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Kinetic and Stoichiometric Analysis of Hairy Roots in a Segmented Bubble Column Reactor Kian H.Kwok and Pauline M.Doran* Department of Biotechnology, University of New South Wales, Sydney, NSW 2052, Australia
Hairy roots of Atropa belladonna were cultured in a modified 2.5-L multicompartment bubble column for analysis of growth kinetics, stoichiometry, and atropine production. Average biomass density reached 9.9 g L-' dry weight after 43 days of batch culture; local root densities in some parts of the vessel were considerably higher, up to 17 g L-l. Bulk mixing in the reactor was very poor: after 14 days of culture, the time taken to reach 95%of the equilibrium value after a concentration pulse in the vessel was 12 min. Growth and specific sugar uptake rates declined continuously throughout the culture even though adequate sugar and nitrogen remained in the medium. The observed biomass yield from sugar was approximately constant at 0.35g g-l; biomass yields from ammonia and nitrate were 0.44 and 0.35 g mmol-l, respectively. Specific atropine content in the roots varied from 4.1 mg g-l dry weight at the beginning of the culture to 1.4 mg g-l after 28 days; 35 mg or 14 mg L-l atropine was produced over the 43-day culture period. Biomass composition was represented by the elemental formula CH1.6300.80N0.13, plus 9.8% (w/w) ash. A balanced stoichiometric equation was developed for hairy root growth; this indicated that 8.3% of carbon supplied to the culture was excreted into the medium as by-products. ~~
~
~
Introduction Hairy roots are induced in susceptible plants by transformation with Agrobacterium rhizogenes. An increasing number of hairy root cultures is known to produce secondary metabolites in quantities much greater than dedifferentiated plant cells (Hamill and Rhodes, 1993). Hairy roots are less prone to genetic variation than callus or suspended cells (Aird et al., 1988) and therefore are more predictable in terms of growth characteristics and product accumulation (Flores, 1987). By virtue of their rapid growth rate, high product yield, simple medium requirements, and culture stability, hairy roots have significant potential as an alternative means for in vitro production of valuable phytochemicals. In recent years, several bioreactor designs have been applied for the culture of hairy roots. These include stirred (Davioud et al., 1989; Kondo et al., 1989; Hilton and Rhodes, 19901, air-driven (Taya et al., 1989; Sharp and Doran, 1990; Rodriguez-Mendiolaet al., 1991),trickle bed (Flores and Curtis, 1992), rotating drum (Kondo et al., 1989), nutrient mist (Wilson et al., 1990; DiIorio et al., 19921, and liquid-impelled external loop (Buitelaar et al., 1991) configurations. A number of studies have identified clumping and poor spatial distribution of roots as a problem in conventional reactors (Sharp and Doran, 1990; Davioud et al., 1989). Clumping results in low overall biomass densities as roots accumulate in one section of the vessel but fail to occupy the remaining volume. Local nutrient depletion a t the core of dense clumps limits biomass expansion and substrate conversion (Sharp and Doran, 1990; Subroto and Doran, 1994; Yu and Doran, 1994) and could also influence the profile of secondary products synthesized. Several investigators have installed support structures inside reactors to promote uniform biomass distribution and higher levels of biomass accumulation; these include wire mesh cages
* Author to whom correspondence
should be addressed.
(Hilton and Rhodes, 1990), a cylindrical mesh column (Rodriguez-Mendiola et al., 19911, and a polyurethane foam rod (Taya et al., 1989). Oxygen transport is an important consideration in the design of root reactors. The effect of inadequate external oxygen mass transfer has been demonstrated with both untransformed (Prince et al., 1991)and transformed (Yu and Doran, 1994) roots. Because of boundary layer effects, the rate of oxygen uptake by hairy roots depends on the velocity of bulk liquid in the reactor and the penetration of convective currents into the root ball. However, poor mixing is a particular problem in root reactors (Flores and Curtis, 1992);liquid entrainment in root clumps means that local fluid velocities are unlikely to reach the levels required to eliminate boundary layer resistance. As a general rule, when bulk mixing in reactors is impeded, the use of multiple entry points for added substrates such as oxygen helps to alleviate concentration gradients. Instead of reliance on a single sparger to supply the entire vessel, oxygen can be delivered closer to where it is needed in the reactor through multiple aeration points. The design of suitable bioreactors that support dense growth of roots is a pressing need that would significantly improve our ability to exploit hairy root cultures in industrial processes. Reactors that eliminate mass transfer limitations in highdensity root culture are yet to be found. Quantitative information about growth kinetics, sugar utilization, respiratory requirements, and biomass and product yields is required t o properly evaluate the potential of hairy root cultures. Despite its biochemical complexity, growth of many organisms is amenable to macroscopic mass balance analysis, where the system is considered to be a black box exchanging a limited number of components with the environment. Measured variables coupled with conservation equations and metabolic stoichiometry are used to obtain simple, unstructured models of growth (Heijnen and Roels, 1981; Roels, 1983; Noorman et al., 1991). Such descriptions of cell activity
8756-7938/95/3011-0429$09.00/00 1995 American Chemical Society and American Institute of Chemical Engineers
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can form the basis of process monitoring and control strategies. Stoichiometric techniques have been applied to suspended plant cell cultures in several studies using data from batch and continuous culture experiments (Pareilleux and Chaubet, 1981; van Gulik et al., 1989, 1992; Taticek et al., 1990; Rho and Andre, 1991). Similar analysis has not been reported for hairy roots. In this study, a novel multicompartment bioreactor was tested for the culture of Atropa belladonna hairy roots. Air was sparged into the reactor from several outlets evenly spaced throughout the vessel. Root growth, biomass composition, and alkaloid production were determined for the reactor as a whole and as a function of position within the vessel. Material balances and measured yield coefficients were applied for stoichiometric analysis of growth, allowing patterns of substrate conversion by hairy roots to be compared with those for dedifferentiated suspension cultures.
To gas analysersf4
-
=
-
p~ Air in
Materials and Methods Hairy Root Initiation and Maintenance. Seeds of Atropa belladonna were obtained from the Royal Botanic Gardens, Kew, UK. Hairy roots were induced by infection of seedlings with Agrobacterium rhizogenes strain A4 provided by Prof A. Ken, Waite Agricultural Institute, Adelaide, Australia. Root cultures were maintained as described previously (Sharp and Doran, 1990) at 25 "C in liquid Murashige and Skoog (MS) medium (Flow Laboratories, UK) containing 30 g L-l sucrose (BDH), with the pH adjusted to 5.7 before autoclaving. Bioreactor Experiments. A 2.5-L bubble column reactor (LH Fermentation, UK) was fitted with a cylindrical stainless steel cage with three horizontal wire mesh screens. The reactor was thus divided into four vertical segments designated 1, 2, 3, and 4, as shown schematically in Figure 1. Air was sparged into the vessel through three 10-pm stainless steel frits located in segments 2, 3, and 4. The total air flow rate of 200 mL min-l was divided equally between the three spargers. Bubbles produced at the spargers were approximately uniform in size with a Sauter mean diameter of 1.6 mm. Except at the beginning of the culture when the root density was very low, these bubbles coalesced almost immediately within the root clumps growing in each section of the reactor. The bioreactor was operated under constant illumination of ca. 1000 lx. Mixing patterns in the vessel varied with culture time. Liquid circulated throughout the entire reactor during the first 10 days when the root density was relatively low; at this stage, small bubbles and entrained liquid passed readily through the wire mesh screens. As the biomass density increased, mixing currents became confined to the separate reactor sections. Characteristic mixing time in the bioreactor was determined by adding pulses of 1M H2S04 or 1M NaOH to the top of the vessel and measuring the time taken for the pH to reach 95% of the equilibrium value. Five separate batch experiments were carried out for periods of 10,20,28,40, and 43 days. The medium used was MS with 30 g L-l sucrose. Each experiment was initiated using the same procedure. Inocula consisted of roots grown in shake flasks; each flask contained ca. 0.05 g dry weight roots produced from a single root tip cultured for 7 days at 25 "C. Segments 2,3, and 4 of the bioreactor were inoculated separately, each with one flask of roots, through wide-bore silicone tubing. The tubing used to inoculate segment 4 was pulled up to inoculate segment 3; segment 2 was inoculated using separate tubing. The initial root density in the fermentor was
Water jacket
Figure 1. Segmented bubble column bioreactor for hairy root culture.
close to 0.06 g L-l dry weight for all experiments. Segment 1 was not inoculated with roots. Electrodes (Ingold, Switzerland) were used for pH and dissolved oxygen measurements. Temperature was maintained at 25 "C. Evaporation was controlled by a condenser operated at 4 "C. Exit gas composition was measured after dehumidification using infrared COZ and paramagnetic 0 2 gas analyzers (Servomex, UK). Samples of culture medium were taken every 2-4 days. At the end of each experiment the bioreactor was drained, the steel cage lifted out, and the biomass harvested separately from each segment for determination of dry weight, elemental composition, and alkaloid content. The residual liquid volume was also measured. Analysis of Biomass and Sugar Concentrations. Biomass dry weight was obtained by freeze-drying roots a t -40 "C to constant weight. Sucrose, glucose, and fructose concentrations in the medium were analyzed by HPLC using a 15 cm x 4.6 mm amino column (Waters, USA), with lactose as the internal standard. The mobile phase was 80:20 acetonitrile/water at a flow rate of 1.4 mL min-l. Total sugar concentration was calculated as sucrose equivalents (Sharp and Doran, 1990). Alkaloid Analysis. Biomass samples were analyzed for alkaloids in triplicate. Freeze-dried roots were ground to a powder, and 0.50-g samples were extracted by standing overnight in 100 mL of basic ethanol consisting of a 19:l mixture of ethanol/28% ammonia (Mano et al., 1986). Each overnight extract was filtered through
Biotechnol. Prog., 1995, Vol. 11, No. 4
Whatman No. 1 paper under vacuum and washed with 20 mL of ethanol. The combined filtrate and washing were evaporated to a dry residue using a rotary evaporator. Procedures for HPLC sample preparation were adapted from Svensson et al. (1982). Residue from the evaporator flask was dissolved in 2 mL of HPLC solvent consisting of 20 mL of low-W PIC-B7 ion-pairing reagent (Waters) per liter of 40:60 methanollwater (pH 4.1) and then passed through a 1-mL C18 Sep-Pak column (Waters). This process was carried out five times per residue sample to progressively collect 10 mL of Sep-Pak filtrate. The solution was then filtered through a 0.45-pm filter (Waters) for injection into the HPLC. Alkaloid recovery from the freeze-dried and ground samples was measured by adding known amounts of atropine (Sigma) and scopolamine (Sigma);recovery measured in this way was high at 94-96%. For analysis of alkaloids in the culture medium, the pH of 10 mL of medium was increased to 9.3 with 0.5 M (NH&SOdNHdOH buffer; the solution was then passed directly through a C18 Sep-Pak cartridge. Alkaloid was retained on the packing by bonding under alkaline aqueous conditions. The column was eluted with 10 mL of HPLC solvent. Sep-Pak eluate was filtered through a 0.45-pm filter (Waters) into HPLC vials. Analysis of atropine and scopolamine was performed using a 10 pm 300 mm x 3.9 mm p-Bondapak C18 column (Waters) with the mobile phase described earlier at a flow rate of 1mL min-l and an injection volume of 15 pL. Detection was by W absorption at 205 nm. Standard solutions of atropine (50-250 mg L-l) and scopolamine (50- 100 mg L-l) were used for calibration. Biomass Elemental Analysis. C, H, and N analysis of dried roots from individual segments of the bioreactor was carried out using a Perkin-Elmer Model 240B elemental analyzer with acetanilide (71.09% carbon, 6.71% hydrogen, 10.36% nitrogen) as the calibrating standard. The analyzer was loaded with 1.2-2 mg of freeze-dried roots; ash was determined after heating in a furnace at 800 "C. The oxygen content was calculated by balance. Ammonia and Nitrate Analyses. Residual concentrations of nitrate and ammonium ions in culture medium were determined using Spectroquant 14773 and Spectroquant 14752 assay kits (Merck, Darmstadt), respectively.
Stoichiometric Analysis The stoichiometry of hairy root growth was examined using data from multiple batch cultures in the segmented bubble column reactor. Batch growth is less well suited to stoichiometric studies than continuous or fed-batch cultures in which steady or quasi steady states can be achieved (van Gulik et al., 1992; Schnapp et al., 1991). However, steady state is impractical or impossible to achieve in hairy root reactors as roots cannot be removed from the vessel during operation. In these circumstances, it is difficult to establish well-defined values of the specific growth rate; determination of yield and kinetic parameters may also be affected by intracellular storage of substrates and transient changes in biomass composition. The mass balance of gaseous components is also troublesome in batch cultures. Many plant cell cultures do not respond well to buffered medium or to control of pH to a constant value (Nesius and Fletcher, 1973; Banthorpe and Brown, 1990);pH control has been found to alter biomass yields and interfere with ammonia and nitrate uptake (Martin and Rose, 1976). Carbon dioxide evolution and apparent respiratory quotients can be
431
loo
0.01
1 0
5
I
I
1
,
10
15
20
25
30
35
,
1
40
45
Time (d)
Figure 2. Growth curve for A. belladonna hairy roots.
altered significantly by the usual fluctuations in broth pH that occur during plant cell growth and expansion phases. Experimental limitations such as these reduce the number of measurable parameters that can be applied in stoichiometric analysis of hairy roots. On the other hand, the relative simplicity of plant culture media and the absence of complex substrates such as yeast extract facilitate elemental balance techniques. In this work, the system was fully determined (van der Heijden et al., 19941, in that all nonmeasured stoichiometric coefficients could be calculated from measured yields. The following general equation is used to describe hairy root growth in Murashige and Skoog medium, with sucrose as the carbon and energy source:
+ aO, + bNH, + cHNO, dCHaOpN, + eCHaOpN, + f CO, + gH,O
C6H,,06
(biomass)
(excreted byproduct)
(1) where a,b, c , d , e , f,andg are stoichiometric coefficients. Substrate taken up by the roots is glucose or fructose (CeH1206); sucrose supplied to the culture is rapidly hydrolyzed in the medium prior to assimilation (Sharp and Doran, 1990). MS medium contains two nitrogen sources: ammonia and nitrate. CH,OpN, is the molecular formula for biomass; values of a, @, and y are obtained using elemental analysis. Macromolecular byproducts accumulate in the medium during hairy root culture as a result of cell lysis and excretion of polysaccharides and protein. The average elemental composition of byproduct is assumed to be same as the biomass (van Gulik et al., 1992). Seven stoichiometric coefficients are needed to complete eq 1. Balance equations for the four major elements C, H, 0,and N must be supplemented by three additional measured values for the system to be fully determined. For hairy roots, the most convenient parameters to measure are biomass yields from the two nitrogen sources and sugar. We assume that nitrogen is not involved in maintenance metabolism; therefore, measured yields from nitrogen compounds can be used directly to calculate corresponding stoichiometric coefficients.
Results Biomass Culture Characteristics. Data for biomass growth are shown in Figure 2. Average root density in the bioreactor reached 9.9 g L-l dry weight after 43 days of culture. Because segment 1was not inoculated with roots, the root densities achieved in individual segments
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0'31
0.0
' 2.0
\ '
I 0
'
I
I
I
5
10
15
'
'
'
'
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20
25
30
35
40
45
I
-
0.0
0 5
0
10
15
20
25
30
40
35
45
Time (d)
Time (d)
Figure 3. Variation in specific growth rate ( 0 )and specific rate of sugar consumption (0) during batch culture ofA. belladonna hairy roots.
Figure 6. Average specific atropine content in the biomass (M) and volumetric atropine accumulation in the reactor ( 0 )during batch culture of A. belladonna hairy roots. No atropine was found in the free medium.
20 25
1
0 0
10
20
30
40
50
70
60
80
Figure 4. Linear relationship between biomass production and sugar consumption for A. belladonna hairy roots. The observed biomass yield coefficient was constant at 0.35 g g-l. 80
n
1
-
60
t
0
5
10
15
O
I 0
Total sugar consumed (9)
20
25
Biomass produced (g dry weight)
Figure 5. Linear relationship between biomass production and consumption of nitrate (M) and ammonium ( 0 ) ions by A. belladonna hairy roots cultured in MS medium. The ratio of millimoles of NO3 to millimoles of NH3 consumed was 1.251. were considerably higher than the average, up to 17 g L-' dry weight. The volume of free liquid decreased from 2.5 to 1.25 L due to absorption of water by new biomass. The growth data in Figure 2 suggest that the roots were close to stationary phase after 43 days; however, ca. 14 g L-' sugar and 24.5 mM nitrogen remained in the medium at this time. The patterns of sucrose hydrolysis and sugar uptake were similar to those reported previously for A. belladonna hairy roots (Sharp and Doran, 1990). Sucrose was completely hydrolyzed
5
10
15
20
Segment biomass density (g C' dry weight)
Figure 7. Correlation between biomass density and specific atropine level in roots in different segments of the reactor. At low biomass density, atropine content was relatively high. At root densities above ca. 5 g L-' dry weight, the specific atropine content at all reactor locations remained roughly constant with an average value of 1.4mg g-l.
to glucose and fructose by day 28. Instantaneous specific rates of growth and total sugar consumption evaluated by graphical differentiation of concentration data are shown in Figure 3. The specific growth rate of roots over the entire reactor declined with culture time from an initial value of 0.29 day-' (doubling time, 2.4 days). The specific rate of total sugar uptake also declined from 1.1 to 0.11 day-' over the culture period. The apparent biomass yield from substrate was determined from a plot of biomass produced versus total sugar consumed. As shown in Figure 4, the observed yield was approximately constant during the culture period at 0.35 g g-' dry weight. Because the yield value did not change significantly with growth rate, substrate requirements for maintenance could be neglected (Pirt, 19651, and the stoichiometric yield for biomass could be considered equal to the observed yield. Murashige and Skoog medium contains two nitrogen sources, nitrate and ammonia, at a molar ratio of 1.91:l. The relationship between consumption of ammonium and nitrate ions and production of hairy root biomass is shown in Figure 5. Yield of biomass from both nitrogen sources remained approximately constant during culture; for each gram dry weight roots produced, 2.87 mmol of NO3 and 2.29 mmol of NHs were consumed. The ratio of millimoles of NO3 to millimoles of NH3 consumed was therefore 1.25:l.
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Table 1. Average Elemental Composition and Ash Content of A. belladonna Hairy Roots as a Function of Batch Culture Time" time (days) 20 28 43 carbon 38.4 f 0.4 38.7 f 0.5 38.2 f 1.5 5.1 f 0.1 5.4 =k 0.2 hydrogen 5.2 f 0.1 39.7 f 1.7 43.5 f 2.2 oxygen 39.5 f 0.6 nitrogen 5.8 f 0.2 6.0 f 0.2 5.0 f 0.7 11.1 f 0.4 10.5 i 1.9 7.9 f 0.3 ash UValues are expressed as w t % dry biomass; f represents standard deviation for four replicate measurements.
The dissolved oxygen tension measured in segment 1 of the reactor declined steadily from 100%air saturation a t the beginning of the culture to ca. 40% after 43 days. The pH varied between 4.8 and 5.5, from an initial value of 5.2. Consumption of oxygen and production of carbon dioxide were evaluated from analysis of the fermentor exit gas; the respiratory quotient (RQ = moles of C02 producedmoles of 02 consumed) remained at about 1.10 for most of the culture period. Mixing time measured at an air flow rate of 200 mL min-' increased from 2 min 1 day after inoculation to 12 min a t day 14. Atropine Production. Roots and residual culture medium were assayed for atropine and scopolamine by HPLC. Only atropine was detected in measurable amounts; free liquid in the reactor contained negligible quantities of alkaloid. Figure 6 shows specific and volumetric alkaloid concentrations averaged over the entire bioreactor as a function of culture time. Specific atropine content in the roots declined steadily from 4.1 mg g-' dry weight to ca. 1.4 mg g-' after 28 days. Volumetric atropine levels continued to increase due to biomass production; 35 mg or 14 mg L-l atropine was produced over the 43-day culture period. In Figure 7, the specific atropine level in the roots is plotted as a function of biomass density in the individual bioreactor
segments. Irrespective of culture time, roots in segments with low biomass density contained relatively high levels of atropine. As local biomass density increased above 5 g L-l, the specific atropine content remained roughly constant with an average value of 1.4 mg g-l. Biomass Composition. "he composition of hairy roots in terms of the major elements (C, H, 0, and N) and ash was determined for each segment of the bioreactor at culture times 20,28, and 43 days. Variation in root composition between segments was negligible. Results for elemental composition and ash content averaged over the entire vessel are listed in Table 1. Biomass composition can be represented by an average formula CH1.6300.8&13 with 9.8 w t % ash; the corresponding biomass molecular weight (cells ash) is 31.3. StoichiometricAnalysis. From Figures 4 and 5, the biomass yield from substrate is 0.35 g g-l, and the biomass yields from nitrate and ammonia are 2.87 and 2.29 mmol g-l, respectively. After these values are converted to molar units and elemental balances are applied, eq 1becomes
+
C,H,,O,
+ 3.40, + 0.15NH3 + 0.18HN03 -
2*0CH1.6300.80N0.13
(biomass)
-k
0.50CH1.6300.80N0.13
+
(excreted byproduct)
3.5c0, i- 4.3H20 (2) From the elemental balance, the respiratory quotient is 1.04, which is close to the measured value of 1.10.
Discussion Division of the bubble column into segments and installation of multiple spargers significantly increased the maximum biomass level from 1.3 g L-' dry weight obtained with a standard 2.5-L air-driven vessel (Sharp
Table 2. Elemental Formulae for Cultured Plant Biomass plant system biomass formula Atropa belladonna hairy roots CHi.6300.soNo.13 (batch bubble column) Catharanthus roseus suspension CHi.soOo.7eNo.iz (stirred batch reactor) CHi.6~00.6sN0.15 (stirred chemostat) CHi.do.62No.11 (shake flasks, active growth) Nicotiana tabacum suspension CHi.7500.74No.13 (stirred batch reactor) CHi.6400.73N0.16 (stirred chemostat) Medicago sativa suspension CH2.ooOo.saNo.i.i (stirred batch reactor)
ash (wt%) 9.8
reference present work
7.6
van Gulik et al., 1992
13.5
van Gulik et al., 1992
15.9
Rho and And& 1991
10.5
van Gulik et al., 1992
16.2
van Gulik et al., 1992 Pareilleux and Chaubet, 1981
Table 3. Stoichiometric Equations for Growth of Plant Cells in Vitro Atropa belladonna hairy roots batch bubble column CsHi206 + 3.402 0.15NH3 0.18HN03 2.OCHi,6300.8oNo.i3+ 0.50CHi.6300.soNo.i3+ 3.5Co2 + 4.3H20 (biomass) (excreted byproduct) Eschscholtzia californica suspension culture shake flasks C6Hi206 2.6902 + 0.032NH4 + 0.388N03 2.55CHz,ooOo.s3No.i7+ 3.46c02 batch bubble column C6Hi206 + 2.1002 0.038NH4 + 0.462N03 3.00CHz.ooOo.s3No.i7+ 3.OOc02 Catharanthus roseus suspension culture shake flasks (active growth) C6Hi206 2.3202 + 0.32N03 2.96CHi.5700.62No.ii + 3.04C02 i3.67H20
+
+
+
+
-
-
+
-
reference present work
Taticek et al., 1990
+ 3.52H20 + 3.08H20 Rho and Andr6, 1991
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and Doran, 1990) to 9.9 g L-l dry weight. However, although the growth of roots had virtually ceased after 43 days, substantial amounts of sugar and nitrogen compounds remained in the medium. Previous work has shown that oxygen supply is a limiting factor in submerged hairy root cultures (McKelvey et al., 1993; Yu and Doran, 1994);further improvements in mass transfer conditions are required to ensure complete conversion of substrates. With mixing severely impeded in the bubble column, lzLa measurements have little meaning as an indicator of mass transfer rate; the environment in the reactor is not uniform, and the values depend on where the measurements are taken. The relationship between specific atropine content and local biomass density in Figure 7 suggests that atropine accumulation may also be affected by oxygen supply. The elemental composition of A. belladonna hairy roots is similar to that of other plant cells cultured in vitro. A comparison of results from this and other work is presented in Table 2. A balanced stoichiometric equation for hairy root growth was obtained using three measured yield coefficients. Solution of the stoichiometric equation was not overdetermined by redundant experimental values; accordingly, it was not possible to check the consistency of the data using statistical analysis or material balance principles (Wang and Stephanopoulos, 1983). Byproduct formation was included in the stoichiometric analysis following work by van Gulik et al. (1989), which showed that the ratio of carbon used for biomass production to carbon used for byproduct synthesis in Catharanthus roseus suspensions averaged about 4:l. In the present work, this ratio was also found to be 4:l. Of the carbon supplied to the roots as sugar, 8.3% was excreted as byproducts; this can be compared with 6-8% in C. roseus and Nicotiana tabacum suspensions (van Gulik et al., 1992). Increased foaming of hairy root broths during culture without biomass disintegration or root necrosis is possible indirect evidence of byproduct accumulation in the reactor. Calculated coefficients in the stoichiometric equation for hairy roots are comparable to those reported for suspension cultures, as indicated in Table 3.
Acknowledgment We are grateful to Malcolm Noble for assistance with the HPLC and to Bill Khoo for performing the elemental analyses. This work was supported by the Australian Research Council (ARC). Literature Cited Aird, E. L. H.; Hamill, J. D.; Rhodes, M. J. C. Cytogenetic analysis of hairy root cultures from a number of plant species transformed by Agrobacterium rhizogenes. Plant Cell, Tissue Organ Cult. 1988, 15, 47-57. Banthorpe, D. V.; Brown, G. D. Growth and secondary metabolism in cell cultures of Tanacetum, Mentha and Anethum species in buffered media. Plant Sci. 1990, 67, 107-113. Buitelaar, R. M.; Langenhoff, A. A. M.; Heidstra, R.; Tramper, J. Growth and thiophene production by hairy root cultures of Tagetes patula in various two-liquid-phase bioreactors. Enzyme Microb. Technol. 1991, 13, 487-494. Davioud, E.; Kan, C.; Hamon, J.; Tempe, J.; Husson, H.-P. Production of indole alkaloids by in vitro root cultures from Catharanthus trichophyllus.Phytochemistry 1989,28,26752680. DiIorio, A. A.; Cheetham, R. D.; Weathers, P. J. Growth of transformed roots in a nutrient mist bioreactor: reactor performance and evaluation. Appl. Microbiol. Biotechnol. 1992,37,457-462. Flores, H. E. In Biotechnology in Agricultural Chemistry; LeBaron, H. M., Mumma, R. O., Honeycutt, R. C., Duesing,
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Abstract published in Advance ACS Abstracts, April 15,1995.
