Large-scale cultivation of H e l i a n t h u s annuus cell suspensions

Alan H. Scragg

D e p a r t m e n t o f Molecu lar Biology and Bio technology , The Unioersity o f Sheff ield, Shef f ie ld, U K

Suspension cultures of Helianthus annuus have been cultivated in volumes of I00 ml to 80 1 in a variety o f vessels, including shake flasks, air-lift, and stirred-tank bioreactors. In general, the growth rates and biomass yields" were similar, with no bioreactor showing a distinct advantage. Scale-up.from 100 ml to 80 I could be achieved within 40 days using three lO-day subcultures. The determination o f oxygen uptake rates indicated that any reduction in growth rate did not appear to be due to oxygen supply.

Keywords: Helianthus annuus suspension cultures; bioreactors; aggregates; oxygen uptake

Introduction

The mass cultivation of plant cells in bioreactors in order to provide an alternative supply of phytochemi- cals has been proposed for some time. 1-4 Phytochemi- cals such as morphine, serpentine, and diosgenin repre- sent high-value, low-volume products and hence are ideal candidates for production by plant cell culture. The announcement by the Mitsui Petrochemical Com- pany Limited of the production of the dye and antibac- terial shikonin using bioreactor-grown cultures o f Lith- o s p e r m u m erythrorhizon has renewed interest. 5

Any commercial process using plant cell suspension for the production of fine chemicals requires the culti- vation of the particular cell line in volumes of 500 1 or more. The mass cultivation of plant cells has been possible for some time, starting with the work of Tu- lecke and Nickell.6 Much of the early work was carried out using modified stirred-tank bioreactors, reducing the stirrer speed and removing the baffles. 7.s This early work concentrated on the growth of Nicot iana taba- cum cells. As the number of cell lines grown in bioreac- tors increased, it became clear that the properties of plant cell suspensions were different from microbial cultures, which had a considerable influence on their mass cultivation. 9 In particular, the apparent shear sen- sitivity of plant cells in suspension meant that the shear developed in the normal stirred-tank bioreactor was too high to allow growth. Therefore , the air-lift bioreac- tor has been increasingly used for the cultivation of plant cell suspensions.~°-12

Address reprint requests to Dr. Scragg at the Department of Molecu- lar Biology and Biotechnology, The University of Sheffield, Sheffield SI0 2TN, UK Received 27 June 1988; revised 20 January 1989

Another important requirement in the development of a plant cell culture process is a high yield of product. Plant cell lines with high yields of secondary product have been isolated, 1'2 but not all have proved to be stable 13-~5 without continued selection. Therefore , many selected lines would have to be scaled up as rapidly as possible in the minimum of subcultures in order to retain their high yielding capacity. Deus- Neumann and Zenk 15 quote a value of 30 generations for the scale-up of C. roseus culture from 100 ml to 20,0001. The process of scale-up may also affect growth and product formation.16 Here I report the scale-up of a Hel ian thus annuus suspension culture from 100-ml cultures to growth in an 80-1 air-lift bioreactor in three subcultures, its growth in a stirred-tank bioreactor , and under draw-fill conditions.

Materials and methods

I s o l a t i o n a n d m a i n t e n a n c e o f c u l t u r e

Callus cultures were established from Hel ian thus an- nuus seeds obtained commercially. The seeds were surface sterilized in 15% Domestos (Lever Limited, UK, 3% available chlorine) for 30 rain, washed several times with sterile distilled water, and transferred to agar-based B5 medium 17 supplemented with coconut milk (10% v/v), 2% (w/v) sucrose, 0.8% (w/v) agar, I mg 1 - ~ 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg 1-1 kinetin. The pH was adjusted to 5.8 prior to autoclaving. Once the callus was initiated, it was sub- cultured every 6 weeks onto fresh medium. Suspension cultures were initiated from the stock callus after 10 subcultures using the B5 medium without agar and coconut milk. The stock suspension cultures were grown in 250-ml Er lenmeyer flasks containing 100 ml medium and shaken at 150 rev min ~ at 25°C. Cells

82 Enzyme Microb. Technol., 1990, vol. 12, February @ 1990 Butterworth Publishers

Cultivation of Helianthus annuus cell suspensions: A. H. Scragg

were subcultured every 10 days at a dilution of 1 • 5 (20 ml of cultured cells added to 100 ml fresh medium).

Growth o f cultures in bioreactors

Larger cultures were initiated by adding the contents of a 250-ml flask to 1 1 B5 medium in a 2-1 Erlenmeyer flask shaken at 150 rev min-i at 25°C. The contents of a 2-1 flask (1,120 ml) were used to inoculate the 7-1 air-lift bioreactor containing the same medium. The construction and operation of the 7-1 bioreactor have been described previously. 18 The air-lift bioreactor was used to inoculate a L.H. Fermentation Ltd. (Bells Hill, Stoke Poges, Slough, UK) 100-1 air-lift bioreactor (working volume 80 1) containing B5 medium. The 100-1 L.H. air-lift bioreactor was operated as described previously.t6 The working temperature was 25°C, with an aeration rate of 10 1 min -~, and samples (250 ml) were removed each day for analysis. The 3-1 stirred- tank bioreactor was constructed according to the stan- dard Porton design as described previously. 19 Agitation was achieved with a 7.3-cm diameter, six-blade impel- ler run at 480 rev rain -I. The temperature was main- tained at 25°C by circulating water from a thermocircu- iator (Circon 3, Baird and Tatlock) through an internal coil. Samples were removed at daily intervals using a pinch clip attachment. 18 The inlet and exit oxygen and carbon dioxide levels could be monitored from each vessel using an on-line process mass spectrometer (V.G. MM80) fitted with a multiport sampler.

Analyt ical methods

Wet weight, dry weight, and cell viability were deter- mined as described previously.2°

The oxygen uptake rate of cell suspension samples was determined using a Clark oxygen electrode.

Results

The H. annuus cell suspension was a recently devel- oped suspension culture when the experiments were started, having been in culture for about a year (subcul- ture number 34). However, it showed a rapid growth rate requiring subculturing every 10 days. If a 14-day subculture routine was used, the cells had reached sta- tionary phase and had begun to lose viability at subcul- ture. The doubling times of the H. annuus cultures in volumes of 100 ml to 80 1 were very similar, with the slowest rate giving a doubling time of about 2.5 days. A typical growth curve of H. annuus cell suspension in a 7-1 air-lift bioreactor is shown in Figure 1. The lag phase varied from 2 days in the shake flasks to 4 days in the 100-1 air-lift bioreactor.

The mean growth rates, doubling times, time to max- imum biomass, and maximum biomass for growth in the various bioreactors are given in Table 1.

Recent progress has indicated that stirred-tank bio- reactors may be more suitable than was at first thought for the culture of plant cell suspensions. ~9,2Lz2 Experi- ence has shown that suspension cultures that have been in culture for a considerable time, for example C. ro-

10 200

"7

&

%

3.5

)4,

0.3 .=_o

-6 0.2 ~,

0.1

5 10 Time days

Figure 1 Growth of Helianthus annuus suspens ion cu l ture in a 7-1 air- l i f t b ioreactor , conta in ing 7 1 med ium. Dry we igh t g I-1 (C)); we t we igh t g I 1 (G); carbon d iox ide evo lu t ion as a percen tage of ex i t gas (0)

seus, became more robust, and were capable of growth in a stirred-tank bioreactor. The H. annuus suspension had been in culture for about a year before its cultiva- tion was attempted in a small stirred-tank bioreactor (Figure 2). The culture grew well and achieved a growth rate of 0.26 per day and doubling time of 2.7 days (Table 1). The final biomass levels were low com- pared with the air-lift yields due to failure of the agita- tion motor. The agitation speed was 450 rev min-t, representing an average shear rate of 73.3 s-J using the formula of Metzner et al.,23 and the aeration rate was 100 ml rain -j.

The evolution of carbon dioxide was followed from both the 7-1 air-lift bioreactor (aerated at 61 min-t) and the 3-1 stirred-tank. In the 7-1 vessel, a peak of carbon dioxide evolution was found at mid-log phase (Figure /). Carbon dioxide evolution for the 3-1 stirred-tank bioreactor increased during the growth phase, but no single peak was observed.

The uptake of oxygen, as measured by the mass spectrometer, gave a less clear-cut peak, than the car- bon dioxide, but the uptake rate was in the region of 1.2-2.8 x 10 -4 moles 0 2 dry weight -t h -t, a value similar to that found for C. roseus culture using a simi- lar method. 24

The development of a plant cell culture process will require as high a productivity as possible, and a key feature of this will be the rapid production of biomass. The systems used here are batch culture, where the bioreactor has to be cleaned, filled, and sterilized be- tween runs. The time taken to carry out these opera- tions can be eliminated by the use of continuous cul- ture. However, because of very slow growth rates and

Enzyme Microb. Technol., 1990, vol. 12, February 83

Papers

Table 1 Growth of suspension cultures of H. Annuus in vessels of various sizes

Culture vessel

Culture Mean Doubling M a x i m u m volume growth time biomass

(liter) rate (# d 1) (Td days) (g I 1)

T ime to Aeration m a x i m u m rate

biomass (days) (vvm)

250 ml f lask 0.1 0.42 1.65 9.8 8 - 2 I f lask 1.0 0.32 2.2 11.3 11 - 7 I a i r - l i f t 7 0.29 2.4 10.3 7 0.86

bioreactor 100 I a i r - l i f t 80 0.30 2.3 9.5 10 0.125 3 I s t i r red- tank 3 0.26 2.7 7.3* 9 0.033

bioreactor

* The low maximum biomass due to stirrer motor failure

meringue or crust formation in the bioreactors, the continuous culture of plant cells is technically difficult. An alternative to continuous culture is the semicontinu- ous or draw-fill method. Figure 3 shows the draw-fill cultivation of H. annuus using a 7-1 air-lift bioreactor. The bioreactor was operated as for the batch culture, except that once the biomass approached a maximum, 6 1 (7 I total) of culture were removed and replaced by fresh medium. For this first cycle, the doubling time was 3.2 days (/z = 0.22), and was the same for the second cycle, but the doubling time increased to 4.5 days (/z = 0.15) at the third cycle. At the fourth cycle, the culture lost viability and failed to grow. The initial growth rates are somewhat lower than those obtained

"7-

, / oJ

ioo k/ / i , .

rW:/ ~0 --or ~

.1"0

o~ L .(}5 =

c~

5 10 Time days

Figure 2 Growth of H e l i a n t h u s a n n u u s suspension culture in a 3-1 stirred-tank bioreactor. The bioreactor was of the standard Porton type using a flat-bladed impeller run at 480 rev min 1. The vessel was inoculated with 500 ml o f a 10-day-o ld culture, incubated at 25°C with an aeration rate of 100 ml min -~. Samples were removed each day for wet weight (not shown) and dry weight determinations. Dry weight g I 1 (©) ; wet weight g 1-1 (A); carbon dioxide evolution as a percentage of exit gas (D)

in the previous batch culture in the 7-1 air-lift biore- actor.

D i s c u s s i o n

In order to utilize plant cell suspensions as an alterna- tive source of natural compounds , the retention of both good growth and product formation is required when cultivated in large volumes. Although a wide range of cell lines have been successfully grown in bioreactors in volumes up to 6,500 I, other cell lines have proved difficult to cultivate in bioreactors. Maintenance of growth and product formation has been achieved with cell lines of Lithosperrnurn 5 and Coleus . 22 However , many high-yielding cell lines frequently lose their pro- ductivity during subsequent cultivation unless selec- tion is continued. 4-~3'14 In these cases the cell line would have to be grown up to the production volumes in the minimum number of subcultures. In addition to these problems, the choice of bioreactor design still remains unresolved. From early experiences with mass cultiva- tion of plant cells, it was believed that the air-lift biore-

I0 T

5

Td: 32days Td:3.2 days Td:45days

5 10 15 20 Time days

Figure 3 The draw-fill culture of 14. a n n u u s . H. a n n u u s suspen- sion cells (1 I) were used to inoculate a 7-1 airqift bioreactor, run at 25°C, aerated at 11 m i n - 1. At days 8, 15, 22, 6 I of culture were removed and replaced by fresh medium. Dry weight g 1-1 (O)

84 Enzyme Microb. Technol., 1990, vol. 12, February

Cultivation of

actor was the only suitable reactor because of its low shear levels. However, it is clear from experience that plant cells that have been in suspension for some time are capable of growing in the conventional stirred-tank bioreactor.~9'22 In order to investigate these questions, a recently developed suspension culture of Helianthus that had not been grown in bioreactors previously was used.

The growth rates and biomass levels achieved in vessels from 250-mi shake flasks to the 80-1 air-lift bio- reactor were very similar. An 80-1 culture could be produced in 40 days or three 10-day subcultures using an approximately l : 10 inoculation regime. The 80-1 culture could act as an inoculum for a 1,000-1 culture or be switched to a production medium as described for shikonin and rosmarinic acid. Therefore, with a rapidly growing culture such as Helianthus, the scale- up of unstable high-producing lines appears possible. The Helianthus cultures contain no commercially use- ful product, and therefore the problems of product sta- bility on scale-up cannot be addressed with this cell line.

The oxygen uptake rates estimated for cultures grown in both the 7-1 air-lift and the 3-1 stirred-tank bioreactors were similar, and of the same order as those for C. roseus cultures. 24

In terms of bioreactor design, both types, air-lift and stirred-tank, gave similar results in terms of growth rate. The Helianthus culture, although highly aggre- gated, grew well in both air-lift and stirred-tank biore- actor, even though the aggregate size was reduced con- siderably in the stirred tank (results not shown). This change in aggregate distribution may have some sig- nificance if some natural products are associated with aggregates of certain sizes. The alternative to batch culture, the semicontinuous or draw-fill method, was tried with H. annuus suspension cultures in a 7-1 air- lift bioreactor. The growth rates were lower than that obtained for a batch culture in a similar bioreactor, but three cycles were obtained before the cells lost viability and failed to grow. The lower growth rate in the draw- fill bioreactors means that over the 22 days of culture the biomass productivity was 0.90 g I i day- i, whereas the batch culture yielded 1.0 g 1-~ day- l , allowing 3 days for cleaning and resterilization. If the growth rate and biomass levels were equivalent, the draw-fill method would give a higher productivity. The loss of viability at the fourth cycle could be due to the buildup of inhibitory substances or to allowing the culture to become too old before replacing the medium. The latter is perhaps more likely, as the culture required a 10-day

Helianthus annuus cell suspensions: A. H. Scragg

subculture regime and any longer resulted in loss of viability. If this was the reason, it would not allow the draw-fill method to harvest the cells at maximum biomass, and therefore would affect the overall produc- tivity.

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