Plant cell and hairy root cultures in bioreactor-based production processes: Dissertation

In this study, laboratory scale biotechnical production processes were developed for different types of plant cell cultures: embryogenic cell culture, undifferentiated cell suspension culture and hairy root culture. The production of somatic embryos, secondary metabolites and biomass were optimized. The growth as well as the production characteristics and the nutrient uptake of the different plant cell systems were determined. The suitabilities of different bioreactor configurations for the various culture types were tested in laboratory scale. In the optimization of growth and production, statistical experimental designs were used in addition to the conventional method of parallel testing of different media were used. The embryo production medium for an embryogenic cell line of birch (Betula pendula Roth) was optimized using a 22-factorial design. Factorial design was also tested for the optimization of alkaloid production medium for a cell suspension of Catharanthus roseus (L.) G. Don, but the conventional method of parallel testing of different media proved to be more successful. Factorial experimental design was used to determine the effects of low phosphate and sugar concentrations on strawberry (Fragaria x ananassa Duch.) hairy roots. In this way a slowly growing root system was obtained for infection studies with the native Finnish arbuscular mycorrhizal fungus, Glomus fistulosum V128. The embryogenic cell line of birch was cultivated in four different types of bioreactors: a sparged blade-stirred bioreactor, a diffusion-aerated bioreactor with a marine impeller (ChemCell, Chemap), an air-lift and a bubble column bioreactor. Embryo development was monitored during the cultivations by computer vision. The highest embryo production was achieved in the bubble column bioreactor. However, this was only 20% of the production in control shake flasks. In the mechanically stirred bioreactors production was even lower, indicating shear damage of the embryos. The suspension culture of Catharanthus roseus L. was cultivated in three different types of bioreactors: a standard aerated and stirred bioreactor (control bioreactor), a recirculation bioreactor in which part of the air is recirculated with a peristaltic pump, and a bubble column bioreactor. The recirculation bioreactor was used to mimic the gaseous regime of a shake flask. When cultured in a turbine stirred tank reactor aerated through a sparger and in the bubble column the cells did not grow well and the production of ajmalicine was low. When cultivated in the recirculation bioreactor the biomass concentration and production of ajmalicine were similar to the level observed in shake flasks. Hairy roots of C. roseus were cultivated in three different types of bioreactors: an air-sparged bioreactor with no stirring, an air-sparged bioreactor in which the medium is slowly circulated through the vessel with a peristaltic pump and a standard air-sparged and stirred bioreactor. The best biomass yield and root morphology were obtained in the air-sparged bioreactor with no stirring. The indole alkaloid contents of the roots cultivated in the bioreactor with stirring and in the bioreactor with medium circulation were significantly lower than that of the roots in the air-sparged bioreactor. Hairy roots of strawberry (Fragaria x ananassa Duch.) were cultivated in an air-sparged bioreactor with no stirring. The biomass yield was good and the roots were long and branching. The roots produced were used as a host for an endophyte, arbuscular mycorrhizal fungus, Glomus fistulosum V128.