Plant Cell, Tissue and Organ Culture 25: 219-224, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.
Application of process mass spectroscopy to the detection of metabolic changes in plant tissue culture Peggy Nikolova ~, Murray Moo-Young & Raymond L. Legge* Biochemical Engineering Group, Department of Chemical Engineering, ~Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2 L 3G1 (*requests for offprints) Received 21 May 1990; accepted in revised form 25 February 1991
Key words: bioreactor, plant cell culture, process mass spectroscopy, respiratory quotient, Syringa vulgar~ Abstract
There is a clear need in the area of plant cell culture for methods of on-line estimation of culture parameters. The introduction of plant cells into culture can result in a loss of their photoautotrophic character so that they are largely heterotrophic. As a result, fermentation off-gas analysis may not be confounded by photosynthetically-related O 2 production. In this study performance of a suspension culture of Syringa vulgaris, in a pneumatically agitated bioreactor of in-house design, was investigated. The effect of light on growth, carbohydrate metabolism and the respiratory quotient (RQ), determined by process mass spectroscopy, was studied. Yield coefficients for cells grown in the light and dark were similar although the patterns of carbohydrate uptake were quite different. Maximum biomass yields were higher in this bioreactor than normally observed in shake flasks. The RQ was dynamic during the course of the fermentation, peaking during the transition from the lag phase to the growth phase. It is suggested that the RQ may prove useful as an on-line parameter for monitoring transitions in cellular metabolism during plant cell culture fermentations.
Abbreviations: RQ - respiratory quotient, v.v.m. - volume of gas fed to fermenter per unit volume per minute, Y x / s - growth yield coefficient based on total carbohydrate
Introduction
Plant cell culture remains largely an untapped reserve for the production of specialty chemicals such as pharmaceuticals, fragrances, flavours, pigments and fine chemicals [1]. One of the reasons for this is our poor understanding of plant secondary metabolism and the factors which regulate it. Recent developments in the area of plant cell elicitation [2] and hairy root culture [3] offer new promise in this respect. These factors are often found to activate secondary metabolism in culture and in some cases cause secretion of the product. In addition to the
problems associated with plant secondary metabolism are those which arise during scale-up [4]. Plant cells are large and possess a fairly rigid cell wall which contributes to their susceptibility to hydrodynamic shear [1]. In addition, their growth rate is slow, which translates into long culture periods and a correspondingly higher risk of contamination. Bioreactors of the pneumatically-agitated type, which have been the focus of our studies, are highly suitable for culturing plant cells due to their simplicity in design and ability to satisfy aeration and mixing needs with relatively low hydrodynamic shear. Most plant cells become heterotrophic when
220 they are introduced into culture. It is not clear to what extent this shift from autotrophic to heterotrophic character modifies cellular metabolism. For example, does the phytochrome system continue to function or does CO 2 continue to play a role in the regulation of metabolism? Studies by Kato et al. [5] and Smart & Fowler [6] have shown that metabolic activity, and hence productivity, are particularly sensitive to alterations in the rate and nature of aeration. This may stem from the importance that carbon dioxide and oxygen play in supporting the growth of non-photosynthetic plant cell cultures [7, 8]. Most studies on the performance of plant cell cultures have been performed at the level of the shake flask which does not simulate the conditions normally experienced under scale-up conditions. Fermentation exhaust gas analysis, using process mass spectroscopy, has been examined as a potential on-line method for following cellular metabolism. This technique, attractive because of its high degree of accuracy and rapid response time, has been applied to microbial fermentations [9] but applications to plant cell culture are few likely because process mass spectrometers are expensive and not commonly available [10]. By analyzing the composition of the inlet and outlet gas, a number of on-line parameters such as the respiratory quotient (RQ), carbon dioxide evolution rate (CER) and oxygen uptake rate (OUR), can be determined. In this study we have investigated the relationship between various cultural characteristics and the respiratory coefficient for a suspension culture of Syringa vulgaris, grown under light and dark conditions, in a simple pneumaticallyagitated bioreactor.
Materials and methods
1-1 of 2,4-dichlorophenoxyacetic acid (Sigma Chemical, St. Louis, MO) and 0.2 mg 1-1 kinetin (Sigma Chemical, St. Louis, MO) as growth factors and 30 gl 1 sucrose as a carbon source. The cultures were subcultured every 7 days using a 10% (v/v) inoculum and were cultivated at room temperature on a shaker (120 rpm) with an 18 h photoperiod. A working volume of 50 ml for 250 ml Erlenmeyer flasks was used for the stock cultures. Results reported are representative of two or more separate experiments.
Bioreactor growth studies A slanted-bottom pneumatically-agitated bioreactor of in-house design with 1-1 working volume (height to diameter ratio H : D = 3 . 7 5 ) , which has been previously described [12], was used in this study. This reactor is a bubble column with an offset sparger and slanted bottom to enhance circulation and mixing. Air was filter sterilized through a 0.22 ~m Millex-FGs0 50 mm filter and flowrates regulated using Gilmont No. 2 or No. 3 shielded flowmeters with micrometer valve assemblies (Cole-Palmer, Chicago, IL). The standard flowrate was 0.25 v.v.m. (250 ml min -1) at room temperature. Light grown cultures were maintained under continuous illumination at 27001ux. For dark grown cultures the inoculum was dark-adapted for a one week subculture and the bioreactor wrapped with several layers of aluminum foil. A 10% (v/v) inoculum was used to initiate the culture. Dimethylpolysiloxan (20% (v/v) General Electric Corp., Waterford, NY) in deionized water was used as an antifoaming agent and added as required. Total additions did not exceed 0.02% (v/v) and were required largely during the first week of the fermentation. Samples were taken aseptically for determination of biomass concentration, cell viability and carbohydrate concentration.
Plant material and culture medium Analytical procedures Plant cell suspension cultures of Syringa vulgaris were obtained from Dr. Brian E. Ellis (University of British Columbia, Vancouver, B.C., Canada). The cultures were maintained on standard B5 medium [11] supplemented with 0.2 mg
Dry weight Samples were collected on preweighed Whatman No. 1 filter paper and dried at 60°C until constant weight. The filtrate was saved for carbohy-
221 drate analysis. Biomass concentrations were determined on both a fresh and dry weight basis.
Assays of carbohydrates The concentrations of sucrose, fructose (J.T. Baker Chemical Co., Phillipsburg, NY) and glucose (Sigma Chemical Co., St. Louis, MO) in the medium were determined using a HPX-47 C Carbohydrate Analysis HPLC Column (Biorad, Richmond, CA) and R401 Differential Refractive Index Detector (Millipore-Waters, Bedford, MA). Peak recording and integration was carried out with a Waters 730 Data Module (MilliporeWaters, Bedford, MA). Reagent grade water prepared using a NANO Pure Water Purification System (Sybrom/Barnstead, Boston, MA) was sterilized and used as eluant (flowrate 0.6ml min- ~; 85°C). Cell viability A 0.5% (w/v) solution of Evans Blue stain (Sigma Chemical, St. Louis, MO) prepared in B5 medium was used for cell viability determinations according to Gaff and Okong 'O-Ogola [131. On-line mass spec measurements On-line off-gas analysis was done using a VG MM8-80F multi-stream inlet mass spectrometer. Calibration was performed twice daily with a multi-component calibration gas containing 300 ppm CO2, 21% 0 2 and the balance N 2. 0 2 and CO 2 concentrations in the inlet and outlet gases to the fermenter were measured and used to determine the respiratory quotient (RQ) online as described by Buckland et al. [10]. RO=
moles CO 2 formed moles 0 2 consumed
Results and discussion
A l l pneumatically-agitated bioreactor of inhouse design was employed in this study. This reactor is characterized by a low shear field and good mixing and has been found to be highly suitable for the cultivation of plant cells [12]. The slanted-bottom feature has been shown to create a flow pattern in the reactor similar to that
of a split-flow airlift with a cross-flow at the top sufficient to reduce wall growth and minimize foaming problems. A 10% inoculum was used to initiate the bioreactor and was intentionally selected on the low side so that the duration of the lag phase would be extended. This was the case and the lag phase lasted about 7 days for the light grown cultures (Fig. 1). This is approximately 2 days longer than the lag phase observed for Eschscholtzia californica grown under similar conditions with a 20% inoculum [12]. The growth phase for both light and dark grown cultures occurred between days 6-11. The maximum biomass concentrations were in the order of 16.5 g d.w.1 1 for both light and dark grown cultures which is slightly better than that observed for E. californica. Cell viability is high for this suspension culture and remained relatively constant between 85 and 95% for the light grown cultures (Fig. 2). In contrast, the initial viability of the dark grown culture was lower (87%) than the light grown and dropped to below 75% during the 2 week course of the fermentation. This reactor has been shown to sustain higher viabilities than normally observed in shake flasks which is attributed to better mixing at high biomass concentrations and the stripping of toxic volatiles that may accumulate under shake flask conditions [12]. This is substantiated based on maximum levels of biomass achievable under the
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Fig. 1. Growth curves for cultures in a 1-I slanted-bottom bioreactor in the light (D) and dark (~,).
222 35
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Fig. 2. Cell viability (%) as a function of time for light ([3) and dark (0) grown cultures in 1-1 slanted-bottom bioreactor. two configurations. Previous reports indicate maximum biomass concentrations in the order of 200 g fr wt 1-1 [14] whereas in this study between 250-270 g fr wt 1- I in the slanted bottom bioreactot were observed. Figures 3 and 4 show the patterns of carbohydrate uptake for light and dark grown cultures, respectively. In the light, sucrose was rapidly and
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Fig. 3. Carbohydrate uptake for cultures grown in the light in a 1-1 slanted-bottom bioreactor. (V?) sucrose concentration, g 1-~; (0) glucose concentration, g 1-1; ( I ) fructose concentration, g I ]; (0) total carbohydrate concentration.
Fig. 4. Carbohydrate uptake for cultures grown in the dark in a 1-1 slanted-bottom bioreactor. ([3) sucrose concentration, g 1 1; (0) glucose concentration, g 1 1; (I) fructose concentration, g 1 1; (0) total carbohydrate concentration. completely hydrolyzed to glucose and fructose during the first 5 days of the fermentation. This hydrolysis can likely be attributed to an acid invertase, which may be associated with the cell wall, as has been found for cultured carrot cells [15]. Sucrose degradation may also involve a novel pathway which is initiated by sucrose synthase [16]. Under dark conditions, the initial rate of hydrolysis is rapid but then tails-off after day 4. Glucose is preferentially utilized under both light and dark conditions although more pronounced for dark grown cultures where fructose utilization is not initiated until glucose is completely exhausted from the medium (day 8). Although the pattern of carbohydrate uptake is different, the overall yield coefficients (Yx/s) are about the same, being 0.66 and 0.69 g/g for the light and dark grown cultures, respectively. These values are comparable to those reported for E. californica and published values for several other species [12]. Although S. vulgaris is green in culture it is apparently non-photosynthetic given the similarity in yield coefficients. On-line estimation of culture parameters is becoming increasingly important for microbial fermentations. These parameters provide on-line information on the performance of fermentation systems and can be used for control purposes. Unfortunately for plant cell culture, these on-
223 line parameters have been largely restricted to pH, temperature and dissolved oxygen. The availability of various types of instrumentation for off-gas analysis, including process mass spectroscopy, should expand the range of feasible on-line measurements. One such measurement is the respiratory coefficient (RQ), which provides an indication of the functionality of the respiratory system. The RQ in actively photosynthesizing material is not easily determined because of the production of oxygen. Mass spectroscopy has been of benefit because 1802 can be used in a closed gas exchange system to determine the RQ [17]. A technical difficulty with RQ measurements is related to the fact that small depletions in carbon dioxide result in very small changes in oxygen. These small changes against a large oxygen concentration in the background make accurate measurements of RQ difficult; the stability and sensitivity of process mass spectroscopy resolves part of this problem. It is evident from Fig. 5 that the RQ is a dynamic parameter for this plant cell culture and that the pattern is similar regardless of whether the cells were grown in the light or dark. An increase in RQ was observed during the initial stages of the growth cycle correlating temporally with the lag phase. The RQ for both cultures peaked at a similar value and declined during the growth phase. The transition appears to corre1.6"
late with the point when the sucrose concentration fell below 5 g1-1 in the medium and towards the end of the lag phase. Presumably this reflects a period of active protein and nucleic acid synthesis in preparation for the ensuing period of rapid growth. It is also possible that this inflection may be related to nitrate assimilation for it has been reported that nitrate assimilation may decrease electron transport [18] and that there is competition for nitrate assimilation and mitochondrial generated reductant [19]. The RQ decreases continually after this point reaching a value comparable to that observed during the original inoculation. This cell line is known to accumulate up to 16% of its dry weight as verbascoside with production independent of the growth stage [14]. Clearly the RQ may be a significant indicator of shifts in metabolism. Comparison to another cell line capable of producing a secondary metabolite would be useful. Bond et al. [20] have reported on RQ dynamics for Catharanthus roseus in an airlift vessel. This culture did not exhibit major shifts in the RQ until much later in the growth cycle. The discrepancy in performance of these two cultures is not clear although the conditions for C. roseus were considerably different than those employed in this study. Other factors, including pH, can dramatically affect RQ [21]. Clearly further work is warranted to determine if process mass spectroscopy can provide an on-line parameter for monitoring shifts in metabolism for plant cell suspension cultures.
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Fig. 5. Respiratory quotient as a function of time for light (D) and dark ( e ) grown cultures in a 1-1 slanted-bottom bioreactor.
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