Plant cells: immobilization and oxygen transfer.

The study described in this thesis is part of the integrated project 'Biotechnological production of non-persistent bioinsecticides by means of plant cells in vitro ' and was done in close cooperation with the research Institute Ital within the framework of NOVAPLANT. The plant cells used in this project were Tagetes species which produce thiophenes, naturally occurring biocides, particularly against nematodes. The objective of the study described in this thesis was to use immobilized plant cells or large plant cell aggregates for secondary metabolite production. In particular the upscaling of immobilization techniques for plant cells, the role of diffusion limitation of oxygen as a substrate on the immobilized plant cells and its effect on secondary metabolite production of the immobilized plant cells were subject of research. A literature survey on immobilized plant cells is presented in Chapter 2. The advantages of immobilized plant cells, several aspects concerning immobilization techniques, consequences of plant cell immobilization, immobilized plant cell reactors, and future prospects of immobilized plant cells are discussed in this Chapter. Chapter 3 deals with the application of the resonance nozzle as an immobilization technique with a high production capacity for plant cells as well as yeast cells in calcium alginate gel beads. It is found that this technique has a production capacity of two orders of magnitude larger than the conventional dripping technique with a needle. The viability of the cells after immobilization with the resonance nozzle was preserved. An extension of the applicability of the nozzle technique for thermogelling gel supports (k-carrageenan, agar and gellan gum) Is described in Chapter 4. Plant cells, yeast cells, bacterial cells and insect cells were used as model systems in the experiments. In Chapter 5, the occurrence of oxygen diffusion limitation of Daucus carota cells in agarose, calcium alginate and (κ-carrageenan, is determined by respiration measurements of the immobilized cells in order to explain the enhanced pronounced secondary metabolite production with alginate immobilized plant cells from in the literature reported experiments. However, in our experiments no differences between the support materials could be observed. The effective diffusion coefficient for oxygen (ID e ) in the gel material is an important factor in mathematical model calculations in order to quantify the occurrence of oxygen diffusion limitation. Chapter 6 deals with the experiments in which (ID e ) was determined in different gel materials (calcium alginate, κ-carrageenan, gellan gum, agar and agarose) by measuring the oxygen diffusion from a well- stirred solution into gel beads, which were initially free of oxygen. A mathematical model was fitted on the experimental data resulting in the value of (ID e ) which was used In the following experiments. In several parts of this thesis a mathematical model was used for calculation of oxygen concentration profiles in gel beads containing plant cells or cell aggregates in order to visualize the occurrence of oxygen diffusion limitation. In Chapter 7 this model is tested on validity by experimental measurement of the oxygen concentration profiles in agarose beads containing respiring plant cells of Tagetes minuta . This was done with the aid of an oxygen microelectrode with a tip of 15 μm. The experimental and calculated oxygen concentration profiles correspond quite well. Chapter 8 deals with the effects of aggregate size and oxygen diffusion limitation on thiophene production and cell growth by cell aggregates of Tagetes patula . It is concluded that aggregate size is related to thiophene production by the observation of an 'optimum' aggregate size where the production is highest. Calculations of the oxygen concentration profiles showed that this could be due to the absence of oxygen In the centre of the aggregates.