Biotechnol. Prog. 1995, 11, 140-1 45
140
Cell Damage and Oxygen Mass Transfer during Cultivation of Nicotiana tabacum in a Stirred-TankBioreactor Chung-Han Ho, Kelley A. Henderson, and Gregory L. Rorrer” Department of Chemical Engineering, Oregon State University, Corvallis, Oregon 97331
Cell damage and oxygen mass transfer were investigated during the batch culture of Nicotiana tabacum (tobacco) cells in a 3-L Applikon stirred-tank bioreactor equipped with a marine-blade impeller. Total cell density, % culture viability, viable cell density, and kLa in the culture were measured over a 24-day cultivation period a t impeller speeds of 100-325 rpm under a fixed air flow of 0.43 w m and a temperature of 27 “C. The maximum total biomass density decreased from 11.8 g of dry cell weight (DCW)/L a t 100 rpm t o 8.6 g of DCW/L a t 325 rpm. The net specific biomass production rate of viable cells during the exponential phase and the viable cell death rate (kd) during the stationary phase were estimated from semilog plots of viable cell density vs cultivation time. Values for pv decreased from 0.175 to 0.077 day-’ whereas values for k d increased from 0.042 to 0.109 day-l with increasing impeller tip speed (from 24 t o 77 c d s ) , clearly showing that increasing agitation intensity increased the rate of cell damage. The effect was most pronounced at tip speeds of 24-60 c d s . With respect to oxygen mass transfer, culture kLa values were about 10-20% higher than their corresponding initial kLao values, but as the cell density increased, the values for kLa ultimately decreased. Despite the reduction in oxygen mass transfer rates, at impeller speeds of 100-325 rpm and a n aeration rate of 0.43 w m , oxygen starvation was not observed. Therefore, the reduction in biomass productivity can be attributed to cell damage by hydrodynamic forces and not by inadequate oxygen mass transfer.
Introduction Plant cells in liquid suspension culture can make valuable secondary metabolites. Often, significant levels of secondary metabolites are expressed only at high cell density during the late exponential and stationary phases of growth (Payne et al., 1987). The large-scale cultivation of aerobic plant cells to high cell density is not easy: nonuniform mixing, hydrodynamic shear damage of fragile plant cells, and low rates of oxygen mass transfer are potential barriers to process development (Tanaka, 1987; Panda et al., 1989). Although plant cell cultures are widely regarded as shear sensitive, there are relatively few reports on the effects of hydrodynamic shear forces on culture growth and secondary metabolite production under actual cultivation conditions, particularly in stirred-tank bioreactors. Meijer et al. (1993)recently reviewed the effects of hydrodynamic shear stress on cultured plant cells and concluded that shear sensitivity is best determined by a scaled-down version of the production system. However, previous studies on the shear sensitivity of plant cell cultures focused on the measurement of culture rheology or cell damage of plant cell suspension culture samples after exposure to a well-defined hydrodynamic shear field for a short length of time (Scragg et al., 1988; Hooker et al., 1989; Ballica et al., 1992). Previous bioreactor cultivation studies attempted to gauge the effect of agitation intensity on dry or wet total biomass density (Kat0 et al., 1975; Hooker et al., 1990; Leckie et al., 1991; Ballica and Ryu, 1993). Unfortunately, these bioreactor cultivation studies did not determine the long-term viability of the culture, nor did they attempt to quantify cell damage during the stationary phase of growth when secondary metabolite expression occurs.
* Author to whom correspondence should be addressed. 8756-7938/95/3011-0140$09.00/0
Cell damage can be avoided by operating the stirredtank bioreactor at low mixing speeds. Although the oxygen demand of plant cell cultures is low relative to that of microorganisms (Payne et al., 19871, high cell densities may lower the oxygen mass transfer coefficient, kLa, which could cause oxygen starvation at low mixing speeds where the initial kLa is also low. Although there are no reported studies on the direct measurement of kLa in a stirred-tank bioreactor culture of actively growing plant cells, both Tanaka (1987) and Jolicoeur et al. (1992) showed that the presence of agar “pseudo cells” in liquid medium within a stirred-tank bioreactor decreased KLa significantly with an increasing solids concentration, whereas Ballica and Ryu (1993) observed that kLa in an airlift bioreactor culture of Datura stramonium cells decreased with increasing loadings of freshly inoculated biomass. This investigation has two objectives. The first objective is to investigate viable cell damage to Nicotiana tabacum (tobacco)cells cultivated in a batch stirred-tank bioreactor during the exponential and stationary phases of growth at impeller speeds of 100-325 rpm. The net specific production rate of viable cells during the exponential phase (pv)and the viable cell death rate during the stationary phase ( k d ) will be correlated to the impeller tip speed. This simple approach to the analysis of shear sensitivity during batch cultivation of plant cells in a stirred-tank bioreactor has not been reported elsewhere to date. The second objective of this study is to characterize oxygen mass transfer to the cell suspension in the stirred-tank bioreactor during cultivation to the final cell density. Specifically, the kLa value of the cell suspension culture will be measured as a function of cultivation time at different impeller speeds in order to determine whether the presence of the cell biomass significantly affects the
0 1995 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1995, Vol. 11, No. 2
oxygen mass transfer rate and the degree of mixing in the stirred-tank bioreactor, particularly in dense cultures. Impeller speed was chosen as the variable to induce cell damage into the culture, as it most strongly determines the hydrodynamic shear forces in a stirred-tank bioreactor (Merchuk, 1991). Although increasing the aeration rate increases hydrodynamic stress, it also increases the likelihood of stripping critical dissolved gases, such as COZ and ethylene, from the medium (Ducos and Pareilleux, 1986; Hegarty et al., 1986; Kim et al., 19911, which could reduce culture growth and cell viability by processes other than hydrodynamic stress. An aeration rate of 0.43 w m was considered suitable cultivation of tobacco cells in the stirred-tank bioreactor, based on a previous study of Gao and Lee (1992).
Materials and Methods Tobacco Cell Suspension Culture. Stock Culture Initiation and Maintenance. Three-week-old callus cultures of Nicotiana tabacum var. Wisconsin 38 were obtained from Dr. Donald Armstrong (Department of Botany, Oregon State University). Cell suspension cultures were initiated by the sterile transfer of about 2 g of friable callus to 40 ml; of liquid medium. The cell suspension cultures were grown on Murashige & Skoog basal nutrient medium (Sigma M5519, basal salts plus vitamins) supplemented with 30 g/L sucrose, 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 30 pg/L kinetin. The medium was also supplemented with 42 mg/L of the antibiotic kanamycin to help prevent culture contamination. The medium was adjusted to pH 5.8 with 1N NaOH prior to autoclaving. Cell suspension cultures were maintained in 125-mL Erlenmeyer flasks on an incubated orbital shaker (orbital displacement, 3/4 in.) a t 27 "C and 150 rpm under diffuse room light. The cell suspensions were subcultured every 7 or 10 days at 25% (v/v) using a 40-mL culture volume per 125-mL flask. Subculturing was performed with a wide-bore pipet (3 mm) to select for a relatively fine cell suspension. To provide large inoculum volumes for bioreactor studies, cell suspension cultures were also maintained in 500mL Erlenmeyer flasks on an incubated orbital shaker at 27 "C and 120 rpm and were subcultured every 7 or 10 days at 25% (v/v). The 500-mL flasks contained 125140 mL of culture. All subcultures were performed using sterile techniques within a laminar flow hood. Following initiation from callus, cell suspension cultures were subcultured for 2 years prior to the bioreactor cultivation experiments described in this work. Stirred-Tank Bioreactor Cultivation. An Applikon 3-L stirred-tank bioreactor (working volume, 2 L) with a jacketed glass vessel and a multiport, stainless steel head plate was used for all cultivation studies. The inner diameter of the round-bottomed vessel was 13 cm, and the height of the vessel was 24 cm. The bioreactor was equipped with a 4.5-cm-diameter, three-blade marine impeller (6-cm height) pitched to an angle of 45". The impeller was positioned 3 cm above the bottom of the vessel. The impeller was driven by a overhead Lipseal stirrer assembly, and constant rotor speed under clockwise rotation was maintained by the stirrer motor control unit. The impeller tip speed, UI,was calculated by
uI = nND, where N is the impeller speed and DIis the impeller diameter (4.5 cm). Ambient air was pumped through a 0.2-pm sterile air filter, bubbled through a humidifier, and then sparged to the culture by an air inlet pipe tube consisting of a row of seven l-mm holes. The air inlet tube was positioned directly beneath the impeller. Heated
141
water from a circulating water bath was pumped through the vessel jacket to maintain a constant cultivation temperature of 27 "C. A steam-sterilizable galvanic oxygen electrode (VirTis, 15.9 mm x 22 cm, Model 2444418) measured the dissolved oxygen (DO) concentration in the culture. Analog readings from the DO meter were sent to a computer data acquisition system. Before each cultivation, 1600 mL of liquid medium (MS basal nutrients plus 30 g/L sucrose, 0.5 m f l 2,4-D, 30 pg/L kinetin, and 42 mg/L kanamycin for contamination control) were loaded into the bioreactor vessel. The bioreactor assembly and medium were autoclaved (121 "C, 45 min). Prior to inoculation, the DO electrode was calibrated to 100% of full scale after the medium had been sparged with air for 15 min at a constant system temperature of 27 "C. The DO electrode zero point was established electrically. The bioreactor was inoculated with tobacco cell suspension cultures from three 7-dayold 500-mL flask cultures (approximately 400 mL total culture volume). The total volume of culture in the bioreactor after inoculation was nominally 2000 mL, and the total liquid height was 15 cm (H/D = 1.15). All inoculation procedures were carried out using sterile techniques in a laminar flow hood. At each impeller speed, the bioreactor culture was maintained at 27 "C and 850 mumin air flow (0.43 wm). At 2-day intervals, duplicate 15-mL culture samples were withdrawn from the bioreactor through an 8-mm-i.d. sample tube under sterile suction. Analysis Procedures. Cell Density. Cell density was measured as the dry cell weight (DCW) per liter of culture. Culture samples (15 mL) were centrifuged at 500g for 15 min. The centrifuged cell pellet was resuspended in distilled water and vacuum filtered on preweighed, dried Whatman No. 1 filter paper disks. The cells were then dried at 70 "C for 48 h, and the dry cell weight (DCW) was determined to a precision of f O . l mg by weight difference. The dry cell weight was then divided by the sample volume to obtain the total dry cell density. All reported cell densities were average values from duplicate measurements. Microscopic Analysis of Cell Viability. Viable cells and nonviable cells were counted microscopically on a Fuchs-Rosenthal deep-well hemocytometer under white light illumination and lOOx magnification. A 0.5-mL sample of the cell suspension was gently swirled and then stained with a drop of 0.25% (w/v) Evan's blue reagent. Viable cells excluded the stain, whereas nonviable or visibly ruptured cells stained dark blue. The sample was loaded into the hemocytometer, and the number of viable and nonviable cells was counted without dilution under 16 triple-ruled squares (one side of the hemocytometer). In general, single cells or small groups of cells were readily observed and counted, and thus no attempts were made to disperse the cell aggregates by enzymic or chemical pretreatment. The number fractions of viable and nonviable cells were determined from the average of three sample measurements. p H . The pH of each culture sample was measured before any analyses were performed. In all cultivation experiments, the pH value was essentially constant at 5.2-5.5 during the entire 24-day cultivation period. Oxygen Mass Transfer Coefficients. The initial oxygen mass transfer coefficient, kLao, in the Applikon 3-L stirred-tank bioreactor was measured at 27 "C in autoclaved liquid medium (MS basal salts plus 30 g of sucrose/L) without any cells present by the Nz gassing out method, as described by Van't Riet (1979). Measurements were obtained for both 1600 and 2000 mL of liquid medium at a constant air flow rate of 850 mumin. All
Biotechnol. Prog., 1995, Vol. 11, No. 2
142 Table 1. Initial kLao vs Impeller Speed at 27 "C and 850 a m i n Air Flow
impeller speed, N (rpm)
initial oxwen mass transfer coeE&nt, kLao(h-l) 1600 mL 2000 mL
100
6.3
150 175 200 250 325
8.0
4.8
5.9 9.8 15.4
11.7 19.2
-+--flask -B-
measurements were repeated in duplicate. Average kLao values at 100-325 rpm are reported in Table 1. Measurements for the volumetric respiration rate, Qo, and volumetric mass transfer coefficient kLa during aerobic, stirred-tank cultivation of tobacco cells were obtained by the dynamic method originally described by Bandyopadhyay et al. (1967). The DO concentration vs time measurements required for the estimation of Qoand kLa in the stirred-tank bioreactor were obtained at regular intervals during the cultivation period, typically every 1 or 2 days. Values for Qo and culture kLa were reported as average values from duplicate measurements.
~
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25
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25
14 12 10
8 6
Results and Discussion
4
Growth curves based on total biomass density, X , (g of DCWL), for tobacco cells cultivated in the Applikon 2-L stirred-tank bioreactor at a fixed aeration rate of 850 mIJ min (0.43 w m ) for impeller speeds of 100, 175,250, and 325 rpm are presented in Figure 1. Increasing the impeller speed depressed the biomass growth rate and the final cell density. In particular, the maximum total biomass density also decreased from 11.8 g of DCWL at 100 rpm to only 8.6 g of DCWL at 325 rpm, a reduction of 27%. These trends suggest that hydrodynamic shear forces at high impeller speeds damage the cells in the culture, leading to a loss of culture viability. To quantify the long-term loss in culture viability, the number fraction of viable cells (I#+) was measured as a function of cultivation time and impeller speed, as shown in Figure 2. Comparison data for the number fraction of viable cells grown in a low-shear environment in 125mL flasks on an orbital shaker at 150 rpm are also shown in Figure 2. As the cultivation proceeded in the stirredtank bioreactor, #v decreased at a nearly constant rate during the early and midexponential phases of growth. During the late exponential and stationary phases of growth, & decreased more rapidly. Also, the values for & measured after the first 2 h of cultivation decreased from 88.0% at 100 rpm to only 66.5% at 325 rpm, implying that a fraction of the cell biomass was very susceptible to damage during the first few hours of cultivation. Microscopic analysis of tobacco cells in stirred-tank bioreactor cultures revealed an increase in the number of visibly ruptured cells, cells with deformed morphology (i.e., serrations), and cell debris relative to the numbers in flask cultures of comparable age and cell density. Although visibly deformed or ruptured cells stained dark blue under Evan's Blue staining, some intact cells stained dark blue as well. Microscopic analysis of tobacco cells cultivated in shake flasks revealed only intact cells and no ruptured cells or cell debris. In order to assess the effect of hydrodynamic forces on the growth of tobacco cell suspension cultures in the stirred-tank bioreactor, the viable cell density (Xc,v)was estimated from the total cell density (X,) and viable cell fraction (&I. Previous studies on stirred-tank bioreactors
2
n
Figure 1. Effect of impeller speed on total biomass density growth curves: (a) flask culture, 100 and 175 rpm; (b) 250 and 325 rpm.
considered the decrease in total biomass density with increasing agitation intensity (Kato et al., 1975; Hooker et al., 1990; Leckie et al., 1991). A simple relationship is proposed for estimating viable cell density (Xc,v):
XC,"= 4 J c This approximate definition of viable cell density was adopted for two reasons. First, it was difficult to count the absolute number of viable and nonviable cells per liter of because of errors associated with obtaining small uniform sample volumes. Expression of the data as number fraction data was much more consistent because it normalized this sampling effect. Second, nonviable cells still possess cell mass prior to complete disintegration. If the cell mass of all cells in the culture is nominally the same, then the number fraction correction for the total cell biomass approximates the viable cell density. Semilog plots of Xc,vvs time reveal two distinct linear regions. Sample data at 175 rpm are presented in Figure 3 to illustrate these two linear regions. Region I represents the exponential phase of growth and has a positive slope pv (day-'), where pv is the net specific production of viable biomass in the exponential phase. Thus, pv is defined as
a,,"
1 pv=x,,dt
(3)
Region I1 represents the stationary phase of growth and has a negative slope - k d (day-I), where k d is the specific death rate of viable cells in the stationary culture phase
Biofechnol. Prog., 1995, Vol. 11, No. 2
143
100 L
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40
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60
exponential phase, on kd during the stationary phase, and on during the exponential phase. the peak
i
0
a,
.-> I
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c
.-0 w
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LL
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10 15 20 Cultivation time (days)
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Figure 2. Number fraction of viable cells vs cultivation time: (a) flask culture, 100 and 175 rpm; (b) 250 and 325 rpm.
I xc
0 X
"
.
10
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A
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B(d
L
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Impeller tip speed, vI (cm/sec)
a,
-
.
- 0 - 175rpm
100 I
--ui
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Cultivation time (days)
Figure 3. Semilog plot of total biomass density (X,) and viable cell density (XCJvs cultivation time at 175 rpm t o illustrate the estimation of p v during the exponential phase and of kd
during the stationary phase.
where cells are not actively dividing. Thus k d is defined as
1
K
- k d = X , , d t
,
V
(4)
The X,,, vs time data points selected for the estimation of pv during the exponential phase and the k d during the stationary phase were based on those values that provided the highest r2value for a minimum of five points. In general, r2for each estimate exceeded 0.92. The hydrodynamic shear forces in a stirred-tank bioreactor are inherently difficult to evaluate. Furthermore, during batch culture the cells are subjected to continu-
ously changing hydrodynamic stress conditions imposed by the changing rheology of the culture as cell biomass increases (Meijer et al., 1993). Despite this complexity, we chose to relate cell damage to the impeller tip speed, UI, a simple measure of agitation intensity. The effects of U I on pv during the exponential phase and on k d during the stationary phase are shown in Figure 4. As the impeller tip speed increased, p, decreased and k d increased, showing that hydrodynamic stress induced cell damage during both the exponential and stationary phases of growth. Both pv and k d were initially sensitive to agitation intensity, but this effect leveled off at impeller tip speeds of about 60 c d s and greater, suggesting that shear damage is more pronounced in a subpopulation of cells. A more careful comparison of the total vs viable cell densities in Figure 3 suggests that the late exponential and early stationary phases of growth may be the most susceptible to hydrodynamic shear forces. This observation is consistent with shortterm shear sensitivity studies of tobacco cells in a Couette flow viscometer conducted by Hooker et al. (1989). Values of k d for bioreactor culture were always much higher than the value of 0.036 day-l for k d in flask culture, whereas p, for flask culture (0.168 day-l) was comparable to p, for bioreactor culture at 100 rpm. The specific cell death rate, kd, quantified the loss in viable cell density due to both hydrodynamic shear damage of cells and natural cell death during the stationary phase. In the low-shear flask cultivation environment, k d represented natural cell death resulting from nutrient depletion. Thus, the increase in k d with increasing agitation intensity in the stirred-tank bioreactor can be attributed to hydrodynamic shear damage. The specific respiration rate of the viable cells in the culture, defined by
qo,v= Qo/X,,v
(5)
is a fundamental measure of metabolic activity. The effect of impeller speed on the specific respiration rate (go,,) is shown in Figures 4 and 5. The peak value for qo,vin the exponential phase increases by a factor of 2, despite the decrease in pv. This increase in is the likely result of a stress-induced increase in culture metabolism. In fact, qo,vcan be related to the specific maintenance coefficient, qO,,,,, for respiration by
40,v = P f l W O 2 + q o , m
(6)
where YWO, is the growth yield coefficient based on 0 2 (g of cells/mol of 0 2 consumed), and p is the specific growth
Biotechnol. Pros., 1995, Vol. 11, No. 2
144 0.5 1
5 a 0
1
I
: + 100rpm
-e,+ 250rpm --A--
175rpm
0.4
325rpm
s
. 0"
0.2
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a -
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P
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Y
0.0'""""""""""""' 0 5 10
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15 20 Cultivation time (days)
, . , . . , . . . , 25
Figure 5. Specific respiration rate of viable cells (so) during batch cultivation of tobacco cells at impeller speeds of 100-325
10 Cultivation time (days)
5
15
Figure 6. Culture kLa during batch cultivation of tobacco cells at impeller speeds of 100-325 rpm.
rpm.
rate based on the total biomass production rate. It can be seen from eq 6 that if YUO,is constant, and qo,v increases while p decreases in response to increased hydrodynamic stress, then qo,mmust likewise increase. Although the estimation of Ymo, is beyond the scope of this study, Gulik et al. (1992) report that YWO,is 2.5 C-mol/mol of 0 2 and qO,mis 0.006 26 mol of OdC-mo1.h for N. tabacum var. White Burley cultivated on glucose as the limiting carbon substrate. Gulik et al. also report that the carbon content is 39.1 wt % for batch culture; thus, estimates for Y U Oand ~ qo,mare 2.4 g of DCW/g of 0 2 and 0.204 mmol of Odgh, respectively. Despite the differences in cultivar and substrate, Gulik et al.'s value for Y ~ owas , used to estimate qo,min the present study. Thus, at 100 rpm, qO,m was 0.115 mmol of Odg-h (52%of total respiration) vs 0.358 mmol of Odgh at 325 rpm (87% of total respiration). Thus, there are significant maintenance energy costs associated with long-term hydrodynamic stress. Oxygen mass transfer to tobacco cell suspension was characterized by measuring the volumetric oxygen mass transfer coefficient kLa in the cell suspension periodically during the entire cultivation period. Values for kLa in the bioreactor culture vs cultivation time for impeller speeds of 100-325 rpm are shown in Figure 6. During the early stages of cultivation, the cell density was relatively dilute, and so kLa in the culture should have approximated the initial kLao values given in Table 1. However, the culture kLa during the early exponential phase of the culture was consistently about 10-20% higher than the corresponding kLao value at a given impeller speed. Possible explanations for this result include (1)enhancement of oxygen mass transfer due to direct contact of cells with air bubbles (Andrews et al., 1984) and (2) reduction in interfacial surface tension between air bubble and liquid medium due to the release of surfactant-like substances (proteins, etc.) from the cell culture. Beyond the early exponential phase of growth, the kLa in the culture decreased before leveling off in the stationary phase. Increasing cell concentrations reduced the oxygen mass transfer coefficient (kLa) in the culture. However, the reduction in culture volume as the result of sampling may have slightly reduced the magnitude of this decrease. Our observed reduction in kLa with increasing biomass concentration is consistent with mass transfer studies of Ballica and Ryu (1993), who showed that kLa in an airlift bioreactor culture of D.stramonium cells at an air velocity of 0.05 d s was equal t o 30 h-' at a PCV of 0.2 mUmL vs 13 h-' at a maximum PCV of 0.8 mUmL, a reduction in kLa of about 60%. However, in Ballica and R p ' s work, the kLa values in airlift cell
Table 2. Initial, Minimum, and Final DO Levels in Stirred-Tank Bioreactor Cultures of Tobacco Cells
impeller speed (rpm)
100 175 250 325
DO level in culture (% saturation) (cultivation time (h)) initial minimum final 72 (2) 22 (143) 56 (597) 77 (2) 41 (89) 78 (546) 43 (212) 88 (2.5) 63 (103) 69 (595) 53 (343) 93 (2) 78 (123) 78 (237)
cultures were determined by adding fresh biomass to the bioreactor at successivelyhigher loadings, whereas in the present study the kLa values were obtained from the same culture over an entire batch cultivation period. The reduction in kLa at high cell density was not severe enough to cause oxygen starvation to the culture, even at a low impeller speed of 100 rpm. During the first 5-8 days of cultivation, the DO level steadily decreased to a minimum value commensurate with the oxygen demand curve. However, as shown in Table 2, at all impeller speeds the DO level was always above 10% of air saturation. Therefore, the reduction in biomass productivity can be attributed to cell damage by hydrodynamic forces and not by inadequate oxygen mass transfer.
Conclusions Cell damage and oxygen mass transfer were investigated during the batch culture of Nicotiana tabacum (tobacco) cells a t 27 "C and 0.43 w m in a 3-L stirredtank bioreactor equipped with a marine-blade impeller. The viable cell density, defined as the product of the total cell density and the viable cell fraction, was used instead of the total cell density to quantify the extent of cell damage. Viable cell density vs cultivation time data were used to determine the net specific growth rate of viable cells during the exponential phase (p,) and the specific death rate of viable cells during the stationary phase ( k d ) in stirred-tank culture. Increasing the impeller tip speed from 24 to 77 c d s (from 100 to 325 rpm) depressed the maximum biomass density and culture viability, reduced pv from 0.175 to 0.077 day-', and increased k d from 0.042 to 0.109 day-l, clearly showing that increasing the hydrodynamic agitation intensity increased cell damage in long-term cultures. However, this effect leveled off a t impeller tip speeds of about 60 c d s and greater, suggesting that cell damage was more pronounced in a subpopulation of cells. Increasing the impeller tip speed from 24 to 77 c d s also doubled the peak 0 2 respiration maintenance coefficient from 0.115 to 0.358 mmol of O,!
Biotechnol. Prog., 1995,Vol. 11, No. 2
gh, showing that this viable cell subpopulation was also under stress from hydrodynamic forces. Oxygen mass transfer rates were measured concurrently with cell damage to determine whether oxygen transfer limitations at relatively high cell densities could account for a n y reduction in biomass growth. Although culture kLa values were initially about 10-20% higher than their corresponding cell-free kLaovalues, as the cell density increased, values for kLa ultimately decreased. Despite the reduction in oxygen mass transfer rates, oxygen starvation w a s not observed even at a low impeller tip speed of 24 c d s . Therefore, the reduction in biomass productivity can be attributed to cell damage by hydrodynamic forces a n d not by inadequate oxygen mass transfer.
Notation impeller diameter (cm) specific death rate of viable cells in the stationary phase (day-l) volumetric oxygen mass transfer coefficient in tobacco cell suspension culture (h-l) initial volumetric oxygen mass transfer coefficient in liquid medium (h-l) impeller rotation rate (rpm) volumetric culture respiration rate (mmol of 021 L-h) maintenance respiration rate (mmol of Oz/gh) specific respiration rate of viable cells in culture (mmol O d g h ) impeller tip speed ( c d s ) total biomass cell density expressed as dry cell weight (g of DCWL) viable cell density (g/L) growth yield coefficient based on 0 2 (g of cells/ mol of 0 2 consumed) net specific production rate of viable cells in the exponential phase (day-l) number fraction of viable cells in culture
Literature Cited Andrews, G. F.; Fonta, J. P.; Marrota, E.; Stroeve, P. The effects of cells on oxygen mass transfer coefficients 11: Analysis of enhancement mechanisms. Chem. Eng. J. 1984,29,B47B55. Ballica, R.; Ryu, D. D. Y. Effects of rheological properties and mass transfer on plant cell bioreactor performance: production of tropane alkaloids. Biotechnol. Bioeng. 1993,42,11811189. Ballica, R.; Ryu, D. D. Y.; Powell, R. L.; Owen, D. Rheological properties of plant cell suspensions. Biotechnol. Prog. 1992, 8,413-420. Bandyopadhyay, B.; Humphrey A. E.; Taguchi, H. Dynamic measurement of the volumetric oxygen mass transfer coefficient in fermentation systems. Biotechnol. Bioeng. 1967,9, 533-544.
145 Ducos, J. P.; Pareilleux, A. Effect of aeration rate and influence of pCOz in large-scale cultures of Catharanthus roseus cells. Appl. Microbiol. Biotechnol. 1986,25,101-105. Gao, J.; Lee, J. M. Effect of oxygen supply on the suspension culture of genetically modified tobacco cells. Biotechnol. Prog. 1992,8,285-290. Hegarty, P.K.;Smart, N. J.; Scragg, A. H.; Fowler, M. W. The aeration of Cutharunthus roseus L. G. Don suspension cultures in airlift bioreactors: the inhibitory effect at high aeration rates on culture growth. J. Exp. Bot. 1986,37,19111920. Hooker, B. S.; Lee, J. M.; An, G. Response of plant tissue culture to a high shear environment. Enyzme Microb. Technol. 1989, 11,484-490. Hooker, B. S.;Lee, J. M.; An, G. Cultivation of plant cells in a stirred vessel: effect of impeller design. Biotechnol. Bioeng. 1990,35,296-304. Jolicoeur, M.; Chavarie, C.; Carreau, P. J.; Archambault, J. Development of a helical-ribbon impeller bioreactor for highdensity plant cell suspension culture. Biotechnol. Bioeng. 1992,39,511-521. Kato, A,; Shimizu, Y.; Nagai, S. Effect of initial kLa on the growth of tobacco cells in batch culture. J. Ferment. Technol. 1975,53,744-751. Kim, D.; Pedersen, H.; Chin, C.-K. Cultivation of Thalictrum rugosum cell suspension in an improved airlift bioreactor: stimulatory effect of carbon dioxide and ethylene on alkaloid production. Biotechnol. Bioeng. 1991,38,331-339. Leckie, F.; Scragg, A. H.; Cliffe, K. C. Effect of impeller design and speed on the large-scale cultivation of suspension cultures of Catharanthus roseus. Enzyme Microb. Technol. 1991,13, 801-810. Meijer, J. J.; ten Hoopen, H. J. G.; Luyben, C. A. M.; Libbenga, K. R. Effects of hydrodynamic stress on cultured plant cells: A literature survey. Enzyme Microb. Technol. 1993,15,234238. Merchuk, J. C. Shear effects on suspended cells. Adv. Biochem. Eng. Technol. 1991,44,65-95. Panda, A. K.;Mishra, S.; Bisaria, V. S.; Bhojwani, S. S. Plant cell reactors- a perspective. Enyzme Microb. Technol. 1989, 11, 386-397. Payne, G. F.; Shuler, M. L.; Brodelius, P. Large scale plant cell culture. In Large Scale Cell Culture Technology; Lydersen, B. K., Ed.; Macmillan Publishing: New York, 1987;pp 195229. Scragg, A. H.; Allan, E. J.; Leckie, F. Effect of shear on the viability of plant cell suspensions. Enyzme Microb. Technol. 1988,10,361-367. Tanaka, H. Large-scale cultivation of plant cells at high cell density: A review. Process Biochem. 1987,22,106-113. van Gulik, W.M.; ten Hoopen, H. J. G.; Heijnen, J. J. Kinetics and stoichiometry of growth of plant cell cultures of Catharanthus roseus and Nicotiana tabacum in batch and continuous fermentors. Biotechnol. Bioeng. 1992,40,863-874. Van't Riet, K. Review of measuring methods and results in nonviscous gas-liquid mass transfer in stirred vessels. Ind. Eng. Proc. Chem. Des. Dev. 1979,18,357-364. Accepted October 5, 1994.@ BP940076X @Abstractpublished in Advance ACS Abstracts, December 1, 1994.
