Release and transport of secondary metabolites from plant cells immobilized in membrane bioreactors

In this work, Catharanthus roseus cells were immobilized in membrane bioreactors provided with a continuous nutrient supply. The release and transport of intracellular alkaloids (ajmalicine and yohimbine) from immobilized cells into the lumen of a membrane bioreactor was experimentally investigated by the permeabilization of cells with dimethylsulfoxide and the iontophoretic technique developed in this study. In the case of Catharanthus roseus cells, alkaloids are mainly stored in an ionic form inside the vacuoles, and the lipidic phase in the cell membranes is the major barrier to the diffusion of the ionic alkaloids. Permeabilization of cells with dimethylsulfoxide caused the delipidation of the cell membranes, thus enabling the passage of alkaloids through the pores (formed by delipidation) in the membrane structure. However, the permeabilization of cells was difficult to achieve without permanently damaging the culture.; The iontophoretic technique developed in this work utilized an electrical field to enable the release and transport of the ionic alkaloids from immobilized cells in the shell side of a bioreactor into its lumen. The electrical current provided not only the driving force to assist in the movement of ionic alkaloids, but also generated an increased permeability of the cell membranes to the positively charged alkaloids. The results revealed that increasing either the current or the time of iontophoresis increased the release of alkaloids. However, the cell viability test using the triphenyltetrazolium chloride showed that an increase in either the current or the time caused a decrease in the cell viability.; The diffusion of yohimbine-HCl into Amicon H1P10-20 and H1P100-43 hollow fibers and a Ceraflo 3mm tube was investigated. A mathematical model which describes the transient diffusion of a solute into a hollow fiber was developed and utilized to estimate the effective diffusivity of yohimbine-HCl. The results revealed that both the Amicon H1P-type hollow fibers and the Ceraflo 3mm tube used as immobilized cell tubular membranes provided significant diffusional resistance to a solute.; The diffusion of sucrose and yohimbine in alginate gel beads entrapped with C. roseus or Malus domestica cells were investigated. It was found that the effective diffusivities of both solutes decreased significantly in the presence of cells inside the beads, and the amount of reduction was proportional to the amount of cell loading.