Response of plant tissue culture to a high shear environment

Response of plant tissue culture to a high shear environment

Brian S. Hooker, James M. Lee and Gynheung An*

Department o f Chemical Engineering and *Institute o f Biological Chemistry, Washington State University, Pullman, WA, USA

Plant cell suspension cultures ofNicotiana tabacum were tested in a Couette-type shearing device to determine the effect of shear rate on culture viability, cell lysis, and the accumulation of secondary metabolites (phenolics). The effects of increasing shear and variable culture age were both investi- gated. Increasing shear caused an increase in cell death, lysis, and phenolics secretion. In testing suspension cultures of various ages, it was found that those cultures in the latter stages of exponential growth and early in the stationary phase were more susceptible to shear damage than cultures in the lag phase, early exponential phase, or later stationary phase. An increase in extracellular phenolics as a response to shear was more apparent with older cultures. The amount ofphenolics secreted from the cells was directly related to the intensity of shear. This may have been due to an increase in cell permeabilization with higher shear rates.

Keywords: Plant cells; shear; plant tissue culture

Introduction

There has been an increase in attention given to the production of primary and secondary metabolites from suspension plant tissue culture in large-scale operations. A wide range of compounds may be produced from plant tissue cultures. J-a Some suspension culture systems can even produce higher concentrations of specific compounds than the intact plant of the same species. 4 However, the use of plant tissue culture in industrial operations has been limited due to the sensitivity of individual cells, in culture, to hydrody- namic shear stress. 2'5'6 Plant cells, unlike microbial cells, have relatively weak, pliable cell walls and, when subjected to higher levels of shear stress, will tend to deform or rupture, causing cell death. The high agitation rates needed for the promotion and growth of plant tissue culture in suspension may also cause decreased culture viability. This presents a severe problem, especially in large-scale production, as more vigorous agitation becomes necessary.

It is important to note that shear and agitation conditions will have an effect not only on culture viability, but also on cell aggregate size, cell lysis,

Address reprint requests to Dr. Lee at the Department of Chemical Engineering, Washington State University, Pullman, WA 99164 Received ll April 1988; revised 15 August 1988

culture growth, and metabolite production. In previous work done by Rosenberg and Dunlop, 5 using cultures of Petunia hybrida cv. Mitchell, it was shown that increasing shear and length of shearing time caused a decrease in cell viability and an increase in cell lysis. Sheared cultures in this investigation were not tested for any changes in metabolite concentration. In additional studies, it has been shown that growth rate, secondary metabolism, and viability are all much lower when cultures are grown in a high shear environment. 5'6 Despite these research efforts, very little is known about shear effects on plant tissue culture, since only a few studies have been completed using selected species of mature cultures.

In this investigation, we have studied the effect of shear on cultures of Nicotiana tabacum, a species not previously tested, using a Couette-type shearing device. Research included monitoring changes in secondary metabolite "product" concentration (phenolics) as well as changes in culture viability and cell lysis. In addition, a portion of our investigation was devoted to the effect of shear on cultures in different stages of growth, using both young and mature cultures. A study was also completed on the initial response of tissue culture to wounding. Wounding is a term used in reference to an incision made on a whole plant. In a general sense, however, the term may be used to describe the response of plant tissue culture to an applied stress.

484 Enzyme Microb. Technol., 1989, vol. 11, August © 1989 Butterworth Publishers

Plant tissue culture in a high shear environment: B. S. Hooker et al.

Materials and methods

Plant material and culture medium

Suspension cultures of Nicotiana tabacum cv. Bright Yellow were used for all experiments. Tobacco cells were grown in a modified Linsmaier and Skoog medium, 7 which contained 4.3 g 1-1 of Murashige and Skoog salt mixture (Gibco), supplemented with 0.2 mg l -l 2,4-dichlophenoxyacetic acid (2,4-D), 0.18 g 1-1 KH2PO4, 0.I g 1-1 inositol, 1 mg 1 -j thiamine HC1, and 30 g 1 -l sucrose. The pH was adjusted to 5.8 before autoclaving. Cultures of N. tabacum were maintained in 125- and 250-ml Erlenmeyer flasks containing 30 and 60 ml, respectively, of modified MS medium and incubated in the dark at 26°C on a gyratory shaker at 150 rev min -~. Subculturing was completed every 7 days, using a 5% (v/v) inoculum.

Cell concentration measurements

The cell concentrations of suspended plant tissue cultures were determined by removing aliquots from well-mixed suspensions and placing the samples into preweighed culture tubes. The samples were centri- fuged; supernatant was withdrawn; and the wet (or fresh) weight was obtained. Dry weight was determined by placing samples in an oven maintained 70°C until the weight no longer changed with time (usually 3 days or more).

Viability assay

The viability of plant tissue culture was measured as the percentage of living cells, as compared to total cells, remaining in the culture. Percent viability was determined using a method developed by Towill and Mazur, 8 which is based on the plant cells' ability to reduce various tetrazolium salts. Reduction occurs when the tetrazolium salt accepts electrons from an electron transport chain in the cell mitochondria. The reduced salt product is a red formazan dye which was extracted from the cells using 95% ethanol. By measuring the change in absorbance at 485 nm of the red

SAMPLE

sos. s,o /_~11 I I l lllli 'k ~ i ~ L

Figure 1 Shear reactor

dye present in a given sample, the percent viability was determined.

Total phenolics assay

The concentration of phenolics secreted from the tobacco cells into the culture medium was determined by a procedure adapted from Singleton and Rossi. 9 This procedure is based on the ability of phenolics to reduce complex polymeric ions formed from phospho- molybdic and phosphotungstic heteropoly acids (Fo- lin-Ciocalteu Reagent). As the phenolics are oxi- dized, Folin-Ciocalteu Reagent forms a blue complex which is stabilized by the addition of sodium carbo- nate. Phenolics concentration was determined from the change in absorbance at 750 nm. Gallic acid (100 mg l -j) was used as the standard.

Cell lysis determination

The relative amount of cell lysis was determined by the change in dry weight concentration of cellular material over the duration of each trial. As lysis occurred, the dry weight concentration decreased steadily while the contents oflysed cells were released into the medium. At complete lysis, the dry weight concentration is approximately 25% of that of unlysed cells. This was determined by sonicating a sample of cells and comparing the dry weight of the sample to a sample with intact cells. Knowing this, the percent lysis was calculated based on changes in dry weight.

Shear reactor

The shear reactor, designed to resemble a Couette viscometer, consisted of a cylindrical vessel, a rotating drum with an attached shaft, and a flanged lid (see Figure 1). The rotating drum fit within the vessel, creating an annulus which contained the suspension culture to be sheared. The volume of culture used for a typical experiment was 240 ml. The shaft attached to the rotating cylinder extended through a hole in the flanged cap and was coupled to a variable-speed motor assembly.

Experimentation using the shear reactor consisted of trials lasting 12 h or less. Because of this short duration, the trials were conducted in a nonsterile environment. Samples were taken hourly and analyzed for cell viability, secondary metabolite (phenolics) concentration, and cell concentration. The shear reactor was used for three different types of investigation: the effect of increasing shear, the effect of plant tissue culture age, and a study of the culture response to wounding.

Several precautions were taken to eliminate experimental error in shear reactor studies. The basic con- cern was that a healthy suspension culture would react differently than an unhealthy culture. A culture which is unhealthy would be more likely to show a decrease in viability in a shear environment. The state of cultures to be used in these studies was evaluated by measuring all concentration, extracellular phenolics

Enzyme Microb. Technol., 1989, vol. 11, August 4 8 5

Papers

concentration, and checking for contamination. A healthy shake flask culture will usually, in the stationary phase, have a wet weight cell concentration of 60% and an extracellular phenolics concentration under 30 mg 1 i. Unhealthy cells will usually have a lower cell concentration and a higher extracellular phenolics concentration. Despite these efforts to improve experimental repeatability, the average discrepancy seen between replicate trials was 17.5% for viability and 31.0% for extracellular phenolics.

The rotating drum was operated at speeds of 400- 800 rev min -1. Although this was not within the laminar region of flow, laminar shear rate was calculated as a guideline for comparison. The laminar shear rate present in the reactor, because of the geometry of the drum, was not constant at each point. However, an average shear rate for the entire reactor was calculated by splitting the reactor into two sections: the annulus and the below-drum space. The annulus, with the stationary wall and the drum rotating at a fixed velocity, was simplified to a model of two flat plates, one fixed and the other moving at a constant velocity. Thus, the shear rate calculation was simplified to

annul = ~ (l)

where Rdr,~ is the drum radius, 6 is the annulus width and N is the rotational speed.

For the below-drum space, the velocity at the drum increased with increasing radius. Thus, rather than calculating a discrete shear rate for this portion of the reactor, the average shear rate was determined by integrating the shear rate over the entire drum bottom surface:

dXX b d - ~ "~ 3~X (2)

where z~x is the gap between the drum bottom and the reactor bottom. The laminar shear rate for the entire reactor was determined by figuring a weighted average based on volume:

dv (~XX)avg -- gbd(dU~ -}- gannul(~-xx)annul (3) gto t \dx/bd gto t

where Vba is the culture volume below the drum, Vannn~ is the culture volume in the annulus, and Vtot is the total culture (working) volume. Using this calculation method, rotational speeds of 400, 600, and 800 rev min -I corresponded to shear rates of 596, 895, and 1193 s -I, respectively.

Results and discussion

The basic objective of the shear reactor studies was to explore the detrimental characteristics of shear on plant tissue culture. The severity of the shear conditions used in this work was much more extreme than that required for normal agitation of suspension cultures. In evaluating the condition and responses of the cultures studied, viability, secondary metabolite pro-

125-

100-

~. 75

5O

25

+ + 4- +

+

C"" ~-------£~ ÷

0 ; 1'0 12 Time (Hrs)

Figure Z Effect of shear on the v iab i l i ty of 10-day-old culture. Drum speed/shear rate: +, 400 rev rain 1/596 s 1; [~, 600 rev min-1/895 s- l ; Q, 800 rev min-1/1193 s 1

duction (phenolics), and cellular lysis were monitored. By measuring viability, the overall health of the culture could be observed. In addition, phenolics concentration measurements (extracellular) gave an indica- tion of the condition of the culture. Usually, an increase in phenolics concentration is a signal that the cells are responding to a type of stress within their environment. Also, an overall increase in concentration may be seen with the occurrence of cell lysis, as the cell contents, rich in phenolics, flow into the medium. This information was used to determine if cell death was accompanied by lysis or if intact cells were dying due to elongation within the shear field. All shear reactor trials were replicated.

Effect of variable shear rate

In this study, shear rate was varied through changing the operating speed of the rotating drum. Mature, 10-day old cultures were used for this phase of experimentation. Preliminary testing showed that drum speeds of 200 rev min -~ or less had no detrimental effects on culture viability. Thus, rotational speeds of 400, 600, and 800 rev min -~ were used for trials of 12-h duration. The shear rates associated with these speeds were calculated, using equation (3), at 596, 895, and 1193 s -l, respectively.

The effect of shear on culture viability and extracellular phenolics concentration is shown in Figures 2 and 3, respectively. As expected, increasing shear rates caused a steady decrease in viability. After 12 h of shearing, the viability of cells at 400 rev min-I did not change. At 600 and 800 rev min -j, the culture viability decreased by about 20% and 95%, respectively. The result at 600 rev min -I may be attributed mostly to experimental variability, since the viability decrease was slight.

The extracellular phenolics concentration increased with time in both the 600 and 800 rev min -l trials (Figure 3). Again, the response observed was more pronounced with increased shear rate, showing an

486 Enzyme Microb. Technol., 1989, vol. 11, August


125-

100- } .

75- o

g 50.

25- O.

0-

Time (Hrs)

Figure 3 Effect of shear on extracel lu lar phenol ics concentra- t ion of 10-day-old culture. Drum speed/shear rate: (3, 800 rev m in -q1193 s-~; l-q, 600 rev ra in-V895 s-~; +, 400 rev min-V596 S - 1

125

100

K

:~ 7 5

5O

0 ¸

0

2 5 ¸

A A

Time (Hre)

Figure 5 Effect of culture age on v iab i l i ty in a shear envi ronment at 400 rev min -~ (596 s-l). Culture age: A, 10 days; +, 3 days; El, 5 days; Q, 7 days

increase in concentration, over 12 h of shearing, of 42 and 80 mg 1-1 for 600 and 800 rev min -j, respectively. During the first 2 h of each run, the concentration of phenolics increased markedly, even for the trial at 400 rev min -1 where there was no observable cell death. This suggests that there was an additional response of the cells, as they were initially subjected to shear. After this initial jump in concentration, any increase in phenolics level seemed to be directly related to a loss in viability. Figure 4 shows percentage cell lysis observed in the shear reactor at 600 and 800 rev min- 1. The increase of the shear rate also caused an increase in cell lysis. At 600 rev min -j, after 12 h of operation, 26% of the total cell matter was lysed; at 800 rev min -1, 81% of the cell matter was lysed. Comparison of Figures 2 and 4 reveals that the majority of cell lysis appears to occur at the end of the runs, while the cell viability decreases progressively throughout each run. This may be due to cells dying while still intact and lysing some time after death has occurred.

Effect of variable culture age

The effect of variable culture age was studied to determine the response by tissue cultures of different ages. Basically, the objective of this study was to determine if there are specific periods in a culture lifetime when cells are more susceptible to shear damage. All cultures used in the shear reactor were maintained at a minimum cell concentration of 40% wet weight. This was done to eliminate any sharp difference in cell concentration between the individual trials.

Figure 5 shows the effect of culture age on viability. Intermediate age cultures, 5 and 7 days old, faired the worst, experiencing a 50% decrease in viability after 12 h of shearing. This may be explained by using a typical culture growth curve, as shown in Figure 6. The 5- and 7-day-old cultures are located in the latter portion of exponential growth and early in the stationary phase. The 3-day-old culture, showing a decrease in viability of 25% after 12 h of shearing, falls just after

¢0

g

13-

100

75

50

25

0 ¸

0

___/j ; 1'o 12

Time (h rs . )

Figure 4 Effect of shear on cell lysis of 10-day-old culture. Drum speed/shear rate: Q, 800 rev min-V1193 s- l ; [S], 600 rev min-~/895 s-~

&

100

80

60

40

20

0

e e

o ; Time (days)

Figure 6

10

Growth curve of the tobacco cell, Nicotiana tabacum

Enzyme Microb. Technol. , 1989, vol. 11, Augus t 487

Papers

exponential growth has begun, while the 10-day old culture, showing no viability decrease during shearing, is located late in the stationary phase.

From this, it appears that cultures in the exponential phase of growth are more susceptible to shear than those in the lag phase or stationary phase. The highest amount of shear damage is seen in the late exponential phase. Although the exact reason for this increased sensitivity to shear is unknown, there are several explanations based on changes occurring in the exponential phase. First, the increase in cell size, associated with growth and expansion, may cause an increase in shear sensitivity. In addition, as cell expansion occurs, the cell wall strength may decrease, causing an increase in susceptibility to shear until the wall components become stronger, accommodating the larger cell size.

Figure 7 shows the effect of variable culture age on extracellular phenolics concentration in the shear reactor. Before the beginning of each run, distinct concentrations were seen, depending upon the age of the culture. This was due to differences in metabolism seen at various culture ages and is not pertinent to this study. What is important here is the increase in phenolics concentration (AC) over the duration of shearing. As seen from the plot, ~C increases with culture age until a maximum is reached with the 7- and 10-day-old cultures.

This response may be explained by several different phenomena which are occurring. The concentration change seems to be increasing as a response to the decrease in viability. Like the concentration increase, the viability decrease was the most pronounced at 7 days. In addition, it is important to note that, although the younger cultures were concentrated by removing part of the medium, the cell concentration of older cultures was higher than that of younger cultures. With more cells responding through increased phenolics secretion and cell lysis, the concentration increase would tend to be higher with older cultures. Also, it may be that cells in older cultures may be

50 ¸

_'Z 4 0

v

6 30 c

20.

~ 1 0 .

tL

®

~ I ZL ~ ZL G

n

f D ~ [3

. . j J ° . + + - - + - - + . p _ _ _ _ ~ 4 - - ~ + + +

0 ' i i [ i

0 4 6 8 10

Time (Hrs)

12

Figure 7 Effect of culture age on extracellular phenolics concentration in a shear env i ronment at 400 rev min -1 (596 s-l). Culture age: A, 10 days; (!), 7 days; [ ] , 4 days; +, 2 days

responding to shear stress differently than cells in younger cultures. It has been reported that mature cells will react differently to stress through wounding than younger cells of certain species.J°

It is also interesting to note the pronounced increase in phenolics concentration observed in more mature cultures during the initial stages of each run. The majority of the overall concentration increase occurred in the first hour of each run. When testing the 7- and 10-day-old cultures, the concentrations actually doubled in the first hour, showing an increase of 12 and 14 g 1 i, respectively. This could be due to a higher amount of secretion due to cell permeabilization caused by shear. The increase in concentration could also have been caused by an increase in secondary metabolite production brought on by wounding. The response to wounding, usually accompanied by cell division, may be to increase other cellular functions, such as phenolics production. George and Sher- rington m have suggested that phenolics in wounded cells may act as inhibitors for indoleacetic acid oxi- dase, and thus stimulate cell division.

Wounding studies

In order to investigate the initial response of plant tissue culture to shear, an additional set of experimental trials was completed. For this particular study, culture age was held constant at 10 days. This age of culture showed the greatest initial increase in phenolics concentration during previous investigations. Again, rotational speeds between 400 and 800 rev min -I were tested. Each tested speed was held constant for the duration of the 2-h trials. Samples, taken throughout each run, were analyzed for both extracellular and total phenolics concentration. The samples tested for total phenolics were first sonicated to ensure complete disruption of cells and release of intracellular phenolics into the medium.

Figure 8 shows the increase in extracellular and total phenolics concentration for each of the speeds tested. In all cases, the concentration of phenolics increased, especially during the initial stages of each run. This increase tapered off, becoming smaller as higher concentrations were attained, demonstrating that individual cells are responding to the shear environment by boosting the concentration level of phenolics rather than merely bolstering the production rate.

Increasing rotational speed had a marked effect on the extracellular concentration, which showed a distinct response for each speed tested. This distinction was seen in all replicate trials. The highest secretion rates corresponded to the highest shear rate (1193 s-l). The extracellular phenolics concentration from this trial increased by 21 mg 1-1 within the first 2 min of reactor operation. Results from trials at 600 and 400 rev min -1 showed a more gradual increase in extracellular phenolics concentration, indicating that permeabilization along with phenolics secretion from the cells may intensify with increasing shear. In contrast,



Table 1 Comparison of phenolics secretion to phenolics production

Length of Shear Secretion Production Ratio of secretion run (h) rate (s -1) (mg 1-1) (mg 1-1) to production

2 1193 32.8 18.0 1.79 2 895 11.5 12.8 0.90 2 596 9.2 33.5 0.28

the effect of the varying speed on the total phenolics concentration is not clearly distinguishable. The total phenolics concentration increased by about 20%, re- gardless of the intensity of shear.

The fact that two mechanisms, cell permeabilization and the cellular response to wounding, may be operating in this environment can be seen by directly comparing the response of extracellular and total phenolics concentrations. Table 1 shows the overall phenolics secretion and production for each shear rate tested. Phenolics secretion was based upon changes in the extracellular phenolics concentration and was calculated by taking the difference between the concentrations measured at the start and finish of each trial. Likewise, phenolics production was based upon changes in the total phenolics concentration and was determined in a similar fashion. Note that the word "secretion" refers to the passive diffusion of phenolics across the cell membrane and wall.

Again it is seen that secretion increases with shear rate, while there is no distinct trend seen in production. At higher shear rates (800 rev min -~, 1193 s-J),

15° I

--, 100 7 - = o c

o 75. 0

o

50- - - _ . . - 6 - . . .

~.4D.g, l~' Q.--- -0 0"

f- 25 . . . . . . . . . . . ~" .......... o ........... o--- 0 "°°''=- . . . . . . . . . i

a o - ~ , - ~ . : : : : + . . . . . + . . . . . . . . . . . + . . . . . . . . . . . +- . . . . . . . . . . . ÷ . . . . . . . . . . . |~.i....,r---

0- I I [

0.0 0.5 1.0 1.5 2.0

Time (hrs)

Figure 8 Extracellular and total phenolics concentration in the presence of various rates of shear for 10-day-old culture. Drum speed/shear rate: G), 800 rev rain-1/1193 s-l; [] 600 rev rain-l/ 895 s-~; +, 400 rev rain-l/596 s-l; .... , extracellular phenolics; - - - , total phenolics

the secretion-to-production ratio was highest, at an average of 1.79, for the 2-h duration of shear. This indicates that, due to high cell wall permeabilization, the amount of phenolics secreted was much higher than the amount produced. The secretion and production became almost equal at 600 rev min -l (895 s-l), showing a ratio of 0.90. At low shear rates (400 rev min -1, 596 s -1) the ratio was much less than one, at an average of 0.28, which shows that cells were not secreting enough to match the production rate. This may be due to a lower amount of permeabilization occurring in the cell walls.

C o n c l u s i o n s

Suspension cultures of N. tabacum were subjected to a shear field in a Couette-type shearing device to determine the effect of changing shear rate on mature cultures and the effect of constant shear on cultures of various ages. As shear was applied, changes in viability, cell lysis, and secondary metabolite concentration were monitored.

By increasing the shear applied to mature suspension cultures, the relative viability was steadily decreased. The majority of the cells dying in the shear reactor had also become lysed. However, contrary to previous studies: the amount of lysis was not propor- tional to the amount of shear. The decrease in viability was accompanied by an increase in extracellular phenolics concentration. This was due to the release of accumulated metabolites in the medium from lysed cells and an increase in phenolics production bolstered by the presence of shear.

Cultures in the latter part of the exponential growth phase and the early stationary phase showed a higher susceptibility to shear damage than cultures in the lag phase, early exponential growth phase, or the latter stages of the stationary phase. This may be due to cell expansion and growth, along with a decrease in cell wall strength. This result can be directly applied to actual fermentations. At different culture ages, lower agitation rates may be used as shear sensitivity increases. Cultures of increasing age showed an increase in the extracellular phenolics secretion caused by shear, due to the increased amount of cells and higher metabolic activity.

When subjected to shear, the cultures also experi- enced what seemed to be an almost immediate increase in cell permeabilization, shown by a large initial increase in the amount of phenolics secretion. The rise

E n z y m e M i c r o b . T e c h n o l . , 1989, vo l . 11, A u g u s t 489

Papers

in phenolics secretion was directly related to the shear rate. The total production rate of phenolics (extracellular and intracellular) did not show a relationship to shear rate. The production of phenolics tended to decrease significantly after a maximum concentration was reached.

A c k n o w l e d g e m e n t

This material is based upon work partially supported by the Washington Technology Center, Seattle, Wash- ington.

References 1 Dziezak, J. D. Food Technol. 1986, 40, 122-129 2 Sahai, O. P. and Knuth, M. Biotechnol. Prog. 1985, 1, I-9 3 Shuler, M. L. Ann. NYAcad. Sci. 1981, 369, 65-79 4 Curtin, M. E. Biotechnology 1983, 1, 649-657 5 Rosenberg, M. Z. and Dunlop, E. H. Proceedings of the 14th

Midwest Biochemical Engineering Conference Columbia, MO, 1984, 69-80

6 Tanaka, H. Biotechnol. Bioeng. 1981, 23, 1203-1218 7 Linsmaier, E. M. and Skoog, F. Physiol. Plant. 1965, 18,

100-127 8 Towill, L. and Mazur, P. Can. J. Bot. 1975, 53, 1097-1102 9 Singleton, V. L. and Rossi, J. A. Am. J. Enol. Vitic. 1965, 163,

144-158 10 George, E. F. and Sherrington, P. D. Plant Propagation by

Tissue Culture Exergetics, Ltd., Hants., England, 1984, p. 328


Documents

Response of plant tissue culture to a high shear environment