Sci. 1987, 8, 390-394 Merck, E. Klinisches Labor 12th Ed., Darmstadt, 1974 (in German) LePage, G. A. in Manometric Techniques--A Manual Describing Methods Applicable to the Study o f Tissue Metabolism (Umbreit, W.W., Burris, R. M. and Stauffer, J. F., eds.) Burgess Publishing, Minneapolis, 1957, pp. 268-281 Brodelius, P. and Mosbach, K. Adv. Appl. Microbiol. 1982, 28, 1-26 Brodelius, P. and Nilsson, K. FEBS Lett. 1980, 122, 312-316 Brodelius, P. in Enzymes and Immobilized Cells in Biotechnology (Laskin, A. I., ed.) Benjamin/Cummings Publishing, Menlo Park, California, 1985, pp. 109-148 Nakajima, H. et al. Appl. Microbiol. Biotechnol. 1986, 24, 266-270 Hahlbrock, K. and Kuhlen, E. Planta (Berl.) 1972, 108, 271-278 Hahlbrock, K., Ebel, J. and Oaks, A. Planta (Berl.) 1974, 118, 75-84