50 100 150 200 AGITATION SPEED ( rpm )

Figure 26. Effect of initial oxygen transfer coefficient and agitation by turbine impeller on cell growth (Kato, 1975).

3.8 Microbial Contamination

According to Manfredini et al. (1982),[23] the most frequent factors causing microbial contamination include: (i) construction materials, (ii) seals and valves, (iii) complexity of the plant, (iv) operator error, (v) instrumentation failure, (vi) process air, (vii) transfer and feed lines, (viii) contamination from vegetative preculture, (ix) critical medium composition, (x) inadequate procedures. As the culture periods of plant cell, tissues, and organs are usually quite long, (particularly for continuous cultures), special designs and operations are necessary to avoid microbial contamination. For example, Hasimoto et al. (1982),t24] reduced contamination in their 20 kl bioreactor by using three air filters in a series; the third filter was a membrane of uniform pore size of 0.4 |am. The design specified aseptic seals in the agitator shaft and exit pipelines for sterilized air after steam sterilization.

3.9 Characteristics

The special characteristics of plant cells that tend to hamper large scale cultivation of the cells are described below.

Bubbling and Adhesion of Cells to the Inner Surface of the Bioreactor. Plant cell cultivation is usually performed by bubbling and agitation which cause foaming and adhesion of the cells to the surface of a bioreactor. Because of this phenomenon, cell growth is inhibited. The authors (Takayama et al., 1977)[25] have examined the possible causes and concluded that the adhesion of cells appeared to be the result of gel formation from pectin and calcium. By reducing the concentration of CaCl^F^O in the medium, the foaming and the number of cells that adhered to the walls was decreased markedly. The cells became easily removable from the inside wall of the fermenter and were returned to the medium. Cell destruction was measured by A660 values which also depend on the CaCl2,2H20 levels. A lower level of CaCl2-2H20 in the medium markedly inhibits cell destruction. These observations are particularly pertinent when large-scale cultivation is being considered.

Cell Morphology and Specific Gravity. According to Tanaka (1982),[26] plant cells have a tendency to grow in aggregates of different sizes. The size distribution of cell aggregates is different from one plant species to another (Tanaka, 1982).[27] Specific gravity of these cells ranges from 1.002 to 1.028. Ifthe diameter ofthe cell aggregate is less than 1 to 2 mm, the cells can be suspended and do not sink to the bottom of the bioreactor (Tanaka, 1982),[26] but, when the specific gravity of the cell is greater than 1.03, the diameter of the aggregate becomes 0.5 to 1.0 cm and the cells sink to the bottom of the bioreactor and cannot be suspended. When the agitation is increased, the size of the cell aggregate becomes smaller (Tanaka, 1981),[28] but the growth of the cell is repressed. In order to separate the cells from the aggregate, the amount of calcium is decreased to suppress the gel formation of pectin which plays an important role in the cell, e.g., cementing plant cells, but has little effect on cell separation (Takayama, 1977).[25J

Viscosity, Fluidity, and Oxygen Supply. When plant cells grow well, they can occupy 40 to 60% of the whole culture volume, and the apparent viscosity becomes very high. Tanaka (1982)'26' examined the relationship between apparent viscosity and concentration of solids in suspension, and concluded that when the cell density exceeds 10 g/1, the slope of the apparent viscosity increases rapidly, and when cell density reaches 30 g/1, the culture medium becomes difficult to agitate and supply with oxygen.


Figure 27. Relationship between apparent viscosity and concentration of cells and pseudocells in culture media (Tanaka, 1982). (□) C. Roseus, (O) C. Tricupsidata B., (A )N. tabacum L., (+) granulated sugar.


Figure 27. Relationship between apparent viscosity and concentration of cells and pseudocells in culture media (Tanaka, 1982). (□) C. Roseus, (O) C. Tricupsidata B., (A )N. tabacum L., (+) granulated sugar.

58 Fermentation and Biochemical Engineering Handbook 3.10 Manipulation

Large Scale Batch Culture. Batch culture systems are in use worldwide and many experimental results have been reported using 101 to 20 klbioreactors. Noguchietal. (1987)[21 ] have examined the growth of tobacco BY-2 cells using a 20 kl aeration-agitation bioreactor with 15klmedium. The medium used was Murashige and Skoog's inorganic nutrients with three times the normal amount of phosphate and 3 % sucrose, incubated at 2 8 °C and aerated at 0.3 wm. The results revealed that the highest growth rate was observed from the incubation time of 45 to 70 hours with a doubling time of about 15 hours, which was almost the same as the growth in flask cultures. Ushiyama etal. (1986)m examined the growth ofPanax ginseng root cultures in 301,2 kl, and 20 kl aeration-agitation bioreactors. The productivity of the cultures in 2 kl and 20 kl bioreactors was 700 and 500 mg/1 /day in dry weight, respectively. Building upon this basic research, large scale batch culture techniques have been developed for the industrial production of cell mass. However, culture conditions suitable for cell mass production are not always suitable for secondary metabolite production. Accordingly, in order to produce both cell mass and metabolites efficiently, two-stage culture techniques have been adopted. This technique uses two batch bioreactors and was first reported by Noguchi et al. (1987)[21] for the production of low nitrogen content tobacco cells. In the 1980's, this technique was widely used for secondary metabolite production such as shikonin (Fujita et al ,),[27] rosmarinic acid (Ulbrich, 1985)[2?1 and digoxin production (Reinhard, 1980).™

For shikonin production by Lithospermum erythrorhizon, two-stage cell culture was used (see Fig. 28). The first stage culture was grown in a MG-5 medium which was suitable for cell mass production. It was then transferred to 2nd-stage culture where it was grown in an M-9 medium, modified by a higher Cu1-1" content and a decreased salt content.

Large Scale Continuous Culture. The growth rate of plant cells is usually low compared to that of microbial organisms. In order to enhance the productivity of cell mass and metabolites, continuous culture methods should be employed (Wilson, 1978;[29] Fig. 29a). In the research laboratories of Japan Tobacco and Salt Co., a pilot plant (1500 1) and an industrial plant system (20 kl) have been used for developing continuous culture techniques (Hashimoto, et al., 1982;[24] Azechi et al., 1983I30!). A 20 kl bioreactor having a working volume of 6.34 kl, was used for the experiment (Fig. 29b) and ran for 66 days of continuous operation. The conditions were: aeration rate, 0.35-0.47 wm; agitation speed, 27.5-35 rpm; dilution rate, 0.28-0.38

days. In this experiment, the residual sugar content was an important index of the operation and, at steady state, its value was maintained above 5 g/1. Other control parameters such as aeration, agitation, and dilution rates were changed gradually. The success of this experiment will soon lead to the establishment of long term industrial continuous culture systems for secondary metabolite production.

Immobilized Culture. Immobilization of plant cells was first reported by Brodelius et al. in 1979,11311 and since then many reports have been published. Unfortunately, an immobilized cell culture technique has not yet been established as an industrial process for secondary metabolite production. However, this technique has many excellent features and should be the subject of future development research.

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