3. Applications of bioreactors in plant propagation

1. Large number of plantlets which are free from physiological disorder can easily be produced in one batch in the bioreactor.
2. Handling of cultures, such as inoculation and harvest, is easy. It also reduces the number of culture vessels and the area of culture space, which further reduces the overall cost of the production.
3. Nutrient uptake and growth rate is increased because the surface of the cultures is always in contact with medium.
4. Forced aeration is performed which improves the growth rate and final biomass.

Many plant species have been cultured in the bioreactor and the responses of cultures in bioreactors may vary from species to species. Following are the list of plant species where bioreactors are used for large scale propagation:

1. Shoots: Atropa belladonna, Chrysanthemum morifolium, Dianthus caryophyllus, Fragaria ananassa, Nicotiana tabacum, Petunia hybrida, Primula obconica, Zoysia japonica, Scopolia japonica, Spathiphyllum, Stevia rebaudiana, etc .
2. Bulbs: Fritillaria thunbergii, Hippeastrum hybridum, Hyacinthus orientalis, Lilium, etc.
3. Corms: Caladium sp., Colocasia esculenta, Pinellia ternata , etc.
4. Tubers: Solanum tuberosum
5. Embryos or adventitious buds: Atropa belladonna

4. Scale-up process

Scale-up generally involves taking a lab-scale bioprocess and replicating it as closely as possible to produce larger amounts of product. A typical scale-up sequence in plant cell and tissue culture studies starts with jars, moves to 1 litre shake flasks, after that to 1-10 litre glass bioreactors, then scale-up through to stainless steel vessels of varying sizes from 30-150 litre to 1000 litre. The large scale cultivation of plant cell and tissue culture is an alternative to the traditional methods of plantation. As compared to microbial cultures, plant cell suspensions, shoot and root cultures pose many different problems in bioreactors during scale-up. Plant cells grow slowly, the cells are large and form clumps, which make them more sensitive to shear associated with agitation and exhibit long processing times. Organ cultures are far more sensitive to shear. These characteristics lead to the necessity to design alternative bioreactor configurations, particularly those that reduce shear within the large scale bioreactor. Various culture conditions must be monitored to control plant morphogenesis and biomass growth in bioreactors, such as the morphology, oxygen supply and CO₂ exchange, mixing, pH and temperature.

5. Process design considerations

5.1. Aggregation

Due to large size (length up to 200 μm) and slow growing nature, compare to the microbial cells, plant cells are although capable of withstanding tensile strain but are sensitive to shear stress. They have a very rigid cell wall and a culture will contain a wide range of cell shapes and sizes. Unlike many microorganisms, plant cells in suspension culture occur as groups or aggregates. Whether these aggregates arise due to failure of the cells to separate after division or by cell aggregation is unknown but they are loose structure whose average size and size distribution vary with culture conditions. Further, the secretion of extracellular polysaccharides, particularly in the later stages of growth, may contribute to increased adhesion. A consequence of these characteristics results in sedimentation, insufficient mixing and diffusion-limited biochemical reaction. On the other hand, the aggregate structure has also been implicated in secondary product accumulation, as it provide cell-cell contact and so form micro-environment within the aggregate, which stimulate secondary product synthesis. Hence controlled aggregation of plant cells is of interest from process engineering point of view.

5.2. Mixing

Mixing favors cell growth by promoting nutrient transfer from liquid and gaseous phases to cells. It also helps dispersion of air bubbles for effective oxygenation. Although plant cells have higher tensile strength, compare to microbial cells, their shear sensitivity towards hydrodynamic stresses restricts the use of high agitation for efficient mixing. Mixing decrease the mean aggregate size but have an unfavorable effect on cell viability. Plant cells are often grown in stirred tank bioreactors at very low agitation speeds. Sufficient mixing can be achieved by proper design of the impeller; helical-ribbon impeller has been reported to enhance mixing at the high density of plant cell suspension cultures.

5.3. Oxygen and aeration effects

Plant cells require comparatively lower oxygen than that of microbial cells due to their low growth rates. High oxygen concentration has proved toxic to the cells, metabolic activities, etc. and may strip nutrients, such as carbon dioxide from the culture broth. Carbon dioxide is often considered as an essential nutrient in the culture of plant cells and has a positive effect on cell growth. Hence, the factors that influence efficient oxygen transfer in plant cell cultures must be carefully analyzed when a bioreactor system is being selected. The intensity of culture broth mixing, the extent of air bubble dispersion, and the hydrodynamic pressure inside the culture vessel influence suitable aeration of the culture.

High aeration may results into severe foaming, which has significant influence on the cell growth and secondary metabolite production. Foaming of plant cell suspensions is associated with aeration rates and extracellular polysaccharides, fatty acids and high sugar concentrations in the plant cell culture medium. This can result in the wall growth phenomenon and clogging of air exhaust filter and lead to high rate of contamination. A number of antifoams such as, polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C have often been employed to control foaming; however, in some cases this resulted in reduction in cell growth and product formation.