PiantCeU Reports
Plant Cell Reports (1992) 11:605-608
9 Springer-Verlag1992
Bioreactor studies on the effect of dissolved oxygen concentrations on growth and differentiation of carrot (Daucus carota L.) cell cultures V~ronique Jay, Simone Genestier, and Jean-Claude Courduroux Laboratoire de Biologic et Physiologic V~g~tales, Universit~ Blaise Pascal, 4 rue Ledru, F-63038 Clermont-Ferrand Cedex 1, France Received March 26, 1992/Revised version received July 1, 1992 - Communicated by A.M. Boudet
ABSTRACT A bioreactor control system w a s used to investigate the effects of t w o dissolved oxygen concentrations (10% and 100%) on the g r o w t h and differentiation of Daucus carota L. cell cultures. The strategy used allowed the dissolved oxygen concentration to be controlled w i t h o u t the need for changing either the agitator speed or the total gas flow rate. During the proliferation phase, reducing oxygen resulted in a lower growth rate and in a delay in sugar uptake kinetics. Nonetheless, varying levels of oxygen were observed to have no effect on the final dry bJomass. The higher alcohol dehydrogenase activity obtained under reduced oxygen conditions suggests that proliferating cultures adapted to the hypoxic environment by inducing alcoholic fermentation. Cell differentiation w a s highly sensitive to reduced oxygen since under this condition, the somatic embryo production w a s inhibited by about 7 5 % . Sugar uptake and embryo formation were also delayed.
Key words: Daucus carota- BJoreactor- Dissolved oxygenSomatic embryogenesis- Growth and differentiation. Abbreviations: ADH: alcohol dehydrogenase; 2,4-D: 2,4dichlorophenoxyacetic acid ; DO2: dissolved oxygen; SE: somatic embryos, Tris: tris(hydroxymethyl)-aminoethane. INTRODUCTION The induction of somatic embryogenesis has been recognized as a technique for producing large numbers of individuals and consequently is of great interest in the development of artificial seeds (Murashige 1980, Redenbaugh et al. 1987). However, for a commercialization of artificial seeds, optimization and control of the process is necessary. Since there is a limited understanding of h o w plant cells are affected by their physical and chemical environment, studies in a bioreactor system are preferred over shaking flask studies as t h e y allow monitoring and control of culture conditions such as temperature, pH, agitation, aeration. In this study, w e used a bioreactor system to investigate the gaseous environment and more particularly oxygen effects on carrot somatic embryogenesis. Several workers have s h o w n a correlation between the culture performance and the oxygen concentration. It is generally agreed that a suboptimal gas transfer m a y reduce the culture g r o w t h of plant cell (Kessel and Carr 1972, Preil et al. 1988, Snape et al. 1989, Tate and Payne 1991). Oxygen also plays a role in e m b r y o differentiation. It has been s h o w n that carrot cells require l o w oxygen levels (below a critical level ~ 16%) for successful somatic embryogenesis (Kessel and Carr 1972). There are several strategies available to alter the gas
Correspondence to: V. Jay
concentration. Most of the experimental methods rely on changing the aeration and/or the agitation speed (Williams et al. 1986, Kessel and Carr 1972). This type of regulation involves modifications in the mass transfer properties of the bioreactor as also in the gaseous compounds and the shear environment. Thus, the effects of oxygen cannot be studied properly since these other parameters can affect embryogenesis. We have used a bioreactor control system which does not rely on changing aeration and agitation. By means of a bio-processor, the oxygen concentration w a s varied under constant agitation and air flow rate. The objectives of this w o r k were to study the effects of t w o dissolved 0 2 concentrations (10% and 100%) during the proliferation (presence of auxin) and the differentiation (auxin free medium) phases of carrot somatic embryogenesis (Daucus carota L.). These oxygen concentrations were meant to encompass the concentrations of control bioreactor culture (without regulation) which were around 5 0 % air saturation at the end of experiment. We investigated biomass kinetics, embryo production, and sugar assimilation in the batch bioreactor system. Furthermore, since the oxygen limitation m a y induce an alcoholic fermentation, alcohol dehydrogenase activity w a s determined. MATERIAL AND METHODS Plant material - Cell suspensions of carrot were initiated from hypocotyls of domestic carrot (Daucus carota L.). The suspensions were subcultured every ten days in a Murashige and Skoog (1962) liquid medium supplemented with adenine (11.7 #M) and 2,4-D (1.8 pM). 2 ml of packed cell volume filtered through steel screens of 500 and 100/Jm pore size were inoculated into 100 ml of fresh medium. All cultures were kept in Erlenmeyer flasks maintained at 26~ on an orbital shaker (100 rpm) under a photoperiod of 16 hours. The fluorescent source (maximum fluence rate 45 pmol m-2s"1) consisted of 58W white daylight tubes (Mazdafluor LJ). The batch fermentor - We used two 3 litre glass vessel bioreactors (Applikon, Holland) with a working volume of 1.7 I for the batch studies. Temperature was maintained at 27 oc with a water jacket. Each bioreactor was equipped with oxygen and pH probes (Ingold, France). The mixing was achieved with four blade impellers at 100 rpm during the growth phase. Depending on the biomass increase, between 50 to 150 rpm was employed for the differentiation phase . The aeration rate was achieved using a compressor. It was maintained constant at 200 ml/min for the proliferation phase and at 150 ml/min during the differentiation phase. The bioreactor was loaded with about 8.5 g (fresh weight) of an inoculum of aggregate cells whose size was between 100 and 500 pm for the undifferentiated phase. For the differentiation phase, we inoculated about 850 mg of 10 day old packed cells between 50 and 100/Jm. The growth medium was identical to that used for the cultures in the Erlenmeyer flasks. The medium employed for the differentiation phase was
606 similar to the proliferation phase except 2,4-D was omitted. All experiments were run under a photoperiod of 16 hours (the maximum fluence rate was 25 ~,mol m-2s -1). During the growth phase, silicon (Bevaloid 6432, Rh0ne-Poulenc, France) was added to avoid formation of foam on the surface of the suspension. Oxygen regulation - Readings from the dissolved 02 probe were reported as the percentage of the oxygen concentration, where 100 % corresponds to the value of a solution saturated with air at 27~ The bio+ processor ADI 1020 (Applikon) converted the milliamps signals from the probe to digital signals which were recorded on the computer (Tandon PC) by an appropriate software (Biowatch - Applikon) - (Fig. 1). Depending on the oxygen concentration required, pure oxygen or pure nitrogen were used as a supplement. The former was employed to maintain oxygen concentration at 100% and the latter to adjust it to 10%. Depending on the control factors selected and the signals from the probe, ADI 1020 was able to open or to close the gas line valve to keep the oxygen concentration at the required level. The valve opening time was short enough not to modify significantly the total air flow held constant by a flow meter. With this type of regulation, the dissolved oxygen concentration remained stable throughout the culture. All the experiments were done in triplicate. Determination of the biomass - Samples of culture were removed and a known volume was filtered through a GF/A filter (Whatman) under a reduced pressure. The recovered cells were weighed, and then were dried 24 h at 80~ to determine the dry weight. The growth rate (/J) was calculated during exponential growth as the slope of a linear regression of the In (dry weight) versus time. The doubling time (Td) was based on the growth rate where Td = 0.693//1. The cell viability was investigated by a fluorescein diacetate staining method (Widholm 1972). Assays of the culture medium - Every few days, a sample of medium was filtered on a cellulose acetate filter of 0.2 pm pore size and sucrose, fructose, glucose were assayed using a diagnostic kit (Boehringer, France). ADH assay - Cells were filtered under vacuum on a GF/A filter (Whatman) and 700 mg were homogenized in liquid nitrogen with a mortar and pestle. The powder obtained was resuspended in 400/JI of Tris-HCI buffer 100 mM (pH 8.4) containing 50 mM sucrose and 2 mM glutathione. Homogenates were centrifuged at 14,000 rpm for 10 rain. The supernatant was used for measuring the ADH activity. Our assay was based on the Bergmeyer (1983) method. The reaction mixture (3 ml) contained 85 mM tris buffer (pH 8,4), 29 mM ethanol, 0.12 mM NAD, 0,023 mM phenazine methosulfate, 0.17 mM p-iodonitrotetrazolium violet and 5 pl of diluted extract. The change in absorbance at 500nm was monitored over a period of 20 min at 37~ One unit of ADH was the amount that produces one pmol NADH min "1 under assay conditions.
The total protein content of cell extracts was measured by a dye binding assay (Bradford 1976). 0v~ntification of somatic embryos - At the end of the growth phase, samples were removed. Embryo differentiation was induced by inoculating screened (50-100 pm) and washed suspensions into Erlenmeyer flasks containing a medium lacking 2,4-D at a density of 0.5 /JI packed cell volume per ml of medium. Embryos were grown in 50 ml of medium. After 20 days, 1 ml was removed and mixed with 4 ml of 2% agar in water in petri dishes (36 mm diameter) to facilitate counting under the microscope. For the differentiation phase, an aliquot was removed after approximately 20 days of the run and then the embryos were counted as described above. We took account of all the embryo stages. If necessary, the counting was done after a ten fold dilution. RESULTS Effects o f t w o dissolved o x v a e n c o n c e n t r a t i o n s on the proliferation phase The e f f e c t s o f t w o dissolved o x y g e n c o n c e n t r a t i o n s ( 1 0 % and 1 0 0 % ) w e r e investigated on the carrot cells' g r o w t h in t e r m s of fresh and dry w e i g h t (Fig.2). The g r o w t h c u r v e s w e r e characterized by a lag phase of a b o u t 1 d a y f o l l o w e d by an e x p o n e n t i a l phase. Based on the dry biomass variations, w e o b s e r v e d a s t a t i o n a r y phase f o r 1 0 0 % DO2 a f t e r d a y 10 while the f r esh w e i g h t continued t o increase. This result coincided w i t h cell enlargement. No s t a t i o n a r y phase w a s o b s e r v e d f o r 1 0 % DO2. The g r o w t h rates w e r e established t o q u a n t i f y the g r o w t h behaviour of _the t w o t y p e s of conditions. For 1 0 0 % DO2, p = 0 . 2 3 d a y -1 ( T d = 3 days) and w a s higher t h a n for 1 0 % D O 2 since the c o r r e s p o n d i n g value w a s p = 0 . 1 8 day-" ( T d = 3 . 9 days). H o w e v e r , e~ven t h o u g h the g r o w t h w a s f a s t e r f o r 1 0 0 % D O 2 , the final dry biomasses w e r e not significantly d i f f e r e n t on d a y 12 (for 1 0 0 % DO2) and d a y 13 (for 1 0 % DO2).
5D
8 0 84 )
/
60
/
.~0
/
w
=_. 4 0 |
/
/
~0 v
2D
..= m ]= ==
20
Probes im O2 pH
l"
I
AD11020
0'01 1
') ?
F I I
1 eiore=ctot
I
I
--
t e'~176 ........
Computer
F']
i,L~u,m
'2'
i.
,m~,
,~,
'4'
6 '8 Time ( days )
.M
:
:
10
: GO 12 13
Fig.2. Influence of the D O 2 c o n c e n t r a t i o n on the g r o w t h o f u n d i f f e r e n t i a t e d c a r r o t cells. Dry w e i g h t (g/I) • and f r esh w e i g h t (g/I) - - e - f o r 1 0 0 % DO2. Dry weight (g/I) I'--I and f r esh w e i g h t (g/l) f o r 1 0 % DO2. (Generally the st andar d d e v i a t i o n s are ~< t o 1 0 % o f the m e a n value e x c e p t f o r the f r e s h w e i g h t m e a s u r e d at t h e end of t h e culture w h e r e t h e y are near 2O%).
_J
i
Gases:
02 or N2
Fig.l. The control system.
T o c h a r a c t e r i z e the culture, the variations in glucose, f r u c t o s e and s u c r o s e w e r e i n v e s t i g a t e d in the culture medium. H y d r o l y s i s o f s u c r o s e rapidly occurred since a t d a y 2, t her e w a s s c a r c e l y a n y s u c r o s e left ( < 2 g / I ) f o r the t w o runs (Fig.3). The sugar u p t a k e w a s d e l a y e d w i t h 1 0 % DO2 . U p t a k e w a s only s e e n a f t e r d a y 6 whi l e it occur r ed a f t e r d a y 4 f o r 1 0 0 % DO2. A s p r e v i o u s l y o b s e r v e d ( M c D o n a l d et al. 1 9 8 9 ) , g l u c o s e w a s utilized pr ef er ent i al l y over f r u c t o s e .
607 2.2 9 20~
--
~
10
lOO~ DO2
1.8_= 1.4 o
==_ r
1.0
f
.-"?+
.IF
.K.
0.6 0.2 0.0 +
2
0
4
6 8 Time ( days)
10
12 13
Fig.4. Specific ADH activity during the proliferation phase for 100% DO2 I ~ and for 10% DO2 F--]. Error bars indicate standard deviation. (* values significantly higher (p~
Plant Cell Reports (1992) 11:605-608
9 Springer-Verlag1992
Bioreactor studies on the effect of dissolved oxygen concentrations on growth and differentiation of carrot (Daucus carota L.) cell cultures V~ronique Jay, Simone Genestier, and Jean-Claude Courduroux Laboratoire de Biologic et Physiologic V~g~tales, Universit~ Blaise Pascal, 4 rue Ledru, F-63038 Clermont-Ferrand Cedex 1, France Received March 26, 1992/Revised version received July 1, 1992 - Communicated by A.M. Boudet
ABSTRACT A bioreactor control system w a s used to investigate the effects of t w o dissolved oxygen concentrations (10% and 100%) on the g r o w t h and differentiation of Daucus carota L. cell cultures. The strategy used allowed the dissolved oxygen concentration to be controlled w i t h o u t the need for changing either the agitator speed or the total gas flow rate. During the proliferation phase, reducing oxygen resulted in a lower growth rate and in a delay in sugar uptake kinetics. Nonetheless, varying levels of oxygen were observed to have no effect on the final dry bJomass. The higher alcohol dehydrogenase activity obtained under reduced oxygen conditions suggests that proliferating cultures adapted to the hypoxic environment by inducing alcoholic fermentation. Cell differentiation w a s highly sensitive to reduced oxygen since under this condition, the somatic embryo production w a s inhibited by about 7 5 % . Sugar uptake and embryo formation were also delayed.
Key words: Daucus carota- BJoreactor- Dissolved oxygenSomatic embryogenesis- Growth and differentiation. Abbreviations: ADH: alcohol dehydrogenase; 2,4-D: 2,4dichlorophenoxyacetic acid ; DO2: dissolved oxygen; SE: somatic embryos, Tris: tris(hydroxymethyl)-aminoethane. INTRODUCTION The induction of somatic embryogenesis has been recognized as a technique for producing large numbers of individuals and consequently is of great interest in the development of artificial seeds (Murashige 1980, Redenbaugh et al. 1987). However, for a commercialization of artificial seeds, optimization and control of the process is necessary. Since there is a limited understanding of h o w plant cells are affected by their physical and chemical environment, studies in a bioreactor system are preferred over shaking flask studies as t h e y allow monitoring and control of culture conditions such as temperature, pH, agitation, aeration. In this study, w e used a bioreactor system to investigate the gaseous environment and more particularly oxygen effects on carrot somatic embryogenesis. Several workers have s h o w n a correlation between the culture performance and the oxygen concentration. It is generally agreed that a suboptimal gas transfer m a y reduce the culture g r o w t h of plant cell (Kessel and Carr 1972, Preil et al. 1988, Snape et al. 1989, Tate and Payne 1991). Oxygen also plays a role in e m b r y o differentiation. It has been s h o w n that carrot cells require l o w oxygen levels (below a critical level ~ 16%) for successful somatic embryogenesis (Kessel and Carr 1972). There are several strategies available to alter the gas
Correspondence to: V. Jay
concentration. Most of the experimental methods rely on changing the aeration and/or the agitation speed (Williams et al. 1986, Kessel and Carr 1972). This type of regulation involves modifications in the mass transfer properties of the bioreactor as also in the gaseous compounds and the shear environment. Thus, the effects of oxygen cannot be studied properly since these other parameters can affect embryogenesis. We have used a bioreactor control system which does not rely on changing aeration and agitation. By means of a bio-processor, the oxygen concentration w a s varied under constant agitation and air flow rate. The objectives of this w o r k were to study the effects of t w o dissolved 0 2 concentrations (10% and 100%) during the proliferation (presence of auxin) and the differentiation (auxin free medium) phases of carrot somatic embryogenesis (Daucus carota L.). These oxygen concentrations were meant to encompass the concentrations of control bioreactor culture (without regulation) which were around 5 0 % air saturation at the end of experiment. We investigated biomass kinetics, embryo production, and sugar assimilation in the batch bioreactor system. Furthermore, since the oxygen limitation m a y induce an alcoholic fermentation, alcohol dehydrogenase activity w a s determined. MATERIAL AND METHODS Plant material - Cell suspensions of carrot were initiated from hypocotyls of domestic carrot (Daucus carota L.). The suspensions were subcultured every ten days in a Murashige and Skoog (1962) liquid medium supplemented with adenine (11.7 #M) and 2,4-D (1.8 pM). 2 ml of packed cell volume filtered through steel screens of 500 and 100/Jm pore size were inoculated into 100 ml of fresh medium. All cultures were kept in Erlenmeyer flasks maintained at 26~ on an orbital shaker (100 rpm) under a photoperiod of 16 hours. The fluorescent source (maximum fluence rate 45 pmol m-2s"1) consisted of 58W white daylight tubes (Mazdafluor LJ). The batch fermentor - We used two 3 litre glass vessel bioreactors (Applikon, Holland) with a working volume of 1.7 I for the batch studies. Temperature was maintained at 27 oc with a water jacket. Each bioreactor was equipped with oxygen and pH probes (Ingold, France). The mixing was achieved with four blade impellers at 100 rpm during the growth phase. Depending on the biomass increase, between 50 to 150 rpm was employed for the differentiation phase . The aeration rate was achieved using a compressor. It was maintained constant at 200 ml/min for the proliferation phase and at 150 ml/min during the differentiation phase. The bioreactor was loaded with about 8.5 g (fresh weight) of an inoculum of aggregate cells whose size was between 100 and 500 pm for the undifferentiated phase. For the differentiation phase, we inoculated about 850 mg of 10 day old packed cells between 50 and 100/Jm. The growth medium was identical to that used for the cultures in the Erlenmeyer flasks. The medium employed for the differentiation phase was
606 similar to the proliferation phase except 2,4-D was omitted. All experiments were run under a photoperiod of 16 hours (the maximum fluence rate was 25 ~,mol m-2s -1). During the growth phase, silicon (Bevaloid 6432, Rh0ne-Poulenc, France) was added to avoid formation of foam on the surface of the suspension. Oxygen regulation - Readings from the dissolved 02 probe were reported as the percentage of the oxygen concentration, where 100 % corresponds to the value of a solution saturated with air at 27~ The bio+ processor ADI 1020 (Applikon) converted the milliamps signals from the probe to digital signals which were recorded on the computer (Tandon PC) by an appropriate software (Biowatch - Applikon) - (Fig. 1). Depending on the oxygen concentration required, pure oxygen or pure nitrogen were used as a supplement. The former was employed to maintain oxygen concentration at 100% and the latter to adjust it to 10%. Depending on the control factors selected and the signals from the probe, ADI 1020 was able to open or to close the gas line valve to keep the oxygen concentration at the required level. The valve opening time was short enough not to modify significantly the total air flow held constant by a flow meter. With this type of regulation, the dissolved oxygen concentration remained stable throughout the culture. All the experiments were done in triplicate. Determination of the biomass - Samples of culture were removed and a known volume was filtered through a GF/A filter (Whatman) under a reduced pressure. The recovered cells were weighed, and then were dried 24 h at 80~ to determine the dry weight. The growth rate (/J) was calculated during exponential growth as the slope of a linear regression of the In (dry weight) versus time. The doubling time (Td) was based on the growth rate where Td = 0.693//1. The cell viability was investigated by a fluorescein diacetate staining method (Widholm 1972). Assays of the culture medium - Every few days, a sample of medium was filtered on a cellulose acetate filter of 0.2 pm pore size and sucrose, fructose, glucose were assayed using a diagnostic kit (Boehringer, France). ADH assay - Cells were filtered under vacuum on a GF/A filter (Whatman) and 700 mg were homogenized in liquid nitrogen with a mortar and pestle. The powder obtained was resuspended in 400/JI of Tris-HCI buffer 100 mM (pH 8.4) containing 50 mM sucrose and 2 mM glutathione. Homogenates were centrifuged at 14,000 rpm for 10 rain. The supernatant was used for measuring the ADH activity. Our assay was based on the Bergmeyer (1983) method. The reaction mixture (3 ml) contained 85 mM tris buffer (pH 8,4), 29 mM ethanol, 0.12 mM NAD, 0,023 mM phenazine methosulfate, 0.17 mM p-iodonitrotetrazolium violet and 5 pl of diluted extract. The change in absorbance at 500nm was monitored over a period of 20 min at 37~ One unit of ADH was the amount that produces one pmol NADH min "1 under assay conditions.
The total protein content of cell extracts was measured by a dye binding assay (Bradford 1976). 0v~ntification of somatic embryos - At the end of the growth phase, samples were removed. Embryo differentiation was induced by inoculating screened (50-100 pm) and washed suspensions into Erlenmeyer flasks containing a medium lacking 2,4-D at a density of 0.5 /JI packed cell volume per ml of medium. Embryos were grown in 50 ml of medium. After 20 days, 1 ml was removed and mixed with 4 ml of 2% agar in water in petri dishes (36 mm diameter) to facilitate counting under the microscope. For the differentiation phase, an aliquot was removed after approximately 20 days of the run and then the embryos were counted as described above. We took account of all the embryo stages. If necessary, the counting was done after a ten fold dilution. RESULTS Effects o f t w o dissolved o x v a e n c o n c e n t r a t i o n s on the proliferation phase The e f f e c t s o f t w o dissolved o x y g e n c o n c e n t r a t i o n s ( 1 0 % and 1 0 0 % ) w e r e investigated on the carrot cells' g r o w t h in t e r m s of fresh and dry w e i g h t (Fig.2). The g r o w t h c u r v e s w e r e characterized by a lag phase of a b o u t 1 d a y f o l l o w e d by an e x p o n e n t i a l phase. Based on the dry biomass variations, w e o b s e r v e d a s t a t i o n a r y phase f o r 1 0 0 % DO2 a f t e r d a y 10 while the f r esh w e i g h t continued t o increase. This result coincided w i t h cell enlargement. No s t a t i o n a r y phase w a s o b s e r v e d f o r 1 0 % DO2. The g r o w t h rates w e r e established t o q u a n t i f y the g r o w t h behaviour of _the t w o t y p e s of conditions. For 1 0 0 % DO2, p = 0 . 2 3 d a y -1 ( T d = 3 days) and w a s higher t h a n for 1 0 % D O 2 since the c o r r e s p o n d i n g value w a s p = 0 . 1 8 day-" ( T d = 3 . 9 days). H o w e v e r , e~ven t h o u g h the g r o w t h w a s f a s t e r f o r 1 0 0 % D O 2 , the final dry biomasses w e r e not significantly d i f f e r e n t on d a y 12 (for 1 0 0 % DO2) and d a y 13 (for 1 0 % DO2).
5D
8 0 84 )
/
60
/
.~0
/
w
=_. 4 0 |
/
/
~0 v
2D
..= m ]= ==
20
Probes im O2 pH
l"
I
AD11020
0'01 1
') ?
F I I
1 eiore=ctot
I
I
--
t e'~176 ........
Computer
F']
i,L~u,m
'2'
i.
,m~,
,~,
'4'
6 '8 Time ( days )
.M
:
:
10
: GO 12 13
Fig.2. Influence of the D O 2 c o n c e n t r a t i o n on the g r o w t h o f u n d i f f e r e n t i a t e d c a r r o t cells. Dry w e i g h t (g/I) • and f r esh w e i g h t (g/I) - - e - f o r 1 0 0 % DO2. Dry weight (g/I) I'--I and f r esh w e i g h t (g/l) f o r 1 0 % DO2. (Generally the st andar d d e v i a t i o n s are ~< t o 1 0 % o f the m e a n value e x c e p t f o r the f r e s h w e i g h t m e a s u r e d at t h e end of t h e culture w h e r e t h e y are near 2O%).
_J
i
Gases:
02 or N2
Fig.l. The control system.
T o c h a r a c t e r i z e the culture, the variations in glucose, f r u c t o s e and s u c r o s e w e r e i n v e s t i g a t e d in the culture medium. H y d r o l y s i s o f s u c r o s e rapidly occurred since a t d a y 2, t her e w a s s c a r c e l y a n y s u c r o s e left ( < 2 g / I ) f o r the t w o runs (Fig.3). The sugar u p t a k e w a s d e l a y e d w i t h 1 0 % DO2 . U p t a k e w a s only s e e n a f t e r d a y 6 whi l e it occur r ed a f t e r d a y 4 f o r 1 0 0 % DO2. A s p r e v i o u s l y o b s e r v e d ( M c D o n a l d et al. 1 9 8 9 ) , g l u c o s e w a s utilized pr ef er ent i al l y over f r u c t o s e .
607 2.2 9 20~
--
~
10
lOO~ DO2
1.8_= 1.4 o
==_ r
1.0
f
.-"?+
.IF
.K.
0.6 0.2 0.0 +
2
0
4
6 8 Time ( days)
10
12 13
Fig.4. Specific ADH activity during the proliferation phase for 100% DO2 I ~ and for 10% DO2 F--]. Error bars indicate standard deviation. (* values significantly higher (p~
